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Journal Articles

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2024

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• R. Qelibari, E. Cruz Ortiz, N. van Treel, F. Lombeck, C. Schare, A. Münchinger, N. Dumbadze, G. Titvinidze, C. Klose, S. Vierrath
  74 µm PEEK-Reinforced Sulfonated Poly(phenylene sulfone)-Membrane for Stable Water Electrolysis with Lower Gas Crossover and Lower Resistance than Nafion N115
  2024 Adv. Energy Mater., page: 2303271
• M.U. Iturritza, P. Mlotshwa, J. Gantelius, T. Alfvén, E. Loh, J. Karlsson, C. Hadjineophytou, K. Langer, K. Mitsakakis, A. Russom, H.N. Jönsson, G. Gaudenzi
  An Automated Versatile Diagnostic Workflow for Infectious Disease Detection in Low-Resource Settings
  2024 Micromachines, volume: 15, issue: 6, page: 708
• V. Zieger, E. Woehr, S. Zimmermann, D. Frejek, P. Koltay, R. Zengerle, S. Kartmann
  Automated Nanodroplet Dispensing for Large-Scale Spheroid Generation via Hanging Drop and Parallelized Lossless Spheroid Harvesting
  2024 Micromachines, volume: 15, page: 231
• D. Straubinger, P. Koltay, R. Zengerle, S. Kartmann, Z. Shu
  Bulge-Free and Homogeneous Metal Line Jet Printing with StarJet Technology
  2024 Micromachines, volume: 15, page: 743
• R. Kronstein-Wiedemann, J. Thiel, D. Sürün, M. Teichert, S. R. Künzel, S. Zimmermann, L. Wagenführ, F. Buchholz, T. Tonn
  Characterization of immortalized bone marrow erythroid progenitor adult (imBMEP-A)—The first inducible immortalized red blood cell progenitor cell line derived from bone marrow CD71-positive cells
  2024 Cytotherapy
• K. Seteiz, J. N. Häberlein, P. A. Heizmann, L. Bohn, S. Vierrath, J. Disch
  Effect of Ionomer Content and Ag/C Catalyst Surface Area on the Performance of CO2 Electrolysis to CO
  2024 ACS Appl. Eng. Mater, volume: 2, issue: 6, page: 1654
• A. Klebes, H. Ceren Ates, Rene D. Verboket, G. A. Urban, F. von Stetten, C. Dincer, S. M. Früh
  Emerging multianalyte biosensors for the simultaneous detection of protein and nucleic acid biomarkers
  2024 Biosensors and Bioelectronics, volume: 244, page: 115800
• L. Niebling, R. Nitzsch, T. Sieksmeyer, V. Haskamp, J. Kissenkötter, A. Abd El Wahed, T. Teufel, H. Hermann, N. Paust, A. R. Homann
  Fast and on-site animal species identification in processed meat via centrifugal microfluidics and isothermal amplification
  2024 LabChip, volume: 24, page: 975
• H. Liepold, H. Nguyen, P. A. Heizmann, C. Klose, S. Vierrath, A. Münchinger
  Gas Transport Resistance of Hydrocarbon-Based Catalyst Layers in Proton-Exchange Membrane Fuel Cells
  2024 Journal of The Electrochemical Society, volume: 171, page: 054509
• O. Rajabnia, A. Ernst, N. Lass, L. Riegger, R. Zengerle
  Highly Parallel Droplet Dispensing Approach to Provide Homogeneous and Controllable Droplet Arrays for Diagnostic Test Manufacturing
  2024 micromachines, volume: 15, page: 824
• Z. Khan, D. Gururajan, P. Koltay, S. Kartmann, R. Zengerle, Z. Shu
  Hybrid 3D Printing of Molten Metal Microdroplets and Polymers for Prototyping of Printed Circuit Boards Featuring Interdigitated 3D Capacitors
  2024 EEJ TRANSACTIONS ON ELECTRICAL AND ELECTRONIC ENGINEERING, IEEJ Trans 2024
• Z. Khan, A. Saphala, S. Kartmann, P. Koltay, R. Zengerle, O. Amft, Z. Shu
  Hybrid Printing of Conductive Traces from Bulk Metal for Digital Signals in Intelligent Device
  2024 Micromachines, volume: 15, page: 750
• J. Stiegeler, T. Mittermeier, L. Tsikonis, T. Lehre, S. Vierrath
  Influence Factors of Platinum Dissolution in Proton Exchange Membrane Fuel Cells: A Sensitivity Study
  2024 J. Electrochem. Soc, volume: 171, page: 054517
• B. Johannsen, D. Baumgartner, M. Karpíšek, D. Stejskal, N. Paust, R. Zengerle, K. Mitsakakis
  Integration of a bead-based immunoassay on a commercial PCR-performing POC device
  2024 Proceedings, volume: 97, page: 166
• Z. Khan, D. Gururajan, S. Kartmann, P. Koltay, R. Zengerle, Z. Shu
  Iterative printing of bulk metal and polymer for additive manufacturing of multi-layer electronic circuits
  2024 npj Adv. Manuf., volume: 1, issue: 1
• R. Weber, M. Klingenhof, S. Koch, L. Metzler, T. Merzdorf, J. Meier-Haack, P. Strasser, S. Vierrath, M. Sommer
  Meta-kinks are key to binder performance of poly(arylene piperidinium) ionomers for alkaline membrane water electrolysis using non-noble metal catalysts
  2024 J. Mater. Chem. A, volume: 12, page: 7826
• L. Bohn, J. Kieninger, S. J. Rupitsch, C. Klose, S. Vierrath, J. Disch
  Reference Electrode Types for Zero-Gap CO2 Electrolyzers: Benefits and Limitations
  2024 Advanced Science, volume: 11, page: 32
• V. Zieger, D. Frejek, S. Zimmermann, G. A. A. Miotto, P. Koltay, R. Zengerle, S. Kartmann
  Towards Automation in 3D Cell Culture: Selective and Gentle High-Throughput Handling of Spheroids and Organoids via Novel Pick-Flow-Drop Principle
  2024 Adv Healthc Mater, page: e2303350
• J. Schlanderer, H. Hoffmann, J. Lüddecke, A. Golubov, W. Grasse, E. V. Kindler, T. A. Kohl, M. Merker, C. Metzger, V. Mohr, S. Niemann, C. Pilloni, S. Plesnik, B. Raya, B. Shresta, C. Utpatel, R. Zengerle, M. Beutlerd, N. Paust
  Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS
  2024 LabChip, volume: 24, page: 74

2023

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• K. Yildirim, H. Fadlullah, C. Schwarz, F. Lombeck, C. Klose, S. Vierrath, M. Breitwieser
  30μm Thin Anode Gas Diffusion Layers for OptimizedPEM Fuel Cell Operation at 120 °C and Low Relative Humidity
  2023 Adv. Energy Sustainability Res, page: 2300179
• F. Fischer, A. Doll, D. Uereyener, S. Roenneberg, C. Hillig, L. Weber, V. Hackert, P. Seiringer, J. Thomas, P. Anand, L. Graner, F. Schlenker, R. Zengerle, M. Jargosch, F. J. Theis, C. B. Schmidt-Weber, T. Biedermann, M. Howell, K. Reich, M. Menden, N. Garzorz-Stark, F. Lauffer, S. Eyerich
  394 Gene expression based molecular test proves clinical validity as diagnostic aid for the differential diagnosis of psoriasis and eczema in formalin fixed and paraffin embedded tissue
  2023 British Journal of Dermatology, volume: 188, issue: 3
• H. Nguyen, J. Stiegeler, H. Liepold, C. Schwarz, S. Vierrath, M. Breitwieser
  A Comparative Study of Conditioning Methods for Hydrocarbon-Based Proton-Exchange Membrane Fuel Cells for Improved Performance
  2023 Energy Technol., page: 2300202
• J. Weygant, F. Koch, K. Adam, K. Tröndle, R. Zengerle, G. Finkenzeller, S. Kartmann, P. Koltay, S. Zimmermann
  A Drop-on-Demand Bioprinting Approach to Spatial Arrangement of Multiple Cell Types and Monitoring Their Cell–Cell Interactions towards Vascularization Based on Endothelial Cells and Mesenchymal Stem Cells
  2023 Cells, volume: 12, issue: 4, page: 646
• P. A. Heizmann, H. Nguyen, M. von Holst, A. Fischbach, M. Kostelec, F. J. Gonzalez Lopez, M. Bele, L. Pavko, T. Đukić, M. Šala, F. Ruiz-Zepeda, C. Klose, M. Gatalo, N. Hodnik, S. Vierrath, M. Breitwieser
  Alternative and facile production pathway towards obtaining high surface area PtCo/C intermetallic catalysts for improved PEM fuel cell performance
  2023 RSC Adv, volume: 13, pages: 4601 - 4611
• J. Disch, S. Ingenhoven, S. Vierrath
  Bipolar Membrane with Porous Anion Exchange Layer for Efficient and Long-Term Stable Electrochemical Reduction of CO2 to CO
  2023 Adv. Energy Mater., page: 2301614
• K. Seteiz, J. N. Häberlein, P. A. Heizmann, J. Disch, S. Vierrath
  Carbon black supported Ag nanoparticles in zero-gap CO2 electrolysis to CO enabling high mass activity
  2023 RSC Advances, volume: 27
• P. Sardana, M. Zolfaghari, G. Miotto, R. Zengerle, T. Brox, P. Koltay, S. Kartmann
  DropletAI: Deep Learning-Based Classification of Fluids with Different Ohnesorge Numbers during Non-Contact Dispensing
  2023 Fluids, volume: 8, page: 183
• A. Klebes, H. C. Ates, R. D. Verboket, G. Urban, F. von Stetten, C. Dincer, S. M. Früh
  Emerging multianalyte biosensors for the simultaneous detection of protein and nucleic acid biomarkers
  2023 Biosensors and Bioelectronics, page: 115800
• T. T. Bachmann, K. Mitsakakis, J. P. Hays, A. van Belkum, A. Russom, G. Luedke, G. Skov Simonsen, G. Abel, H. Peter, H. Goossens, J. Moran-Gilad, J. Vila, K. Becker, P. Moons, R. Sampath, R. W Peeling, S. Luz, T. van Staa, V. Di Gregori, JPIAMR AMR-RDT Working Group
  Expert guidance on target product profile development for AMR diagnostic tests
  2023 BMJ Glob Health, volume: 18, issue: 8, page: e012319
• F. Fischer, A. Doll, D. Uereyener, S. Roenneberg, C. Hillig, L. Weber, V. Hackert, M. Meinel, A. Farnoud, P. Seiringer, J. Thomas, P. Anand, L. Graner, F. Schlenker, R. Zengerle, P. Jonsson, M. Jargosch, FJ Theis, CB Schmidt-Weber, T. Biedermann, M. Howell, K. Reich, K. Eyerich, M. Menden, N. Garzorz-Stark, F. Lauffer, S. Eyerich
  Gene expression based molecular test as diagnostic aid for the differential diagnosis of psoriasis and eczema in formalin fixed and paraffin embedded tissue, microbiopsies and tape strips
  2023 The Journal of Investigative Dermatology
• G. Miotto, K. Thiemann, M. Rombach, R. Zengerle, S. Kartmann
  Holographic PIV/PTV for nano flow rates - a study in the 70 to 200 nL/min range
  2023 Biomedical engineering, volume: 68, issue: 1, pages: 97 - 107
• Y.-K. Lai, Y.-T. Kao, J. Hess, S. Calabrese, F. von Stetten, N. Paust
  Interfacing centrifugal microfluidics with linear-oriented 8-tube strips and multichannel pipettes for increased throughput of digital assays
  2023 Lab on a Chip, volume: 23, pages: 2623 - 2632
• F. Schlenker, P. Juelg, J. Lüddecke, N. Paust, R. Zengerle, T. Hutzenlaub
  Nanobead handling on a centrifugal microfluidic LabDisk for automated extraction of cell-free circulating DNA with high recovery rates
  2023 Analyst
• D. Grijalva Garces, S. Strauß, S. Gretzinger, B. Schmieg, T. Jüngst, J. Groll, L. Meinel, I. Schmidt, H. Hartmann, K. Schenke-Layland, N. Brandt, M. Selzer, S. Zimmermann, P. Koltay, A. Southan, G. E. M. Tovar, S. Schmidt, A. Weber, T. Ahlfeld, M. Gelinsky, T. Scheibel, R. Detsch, A. R. Boccaccini, T. Naolou, C. Lee-Thedieck, C. Willems, T. Groth, S. Allgeier, B. Köhler, T. Friedrich, H. Briesen, J. Buchholz, D. Paulus, A. von Gladiss, J. Hubbuch
  On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field
  2023 Biofabrication, volume: 16, page: 015002
• Z. Khan, P. Koltay, R. Zengerle, S. Kartmann, Z. Shu
  One-Stop Hybrid Printing of Bulk Metal and Polymer for 3D Electronics
  2023 Advanced Engineering Materials, page: 2300922
• B. Johannsen, D. Baumgartner, M. Karpíšek, D. Stejskal, N. Boillat-Blanco, J. Knüsli, M. Panning, N. Paust, R. Zengerle, K. Mitsakakis
  Patient Stratification for Antibiotic Prescriptions Based on the Bound-Free Phase Detection Immunoassay of C-Reactive Protein in Serum Samples
  2023 Biosensors, volume: 13, page: 1009
• S. Calabrese, A. M. Markl, M. Neugebauer, S. J. Krauth, N. Borst, F. von Stetten, M. Lehnert
  Reporter emission multiplexing in digital PCRs (REM-dPCRs): direct quantification of multiple target sequences per detection channel by population specific reporters
  2023 Analyst, volume: 148, pages: 5243 - 5254
• S. Koch, L. Metzler, S. Kilian, P. A. Heizmann, F. Lombeck, M. Breitwieser, S. Vierrath
  Toward scalable production: catalyst‐coated membranes (CCMs) for anion‐exchange membrane water electrolysis via direct bar coating
  2023 Advanced sustainable systems, volume: 7, page: 2200332
• V. Zieger, D. Frejek, S. Zimmermann, G. A. A. Miotto, P. Koltay, R. Zengerle, S. Kartmann
  Towards Label-Free Standardization in 3d Cell Culture: Automated, Selective and Gentle High-Throughput Handling of Spheroids and Organoids via Novel Pick-Flow-Drop Principle
  2023 Adv Healthc Mater, page: e2303350

2022

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• E. Kipf, F. Schlenker, N. Borst, M. Fillies, R. Kirschner-Schwabe, R. Zengerle, C. Eckert, F. von Stetten, M. Lehnert
  Advanced minimal residual disease monitoring for acute lymphoblastic leukemia with multiplex mediator probe PCR
  2022 J. Mol. Diag, volume: 24, issue: 1, pages: 57 - 68
• M. Neugebauer, C. E. Grundmann, M. Lehnert, F. von Stetten, S. M. Früh, R. Süss
  Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse Transcription-qPCR
  2022 Pharmaceutics, volume: 2022, issue: 14, page: 1348
• Dornhof J, Zieger V, Kieninger J, Frejek D, Zengerle R, Urban GA, Kartmann S, Weltin A
  Bioprinting-based automated deposition of single cancer cell spheroids into oxygen sensor microelectrode wells
  2022 LabChip, volume: 8, issue: 22, pages: 4369 - 4381
• C. Mills, E. Batista, H. Bissig, F. Ogheard, A. W. Boudaoud, O. Büker, K. Stolt, J. Morgan, S. Kartmann, K. Thiemann, G. Miotto, A. Niemann, S. Klein, G. Ratering, J. Lötters
  Calibration methods for flow rates down to 5 nL/min and validation methodology
  2022 Biomed. Eng.-Biomed. Tech.
• E. Balogun, P. Mardle, H. Nguyen, M. Breitwieser, S. Holdcroft
  Catalyst layers for fluorine‐free hydrocarbon PEMFCs
  2022 Electrochimica Acta, volume: 401, page: 139479
• K. Tröndle, G. Miotto, L. Rizzo, R. Pichler, F. Koch, P. Koltay, R. Zengerle, S. S. Lienkamp, S. Kartmann, S. Zimmermann
  Deep Learning-Assisted Nephrotoxicity Testing with Bioprinted Renal Spheroids
  2022 Int. J. Bioprint, volume: 8, issue: 2, page: 528
• V. Burchak, F. Koch, L. Siebler, S. Haase, V. K. Horner, X. Kempter, G. B. Stark, U. Schepers, A. Grimm, S. Zimmermann, P. Koltay, S. Strassburg, G. Finkenzeller, F. Simunovic, F. Lampert
  Evaluation of a Novel Thiol–Norbornene-Functionalized Gelatin Hydrogel for Bioprinting of Mesenchymal Stem Cells
  2022 Int. J. Mol. Sci., volume: 23, page: 7939
• H. Nguyen, C. Klose, L. Metzler, S. Vierrath, M. Breitwieser
  Fully Hydrocarbon Membrane Electrode Assemblies for Proton Exchange Membrane Fuel Cells and Electrolyzers: An Engineering Perspective
  2022 Adv. Energy Mater., volume: 2022, page: 2103559
• J. Disch, L. Bohn, S. Koch, M. Schulz, Y. Han, A. Tengattini, L. Helfen, M. Breitwieser, S. Vierrath
  High-resolution neutron imaging of salt precipitation and water transport in zero-gap CO2 electrolysis
  2022 nature communications, volume: 13, page: 6099
• G. Miotto, K. Thiemann, M. Rombach, R. Zengerle, S. Kartmann
  Holographic PIV/PTV for nano flow rates–A study in the 70 to 200 nL/min range
  2022 Biomedical Engineering
• B. Johannsen, D. Baumgartner, L. Karkossa, N. Paust, M. Karpíšek, N. Bostanci, R. Zengerle, K. Mitsakakis
  ImmunoDisk - a fully automated bead-based immunoassay cartridge with all reagents pre-stored
  2022 Biosensors, volume: 12, issue: 6, page: 413
• H. Nguyen, D. Sultanova, P. A. Heizmann, S. Vierrath, M. Breitwieser
  Improving the efficiency of fully hydrocarbon-based proton-exchange membrane fuel cells by ionomer content gradients in cathode catalyst layers
  2022 Materials Advances, volume: 23
• S. Hin, N. Paust, M. Rombach, J. Lüddecke, M. Specht, R. Zengerle, K. Mitsakakis
  Magnetophoresis in Centrifugal Microfluidics at Continuous Rotation for Nucleic Acid Extraction
  2022 Micromachines, volume: 13, page: 2112
• F. Koch, O. Thaden, S. Conrad, K. Tröndle, G. Finkenzeller, R. Zengerle, S. Kartmann, S. Zimmermann, P. Koltay
  Mechanical properties of polycaprolactone (PCL) scaffolds for hybrid 3D-bioprinting with alginate-gelatin hydrogel
  2022 J. Mech. Behav. Biomed. Mat., volume: 130, page: 105219
• L. Bohn, M. von Holst, E. Cruz Ortiz, M. Breitwieser, S. Vierrath, C. Klose
  Methods— A Simple Method to Measure In-Plane Electrical Resistance of PEM Fuel Cell and Electrolyzer Catalyst Layers
  2022 J.Electrochem. Soc., volume: 169, issue: 5, page: 054518
• Y.-T. Kao, S. Calabrese, N. Borst, M. Lehnert, Y.-K. Lai, F. Schlenker, P. Juelg, R. Zengerle, P. Garstecki, F. von Stetten
  Microfluidic One-Pot Digital Droplet FISH Using LNA/DNA Molecular Beacons for Bacteria Detection and Absolute Quantification
  2022 Biosensors, volume: 12, issue: 4, page: 237
• A. Klebes, A.-S. Kittel, R. D. Verboket, F. von Stetten, S. M. Früh
  Multianalyte lateral flow immunoassay for simultaneous detection of protein-based inflammation biomarkers and pathogen DNA
  2022 Sensors and Actuators B: Chemical, volume: 355, page: 131283
• S. Hennig, Z. Shu, L. Gutzweiler, P. Koltay, F. von Stetten, R. Zengerle, S. M. Früh
  Paper-based open microfluidic platform for protein electrophoresis and immunoprobing
  2022 Electrophoresis, volume: 43, issue: 4, pages: 621 - 631
• B. Afacan, V. Özgen Öztürk, G. Emingil, T. Köse, K. Mitsakakis, N. Bostanci
  Salivary secretory leukocyte protease inhibitor levels in patients with stage 3 grade C periodontitis: a comparative cross-sectional study
  2022 Scientific Reports, volume: 12, issue: 1, page: 21267
• S. Koch, L. Metzler, S. K. Kilian, P. A. Heizmann, F. Lombeck, M. Breitwieser, S. Vierrath
  Toward Scalable Production: Catalyst-Coated Membranes (CCMs) for Anion-Exchange Membrane Water Electrolysis via Direct Bar Coating
  2022 Adv. Sustainable Syst., page: 2200332
• S. Koch, J. Disch, S. K. Kilian, Y. Han, L. Metzler, A. Tengattini, L. Helfen, M. Schulz, M. Breitwieser, S. Vierrath
  Water management in anion-exchange membrane water electrolyzers under dry cathode operation
  2022 RSC Adv, volume: 12, pages: 20778 - 20784

2021

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• A. Homann, L. Niebling, S. Zehnle, M. Beutler, L. Delamotte, M.C. Rothmund, D. Czurratis, K.-D. Beller, R. Zengerle, H. Hoffmann, N. Paust
  A microfluidic cartridge for fast and accurate diagnosis of Mycobacterium tuberculosis infections on standard laboratory equipment
  2021 Lab on a Chip, volume: 21, issue: 8, pages: 1540 - 1548
  » show abstract« hide abstract
  Abstract

  We present a novel centrifugal microfluidic approach for fast and accurate tuberculosis (TB) diagnosis based on the use of standard laboratory equipment. The herein presented workflow can directly be integrated into laboratories with standard equipment and automates complex sample preparation. The system consists of a microfluidic cartridge, a laboratory centrifuge and a standard PCR cycler. The cartridge includes all required reagents and automates collection of bacteria on filter membranes, bacterial lysis, nucleic acid extraction and aliquoting of the DNA extract for PCR analysis. We show that storage of the reagents in aluminium-coated pouches is stable during accelerated storage and transport tests. When the limit of detection was assessed, we found that the cartridge-automated workflow consistently detected 10 CFU ml−1 of mycobacteria in spiked sputum samples. First tests with clinical samples showed a 100% specificity for non-TB specimens. In addition, Mycobacterium tuberculosis (MTB) was re-found in pre-characterized smear microscopy and culture positive sputum samples suggesting a high diagnostic sensitvity. In summary, the novel cartridge-automated workflow enables a flexible and sensitive TB diagnosis without the need to invest in specialized instrumentation.
• J.F.Hess, M.E.Hess, R.Zengerle, N.Paust, M.Boerries, T.Hutzenlaub
  Automated library preparation for whole genome sequencing by centrifugal microfluidic
  2021 Analytica Chimica Acta, page: 338954
• J.-N. Klatt, T.J. Dinh, O. Schilling, R. Zengerle, F. Schmidt, T. Hutzenlaub, N. Paust
  Automation of peptide desalting for proteomic liquid chromatography–tandem mass spectrometry by centrifugal microfluidics
  2021 Lab on a Chip, volume: 21, issue: 21, page: 2255
  » show abstract« hide abstract
  Abstract

  For large-scale analysis of complex protein mixtures, liquid chromatography – tandem mass spectrometry (LC-MS/MS) has been proven to be one of the most versatile tools due to its high sensitivity and ability to both identify and quantify thousands of proteins in a single measurement. Sample preparation typically comprises site-specific cleavage of proteins into peptides, followed by desalting and concomitant peptide enrichment, which is commonly performed by solid phase extraction. Desalting workflows may include multiple liquid handling steps and are thus error prone and labour intensive. To improve the reproducibility of sample preparation for low amounts of protein, we present a centrifugal microfluidic disk that automates all liquid handling steps required for peptide desalting by solid phase extraction (DesaltingDisk). Microfluidic implementation was enabled by a novel centrifugal microfluidic dosing on demand structure that enabled mapping multiple washing steps onto a microfluidic disk. Evaluation of the microfluidic disk was performed by LC-MS/MS analysis of tryptic HEK-293 eukaryotic cell peptide mixtures desalted either using the microfluidic disk or a manual workflow. A comparable number of peptides were identified in the disk and manual set with 19 775 and 20 212 identifications, respectively. For a core set of 10 444 peptides that could be quantified in all injections, intensity coefficients of variation were calculated based on label-free quantitation intensities. The disk set featured smaller variability with a median CV of 9.3% compared to the median CV of 12.6% for the manual approach. Intensity CVs on protein level were lowered from 5.8% to 4.2% when using the LabDisk. Interday reproducibility for both workflows was assessed by LC-SRM/MS analysis of samples that were spiked with 11 synthetic peptides of varying hydrophobicity. Except for the most hydrophilic and hydrophobic peptides, the average CV was lowered to 3.6% for the samples processed with the disk compared to 7.2% for the manual workflow. The presented centrifugal microfluidic DesaltingDisk demonstrates the potential to improve reproducibility in the sample preparation workflow for proteomic mass spectrometry, especially for application with limited amount of sample material.
• J.-N. Klatt, T. Hutzenlaub, T. Subkowski, T. Müller, S. Hennig, R. Zengerle, N. Paust
  Blocking Protein Adsorption in Microfluidic Chips by a Hydrophobin Coating
  2021 ACS Appl. Polym. Mater., volume: 3, issue: 7, pages: 3278 - 3286
  » show abstract« hide abstract
  Abstract

  Microfluidics allows the miniaturization of biochemical analyses. Small dimensions reduce sample and reagent consumption and enhance reaction rates. A downside is that high surface-to-volume ratios increase the unspecific binding of proteins to the substrate material. The resulting sample loss and reagent depletion decrease the sensitivity and specificity of protein-based assays, especially if low concentrations are analyzed. Here, we introduce the hydrophobin coating of microfluidic chips made of cyclic olefin copolymers (COC). The recombinant hydrophobin H*Protein B self-assembles into stable monolayers on hydrophobic surfaces, making them hydrophilic and thus reducing hydrophobic interactions between the chip surfaces and proteins. The substrate and sealing layers of the microfluidic chip were simply dip-coated and subsequently assembled by thermodiffusion bonding, which renders our coating procedure compatible with mass fabrication. Contact angle measurements and atomic force microscopy were used to evaluate the effect of high temperatures (107 °C) on COC substrates coated with H*Protein B. The efficiency of the protein-repellent coating was evaluated by depletion experiments with bovine serum albumin, human serum, and cerebrospinal fluid in microfluidic chips. Protein recovery was investigated down to protein concentrations of 0.3 μg/mL. Recoveries of 90% were observed with total protein amounts of 10 ng, even for microfluidic channels up to 835 mm in length and with a cross section of 80 μm × 230 μm in a COC 6013/8007 foil. For comparison, only 30 and 60% of the protein was recovered in uncoated microfluidic channels with lengths of 835 and 128 mm, respectively. The long-term stability of the hydrophobin-coated chips for 8 weeks was demonstrated.
• F. Schlenker, E. Kipf, N. Borst, N. Paust, R. Zengerle, F. von Stetten, P. Juelg, T. Hutzenlaub
  Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations
  2021 Processes, volume: 2021, issue: 9, supplement: 97
  » show abstract« hide abstract
  Abstract

  We present the centrifugal microfluidic implementation of a four-plex digital droplet polymerase chain reaction (ddPCR). The platform features 12 identical ddPCR units on a LabDisk cartridge, each capable of generating droplets with a diameter of 82.7 +/- 9 µm. By investigating different oil–surfactant concentrations, we identified a robust process for droplet generation and stabilization. We observed high droplet stability during thermocycling and endpoint fluorescence imaging, as is required for ddPCRs. Furthermore, we introduce an automated process for fourcolor fluorescence imaging using a commercial cell analysis microscope, including a customized software pipeline for ddPCR image evaluation. The applicability of ddPCRs is demonstrated by the quantification of three cancer-associated KRAS point mutations (G12D, G12V and G12A) in a diagnostically relevant wild type DNA background. The four-plex assay showed high sensitivity (3.5–35 mutant DNA copies in 15,000 wild type DNA copies) and linear performance (R2 = 0.99) across all targets in the LabDisk.
• T. Gross, C. Jeney, D. Halm, G. Finkenzeller, G. B. Stark, R. Zengerle, P. Koltay, S. Zimmermann
  Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and Emulsion Coupling assay as a low-cellularity workflow for single-cell cloning
  2021 Plos One, volume: 16, issue: 3, page: e0238330
  » show abstract« hide abstract
  Abstract

  The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (Surveyor assay), mRNA RT-PCR assessing altered gene expression, and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.
• D. Kainz, B. Breiner, S. M. Früh, T. Hutzenlaub, R. Zengerle, N. Paust
  Eliminating viscosity bias in lateral flow tests
  2021 Microsystems & Nanoengineering, volume: 7, page: 72
• S. Hin, B. Lopez-Jimena, M. Bakheit, V. Klein, S. Stack, C. Fall, A. Sall, K. Enan, M. Mustafa, V. Rusu, S. Goethel, N. Paust, R. Zengerle, L. Gillies, S. Frischmann, M. Weidmann, K. Mitsakakis
  Fully automated point-of-care differential diagnosis of acute febrile illness
  2021 PLoS Negl. Trop. Dis., volume: 15, issue: 2, page: e0009177
  » show abstract« hide abstract
  Abstract

  In this work, a platform was developed and tested to allow to detect a variety of candidate viral, bacterial and parasitic pathogens, for acute fever of unknown origin. The platform is based on a centrifugal microfluidic cartridge, the LabDisk (“FeverDisk” for the specific application), which integrates all necessary reagents for sample-to-answer analysis and is processed by a compact, point-of-care compatible device...
• M. Schulz, J. Ruediger, E. Landmann, M. Bakheit, S. Frischmann, D. Rassler, A. Homann, F. von Stetten, R. Zengerle, N. Paust
  High Dynamic Range Digital Assay Enabled by Dual-Volume Centrifugal Step Emulsification
  2021 Analytical Chemistry, volume: 93, issue: 5, pages: 2854 - 2860
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  Abstract

  We implement dual-volume centrifugal step emulsification on a single chip to extend the dynamic range of digital assays. Compared to published single-volume approaches, the range between the lower detection limit (LDL) and the upper limit of quantification (ULQ) increases by two orders of magnitude. In comparison to existing multivolume approaches, the dual-volume centrifugal step emulsification requires neither complex manufacturing nor specialized equipment. Sample metering into two subvolumes, droplet generation, and alignment of the droplets in two separate monolayers are performed automatically by microfluidic design. Digital quantification is demonstrated by exemplary droplet digital loop-mediated isothermal amplification (ddLAMP). Within 5 min, the reaction mix is split into subvolumes of 10.5 and 2.5 μL, and 2,5k and 176k droplets are generated with diameters of 31.6 ± 1.4 and 213.9 ± 7.5 μm, respectively. After 30 min of incubation, quantification over 5 log steps is demonstrated with a linearity of R2 ≥ 0.992.
• H. Nguyen, F. Lombeck, C. Schwarz, P. A. Heizmann, M. Adamski, H-F. Lee, B. Britton, S. Holdcroft, S. Vierrath, M. Breitwieser
  Hydrocarbon-based Pemion™ proton exchange membrane fuel cells with state-of-the-art performance
  2021 Sustainable Energy and Fuels, volume: 5, pages: 3687 - 3699
  » show abstract« hide abstract
  Abstract

  Non-fluorinated hydrocarbon ionomers feature distinct technical, cost, and environmental advantages over incumbent perfluorinated sulfonic acid (PFSA)-based ionomers: they offer improved thermo-mechanical properties at temperatures beyond 90 °C, likelihood of lower material cost, lower gas cross-over, and facile recycling of platinum group metals. In addition, fluorine-free hydrocarbon ionomers are less hazardous to the environment owing to the lack of (per)fluorinated precursors. Yet, the performance of hydrogen fuel cells with hydrocarbon-based ionomers and membranes has been historically largely inferior to the PFSA-based state of the art. In this study, we present wholly hydrocarbon fuel cells exceeding previous literature landmarks by a factor of nearly two, with peak power densities of 2.1 W cm−2 under H2/O2 (atmospheric pressure and 95% relative humidity), and 1.1 W cm−2 under H2/air (250 kPaabs and 50% relative humidity). This improvement was achieved by the use of Pemion™ – a sulfo-phenylated polyphenylene-based cation exchange material – as ionomer in the catalyst layer and as proton exchange membrane with a low thickness of 7 μm, and an optimization of cathode catalyst layer based on PtCo/C. Based on an in-depth study of electrochemical performance under various conditions vs. a state-of-the-art PFSA reference cell, the performances of Pemion™-based cells were found to be more sensitive to changes in relative humidity of inlet gases, but the detrimental influence of high temperatures on performance was significantly reduced. At an operation temperature of 110 °C, 250 kPaabs, and 50% relative humidity, the peak power density (0.96 W cm−2) was 8% higher than the short-side chain PFSA-based reference cell (0.89 W cm−2), highlighting the potential of Pemion™ for next-generation fuel cells for heavy-duty or aeronautic applications.
• J.-N. Klatt, I. Schwarz, T. Hutzenlaub, R. Zengerle, F. Schwemmer, D. Kosse, J. Vincent, M. Scaer, K. Franaszczuk, D. Wadsworth, N. Paust
  Miniaturization, Parallelization, and Automation of Endotoxin Detection by Centrifugal Microfluidics
  2021 Analytical Chemistry, volume: 93, issue: 24
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  Abstract

  We demonstrate microfluidic automation and parallelization of Limulus amebocyte lysate (LAL)-based bacterial endotoxin testing using centrifugal microfluidics. LAL is the standard reagent to test for endotoxin contaminations in injectable pharmaceuticals. The main features of the introduced system are more than 90% reduction of LAL consumption, from 100 μL/reaction to 9.6 μL/reaction, automated liquid handling to reduce opportunities for contamination and manual handling errors, and microfluidic parallelization by integrating 104 reactions into a single centrifugal microplate. In a single Eclipse microplate, 21 samples and their positive product controls are tested in duplicate. In addition, a standard curve with up to five points is generated, resulting in a total of 104 reactions. Test samples with a defined concentration of 0.5 endotoxin units per milliliter were tested, resulting in a coefficient of variation below 0.75%. A key feature for achieving a small coefficient of variation is ensuring the same path length along the microfluidic channels to the final reaction chambers for each sample and the reagent, so that any unspecific adsorption to the polymer surfaces does not affect the accuracy and precision. Analysis of a sample containing naturally occurring endotoxin with the developed microfluidic microplate yielded comparable results to the conventional testing method. A test with eight commercially available pharmaceuticals was found to pass all requirements for bacterial endotoxin testing as specified in the United States Pharmacopeia. The automated endotoxin testing system reveals specific advantages of centrifugal microfluidics for analytical biochemistry applications. Small liquid volumes are handled (metered, mixed, and aliquoted) in a very precise, highly integrated, and highly parallel manner within mass-fabricated microplates.
• K. Mitsakakis
  Novel lab-on-a-disk platforms: a powerful tool for molecular fingerprinting of oral and respiratory tract infections
  2021 Expert Rev. Mol. Diagn., volume: 21, issue: 6, pages: 523 - 526
• B. Johannsen, M. Karpíšek, D. Baumgartner, V. Klein, N. Bostanci, N. Paust, S. M. Früh, R. Zengerle, K. Mitsakakis
  One-step, wash-free, bead-based immunoassay employing bound-free phase detection
  2021 Analytica Chimica Acta, volume: 1153, page: 338280
  » show abstract« hide abstract
  Abstract

  We present a simple and fast one-step heterogeneous immunoassay, with performance characteristics that can enable easy and versatile adaptation to miniaturized, automated point-of-care systems. This novel analytical method uses magnetic and fluorescent beads as capture and detection agents respectively. Its main feature is the measurement of the fluorescent signal in the bound-free phase for (semi-)quantitative detection of analytes. Thus, no washing is required and the workflow consists only of sample and reagent supply, incubation, separation and detection. The immunoassay concept is demonstrated with C-reactive protein (CRP), a systemic inflammation marker. CRP in only 5 μL of undiluted serum was measured in the range 20-140 mg L-1 (includes clinically relevant cut-off values). The limit of detection (LOD) was 22.1 ± 6.3 mg L-1 (incubation 15 min). A CRP certified reference material was measured on five different days. Intra- and inter-assay coefficients of variation were 4.6 % ± 1.9 % and 5.6 % respectively. To demonstrate the compatibility of the assay concept with additional matrices and concentration ranges, three oral inflammation markers, namely matrix metalloproteinases 8 and 9 (MMP-8, MMP-9) and tissue inhibitor of metalloproteinases 1 (TIMP-1), were measured in saliva in the ranges 0.47-30 ng mL-1 for MMP-8 and MMP-9, and 0.69-44 ng mL-1 for TIMP-1. LODs were 0.24 ng mL-1, 0.38 ng mL-1 and 0.39 ng mL-1 respectively (incubation 20 min). Multiplexing capacity of the assay concept was also shown with these markers. The demonstrated excellent reproducibility of the results, combined with the versatility and low complexity of the introduced immunoassay concept, make it an attractive candidate for applied analytical chemistry and automated point-of-care testing.
• F. Koch, O. Thaden, K. Tröndle, R. Zengerle, S. Zimmermann, P. Koltay
  Open-source hybrid 3D-bioprinter for simultaneous printing of thermoplastics and hydrogels
  2021 HardwareX, volume: 10, page: e00230
• D. Baumgartner, B. Johannsen, M. Specht, J. Lüddecke, M. Rombach, S. Hin, N. Paust, F. von Stetten, R. Zengerle, C. Herz, J. R. Peham, P. N. Paqué, T. Attin, J. S. Jenzer, P. Körner, P. R. Schmidlin, T. Thurnheer, F. J. Wegehaupt, W. E. Kaman, A. Stubbs, J. P. Hays, V. Rusu, A. Michie, T. Binsl, D. Stejskal, M. Karpíšek, K. Bao, N. Bostanci, G. N. Belibasakis, K. Mitsakakis
  OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice
  2021 Biosensors, volume: 11, issue: 11, page: 423
• B. Shanahan, B. Britton, A. Belletti, S. Vierrath, M. Breitwieser
  Performance and stability comparison of Aemion™ and Aemion+™ membranes for vanadium redox flow batteries
  2021 RSC Advances, volume: 11, issue: 22, pages: 13077 - 13084
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  Abstract

  Anion exchange membranes (AEMs) have shown a significant rise in performance and durability within recent years for applications such as electrolysis and fuel cells. However, in vanadium redox-flow batteries, their use is of particular interest to lower costs and self-discharge rates compared to conventional perfluorinated sulfonic acid-based ionomers such as Nafion. In this work we evaluate the properties of two commercial AEMs, Aemion™ and Aemion+™, based on ex situ characterizations, an accelerated stress test degradation study (>1000 hours storage in highly oxidizing VO2+ electrolyte at 35 °C) and electrochemical battery cycle tests. All membranes feature low ionic resistances of below 320 mΩ cm2, enabling battery cycling at 100 mA cm−2. Aemion shows considerable VO2+ formation within a VO2+ stress test, whereas Aemion+ remains almost unaffected in the 1058 h stress test. Evaluating self-discharge data, cycling performance and durability data, Aemion+™ (50 μm thickness) features the best properties for vanadium redox-flow battery operation.
• L. Becherer, J. F. Hess, S. Frischmann, M. Bakheit, H. Nitschko, S. Stinco, F. Zitz, H. Hofer, G. Porro, F. Hausladen, K. Stock, D. Drossart, H. Wurm, H. Kuhn, D. Huber, T. Hutzenlaub, N. Paust, M. Keller, O. Strohmeier, S. Wadle, N. Borst, R. Zengerle, F. von Stetten
  Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP
  2021 Micromachines, volume: 12, issue: 2, page: 159
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  Abstract

  This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ Tlymphocytes (MT-2 cells) yielded 8 +/- 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 +/- 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 +/- 3 copies per MT-2 cell after a run time of two hours and 10 min.
• A. Brunauer, R. D. Verboket, D. M. Kainz, F. von Stetten, S. M. Früh
  Rapid Detection of Pathogens in Wound Exudate via Nucleic Acid Lateral Flow Immunoassay
  2021 Biosensors, volume: 11, issue: 3, page: 74
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  Abstract

  The rapid detection of pathogens in infected wounds can significantly improve the clinical outcome. Wound exudate, which can be collected in a non-invasive way, offers an attractive sample material for the detection of pathogens at the point-of-care (POC). Here, we report the development of a nucleic acid lateral flow immunoassay for direct detection of isothermally amplified DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with the opportunistic pathogen Pseudomonas aeruginosa that cause severe health issues upon wound colonization. A detection limit of 2.1 × 105 CFU per mL of wound fluid was achieved, and no cross-reaction with other pathogens was observed. Furthermore, we integrated an internal amplification control that excludes false negative results and, in combination with the flow control, ensures the validity of the test result. The paper-based approach with only three simple hands-on steps has a turn-around time of less than 30 min and covers the complete analytical process chain from sample to answer. This newly developed workflow for wound fluid diagnostics has tremendous potential for reliable pathogen POC testing and subsequent target-oriented therapy.
• B. Shanahan, K. Seteiz, P. A. Heizmann, S. Koch, J. Büttner, S. Ouardi, S. Vierrath, A. Fischer, M. Breitwieser
  Rapid wet-chemical oxidative activation of graphite felt electrodes for vanadium redox flow batteries
  2021 RSC Advances, volume: 11, issue: 51, pages: 32095 - 32105
• A. Lux, H. Bott, N. P. Malek, R. Zengerle, T. Maucher, J. Hoffmann, 1
  Real-Time Detection of Tumor Cells during Capture on a Filter Element Significantly Enhancing Detection Rate
  2021 Biosensors, volume: 11, issue: 9, page: 312
• P.N. Paqué, C. Herz, D.B. Wiedemeier, K. Mitsakakis, T. Attin, K. Bao, G.N. Belibasakis, J.P. Hays, J.S. Jenzer, W.E Kaman, M. Karpíšek, P. Körner, J.R. Peham, P.R. Schmidlin, T. Thurnheer, F.J. Wegehaupt, N. Bostanci
  Salivary Biomarkers for Dental Caries Detection and Personalized Monitoring
  2021 J. Pers. Med., volume: 11, issue: 3, page: 235
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  Abstract

  This study investigated the potential of salivary bacterial and protein markers for evaluating the disease status in healthy individuals or patients with gingivitis or caries. Saliva samples from caries- and gingivitis-free individuals (n = 18), patients with gingivitis (n = 17), or patients with deep caries lesions (n = 38) were collected and analyzed for 44 candidate biomarkers (cytokines, chemokines, growth factors, matrix metalloproteinases, a metallopeptidase inhibitor, proteolytic enzymes, and selected oral bacteria). The resulting data were subjected to principal component analysis and used as a training set for random forest (RF) modeling. This computational analysis revealed four biomarkers (IL-4, IL-13, IL-2-RA, and eotaxin/CCL11) to be of high importance for the correct depiction of caries in 37 of 38 patients. The RF model was then used to classify 10 subjects (five caries-/gingivitis-free and five with caries), who were followed over a period of six months. The results were compared to the clinical assessments of dental specialists, revealing a high correlation between the RF prediction and the clinical classification. Due to the superior sensitivity of the RF model, there was a divergence in the prediction of two caries and four caries-/gingivitis-free subjects. These findings suggest IL-4, IL-13, IL-2-RA, and eotaxin/CCL11 as potential salivary biomarkers for identifying noninvasive caries. Furthermore, we suggest a potential association between JAK/STAT signaling and dental caries onset and progression.
• S. M. Früh, U. Matti, P. R. Spycher, M. Rubini, S. Lickert, T. Schlichthaerle, R. Jungmann, V. Vogel, J. Ries, I. Schoen
  Site-Specifically-Labeled Antibodies for Super-Resolution Microscopy Reveal In Situ Linkage Errors
  2021 ACS Nano, volume: 15, issue: 7, pages: 12161 - 12170
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  Abstract

  The precise spatial localization of proteins in situ by super-resolution microscopy (SRM) demands their targeted labeling. Positioning reporter molecules as close as possible to the target remains a challenge in primary cells or tissues from patients that cannot be easily genetically modified. Indirect immunolabeling introduces relatively large linkage errors, whereas site-specific and stoichiometric labeling of primary antibodies relies on elaborate chemistries. In this study, we developed a simple two-step protocol to site-specifically attach reporters such as fluorophores or DNA handles to several immunoglobulin G (IgG) antibodies from different animal species and benchmarked the performance of these conjugates for 3D STORM (stochastic optical reconstruction microscopy) and DNA-PAINT (point accumulation in nanoscale topography). Glutamine labeling was restricted to two sites per IgG and saturable by exploiting microbial transglutaminase after removal of N-linked glycans. Precision measurements of 3D microtubule labeling shell dimensions in cell lines and human platelets showed that linkage errors from primary and secondary antibodies did not add up. Monte Carlo simulations of a geometric microtubule-IgG model were in quantitative agreement with STORM results. The simulations revealed that the flexible hinge between Fab and Fc segments effectively randomized the direction of the secondary antibody, while the restricted binding orientation of the primary antibody’s Fab fragment accounted for most of the systematic offset between the reporter and α-tubulin. DNA-PAINT surprisingly yielded larger linkage errors than STORM, indicating unphysiological conformations of DNA-labeled IgGs. In summary, our cost-effective protocol for generating well-characterized primary IgG conjugates offers an easy route to precise SRM measurements in arbitrary fixed samples.
• M. Drews, J. Büttner, M. Bauer, J. Ahmed, R. Sahu, S. Vierrath, C. Scheu, A. Fischer, D. Biro
  Spruce Hard Carbon Anodes for Lithium-Ion Batteries
  2021 ChemElectroChem, volume: 8, pages: 4750 - 4761
• F. Schlenker, E. Kipf, M. Deuter, I. Höffkes, M. Lehnert, R. Zengerle, F von Stetten, F. Scherer, J. Wehrle, N. von Bubnoff, P. Juelg, T. Hutzenlaub, N. Borst
  Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA
  2021 cancers, volume: 13, issue: 22, page: 5742
• P. Juelg, E. Kipf, M. Specht, M. Fillies, C. Eckert, N. Paust, R. Zengerle, M. Lehnert, T. Hutzenlaub
  The MRD disk: automated minimal residual disease monitoring by highly sensitive centrifugal microfluidic multiplex qPCR
  2021 Lab Chip, volume: 21, pages: 558 - 570
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  Abstract

  We present a proof-of-principle study on automated, highly sensitive and multiplexed qPCR quantification by centrifugal microfluidics. The MRD disk can be used for standardisation of repetitive, longitudinal assays with high requirements on reproducibility and sensitivity, such as cancer monitoring. In contrast to high-throughput qPCR automation by bulky and expensive robotic workstations we employ a small centrifugal microfluidic instrument, addressing the need of low- to mid-throughput applications. As a potential application we demonstrate automated minimum residual disease (MRD) monitoring of prognostic markers in patients with acute lymphoblastic leukaemia (ALL). The disk-workflow covers all aspects of clinical gold standard MRD quantification: generation of standard curves, specificity controls, no template controls and quantification of the ALL patient sample. We integrated a highly sensitive, colorimetric 2-plex analysis of MRD targets, as well as a 2-plex analysis of reference genes, both in parallel and in a single LabDisk cartridge. For this purpose, a systematic procedure for crosstalk- and signal-to-noise-optimisation is introduced, providing a guideline for efficient multiplex readout inside microfluidic platforms. The qPCR standard curves (n = 12/12) generated on-disk reach clinically required linearity (R2 = 98.1% to R2 = 99.8%). In three consecutive MRD disk runs with an ALL patient sample containing the two representative MRD targets VH3D3D5JH3 and VkIkde, we observe high accordance between the on-disk quantifications (48 ± 6 copies/reaction and 69 ± 6 copies/reaction) and the expected concentrations (57 copies/reaction for both targets). In comparison to the clinical gold standard of manually pipetted, singleplex assays, the MRD disk yields comparable limit of quantification (1 × 10−4) in n = 6/6 analyses (vs. n = 4/4 in gold standard) and a limit of detection (1 × 10−5) in n = 6/6 analysis (vs. n = 2/4 in gold standard). The automation reduces the risk of manual liquid handling errors, making the MRD disk an attractive solution to assure reproducibility in moderate-throughput, longitudinal gene quantification applications.
• S. Koch, P. A. Heizmann, S. K. Kilian, B. Britton, S. Holdcroft, M. Breitwieser, S. Vierrath
  The effect of ionomer content in catalyst layers in anion-exchange membrane water electrolyzers prepared with reinforced membranes (Aemion+™)
  2021 J. Mater. Chem. A, volume: 9, pages: 15744 - 15754
• M. Tsagiopoulou, M. C. Maniou, N. Pechlivanis, A. Togkousidis, M. Kotrová, T. Hutzenlaub, I. Kappas, A. Chatzidimitriou, F. Psomopoulos
  UMIc: A Preprocessing Method for UMI Deduplication and Reads Correction
  2021 Front. Genet., volume: May, 28
• N. Bostanci, K. Mitsakakis, B. Afacan, K. Bao, B. Johannsen, D. Baumgartner, L. Müller, H. Kotolová, G. Emingil, M. Karpíšek
  Validation and verification of predictive salivary biomarkers for oral health
  2021 Scientific Reports, volume: 11, page: 6406
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  Abstract

  Oral health is important not only due to the diseases emerging in the oral cavity but also due to the direct relation to systemic health. Thus, early and accurate characterization of the oral health status is of utmost importance. There are several salivary biomarkers as candidates for gingivitis and periodontitis, which are major oral health threats, affecting the gums. These need to be verified and validated for their potential use as differentiators of health, gingivitis and periodontitis status, before they are translated to chair-side for diagnostics and personalized monitoring. We aimed to measure 10 candidates using high sensitivity ELISAs in a well-controlled cohort of 127 individuals from three groups: periodontitis (60), gingivitis (31) and healthy (36). The statistical approaches included univariate statistical tests, receiver operating characteristic curves (ROC) with the corresponding Area Under the Curve (AUC) and Classification and Regression Tree (CART) analysis. The main outcomes were that the combination of multiple biomarker assays, rather than the use of single ones, can offer a predictive accuracy of > 90% for gingivitis versus health groups; and 100% for periodontitis versus health and periodontitis versus gingivitis groups. Furthermore, ratios of biomarkers MMP-8, MMP-9 and TIMP-1 were also proven to be powerful differentiating values compared to the single biomarkers
• F. Schlenker, E. Kipf, N. Borst, T. Hutzenlaub, R. Zengerle, F. von Stetten, P. Juelg
  Virtual Fluorescence Color Channels by Selective Photobleaching in Digital PCR Applied to the Quantification of KRAS Point Mutations
  2021 Analytical Chemistry, volume: 93, issue: 30, pages: 10538 - 10545
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  Abstract

  Multiplexing of analyses is essential to reduce sample and reagent consumption in applications with large target panels. In applications such as cancer diagnostics, the required degree of multiplexing often exceeds the number of available fluorescence channels in polymerase chain reaction (PCR) devices. The combination of photobleaching-sensitive and photobleaching-resistant fluorophores of the same color can boost the degree of multiplexing by a factor of 2 per channel. The only additional hardware required to create virtual fluorescence color channels is a low-cost light-emitting diode (LED) setup for selective photobleaching. Here, we present an assay concept for fluorescence color multiplexing in up to 10 channels (five standard channels plus five virtual channels) using the mediator probe PCR with universal reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching characteristic of 21 URs, which cover the whole spectral range from blue to crimson. This comprehensive UR data set is employed to demonstrate the use of three virtual channels in addition to the three standard channels of a commercial dPCR device (blue, green, and red) targeting cancer-associated point mutations (KRAS G12D and G12V). Moreover, a LOD (limit of detection) analysis of this assay confirms the high sensitivity of the multiplexing method (KRAS G12D: 16 DNA copies/reaction in the standard red channel and KRAS G12V: nine DNA copies/reaction in the virtual red channel). Based on the presented data set, optimal fluorogenic reporter combinations can be easily selected for the application-specific creation of virtual channels, enabling a high degree of multiplexing at low optical and technical effort.

2020

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• C. Klose, T. Saatkamp, A. Münchinger, L. Bohn, G. Titvinidze, M. Breitwieser, K.-D. Kreuer, S. Vierrath
  All-Hydrocarbon MEA for PEM Water Electrolysis Combining Low Hydrogen Crossover and High Efficiency
  2020 Adv Energy Mater, page: 1903995
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  Abstract

  Hydrocarbon ionomers bear the potential to significantly lower the material cost and increase the efficiency of proton-exchange membrane water electrolyzers (PEMWE). However, no fully hydrocarbon membrane electrode assembly (MEA) with a performance comparable to Nafion-MEAs has been reported. PEMWE-MEAs are presented comprising sPPS as membrane and electrode binder reaching 3.5 A cm−2 at 1.8 V and thus clearly outperforming state-of-the-art Nafion-MEAs (N115 as membrane, 1.5 A cm−2 at 1.8 V) due to a significantly lower high frequency resistance (57 ± 4 mΩ cm² vs 161 ± 7 mΩ cm²). Additionally, pure sPPS-membranes show a three times lower gas crossover (<0.3 mA cm−2) than Nafion N115-membranes (>1.1 mA cm−2) in a fully humidified surrogate test. Furthermore, more than 80 h of continuous operation is shown for sPPS-MEAs in a preliminary durability test (constant current hold at 1 A cm−2 at 80 °C). These results rely on the unique transport properties of sulfonated poly(phenylene sulfone) (sPPS) that combines high proton conductivity with low gas crossover.
• D. Podbiel, R. Zengerle, J. Hoffmann
  An analytical model for void-free priming of microcavities
  2020 Microfluid Nanofluid, volume: 24, issue: 16
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  Abstract

  The present work deals with the microfluidic evolution of capillary surfaces that are formed during the priming of microcavity structures with a non-wetting liquid. Due to the large contact angle of the priming liquid, a trapping of air within the microcavities poses a major impediment to a complete filling. We tackle this issue by developing a two-dimensional analytical model describing the geometrical shape of capillary surfaces formed in microcavity structures. In particular, the model is employed to derive two quantitative conditions for a void-free priming of a microcavity structure in terms of its aspect ratio, rounding parameters and the channel width. Microfluidic experiments are performed to verify the analytical results. Finally, we make use of the model to demonstrate a pressure-driven aliquoting of a non-wetting sample liquid in a flow chamber with an array of 55 microcavities by introducing a second immiscible liquid acting as a sealant. In this way, our work constitutes a basis for the design of microcavity-based liquid aliquoting structures that are used in various fields of application like PCR arrays, cell culture chips or digital reaction arrays.
• J. F. Hess, M. Kotrová, S. Calabrese, N. Darzentas, T. Hutzenlaub, R. Zengerle, M. Brüggemann, N. Paust
  Automation of amplicon-based library preparation for next generation sequencing by centrifugal microfluidics
  2020 Anal Chem, volume: 92, issue: 19, pages: 12833 - 12841
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  Abstract

  Next generation sequencing has become a mainstream method in bioanalysis. Improvements in sequencing and bioinformatics turned the complex and cumbersome library preparation to the bottle neck in terms of reproducibility and costs in the complete NGS workflow. Here, we introduce an automated library preparation approach based on a generic centrifugal microfluidic car-tridge. Multiplex polymerase chain reaction amplification and subsequent clean-up were processed with all reagents pre-stored on disk, including cell line-based DNA as quality control. Exchange of pre-stored reagents allows to apply the cartridge to different target genes. Sequencing of automatically prepared libraries from T-cell receptor and immunoglobulin gene rearrangements in context of lymphoproliferative disorders demonstrated excellent clean-up performance between 91.9% and 99.9% of target DNA reads and successful amplification of all target regions by up to 15 forward combined with four reverse primers. The fully auto-mated library preparation by centrifugal microfluidics thus offers attractive automation options in diagnostic settings.
• Z. Shu, M. Fechtig, F. Lombeck, M. Breitwieser, Roland Zengerle, Peter Koltay
  Direct Drop-on-Demand Printing of Molten Solder Bumps on ENIG Finishing at Ambient Conditions through StarJet Technology
  2020 IEEE Access, volume: 8, pages: 210225 - 210233
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  Abstract

  In this paper, we report on a detailed experimental study carried out with the StarJet technology to investigate the mechanical adhesion properties of directly printed solder bumps on electroless nickel immersion gold (ENIG) plated PCB boards. The aim of this study is to determine the maximum bond strength achievable by this method and to find suitable printing parameters that allow for the production of reliable and consistent solder bumps by non-contact printing of molten solder (type SAC305). Molten solder droplets of about 250 μm diameter were printed at melt temperatures between 250 and 400 °C onto ENIG surfaces kept at temperatures in the range of 100 to 200 °C. Using shear force tests, the adhesion of the printed bumps was investigated as a function of the main process parameters: 1. printhead temperature, 2. substrate temperature, and 3. substrate preheating time. The formation of an intermetallic compound (IMC) between the solder and the ENIG was confirmed by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) measurements. As a result of the comprehensive experimental parameter study, suitable printing parameters for establishing bond strengths corresponding to maximum shear force values of 3000 to 4000 mN could be found, i.e. high printhead temperature of 400 °C, short preheating and time of < 2 min, and substrate heating at 180 °C The use of flux was found to slightly improve the bond strength and to improve the consistency of the printing results for extended operation times. The achieved high bond strength and the reasonable reproducibility of the printing results qualify the StarJet technology for further investigations regarding applications in the field of direct soldering of microelectronic chips and devices to PCB boards as well as other micro-assembly tasks in the future.
• F. Hegge, F. Lombeck, E. Cruz Ortiz, L. Bohn, M. von Holst, M. Kroschel, J. Hübner, M. Breitwieser, P. Strasser, S. Vierrath
  Efficient and Stable Low Iridium Loaded Anodes for PEM Water Electrolysis Made Possible by Nanofiber Interlayers
  2020 ACS Appl. Energy Mater, volume: 3, issue: 9, pages: 8276 - 8284
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  Abstract

  Significant reduction of the precious metal catalyst loading is one of the key challenges for the commercialization of proton-exchange membrane water electrolyzers. In this work we combine IrOx nanofibers with a conventional nanoparticle-based IrOx anode catalyst layer. With this hybrid design we can reduce the iridium loading by more than 80% while maintaining performance. In spite of an ultralow overall catalyst loading of 0.2 mgIr/cm2, a cell with a hybrid layer shows similar performance compared to a state-of-the-art cell with a catalyst loading of 1.2 mgIr/cm2 and clearly outperforms identically loaded reference cells with pure IrOx nanoparticle and pure nanofiber anodes. The improved performance is attributed to a combination of good electric contact and high porosity of the IrOx nanofibers with high surface area of the IrOx nanoparticles. Besides the improved performance, the hybrid layer also shows better stability in a potential cycling and a 150 h constant current test compared to an identically loaded nanoparticle reference.
• R. Moroni, S. Thiele
  FIB/SEM tomography segmentation by optical flow estimation
  2020 Ultramicroscopy, volume: 219, page: 113090
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  Abstract

  Focused ion beam/scanning electron microscopy tomography (FIB/SEM tomography) is the method of choice for the tomographic reconstruction of mesoporous materials systems in various fields such as batteries, fuel cells, filter applications or composite materials. However, due to so called shine-through artifacts in FIB/SEM tomographies of porous materials, their segmentation into pore space and solid material is a nontrivial task. Here, an optical flow-based method that utilizes shine-through artifacts for segmentation is introduced. Subsequently, the performance of the method is discussed by means of tomographic datasets of a polymer electrolyte fuel cell catalyst layer and a lithium ion battery composite electrode. Previous, manual segmentations of these datasets allow the evaluation of the results – for the catalyst layer an accuracy of 86.6% and a precision of 84.0% is reached. In both cases, the optical flow-based approach gives significantly better results than comparable segmentations obtained from gray-value threshold binarization.
• P. Holzapfel, M. Bühler, D. Escalera-López, M. Bierling, F. Speck, K. Mayrhofer, S. Cherevko, C. V. Pham, S. Thiele
  Fabrication of a Robust PEM Water Electrolyzer Based on Non-Noble Metal Cathode Catalyst: [Mo3S13]2− Clusters Anchored to N-Doped Carbon Nanotubes
  2020 Small, page: 2003161
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  Abstract

  High investment costs and a dependence on noble metal catalysts currently obstruct the large-scale implementation of proton exchange membrane water electrolyzers (PEMWEs) for converting fluctuating green electricity into chemical energy via water splitting. In this context, this work presents a high-performing and stable non-noble metal catalyst for the hydrogen evolution reaction (HER), consisting of [Mo3S13]2− clusters supported on nitrogen doped carbon nanotubes (NCNTs). Strikingly, a significant electrochemically induced activation of the Mo3S13-NCNT catalyst at high current densities is observed in full cell configuration, enabling a remarkable current density of 4 A cm−2 at a cell voltage of 2.36 V. To the authors’ knowledge, this is the highest reported value to date for a PEMWE full cell using a non-noble metal HER catalyst. Furthermore, only a minor degradation of 83 μV h−1 is observed during a stability test of 100 h constant current at 1 A cm−2, with a nearly unchanged polarization behavior after the current hold. Catalyst stability and activity are additionally analyzed via online dissolution measurements. X-ray photoelectron spectroscopy examination of the catalyst before and after electrochemical application reveals a correlation between the electrochemical activation occurring via electrodissolution with changes in the molecular structure of the Mo3S13-NCNT catalyst.
• D. Podbiel, L. Boecking, H. Bott, J. Kassel, D. Czurratis, F. Laermer, R. Zengerle, J. Hoffmann
  From CAD to microfluidic chip within one day: rapid prototyping of lab-on-chip cartridges using generic polymer parts
  2020 J Micromech Microeng, volume: 30, issue: 11, page: 5012
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  Abstract

  We report on a novel rapid prototyping approach for the manufacturing of highly individualized lab-on-chip (LoC) cartridges from generic polymer parts by laser micromachining and laser welding. The approach allows an immediate implementation of microfluidic networks, components, and functionalities into an existing LoC platform without the need for an expensive and time-consuming fabrication of production tools like molds or masks. We comprehensively describe the individual process steps of the rapid prototyping procedure including a wet-chemical treatment for an easy and effective surface polishing of laser micromachined polymer parts. For laying out, we introduce a generalized diagrammatic description of microfluidic functional units in order to design application-specific cartridges for molecular diagnostic workflows. We demonstrate the usability of our prototyped cartridges by performing microfluidic experiments within. Due to the use of generic polymer parts, our rapid prototyping approach combines a high degree of freedom with an intrinsic compatibility to an established and highly developed LoC system. By enabling an experimental testing within one day, the rapid prototyping procedure shortens development cycles and boosts the evolution of microfluidic networks as well as the implementation of novel microfluidic components and functionalities.
• D. Podbiel, F. Laermer, R. Zengerle, J. Hoffmann
  Fusing MEMS technology with lab-on-chip: nanoliter-scale silicon microcavity arrays for digital DNA quantification and multiplex testing
  2020 Microsyst Nanoeng, volume: 6, page: 82
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  Abstract

  We report on the development of a microfluidic multiplexing technology for highly parallelized sample analysis via quantitative polymerase chain reaction (PCR) in an array of 96 nanoliter-scale microcavities made from silicon. This PCR array technology features fully automatable aliquoting microfluidics, a robust sample compartmentalization up to temperatures of 95 °C, and an application-specific prestorage of reagents within the 25 nl microcavities. The here presented hybrid silicon–polymer microfluidic chip allows both a rapid thermal cycling of the liquid compartments and a real-time fluorescence read-out for a tracking of the individual amplification reactions taking place inside the microcavities. We demonstrate that the technology provides very low reagent carryover of prestored reagents < 6 × 10−2 and a cross talk rate < 1 × 10−3 per PCR cycle, which facilitate a multi-targeted sample analysis via geometric multiplexing. Furthermore, we apply this PCR array technology to introduce a novel digital PCR-based DNA quantification method: by taking the assay-specific amplification characteristics like the limit of detection into account, the method allows for an absolute gene target quantification by means of a statistical analysis of the amplification results.
• F. Koch, K. Tröndle, G. Finkenzeller, R. Zengerle, S. Zimmermann, P. Koltay
  Generic method of printing window adjustment for extrusion-based 3D-bioprinting to maintain high viability of mesenchymal stem cells in an alginate-gelatin hydrogel
  2020 Bioprinting, page: e00094
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  Abstract

  Over the last decade, bioprinting of artificial tissues has been developed into a significant field of research. With an increasing number of printing technologies and bioinks used in bioprinting, its complexity increases as both the printing technology and the properties of the bioink influence the cell biological functionality and printing accuracy of the printed tissue. Therefore, optimization of bioprinting processes often remains a challenge, which could be solved by a smart fine-tuning of the process parameters. We present a novel method to adjust the printing window for extrusion-based bioprinting on the basis of a two-step assessment to determine process parameters such as nozzle size, extrusion flow rate, and printing temperature. First, a suitable printing temperature is deduced from the bioink properties and second nozzle size and extrusion flow rate is selected in a way that the immediate cell damage after printing is reduced. For both steps only basic rheological properties of the bioinks need to be known as well as detailed knowledge of the cell survival in the bioink for different shear stresses. This method is applied to an exemplary alginate-gelatin hydrogel to show how the printing temperature affects the achievable printing accuracy. For this bioink, viability of immortalized mesenchymal stem cells (iMSC) decreases with about 4% per thousand Pascal increase in maximum shear stress. For different combinations of flow rate, nozzle size and nozzle shape it is shown, that only the maximum shear stress experienced by the iMSCs influences average cell viability. Factors like flow rate, nozzle size and shape only play an indirect role by influencing the maximum shear stress and individually have no significant influence on cell viability. The experimental results allow a direct adjustment of printing parameters for the presented combination of hydrogel and cell type but are not limited to it. For other bioinks, the described generic method can be easily used to systematically adjust the printing parameters. For this purpose, only the basic rheological properties and the influence of shear stress on cell survival need to be known and process parameters can be set concerning the respective application.
• Y.-T. Kao, T. S. Kaminski, W. Postek, J. Guzowski, K. Makuch, A. Ruszczak, F. von Stetten, R. Zengerle, P. Garstecki
  Gravity-driven microfluidic assay for digital enumeration of bacteria and for antibiotic susceptibility testing
  2020 Lab Chip, volume: 20, pages: 54 - 63
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  Abstract

  The alarming dynamics of antibiotic-resistant infections calls for the development of rapid and point-of-care (POC) antibiotic susceptibility testing (AST) methods. Here, we demonstrated the first completely stand-alone microfluidic system that allowed the execution of digital enumeration of bacteria and digital antibiograms without any specialized microfluidic instrumentation. A four-chamber gravity-driven step emulsification device generated ∼2000 monodisperse 2 nanoliter droplets with a coefficient of variation of 8.9% of volumes for 95% of droplets within less than 10 minutes. The manual workload required for droplet generation was limited to the sample preparation, the deposition into the sample inlet of the chip and subsequent orientation of the chip vertically without an additional pumping system. The use of shallow chambers imposing a 2D droplet arrangement provided superior stability of the droplets against coalescence and minimized the leakage of the reporter viability dye between adjacent droplets during long-term culture. By using resazurin as an indicator of the growth of bacteria, we were also able to reduce the assay time to ∼5 hours compared to 20 hours using the standard culture-based test.
• G.N. Belibasakis, B. Lund, C. Krüger Weiner, B. Johannsen, D. Baumgartner, D. Manoil, M. Hultin, K. Mitsakakis
  Healthcare Challenges and Future Solutions in Dental Practice: Assessing Oral Antibiotic Resistances by Contemporary Point-of-Care Approaches
  2020 Antibiotics, volume: 9, page: 810
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  Abstract

  Antibiotic resistance poses a global threat, which is being acknowledged at several levels, including research, clinical implementation, regulation, as well as by the World Health Organization. In the field of oral health, however, the issue of antibiotic resistances, as well as of accurate diagnosis, is underrepresented. Oral diseases in general were ranked third in terms of expenditures among the EU-28 member states in 2015. Yet, the diagnosis and patient management of oral infections, in particular, still depend primarily on empiric means. On the contrary, on the global scale, the field of medical infections has more readily adopted the integration of molecular-based systems in the diagnostic, patient management, and antibiotic stewardship workflows. In this perspective review, we emphasize the clinical significance of supporting in the future antibiotic resistance screening in dental practice with novel integrated and point-of-care operating tools that can greatly support the rapid, accurate, and efficient administration of oral antibiotics.
  
  
   
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• E.C. Ortiz, F. Hegge, M. Breitwieser, S. Vierrath
  Improving the performance of proton exchange membrane water electrolyzers with low Ir-loaded anodes by adding PEDOT: PSS as electrically conductive binder
  2020 Rsc Adv, volume: 10, issue: 62, pages: 37329 - 37927
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  Abstract

  Reducing the iridium catalyst loading in the anode of polymer electrolyte membrane electrolyzers is a major goal to bring down the cost. However, anodes with low Ir-loading can suffer from poor electrical connectivity and hence lower the efficiency of the electrolyzer. In this work, we replace parts of the Nafion binder in the anode with an electrically conductive polymer (poly-3,4-ethylenedioxythiophene and polystyrene sulfonate acid complex, PEDOT:PSS) to counter this effect. At the optimal 50 : 50 blend we achieve a 120 mV lower overpotential (2.02 V) at 3 A cm−2 compared to a pure Nafion reference (2.14 V). This corresponds to a 6% better efficiency. Ex situ resistivity measurements and high frequency resistance measurements indicate that the major cause for this improvement lies in the reduced electrical in-plane resistance due to the electrical conductivity of PEDOT:PSS.
• P. Veh, B. Britton, S. Holdcroft, R. Zengerle, S. Vierrath, M. Breitwieser
  Improving the water management in anion-exchange membrane fuel cells via ultra-thin, directly deposited solid polymer electrolyte
  2020 Rsc Adv, volume: 10, pages: 8645 - 8652
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  Abstract

  Thin ionomer membranes are considered key to achieve high performances in anion exchange membrane fuel cells. However, the handling of unsupported anion exchange membranes with thicknesses below 15 μm is challenging. Typical pre-treatments of KOH-soaking, DI-water rinsing and/or wet assembly with sub-15 μm thin films are particularly problematic. In this work, we report configurations of membrane electrode assemblies with solid polymer electrolyte thicknesses equivalent to 3, 5 and 10 μm, made possible by direct coating of the ionomer onto gas diffusion electrodes (direct membrane deposition). The anion-conducting solid polymer electrolyte employed is hexamethyl-p-terphenyl poly(benzimidazolium) (HMT-PMBI), which is known for its high mechanical stability and low rate of gas crossover. By fabricating membrane-electrode-assemblies with PtRu/C anodes and Pt/C cathodes with a low precious metal loading of <0.3 mg cm−2, reproducible performances beyond 1 W cm−2 in H2/O2 atmosphere are achieved. The thin membranes enable excellent performance robustness towards changes in relative humidity, as well as low ionic resistances (<40 mOhm cm2).
• P. Rukavina, F. Koch, M. Wehrle, K. Tröndle, G. B. Stark, P. Koltay, S. Zimmermann, R. Zengerle, F. Lampert, S. Strassburg, G. Finkenzeller, F. Simunovic
  In vivo evaluation of bioprinted prevascularized bone tissue
  2020 Biotechnol Bioeng, pages: 1 - 10
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  Abstract

  Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three‐dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose‐derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion‐based bioprinting and drop‐on‐demand (DoD) bioprinting, respectively. The computer‐generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical‐sized bone defects.
• M. Trotter, D. Juric, Z. Bagherian, N. Borst, K. Gläser, T. Meissner, F. von Stetten, A. Zimmermann
  Inkjet-Printing of Nanoparticle Gold and Silver Ink on Cyclic Olefin Copolymer for DNA-Sensing Applications
  2020 Sensors, volume: 20, issue: 5, page: 1333
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  Abstract

  Inkjet technology as a maskless, direct-writing technology offers the potential for structured deposition of functional materials for the realization of electrodes for, e.g., sensing applications. In this work, electrodes were realized by inkjet-printing of commercial nanoparticle gold ink on planar substrates and, for the first time, onto the 2.5D surfaces of a 0.5 mm-deep microfluidic chamber produced in cyclic olefin copolymer (COC). The challenges of a poor wetting behavior and a low process temperature of the COC used were solved by a pretreatment with oxygen plasma and the combination of thermal (130 °C for 1 h) and photonic (955 mJ/cm²) steps for sintering. By performing the photonic curing, the resistance could be reduced by about 50% to 22.7 μΩ cm. The printed gold structures were mechanically stable (optimal cross-cut value) and porous (roughness factors between 8.6 and 24.4 for 3 and 9 inkjet-printed layers, respectively). Thiolated DNA probes were immobilized throughout the porous structure without the necessity of a surface activation step. Hybridization of labeled DNA probes resulted in specific signals comparable to signals on commercial screen-printed electrodes and could be reproduced after regeneration. The process described may facilitate the integration of electrodes in 2.5D lab-on-a-chip systems.
• C. Van Pham, M. Bühler, J. Knöppel, M. Bierling, D. Seeberger, D. Escalera-López, K. Mayrhofer, S. Cherevko, S. Thiele
  IrO2 coated TiO2 core-shell microparticles advance performance of low loading proton exchange membrane water electrolyzers
  2020 Applied Catalysis B: Environmental, volume: 269, page: 118762
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  Abstract

  Herein we present novel IrO2 coated TiO2 core-shell microparticles (IrO2@TiO2) as an oxygen evolution reaction catalyst. We compare the IrO2@TiO2 catalyst to commercial TiO2 supported IrO2 catalyst (IrO2/TiO2) and pure IrO2 catalyst powder. A stability analysis via on-line inductively coupled plasma mass spectrometry based on the S-number, a descriptor considering both energy efficiency and catalyst utilization efficiency, shows that the IrO2@TiO2 catalyst shows high potential for practical applications. This was further confirmed by full-cell tests showing superior performance of the IrO2@TiO2 catalyst with moderate and low loadings of 1.2 mgIr cm−2 and 0.4 mgIr cm−2, respectively. The core-shell catalyst is synthesized via facile route suitable for large quantities. Moreover, stable inks from the synthesized catalyst powder make this system appealing for large scale manufacturing of cells. Given the facile synthesis route, high activity, and good stability, the IrO2@TiO2 catalyst is potentially suitable for industry proton exchange membrane water electrolysis application.
• L. Becherer, N. Borst, M. Bakheit, S. Frischmann, R. Zengerle, F. von Stetten
  Loop-mediated isothermal amplification (LAMP) – review and classification of methods for sequence-specific detection
  2020 Anal Methods-uk, volume: 12, pages: 717 - 746
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  Abstract

  In the course of the last 20 years, isothermal nucleic acid amplification tests have emerged as an important diagnostic tool, not only for clinical applications, but also for food quality control and environmental monitoring. Loop-mediated isothermal amplification (LAMP) is well known for its robust and highly sensitive and specific amplification of target DNA, which is achieved by utilizing up to six primers. Moreover, LAMP excels through its isothermal and energy efficient amplification requirements, rendering it a prime candidate for low-cost diagnostics and analysis at the point of need. Recently, methods for sequence-specific detection have gained more importance because, unlike sequence-independent detection methods, they are highly specific towards the target DNA. In the last 13 years, a variety of sequence-specific methods have emerged, based on a very diverse range of sensing techniques, including optical, magnetic, piezoelectric, electrochemical and magnetoresistive sensing. To give structure to the diverse multitude of sequence-specific methods, we created a systematic classification and provide a critical comparative evaluation according to a catalogue of criteria (analytical performance, multiplexing, quantification and instrumental requirements). Fluorescence-based detection, making up half of the methods, can be processed on open platforms and satisfies all the criteria listed before. Instrumental requirements are discussed in terms of complexity, portability and fluidic cartridges. In addition, the technological readiness level and the kind of platform (open versus method-tailored) are evaluated, the latter playing an important role in the miniaturization and automation of operational process steps. We also observe an increase in the use of smartphone-integrated sensors to improve LAMP-based point-of-need testing. In summary, recent developments in methods for the sequence-specific detection of LAMP demonstrate high potential for many future applications.
• P.N. Paqué, C. Herz, J.S. Jenzer, D.B. Wiedemeier, T. Attin, N. Bostanci, G.N. Belibasakis, K. Bao, P. Körner, T. Fritz, J. Prinz, P.R. Schmidlin, T. Thurnheer, F.J. Wegehaupt, K. Mitsakakis, J.R. Peham
  Microbial Analysis of Saliva to Identify Oral Diseases Using a Point-of-Care Compatible qPCR Assay
  2020 J Clin Med, volume: 9, page: 2945
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  Abstract

  Oral health is maintained by a healthy microbiome, which can be monitored by state-of-the art diagnostics. Therefore, this study evaluated the presence and quantity of ten oral disease-associated taxa (P. gingivalis, T. forsythia, T. denticola, F. nucleatum, C. rectus, P. intermedia, A. actinomycetemcomitans, S. mutans, S. sobrinus, oral associated Lactobacilli) in saliva and their clinical status association in 214 individuals. Upon clinical examination, study subjects were grouped into healthy, caries and periodontitis and their saliva was collected. A highly specific point-of-care compatible dual color qPCR assay was developed and used to study the above-mentioned bacteria of interest in the collected saliva. Assay performance was compared to a commercially available microbial reference test. Eight out of ten taxa that were investigated during this study were strong discriminators between the periodontitis and healthy groups: C. rectus, T. forsythia, P. gingivalis, S. mutans, F. nucleatum, T. denticola, P. intermedia and oral Lactobacilli (p < 0.05). Significant differentiation between the periodontitis and caries group microbiome was only shown for S. mutans (p < 0.05). A clear distinction between oral health and disease was enabled by the analysis of quantitative qPCR data of target taxa levels in saliva.
• L. Becherer, S. Knauf, M. Marks, S. Lueert, S. Frischmann, N. Borst, F. von Stetten, S. Bieb, Y. Adu-Sarkodie, K. Asiedu, O. Mitjà, M. Bakheit
  Multiplex Mediator Displacement Loop-Mediated Isothermal Amplification for Detection of Treponema pallidum and Haemophilus ducreyi
  2020 Emerg Infect Dis, volume: 26, pages: 282 - 288
• M. Schulz, S. Calabrese, F. Hausladen, H. Wurm, D. Drossart, K. Stock, A. M. Sobieraj, F. Eichenseher, M. J. Loessner, M. Schmelcher, A. Gerhardts, U. Goetz, M. Handel, A. Serr, G. Haecker, J. Li, M. Specht, P. Koch, M. Meyer, P. Tepper, R. Rother, M. Jehle, S. Wadle, R. Zengerle, F. von Stetten, N. Paust, N. Borst
  Point-of-care testing system for digital single cell detection of MRSA directly from nasal swabs
  2020 Lab Chip, volume: 20, pages: 2549 - 2561
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  Abstract

  We present an automated point-of-care testing (POCT) system for rapid detection of species- and resistance markers in methicillin-resistant Staphylococcus aureus (MRSA) at the level of single cells, directly from nasal swab samples. Our novel system allows clear differentiation between MRSA, methicillin-sensitive S. aureus (MSSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS), which is not the case for currently used real-time quantitative PCR based systems. On top, the novel approach outcompetes the culture-based methods in terms of its short time-to-result (1 h vs. up to 60 h) and reduces manual labor. The walk-away test is fully automated on the centrifugal microfluidic LabDisk platform. The LabDisk cartridge comprises the unit operations swab-uptake, reagent pre-storage, distribution of the sample into 20 000 droplets, specific enzymatic lysis of Staphylococcus spp. and recombinase polymerase amplification (RPA) of species (vicK) – and resistance (mecA) -markers. LabDisk actuation, incubation and multi-channel fluorescence detection is demonstrated with a clinical isolate and spiked nasal swab samples down to a limit of detection (LOD) of 3 ± 0.3 CFU μl−1 for MRSA. The novel approach of the digital single cell detection is suggested to improve hospital admission screening, timely decision making, and goal-oriented antibiotic therapy. The implementation of a higher degree of multiplexing is required to translate the results into clinical practice.
• B. Johannsen, D. Mark, N. Boillat-Blanco, A. Fresco, D. Baumgartner, R. Zengerle, K. Mitsakakis
  Rapid diagnosis of respiratory tract infections using a point-of-care platform incorporating a clinical decision support algorithm
  2020 Stud. in Health Technol. Inform, volume: 273, pages: 234 - 239
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  Abstract

  Respiratory Tract Infections (RTIs) are among the top reasons for visiting a General Practitioner (GP) and the main cause of unnecessary antibiotic prescriptions. Reducing inappropriate use is essential to decrease antibiotic resistance and adverse events. The goal of the Eurostars project “Respiotic” is to develop a new point-of-care (POC) platform based on the centrifugal microfluidic LabDisk that will detect the main responsible viruses and bacteria for community-acquired RTIs, including associated resistances and host biomarkers. The diagnostic platform will use a Polymerase Chain Reaction (PCR) and an immunoassay cartridge on the same instrument and provide the combined analysis within less than 1 h. An electronic clinical algorithm will co-assess the test results and act as a decision support tool for the GPs’ patient management and prescriptions.
• M. Rombach, S. Hin, M. Specht, B. Johannsen, J. Lüddecke, N. Paust, R. Zengerle, L. Roux, T. Sutcliffe, J. Peham, C. Herz, M. Panning, O. Donoso Mantke, K. Mitsakakis
  RespiDisk: a point-of-care platform for fully automated detection of respiratory tract infection pathogens in clinical samples
  2020 Analyst, volume: 145, pages: 7040 - 7047
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  Abstract

  We present the RespiDisk enabling the fully automated and multiplex point-of-care (POC) detection of (currently) up to 19 respiratory tract infection (RTI) pathogens from a single sample based on reverse transcriptase polymerase chain reaction (RT-PCR). RespiDisk comprises a RTI-specific implementation of the centrifugal microfluidic LabDisk platform and combines new and existing advanced unit operations for liquid control, thereby automating all assay steps only by a spinning frequency and temperature protocol in combination with the use of a permanent magnet for in situ bead handing. The capabilities of the system were demonstrated with 36 tested quality samples mimicking clinical conditions (clinical and/or cultured material suspended in transport medium or synthetic bronchoalveolar lavage (BAL)) from past external quality assessment (EQA) panels covering 13 of the 19 integrated RTI detection assays. In total, 36 samples × 19 assays/sample resulting in 684 assays were performed with the RespiDisk, and its analytical performance was in full agreement with the routine clinical workflow serving as reference. A strong feature of the platform is its universality since its components allow the simultaneous detection of a broad panel of bacteria and viruses in a single run, thereby enabling the differentiation between antibiotic-treatable diseases. Furthermore, the full integration of all necessary biochemical components enables a reduction of the hands-on time from manual to automated sample-to-answer analysis to about 5 min. The study was performed on an air-heated LabDisk Player instrument with a time-to-result of 200 min.
• J. Riba, J. Schoendube, S. Zimmermann, P. Koltay, R. Zengerle
  Single-cell dispensing and ‘realtime’cell classification using convolutional neural networks for higher efficiency in single-cell cloning
  2020 nature scientific reports, volume: 10, page: 1193
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  Abstract

  Single-cell dispensing for automated cell isolation of individual cells has gained increased attention in the biopharmaceutical industry, mainly for production of clonal cell lines. Here, machine learning for classification of cell images is applied for ‘real-time’ cell viability sorting on a single-cell printer. We show that an extremely shallow convolutional neural network (CNN) for classification of lowcomplexity cell images outperforms more complex architectures. Datasets with hundreds of cell images from four different samples were used for training and validation of the CNNs. The clone recovery, i.e. the fraction of single-cells that grow to clonal colonies, is predicted to increase for all the samples investigated. Finally, a trained CNN was deployed on a c.sight single-cell printer for ‘real-time’ sorting of a CHO-K1 cells. On a sample with artificially damaged cells the clone recovery could be increased from 27% to 73%, thereby resulting in a significantly faster and more efficient cloning. Depending on the classification threshold, the frequency at which viable cells are dispensed could be increased by up to 65%. This technology for image-based cell sorting is highly versatile and can be expected to enable cell sorting by computer vision with respect to different criteria in the future.
• M. Drews, S. Tepner, P. Haberzettl, H. Gentischer, W. Beichel, M. Breitwieser, S. Vierrath, D. Biro
  Towards 3D-lithium ion microbatteries based on silicon/graphite blend anodes using a dispenser printing technique
  2020 RSC Adv., volume: 10, pages: 22440 - 22448
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  Abstract

  In this work we present for the first time high capacity silicon/carbon–graphite blend slurries designed for application in 3D-printed lithium ion microbatteries (3D-MLIBs). The correlation between electrochemical and rheological properties of the corresponding slurries was systematically investigated with the prospect of production by an automated dispensing process. A variation of the binder content (carboxymethyl cellulose/styrene–butadiene rubber, CMC/SBR) between 6 wt%, 12 wt%, 18 wt% and 24 wt% in the anode slurry proved to be crucial for the printing process. Regarding the rheological properties increasing binder content leads to increased viscosity and yield stress values promising printed structures with high aspect ratios. Consequently, interdigital 3D-printed micro anode structures with increasing aspect ratios were printed with increasing binder content. For printed 6-layer structures aspect ratios of 6.5 were achieved with anode slurries containing 24 wt% binder. Electrochemical results from planar coin cell measurements showed that anodes containing 12 wt% CMC/SBR binder content exhibited stable cycling at the highest charge capacities of 484 mA h g−1 at a current rate of C/4. Furthermore, at 4C the cells showed high capacity retention of 89% compared to cycling at C/4. Based on this study and the given material formulation we recommend 18 wt% CMC/SBR as the best trade-off between electrochemical and rheological properties for future work with fully 3D-printed MLIBs.
• J.-N. Klatt, M. Depke, N. Goswami, N. Paust, R. Zengerle, F. Schmidt, T. Hutzenlaub
  Tryptic digestion of human serum for proteomic mass spectrometry automated by centrifugal microfluidics
  2020 Lab Chip, volume: 20, page: 2937
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  Abstract

  Mass spectrometry has become an important analytical tool for protein research studies to identify, characterise and quantify proteins with unmatched sensitivity in a highly parallel manner. When transferred into clinical routine, the cumbersome and error-prone sample preparation workflows present a major bottleneck. In this work, we demonstrate tryptic digestion of human serum that is fully automated by centrifugal microfluidics. The automated workflow comprises denaturation, digestion and acidification. The input sample volume is 1.3 μl only. A triplicate of human serum was digested with the developed microfluidic chip as well as with a manual reference workflow on three consecutive days to assess the performance of our system. After desalting and liquid chromatography tandem mass spectrometry, a total of 604 proteins were identified in the samples digested with the microfluidic chip and 602 proteins with the reference workflow. Protein quantitation was performed using the Hi3 method, yielding a 7.6% lower median intensity CV for automatically digested samples compared to samples digested with the reference workflow. Additionally, 17% more proteins were quantitated with less than 30% CV in the samples from the microfluidic chip, compared to the manual control samples. This improvement can be attributed to the accurate liquid metering with all volume CVs below 1.5% on the microfluidic chip. The presented automation solution is attractive for laboratories in need of robust automation of sample preparation from small volumes as well as for labs with a low or medium throughput that does not allow for large investments in robotic systems.
• S. Hin, D. Baumgartner, M. Specht, J. Lüddecke, E. M. Arjmand, B. Johannsen, L. Schiedel, M. Rombach, N. Paust, F. von Stetten, R. Zengerle, N. Wipf, P. Müller, K. Mavridis, J. Vontas, K. Mitsakakis, * Indicates equally contributing authors
  VectorDisk: A Microfluidic Platform Integrating Diagnostic Markers for Evidence-Based Mosquito Control
  2020 Processes, volume: 8, issue: 12, page: 1677
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  Abstract

  Effective mosquito monitoring relies on the accurate identification and characterization of the target population. Since this process requires specialist knowledge and equipment that is not widely available, automated field-deployable systems are highly desirable. We present a centrifugal microfluidic cartridge, the VectorDisk, which integrates TaqMan PCR assays in two feasibility studies, aiming to assess multiplexing capability, specificity, and reproducibility in detecting disk-integrated vector-related assays. In the first study, pools of 10 mosquitoes were used as samples. We tested 18 disks with 27 DNA and RNA assays each, using a combination of multiple microfluidic chambers and detection wavelengths (geometric and color multiplexing) to identify mosquito and malaria parasite species as well as insecticide resistance mechanisms. In the second study, purified nucleic acids served as samples to test arboviral and malaria infective mosquito assays. Nine disks were tested with 14 assays each. No false positive results were detected on any of the disks. The coeffcient of variation in reproducibility tests was <10%. The modular nature of the platform, the easy adaptation of the primer/probe panels, the cold chain independence, the rapid (2–3 h) analysis, and the assay multiplexing capacity are key features, rendering the VectorDisk a potential candidate for automated vector analysis.
• M. Schulz, S. Probst, S. Calabrese, A. Homann, N. Borst, M. Weiss, F. von Stetten, R. Zengerle, N. Paust
  Versatile Tool for Droplet Generation in Standard Reaction Tubes by Centrifugal Step Emulsification
  2020 Molecules, volume: 25, issue: 8, page: 1914
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  Abstract

  We present a versatile tool for the generation of monodisperse water-in-fluorinated-oil droplets in standard reaction tubes by centrifugal step emulsification. The microfluidic cartridge is designed as an insert into a standard 2 mL reaction tube and can be processed in standard laboratory centrifuges. It allows for droplet generation and subsequent transfer for any downstream analysis or further use, does not need any specialized device, and manufacturing is simple because it consists of two parts only: A structured substrate and a sealing foil. The design of the structured substrate is compatible to injection molding to allow manufacturing at large scale. Droplets are generated in fluorinated oil and collected in the reaction tube for subsequent analysis. For sample sizes up to 100 µL with a viscosity range of 1 mPa·s–4 mPa·s, we demonstrate stable droplet generation and transfer of more than 6 × 105 monodisperse droplets (droplet diameter 66 µm ± 3 µm, CV ≤ 4%) in less than 10 min. With two application examples, a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP), we demonstrate the compatibility of the droplet production for two main amplification techniques. Both applications show a high degree of linearity (ddPCR: R2 ≥ 0.994; ddLAMP: R2 ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance.
• C. Klose, T. Saatkamp, A. Münchinger, L. Bohn, G. Titvinidze, M. Breitwieser, K.-D. Kreuer, S. Vierrath
  Water Electrolyzers: All‐Hydrocarbon MEA for PEM Water Electrolysis Combining Low Hydrogen Crossover and High Efficiency
  2020 Adv Energy Mater
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  Abstract

  Hydrocarbon ionomers bear the potential to significantly lower the material cost and increase the efficiency of proton‐exchange membrane water electrolyzers (PEMWE). However, no fully hydrocarbon membrane electrode assembly (MEA) with a performance comparable to Nafion‐MEAs has been reported. PEMWE‐MEAs are presented comprising sPPS as membrane and electrode binder reaching 3.5 A cm−2 at 1.8 V and thus clearly outperforming state‐of‐the‐art Nafion‐MEAs (N115 as membrane, 1.5 A cm−2 at 1.8 V) due to a significantly lower high frequency resistance (57 ± 4 mΩ cm² vs 161 ± 7 mΩ cm²). Additionally, pure sPPS‐membranes show a three times lower gas crossover (<0.3 mA cm−2) than Nafion N115‐membranes (>1.1 mA cm−2) in a fully humidified surrogate test. Furthermore, more than 80 h of continuous operation is shown for sPPS‐MEAs in a preliminary durability test (constant current hold at 1 A cm−2 at 80 °C). These results rely on the unique transport properties of sulfonated poly(phenylene sulfone) (sPPS) that combines high proton conductivity with low gas crossover.

2019

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• B. Shanahan, T. Böhm, B. Britton, S. Holdcroft, R. Zengerle, S. Vierrath, S. Thiele, M. Breitwieser
  30 μm thin hexamethyl-p-terphenyl poly(benzimidazolium) anion exchange membrane for vanadium redox-flow batteries
  2019 Electrochem Commun, volume: 102, pages: 37 - 40
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  Abstract

  We present the first results of an anion exchange ionomer membrane, hexamethyl-p-terphenyl poly(benzimidazolium) (HMT-PMBI), in a vanadium redox flow battery. Anion exchange membranes exhibit superior vanadium crossover suppression compared to proton exchange membranes due to the Gibbs–Donnan effect. HMT-PMBI was benchmarked against a similarly thin Nafion XL membrane which allowed us to compare differences based solely on chemical properties of the ionomer materials. We report cycling data of 45 cycles at a current density of 150 mA/cm2 with excellent coulombic efficiency of >99.4%, energy efficiency of 80.6–74.2% and a low ohmic resistance of 0.219–0.255 Ω cm2. In addition, a three times lower self-discharge rate is obtained for the HMT-PMBI membrane compared to Nafion XL. HMT-PMBI is therefore a potential alternative for PFSA based ionomers in VRFB applications.
• B. Johannsen, L. Müller, D. Baumgartner, L. Karkossa, S. M. Früh, N. Bostanci, M. Karpíšek, R. Zengerle, N. Paust, K. Mitsakakis
  Automated Pre-Analytic Processing of Whole Saliva Using Magnet-Beating for Point-of-Care Protein Biomarker Analysis
  2019 Micromachines, volume: 10, issue: 12, page: 833
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  Abstract

  Saliva offers many advantages for point-of-care (PoC) diagnostic applications due to non-invasive, easy, and cost-effective methods of collection. However, the complex matrix with its non-Newtonian behavior and high viscosity poses handling challenges. Several tedious and long pre-analytic steps, incompatible with PoC use, are required to liquefy and homogenize saliva samples before protein analysis can be performed. We apply magnet-beating to reduce hands-on time and to simplify sample preparation. A magnet in a chamber containing the whole saliva is actuated inside a centrifugal microfluidic cartridge by the interplay of centrifugal and magnetic forces. Rigorous mixing, which homogenizes the saliva sample, is then initiated. Consequently, fewer manual steps are required to introduce the whole saliva into the cartridge. After 4 min of magnet-beating, the processed sample can be used for protein analysis. The viscosity of whole saliva has been reduced from 10.4 to 2.3 mPa s. Immunoassay results after magnet-beating for three salivary periodontal markers (MMP-8, MMP-9, TIMP-1) showed a linear correlation with a slope of 0.99 when compared to results of reference method treated samples. Conclusively, magnet-beating has been shown to be a suitable method for the pre-analytic processing of whole saliva for fully automated PoC protein analysis.
• P. Juelg, M. Specht, E. Kipf, M. Lehnert, C. Eckert, M. Keller, T. Hutzenlaub, F. von Stetten, R. Zengerle, N. Paust
  Automated serial dilutions for high-dynamic-range assays enabled by fill-level-coupled valving in centrifugal microfluidics
  2019 Lab Chip, volume: 19, pages: 2205 - 2219
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  Abstract

  We introduce a new concept for centrifugal microfluidics that enables fully automated serial dilution generation without any additional means besides temperature control. Key feature is time-independent, serial valving of mixing chambers by fill-level-coupled temperature change rate (FLC-TCR) actuated valving. The automated dilution is realized under continuous rotation which enables reliable control of wetting liquids without the need of any additional fabrication steps such as hydrophobic coatings. All fluidic features are implemented in a monolithic fashion and disks are manufactured by foil thermoforming for scalable manufacturing. The new valving concept is demonstrated to reliably prevent valving if the diluted sample is not added to the mixing chamber (n = 30) and ensures valving if the dilution stage is completed (n = 15). Accuracy and precision of the automated serial dilution are verified by on-disk generation of qPCR standard curve dilutions and compared with manually generated reference dilutions. In a first step, the 5-log-stages standard curves are evaluated in a commercial qPCR thermocycler revealing a linearity of R² ≥ 99.92 % for the proposed LabDisk method vs. R² ≥ 99.67 % in manual reference dilutions. In a second step, the disk automated serial dilution is combined with on-disk qPCR thermocycling and readout, both inside a LabDisk Player. A 4-log-stages linearity of R² ≥ 99.81 % and a sensitivity of one leukemia associated ETV6-RUNX1 mutant DNA copy in a background of 100,000 wild-type DNA copies is achieved.
• K. Tröndle, F. Koch, G. Finkenzeller, G. B. Stark, R. Zengerle, P. Koltay, S. Zimmermann
  Bioprinting of high cell density constructs leads to controlled lumen formation with self‐assembly of endothelial cells
  2019 Journal of Tissue Engineering and Regenerative Medicine, volume: 13, issue: 10, pages: 1883 - 1895
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  Abstract

  Active nutrient supply and waste product removal are key requirements for the fabrication of long term viable and functional tissue constructs of considerable size. This work aims to contribute to the fabrication of artificial perfusable networks with a bioprinting process, based on drop‐on‐demand (DoD) printing of primary endothelial cell (EC) suspension bioink (25 · 106 ± 3 · 106 cells/ml). The process results in prescribed lumen between two hydrogel layers, allowing its integration in common layering based bioprinting processes. Low volume bioink droplets (appr. 10 nl) as building blocks, were deposited between two fibrin or collagen I layers to realize shapeable, cell‐rich aggregates. Unattainable with manual positioning, DoD printing allowed precise fabrication of various designs, such as spheroidal‐, line‐shaped and Y‐branch cellular structures, with a mean lateral extension of 285 ± 81 μm. For basic characterization, the cell suspension building blocks were systematically compared to preformed spheroids of the same cell type, passage and number. Post printing investigations of initially loose cell arrangements showed self‐assembly and formation of central lumen with a mean cross‐sectional area of Ølumen = 6400 μm2 at day 3, lined by a single layer of CD31 positive ECs, as evaluated by confocal microscopy. Originating from this main lumen smaller, undirected side‐branches (Øbranches = 740 μm2) were formed by sprouting cells, inducing a first step towards a simplistic hierarchically organized network. These lumen could prospectively help for tissue construct perfusion in vitro or, potentially, as niche for angiogenesis of host vascularization in implants.
• M. Schulz, F. von Stetten, R. Zengerle, N. Paust
  Centrifugal Step Emulsification: How Buoyancy Enables High Generation Rates of Monodisperse Droplets
  2019 Langmuir : the ACS Journal of Surfaces and Colloids, volume: 35, issue: 30, pages: 9809 - 9815
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  Abstract

  We demonstrate that buoyancy in centrifugal step emulsification enables substantially higher generation rates of monodisperse droplets compared to pressure driven set-ups. Step emulsification in general can produce droplets in comparatively simple systems (only one moving liquid) with a low CV of <5% in droplet diameter and with a minimum dead volume. If operated below a critical capillary number, the droplet diameter is defined by geometry and surface forces only. Above that critical capillary number, however, jetting occurs, leading to an increased droplet diameter and CV. Consequently, generation rates of monodisperse droplets are limited in pressure-driven systems. In this paper, we show that centrifugal step emulsification can overcome this limitation by applying sufficient buoyancy to the system. The buoyancy, induced by the centrifugal field and a density difference of the continuous and disperse phase, supports droplet necking by pulling the forming droplet away from the nozzle. The influence of buoyancy is studied using specific microfluidic designs that allow for supplying different buoyancies to the same droplet generation rates. For a droplet diameter of 100 μm, droplet generation at rates above 2.8k droplets per second and nozzle were reached, which is an increase of more than a factor of 8 in comparison to pressure-driven systems.
• M. Wehrle, F. Koch, S. Zimmermann, P. Koltay, R. Zengerle, G. B. Stark, S. Strassburg, G. Finkenzeller
  Examination of Hydrogels and Mesenchymal Stem Cell Sources for Bioprinting of Artificial Osteogenic Tissues
  2019 Cellular and Molecular Bioengineering, pages: 1 - 15
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  Abstract

  Mesenchymal stem cells (MSCs) represent a very important cell source in the field of regenerative medicine and for bone and cartilage tissue engineering applications. Three-dimensional (3D) bioprinting has the potential to improve the classical tissue engineering concept as this technique allows the printing of cells with high spatial control of cell allocation within a 3D construct. In this study, we systematically compared different hydrogel blends for 3D bioprinting of MSCs by testing their cytocompatibility, ability to support osteogenic differentiation and their mechanical properties. In addition, we compared four different MSC populations isolated from different human tissues for their osteogenic differentiation capacity in combination with different hydrogels. The aim of this study was to identify the best MSC source and the most suitable hydrogel blend for extrusion-based bioprinting of 3D large-scaled osteogenic constructs.
• D. Kainz, S. M. Früh, T. Hutzenlaub, R. Zengerle, N. Paust
  Flow control for lateral flow strips with centrifugal microfluidics
  2019 Lab Chip, pages: 2718 - 2727
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  Abstract

  Lateral flow strips (LFSs) are widely used for clinical diagnostics. The restricted flow control of the current designs is one challenge to the development of quantitative and highly sensitive LFSs. Here, we present a flow control for LFSs using centrifugal microfluidics. In contrast to previously presented implementations of lateral flow membranes into centrifugal microfluidic cartridges, we direct the flow radially outwards through the membrane. We control the flow using only the centrifugal force, thus it is independent of membrane wetting properties and permeability. The flow rate can be decreased and increased, enabling control of incubation times for a wide variety of samples. We deduced a formula as a guideline for the integration of chromatographic membranes into centrifugal microfluidic disks to ensure that all the sample liquid flows through the membrane, hence safely avoiding bypass flow around the membrane. We verified the calculated operation conditions using different membranes, different flow rates, and different sample viscosities.
• M. Bühler, P. Holzapfel, D. McLaughlin, S. Thiele
  From catalyst coated membranes to porous transport electrode based configurations in PEM water electrolyzers
  2019 J Electrochem Soc, volume: 166, page: F1070
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  Abstract

  So far the superior cell polarization behavior of membrane electrode assemblies (MEAs) using catalyst coated membranes (CCMs) as compared to those using porous transport electrodes (PTEs) was a paradigm in proton exchange membrane water electrolyzers (PEMWEs). However, this paradigm was so far neither systematically investigated nor understood. In this study, we investigate the changes in PEMWE polarization behavior upon gradually changing the MEA from a full CCM toward a full PTE-type configuration. We explain all observed findings based on the idea for a structural model of discontinuous catalyst layers. Our results show, that for current densities above 750 mA cm−2, PTE-based MEAs can result in a better polarization behavior than CCMs. Therefore, the prevailing paradigm was disproved. CCMs showed better kinetics, while PTE-type configurations performed more reproducible than CCMs despite rougher surfaces. Due to the trend of a stabilizing HFR-free cell voltage, an improved mass transport behavior of the PTE-type configurations at high current densities is assumed. Within the error-tolerance, no clear differences between PTE and CCM-based configurations in ohmic resistance could be determined. We conclude that PTE-based configurations for PEMWE, as alternatives to standard CCM-configurations, could be highly important for future manufacturing techniques depending on the application's needs.
• F. Hegge, J. Sharman, R. Moroni, S. Thiele, R. Zengerle, M. Breitwieser, S. Vierrath
  Impact of Carbon Support Corrosion on Performance Losses in Polymer Electrolyte Membrane Fuel Cells
  2019 J Electrochem Soc, volume: 166, pages: F956 - F962
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  Abstract

  Corrosion of the carbon support leads to a severe decay in the performance of PEM fuel cells, mainly due to an increase in the oxygen transport resistance. To investigate the effect of degradation on oxygen transport, we cycled MEAs between 1−1.5 V and analyzed the electrode structure with FIB-SEM tomography at various ageing states. The tomography results show that the electrode structure changes over 1000 cycles in terms of thickness (7.8 to 6.5 μm), porosity (44 to 38%) and diffusivity (9 to 8 105 m2s−1). Limiting current measurements in the wet (hydrogen/air) and dry state (hydrogen pumping) allowed the pressure dependent and pressure independent mass transport resistances to be distinguished and to quantify the impact of product water. The pressure independent resistance increased from 24 to 41 sm−1. Considering the marginal contribution of the catalyst pore space resistance (3 to 4 sm−1) it is concluded that the largest portion of the increase (50%) is caused by an increased local mass transport resistance. This is due to a decrease of the electrode roughness factor (282 to 169). The limiting current under wet conditions shows that another 44% could stem from a change in the wetting behavior, while 6% remains unexplained.
• M. Solihul Mu’min, T. Böhm, R. Moroni, R. Zengerle, S. Thiele, S. Vierrath, M. Breitwieser
  Local hydration in ionomer composite membranes determined with confocal Raman microscopy
  2019 Journal of Membrane Science, volume: 585, pages: 126 - 135
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  Abstract

  Water management in electrochemical energy applications like fuel cells has a crucial impact on performance, in particular on the ionic conduction of ionomer membranes. To strengthen the understanding of water management in such devices, we report a novel method for non-destructive measurements of the hydration of composite membranes based on confocal Raman microscopy. Composite membranes were produced by spray-coating of Nafion into a mesh of electrospun poly(vinylidene fluoride-co-hexafluoropropylene)/polyvinylpyrrolidone (PVDF-HFP/PVP) blend nanofibers. Hydration levels of several pure nanofiber meshes and nanofiber/Nafion composites were evaluated by linear least squares fitting of reference Raman spectra to hyperspectral images. We found that spectral contribution of water to nanofiber spectra depends on the PVDF-HFP/PVP ratio and is independent from fiber diameter. Further, we were able to reliably determine nanofiber polymer composition of single fibers based on Raman spectroscopy. Raman imaging of composite membranes was performed at ambient air and fully hydrated conditions to study the local hydration in PVDF-HFP/PVP/Nafion composites as well as in a Nafion XL membrane. 2D through-plane mappings revealed that the nanofiber hydration positively correlated with PVP content. In the Nafion XL membrane, the polytetrafluoroethylene-based reinforcement was verified as a hydrophobic layer sandwiched between Nafion ionomer, which showed a more than 10% reduced hydration compared to the outer Nafion layers. These results motivate the use of confocal Raman microscopy as a novel method to investigate the local water distribution in ionomer composite membranes that are widely used in electrochemical energy conversion.
• U. Karakus, T. Thamamongood, K. Ciminski, W. Ran, S. C. Günther, M. O. Pohl, D. Eletto, C. Jeney, D. Hoffmann, S. Reiche, J. Schinköthe, R. Ulrich, J. Wiener, M. G. B. Hayes, M. W. Chang, A. Hunziker, E. Yángüez, T. Aydillo, F. Krammer, J. Oderbolz, M. Meier, A. Oxenius, A. Halenius, G. Zimmer, C. Benner, B. G. Hale, A. García-Sastre, M. Beer, M. Schwemmle, S. Stertz
  MHC class II proteins mediate cross-species entry of bat influenza viruses
  2019 Nature, volume: 567, pages: 109 - 112
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  Abstract

  Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR–Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.
• M. Bühler, P. Holzapfel, F. Hegge, M. Bierling, S. Vierrath, S. Thiele
  Optimization of porous transport electrodes for PEM water electrolysis
  2019 J Mater Chem A, volume: 165, page: F305
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  Abstract

  In this study we investigate the potential of porous transport electrode (PTE) based membrane electrode assemblies (MEAs) for proton exchange membrane water electrolysis. The focus is on the overpotential determining anodic PTE for the oxygen evolution reaction. The influences of catalyst loading, ionomer content and porous titanium substrate on the polarization behavior are analyzed. The comparison of a porous fiber-sintered substrate with a powder-sintered substrate shows no significant differences in the kinetic and mass transport regions. Ohmic losses, however, are lower for fiber PTEs above a catalyst loading of 1.0 mgIrO2 cm−2. Variations of the Nafion content in the catalyst layer reveal changes of mass transport and ohmic losses and have an influence on the reproducibility. Varying the noble metal loading and therefore the thickness of the applied catalyst layer influences the kinetic region and ohmic resistance of the MEAs. The best compromise between reproducibility and performance is found for a loading of 1.4 mgIrO2 cm−2 and 9 wt% Nafion. The stable operation of the aforementioned PTE is shown in a 200 h durability test at 2 A cm−2.
• J. F. Cahill, J. Riba, V. Kertesz
  Rapid, Untargeted Chemical Profiling of Single Cells in Their Native Environment
  2019 Anal Chem, volume: 91, pages: 6118 - 6126
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  Abstract

  We report a method that enables untargeted, high throughput, and quantitative mass spectrometric analysis of single cells from cell suspension without needing additional sample preparation procedures (e.g., molecular tagging) through the combination of single-cell printer technology and liquid vortex capture−mass spectrometry (SCP-LVC-MS). The operating principle behind the SCP-LVC-MS technology is single cell isolation via small droplet piezoelectric ejection followed by capture of the droplet into an LVC-MS sampling probe. Once exposed to an appropriate solvent, the cell is lysed, extracted, and analyzed by MS. The SCP-LVC-MS approach was validated by measuring the lipid composition of microalgae, Chlamydomonas reinhardtii (ChRe) and Euglena gracilis (EuGr), and HeLa cells in their native growth media. Numerous diacylglyceryltrimethylhomo-Ser (DGTS), phosphatidylcholine (PC), monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) lipids were observed in single cells. Continuous solvent flow ensures that cells are analyzed rapidly, and no signal carryover between cells is observed. ChRe and EuGr microalgae mixed together in the same solution were differentiated cell-by-cell in real-time based on differences between levels of diacylglyceryltrimethylhomo-Ser (DGTS) and phosphatidylcholine (PC) lipids measured in each cell. Several DGTS lipids present in ChRe were quantified with single-cell resolution by normalizing to a DGTS(32:0) internal standard added to the LVC probe solvent during analysis. Quantitative peak areas were validated by comparing to bulk lipid extracts. Lastly, peak area distributions comprised of hundreds of cells were compared for ChRe after 5 days of nitrogen-limited and normal growth conditions, which show clear differences and the ability to resolve cellular population differences with single-cell resolution.
• T. Boehm, R. Moroni, M. Breitwieser, S. Thiele, S. Vierrath
  Spatially Resolved Quantification of Ionomer Degradation in Fuel Cells by Confocal Raman Microscopy
  2019 J Electrochem Soc, volume: 166, issue: 7, pages: F3044 - F3051
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  Abstract

  Ionomer membranes are crucial components of many electrochemical devices. In this work, confocal Raman microscopy is employed to characterize Nafion ionomers quantitatively in pristine status and after usage as a proton exchange membrane in a fuel cell. Confocal Raman microscopy allows non-destructive thickness and equivalent weight measurements of Nafion with a 95% confidence interval of ±13 g mol−1 at an equivalent weight of 1000 g mol−1, which is significantly more accurate than previously reported methods. Characterization can be performed at a spatial resolution better than 2 μm, providing insights into local membrane degradation after fuel cell operation. Membrane thinning to less than 40% of the initial thickness of Nafion NR-211 occurs after a 100 h open circuit voltage hold, accompanied by an anisotropic increase of the equivalent weight from 1035 g mol−1 to an average of 1200 g mol−1. Most pronounced increases are found close to the anode. Further, the characterization of a Nafion XL membrane shows that its microporous reinforcement is represented as increased equivalent weight with local heterogeneities within the membrane. These results show that confocal Raman microscopy is a valuable tool to investigate ionomers that are used as ion exchange membranes in electrochemical devices.
• J.P. Hays, K. Mitsakakis, S. Luz, A. van Belkum, K. Becker, A. van den Brue, S. Harbarth, J. H. Rex, G. Skov Simonsen, G. Werner, V. Di Gregori, G. Lüdke, T. van Staa, J. Moran-Gilad, T. T. Bachmann, on behalf of the JPIAMR AMR-RDT
  The successful uptake and sustainability of rapid infectious disease and antimicrobial resistance point-of-care testing requires a complex ‘mix-and-match’ implementation package
  2019 Eur J Clin Microbiol, volume: 38, pages: 1015 - 1022
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  Abstract

  The emergence and spread of antimicrobial resistance is one of the major global issues currently threatening the health and wealth of nations, with effective guidelines and intervention strategies urgently required. Such guidelines and interventions should ideally be targeted at individuals, communities, and nations, requiring international coordination for maximum effect. In this respect, the European Joint Programming Initiative on Antimicrobial Resistance Transnational Working Group ‘Antimicrobial Resistance - Rapid Diagnostic Tests’ (JPIAMR AMR-RDT) is proposing to consider a ‘mix-and-match’ package for the implementation of point-of-care testing (PoCT), which is described in this publication. The working group was established with the remit of identifying barriers and solutions to the development and implementation of rapid infectious disease PoCT for combatting the global spread of antimicrobial resistance. It constitutes a multi-sectoral collaboration between medical, technological, and industrial opinion leaders involved in in vitro diagnostics development, medical microbiology, and clinical infectious diseases. The mix-and-match implementation package is designed to encourage the implementation of rapid infectious disease and antimicrobial resistance PoCT in transnational medical environments for use in the fight against increasing antimicrobial resistance.

2018

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• E. Kipf, S. Sané, D. Morse, T. Messinger, R. Zengerle, S. Kerzenmacher
  An air-breathing enzymatic cathode with extended lifetime by continuous laccase supply
  2018 Bioresource Technol, volume: 264, pages: 306 - 310
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  Abstract

  We present a novel concept of an air-breathing enzymatic biofuel cell cathode combined with continuous supply of unpurified laccase-containing supernatant of the white-rot fungus Trametes versicolor for extended lifetime. The air-breathing cathode design obviates the need for energy-intensive active aeration. In a corresponding longterm experiment at a constant current density of 50 μA cm−2, we demonstrated an increased lifetime of 33 days (cathode potential above 0.430 V vs. SCE), independent of enzyme degradation. The obtained data suggest that theoretically a longer lifetime is feasible. However, further engineering efforts are required to prevent clogging and fouling of the supply tubes. These results represent an important step towards the realization of enzymatic biofuel cell cathodes with extended lifetime and enhanced performance.
• B. Gerdes, M. Breitwieser, T. Kaltenbach, M. Jehle, J. Wilde, R. Zengerle, P. Koltay, L. Riegger
  Analysis of the metallic structure of microspheres produced by printing of aluminum alloys from the liquid melt
  2018 Mater Res Express, volume: 6, page: 036514
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  Abstract

  This work presents an analysis of the metallic structure of microspheres produced by drop-on-demand printing of the aluminum alloy AlSi12 directly printed from the liquid melt via StarJet technology. AlSi alloys are commonly used in casting processes, but microdroplets from these materials could potentially be used for additive manufacturing of metal and composite parts. Recently, several printing technologies were presented that enable the drop-on-demand printing of Al-alloy microdroplets. However, the material distribution and metallic structure inside of printed droplets is expected to be significantly different from the bulk material properties, and hardly any data on the microscopic structure of small droplets that have undergone rapid solidification has been published so far. Therefore, a microscopic in-depth study of microdroplets printed directly from the metal melt has been carried out: By the means of energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), Auger electron spectroscopy (AES) and optical microscopy the material properties as well as the droplet morphology are investigated for the first time. The analysis demonstrates that the Al alloy droplets printed via StarJet technology exhibit almost no oxidation during the printing process and can therefore potentially be used for additive manufacturing of metal parts. Moreover, the metallurgical structure inside the droplets is analyzed. It exhibits significant difference to the bulk material in terms of the average secondary dendrite arm spacing.
• J. Streeck, C. Hank, M. Neuner, L. Gil-Carrera, M. Kokko, S. Pauliuk, A. Schaadt, S. Kerzenmacher, R. J. White
  Bio-electrochemical conversion of industrial wastewater-COD combined with downstream methanol synthesis – an economic and life cycle assessment
  2018 Green Chem, volume: 20, pages: 2742 - 2762
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  Abstract

  Herein, a techno-economic and environmental performance evaluation (i.e. Life Cycle Assessment (LCA)) of a 45 kW Microbial Electrolysis Cell (MEC) system is presented in the context of industrial wastewater remediation. This system produces H2 and CO2 – suitable for downstream CH3OH synthesis – based on the bio-electrochemical conversion of chemical industry wastewater with an organic content of 3.9 g(COD) L−1. A cost–benefit analysis indicates that the MEC system hardware costs, share of CO2 captured from the MEC and MEC operating current density (i.e. 1.0 mA cm−2) are crucial parameters influencing the total cost and represent areas for potential cost reductions. It was established based on the present study that MEC system operation with renewable electricity leads to H2 production costs of 4–5.7€ kg(H2) −1 (comparable to H2O electrolysis) and CH3OH production costs of 900€ t(CH3OH) −1. At the current CH3OH market prices, however, the production is currently not profitable. In turn, the cost-efficient construction of the MEC system and the use of less expensive materials could lead to improved CH3OH production economics based on this route. Our results indicate that the use of low-cost materials has greater potential with regard to cost reduction compared to reducing the internal resistance and polarization losses via the use of expensive high-performance materials in MEC construction. A complementary LCA of the proposed system, based on a “cradle-to-gate” definition, indicates that waste-based is superior to fossil-based CH3OH production with respect to global warming potential and cumulated fossil energy demand, provided the system is operated with 100% renewable electricity and CO2 sourced only from the MEC. However, with regard to the impact categories Metal Depletion and Freshwater Eutrophication Potential, the system was found to perform less satisfactorily (i.e. in comparison with fossil-based CH3OH production).
• K. Mitsakakis, W.E. Kaman, G. Elshout, M. Specht, J.P. Hays
  Challenges in identifying antibiotic resistance targets for point-of-care diagnostics in general practice
  2018 Future Microbiol, volume: 13, issue: 10, pages: 1157 - 1164
  » show abstract« hide abstract
  Abstract

  General practitioners stand at the front line of healthcare provision and have a pivotal role in the fight against increasing antibiotic resistance. In this respect, targeted antibiotic prescribing by general practitioners would help reduce the unnecessary use of antibiotics, leading to reduced treatment failures, fewer side-effects for patients and a reduction in the (global) spread of antibiotic resistances. Current ‘gold standard’ antibiotic resistance detection strategies tend to be slow, taking up to 48 h to obtain a result, although the implementation of point-of-care testing by general practitioners could help achieve the goal of targeted antibiotic prescribing practices. However, deciding on which antibiotic resistances to include in a point-of-care diagnostic is not a trivial task, as outlined in this publication.
• S. H. Moosavi, M. Kroener, M. Frei, F. Frick, S. Kerzenmacher, P. Woias
  Development of a TEM Compatible Nanowire Characterization Platform With Self-forming Contacts
  2018 Ieee T Semiconduct M, volume: 31, issue: 1, pages: 1 - 10
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  Abstract

  A nanowire characterization platform is designed and fabricated in MEMS-technology for the thermoelectric and structural characterization of single nanowires. The latter is achieved by making the chip compatible to TEM holder, by restricting its thickness to less than 160μm and its diagonal dimensions to less than 3mm. Two different fabrication technologies are presented for the realization of such platform, based on a design reproducing the functional requirements. Our first fabrication technique is based on ICP etching, using (100)-silicon wafers, and a subsequent rear grinding. However, as the design includes a vertical wall trench structures, ICP etching of such deep recesses is time-consuming and expensive. Therefore, a second fabrication process is developed, making use of wet-etched (110)-silicon. With these substrates a challenge arises from the intersection of inclined {111} facets. To solve this natural limitation, we introduce a novel fabrication process. Our technique relies on damaging the intersecting {111} planes, to make them etchable again and to produce deeper trenches with vertical walls in a wet-chemical etching process. Additionally, the electrical contacts at the platform are made from porous metal to increase the surface-to-volume ratio, to increase the possibility of a spontaneous electrical contact between the electrodes and nanowires.
• C.H. Tsai, X. Wu, D.H. Kuan, S. Zimmermann, R. Zengerle, P. Koltay
  Digital hydraulic drive for microfluidics and miniaturized cell culture devices based on shape memory alloy actuators
  2018 J Micromech Microeng, volume: 28, page: 084001
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  Abstract

  In order to culture and analyze individual living cells, microfluidic cell culture and manipulation of cells becomes an increasingly important topic. Such microfluidic systems allow for exploring the phenotypic differences between thousands of genetically identical cells or pharmacological tests in parallel, which is impossible to achieve by traditional macroscopic cell culture methods. Therefore, plenty of microfluidic devices have been developed for cell biological studies like cell culture, cell sorting, and cell lysis in the past. However, these devices are still limited by the external pressure sources which most of the time are large in size and have to be connected by fluidic tubing leading to complex and delicate systems.&#13; In order to provide a miniaturized, more robust actuation system a novel, compact and low power consumption Digital Hydraulic Drive (DHD) has been developed that is intended for use in portable and automated systems for microfluidic applications. The DHD consists of a shape memory alloy (SMA) actuator and a pneumatic cylinder. The switching time of the digital modes (pressure ON vs. OFF) can be adjusted from 1 second to minutes. Thus, the DHDs might have many applications for driving microfluidic devices. In this work different implementations of DHDs are presented and their performance is characterized by experiments. In particular, it will be shown that DHDs can be used for microfluidic large-scale integration (mLSI) valve control (256 valves in parallel) as well as potentially for droplet-based microfluidic systems. As further application example high-throughput mixing of cell cultures (96 wells in parallel) are demonstrated employing the DHD and a so called "functional lid" (FL) approach, to enable a miniaturized microbioreactor in a regular 96-well micro well plate.
• B. Gerdes, R. Zengerle, P. Koltay, L. Riegger
  Direct printing of miniscule aluminum alloy droplets and 3D structures by StarJet technology
  2018 J Micromech Microeng, volume: 28, page: 074003
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  Abstract

  Drop-on demand printing of molten metal droplets could be used for prototyping of 3D objects as promising alternative to laser melting technologies. However, up to date only few printheads were investigated for this purpose using only a limited range of materials. The pneumatically actuated StarJet technology presented previously enables the direct and non-contact printing of molten metal microdroplets from metal melts at high temperatures. Printheads according to the StarJet technology utilize nozzle chips featuring a star-shaped orifice geometry that leads to a formation of the droplets inside the nozzle with high precision. In this paper we present a novel StarJet printhead for printing aluminum alloys featuring a hybrid design with a ceramic reservoir for the molten metal and an outer shell fabricated from stainless steel. The micro machined nozzle chip is made from silicon carbide (SiC). This printhead can be operated at up to 950 °C and is capable of printing high melting metals like aluminum (Al) alloys in standard laboratory conditions. In this work, an aluminum-silicon alloy that features 12 % silicon (AlSi12) is printed. The printhead, the nozzle and the peripheral actuation system have been optimized for stable generation of AlSi12 droplets with high monodispersity, low angular deviation and miniaturized droplet diameters. As main result, a stable drop-on-demand printing of droplets exhibiting diameters of ddroplet = 702 µm +/- 1 % was demonstrated at 5 Hz with a low angular deviation of 0.3°, when a nozzle chip with 500 µm orifice diameter was used. Further, AlSi12 droplets featuring ddroplet = 176 µm ± 7 % were printed when using a nozzle chip with an orifice diameter of 130 µm. Moreover, we present directly printed objects from molten aluminum alloy droplets such as high aspect ratio, free-standing walls (aspect ratio 12:1) and directly printed, flexible springs to demonstrate the principle of 3D printing with molten metal droplets.
• C. Van Pham, B. Britton, T. Böhm, S. Holdcroft, S. Thiele
  Doped, Defect-Enriched Carbon Nanotubes as an Efficient Oxygen Reduction Catalyst for Anion Exchange Membrane Fuel Cells
  2018 Adv Mater Interfaces, page: 1800184
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  Abstract

  Bond polarization of doped atoms and carbon and lattice defects are con- sidered important aspects in the catalytic mechanisms of oxygen reduction reaction (ORR) on heteroatom-doped carbon catalysts. Previous work on metal-free catalysts has focused either on bond polarization or lattice defects. Here multi-heteroatom doped defect-enriched carbon nanotubes (MH-DCNTs) that combine both effects to enhance ORR activity are designed. Lattice defects in MH-DCNTs are enriched by unzipping and length-shortening of carbon nanotubes, and also by creating carbon vacancies via decomposition of doped F atoms. Electrochemical analysis using rotating disc electrode voltam- metry shows that the ORR kinetic current density of MH-DCNT increases with lattice-defect density, the latter of which is verified by Raman spectroscopy, while the onset potential increases with annealing temperatures. An optimized MH-DCNT ORR catalyst exhibits a half-wave potential of 0.81 V versus revers- ible hydrogen electrode and limiting current density of 5.0 mA cm −2 at an electrode rotation speed of 1600 rpm in 0.1 M KOH. Further, it is demonstrated that MH-DCNT, as a cathode catalyst layer in an anion exchange membrane fuel cell (AEMFC), delivers a peak power density of 250 mW cm −2 , which is ≈70% the performance of an AEMFC using a conventional Pt/C catalyst.
• M. Lehnert, E. Kipf, F. Schlenker, N. Borst, R. Zengerle, F. von Stetten
  Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides
  2018 Anal Methods-uk
  » show abstract« hide abstract
  Abstract

  In this study we optimised the fluorescence signal generation of contact quenched universal reporter oligonucleotides. These are used as secondary probes in real-time Mediator Probe PCR to detect the sequence-specific cleavage of label-free primary mediator probes. Since the fluorescence signal generation of a universal reporter is not influenced by the target DNA sequence, optimisation of the fluorescence signal-to-noise ratio will improve the performance of all Mediator Probe PCRs that are based on this type of universal reporter. To determine the critical factors influencing signal-to-noise optimisation, we systematically analysed four parameters. These parameters were type of fluorophore, type of quencher molecule, intramolecular orientation of both residuals, and the number of quencher labels. In total, more than 30 different fluorogenic universal reporter structures were analysed, covering the whole fluorescence spectrum from green to crimson. From our results, we deduced a novel set of guidelines for signal-to-noise optimisation in the design of contact quenched, fluorogenic universal reporter oligonucleotides. We confirmed these guidelines in a different thermocycler, and by designing a second set of universal reporters, which were used for multiplex real-time PCR quantification of acute lymphoblastic leukaemia marker sequences. This optimised biplex Mediator Probe PCR showed an improved performance under clinical conditions, with a 10 times higher resolution regarding the limit of quantification. In addition to Mediator Probe PCR, these guidelines may also prove useful in signal-to-noise optimisation of other fluorescence-based assays where contact quenched oligonucleotides or secondary reporter molecules are used.
• B. Gerdes, M. Jehle, N. Lass, L. Riegger, A. Spribille, M. Linse, F. Clement, R. Zengerle, P. Koltay
  Front side metallization of silicon solar cells by direct printing of molten metal
  2018 Solar Energy Materials and Solar Cells, volume: 180, pages: 83 - 90
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  Abstract

  In this work, a new approach for the front side metallization of silicon solar cells is presented. Molten solder (Sn96Ag3Cu) is directly printed in a non-contact manner on solar cell precursors via StarJet technology. The StarJet technology features a pneumatically driven, heatable printhead with a reservoir of molten metal and a star-shaped silicon nozzle chip. Using this printhead, a jet of molten metal with 55 μm ± 5 μm diameter is generated and used to apply busbars as well as contact fingers on prefabricated electroplated seed layers. After deposition via StarJet, printed fingers have a minimum width of 70 μm and a mean aspect ratio of 0.94. The printed metallization is evaluated optically and electrically. Aluminum back surface field silicon solar cells with front side electroplated NiAg seed layers and StarJet metallization (busbars and fingers) show efficiencies of up to 18.1% after degradation. Solder is about 30–40 times cheaper than silver and therefore may allow costefficient solar cell metallization. The StarJet metallization on electroplated NiAg seed layers is fully functional and requires no additional post-processing steps. Only 6 mg of Ag per cell are consumed for the seed layer. As a proof-of-principle, a module is demonstrated, which consists of four solar cells that are metallized via StarJet.
• K. Kraiczek, G. Rozing, R. Zengerle
  G-Index: A New Metric to Describe Dynamic Refractive Index Effects in HPLC Absorbance Detection
  2018 Talanta, volume: 187, pages: 200 - 206
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  Abstract

  High performance liquid chromatography (HPLC) with a solvent gradient and absorbance detection is one of the most widely used methods in analytical chemistry. The observed absorbance baseline is affected by the changes in the refractive index (RI) of the mobile phase. Near the limited of detection, this complicates peak quantitation. The general aspects of these RI-induced apparent absorbance effects are discussed. Two different detectors with fundamentally different optics and flow cell concepts, a variable-wavelength detector equipped with a conventional flow cell and a diode-array detector equipped with a liquid core waveguide flow cell, are compared with respect to their RI behavior. A simple method to separate static – partly unavoidable – RI effects from dynamic RI effects is presented. It is shown that the dynamic RI behavior of an absorbance detector can be well described using a single, relatively easy-to-determine metric called the G-index. The G-index is typically in the order of a few seconds and its sign depends on the optical flow cell concept.
• J. M. Stosch, A. Heumüller, C. Niemöller, S. Bleul, M. Rothenberg-Thurley, J. Riba, N. Renz, K. Szarc vel Szic, D. Pfeifer, M. Follo, H. L. Pahl, S. Zimmermann, J. Duyster, J. Wehrle, M. Lübbert, K. H. Metzeler, R. Claus, H. Becker
  Gene mutations and clonal architecture in myelodysplastic syndromes and changes upon progression to acute myeloid leukaemia and under treatment
  2018 Brit J Haematol, volume: 182, pages: 830 - 842
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  Abstract

  Knowledge of the molecular and clonal characteristics in the myelodysplastic syndromes (MDS) and during progression to acute myeloid leukaemia (AML) is essential to understand the disease dynamics and optimize treatment. Sequencing serial bone marrow samples of eight patients, we observed that MDS featured a median of 3 mutations. Mutations in genes involved in RNA-splicing or epigenetic regulation were most frequent, and exclusively present in the major clone. Minor subclones were distinguishable in three patients. As the MDS progressed, a median of one mutation was gained, leading to clonal outgrowth. No AML developed genetically independent of a pre-existing clone. The gained mutation mostly affected genes encoding signalling proteins. Additional acquisition of genomic aberrations frequently occurred. Upon treatment, emergence of new clones could be observed. As confirmed by single-cell sequencing, multiple mutations in identical genes in different clones were present within individual patients. DNA-methylation profiling in patients without identification of novel mutations in AML revealed methylation changes in individual genes. In conclusion, our data complement previous observations on the mutational and clonal characteristics in MDS and at progression. Moreover, DNA-methylation changes may be associated with progression in single patients. Redundancy of mutated genes in different clones suggests fertile grounds promoting clonal selection or acquisition.
• C. Klose, P. Trinke, T. Böhm, B. Bensmann, S. Vierrath, R. Hanke-Rauschenbach, S. Thiele
  Membrane Interlayer with Pt Recombination Particles for Reduction of the Anodic Hydrogen Content in PEM Water Electrolysis
  2018 J Electrochem Soc, volume: 165, pages: F1271 - F1277
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  Abstract

  Polymer electrolyte membrane (PEM) water electrolysis is a key technology for sustainable hydrogen based energy supply. Gaspermeation through the PEM leads to hydrogen in oxygen at the anode side posing a safety hazard and therefore restricting theoperation window of PEM water electrolysis, especially when operating under pressure. In this work the hydrogen in oxygen contentat the anode is significantly reduced when a recombination interlayer is integrated into the membrane electrode assemblies (MEAs)compared to reference MEAs without interlayer. The recombination interlayer with a platinum loading of 0.02 mg cm−2is sprayedbetween two membranes that are coated with anode and cathode catalysts on the outside. The permeating H2and O2forms waterat the recombination interlayer, leading to higher gas purity and resolving safety issues. In case of the MEAs with interlayer alsoa constant current hold at 1 A cm−2for 245 h revealed only a slight increase of the hydrogen in oxygen content (below 140·10−6vol.% h−1) whereas for the reference MEAs without interlayer a stronger increase was observed (above 1250·10−6vol.% h−1).Furthermore, the long-term experiments showed no increased degradation rates compared to the reference MEAs.
• S. Hin, M. Loskyll, V. Klein, M. Keller, O. Strohmeier, F. von Stetten, R. Zengerle, K. Mitsakakis
  Membrane-based sample inlet for centrifugal microfluidic cartridges
  2018 Microelectron Eng, volume: 187-188, pages: 78 - 83
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  Abstract

  Centrifugal microfluidics enables the rapid execution of complex blood sample analyses in a fully automated manner at the point of care. However, during blood sample addition, the cartridge is at rest and centrifugal forces are not present to allocate the sample in a controlled way. We present a versatile approach for the user-friendly, well-controlled and safe sample addition into a centrifugal microfluidic cartridge for use in serology. It features a commercial (plasma separation) membrane stacked into the inlet chamber of the cartridge. This combination of sample inlet and plasma separation in one structural unit brings the advantage of reducing footprint in highly integrated point-of-care testing. By using a pipette the user may add a blood sample (90 μL) to the membrane holding the liquid by capillary forces. Flowing across the membrane, cells separate from blood plasma. The blood plasma releases into the downstream structure upon centrifugation. The mean plasma recovery rate was 57.3 (± 4.7) % and mean plasma purity 99.5 (± 0.6) % from samples of varying hematocrit (36%–59%). Furthermore, the analyte C-reactive protein (CRP) did not significantly adsorb to the membrane. This was concluded, since CRP immunoassay results with plasma from spiked whole blood, obtained from membrane-based plasma separation and from plasma separation on a standard laboratory centrifuge, did not significantly differ. Thus, the suggested approach is promising for simultaneous application as sample inlet holding the sample by capillary forces and as a plasma separation module for centrifugal microfluidics. Potential future applications may include other sample matrices. The use with further blood transfer devices (capillary, directly from fingertip) seems possible, yet requiring further evaluation.
• M. Frei, J. Martin, S. Kindler, G. Cristiano, R. Zengerle, S. Kerzenmacher
  Power supply for electronic contact lenses: Abiotic glucose fuel cells vs. Mg/air batteries
  2018 J Power Sources, volume: 401, pages: 403 - 414
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  Abstract

  Electronic contact lenses are a promising platform for medical sensors. With these devices a variety of vital signs and medical parameters can be monitored noninvasively and without the risk of foreign body response. However, one current limitation of this technology is the need for an external power supply, resulting in bulky, multi component devices. In this paper, we for the first time investigate and compare the application of abiotic glucose fuel cells and Mg/air batteries as alternative power supply technologies for electronic contact lenses. While abiotic glucose fuel cells harvest energy from metabolites present in tear fluid, Mg/air batteries provide electricity by the oxidation of a sacrificial anode. Considering the space available on standard contact lenses, our results indicate that approx. 40 μW and 2 μW can be generated by Mg/air batteries and glucose fuel cells for a period of at least 24 h, respectively. However, coating galvanic cells with the commonly used contact lens material pHEMA, results in drastically reduced performance, presumably due to hindered mass transport. Nevertheless, even under those circumstances a Mg/air battery can still provide about 7 μW for 24 h, which would already be sufficient for many electronic contact lens applications.
• M. Frei, C. Köhler, L. Dietel, J. Martin, F. Wiedenmann, R. Zengerle, S. Kerzenmacher
  Pulsed electro-deposition of highly porous Pt-alloys for the use in methanol, formic acid, and glucose fuel cells
  2018 Chemelectrochem, pages: 1013 - 1023
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  Abstract

  We demonstrate an electro-deposition process for the fabrication of highly porous PtCu-alloy anodes. In the fabrication process, Pt and different amounts of a second noble metal (Pd, Ru, Au) are repeatedly co-deposited with Cu from an aqueous electrolyte, followed by selective dealloying of Cu. This way, highly porous PtCu alloys with roughness factors ranging from 400 to 4000 can be obtained. In all cases both noble metal partners are present on the electrode surface, whereas the majority of copper is likely buried underneath. In addition, we can show that H-desorption and COstripping yield substantially different roughness factors, even when applied to PtCu anodes. Hence, when using or comparing results from different stripping methods a calibration is required. Compared to PtCu anodes, small additions of Ru (~3 at% Ru) lead to significantly enhanced catalytic activity for the electro-oxidation of formic acid and methanol, whereas Au-rich PtCu-Au alloys (~75 at% Au) exhibit significantly improved electro-catalytic activity for glucose oxidation. In some cases, large variations impede the identification of significant differences in electro-catalytic activity. To reduce process variability and to increase the specific surface area further optimization of the fabrication process is required. Similarly, the deposition of defined alloy compositions will require further investigation since the composition of electrolyte and deposited alloy do not directly correspond.
• L. Becherer, M. Bakheit, S. Frischmann, S. Stinco, N. Borst, R. Zengerle, F. von Stetten
  Simplified real-time multiplex detection of loop-mediated isothermal amplification (LAMP) using novel mediator displacement probes with universal reporters
  2018 Anal Chem, volume: 90, pages: 4741 - 4748
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  Abstract

  A variety of real-time detection techniques for LAMP based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel Mediator Displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3-4 h), shorter times to positive (MD 4.1±0.1 min shorter than MB, n = 36), improved signal to noise fluorescence ratio (MD: 5.9±0.4, MB: 2.7±0.4; n = 15) and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 & HTLV-1 and a biplex LAMP of Haemophilus ducreyi & Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels.
• J. Madjarov, A. Götze, R. Zengerle, S. Kerzenmacher
  Simultaneous use of a crossflow filtration membrane as microbial fuel cell anode – permeate flow leads to 4-fold increased current densities
  2018 Bioresource Technol, volume: 257, pages: 274 - 280
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  Abstract

  A new concept for the combination of membrane bioreactors and microbial fuel cells is introduced, that aims at the production of electricity for reducing the overall energy consumption of wastewater treatment. In contrast to previous approaches, the anode is integrated as microfiltration membrane in sidestream crossflow configuration. Using a stainless steel filtration membrane with G. sulfurreducens and an acetatebased synthetic medium, up to 4-fold higher current densities are achieved. In a standard setup without filtration, a membrane of filter grade 1 μm shows current densities of 5.8 A m-2 ± 0.5 A m-2 compared to >11 A m-2 when it is used simultaneously as membrane filter. With smaller pore sizes of filter grade 0.5 μm 4.4 A m-2 ± 0.5 A m-2 in a standard setup and >15 A m-2 in a filtration setup are achieved. The permeate flow was identified as the main parameter leading to increased current densities.
• E. Kipf, J. Erben, R. Zengerle, J. Gescher, S. Kerzenmacher
  Systematic investigation of anode materials for microbial fuel cells with the model organism G. sulfurreducens
  2018 Bioresource Technology Reports, volume: 2, pages: 29 - 37
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  Abstract

  Different carbon and metal-based anode materials for microbial fuel cells were systematically investigated with a pure culture of the model organism G. sulfurreducens. The highest limiting current density of 756 ± 15 μA cm-2 at -0.253 ± 0.037 V vs. SCE was achieved with graphite foil using a step-wise galvanostatic technique. But also the application of completely different anode materials such as activated carbon cloth, stainless steel and graphite felt led to similar high limiting current densities, suggesting that G. sulfurreducens is able to use a large range of substantially different anode materials as external electron acceptor. Additionally, we could show that a step-wise galvanostatic technique to record polarization curves yields similar current densities as potentiostatic control at -0.400 V vs. SCE with the investigated carbon-based materials. In case of stainless steel these techniques yield slightly different results, presumably due to an effect related to the material’s surface properties.
• S. Hin, N. Paust, M. Keller, M. Rombach, O. Strohmeier, R. Zengerle, K. Mitsakakis
  Temperature change rate actuated bubble mixing for homogeneous rehydration of dry pre-stored reagents in centrifugal microfluidics
  2018 Lab Chip, volume: 18, pages: 362 - 370
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  Abstract

  In centrifugal microfluidics, dead volumes in valves downstream of mixing chambers can hardly be avoided. These dead volumes are excluded from mixing processes and hence cause a concentration gradient. Here we present a new bubble mixing concept which avoids such dead volumes. The mixing concept employs heating to create a temperature change rate (TCR) induced overpressure in the air volume downstream of mixing chambers. The main feature is an air vent with a high fluidic resistance, representing a low pass filter with respect to pressure changes. Fast temperature increase causes rapid pressure increase in downstream structures pushing the liquid from downstream channels into the mixing chamber. As air further penetrates into the mixing chamber, bubbles form, ascend due to buoyancy and mix the liquid. Slow temperature/ pressure changes equilibrate through the high fluidic resistance air vent enabling sequential heating/cooling cycles to repeat the mixing process. After mixing, a complete transfer of the reaction volume into the downstream fluidic structure is possible by a rapid cooling step triggering TCR actuated valving. The new mixing concept is applied to rehydrate reagents for loop-mediated isothermal amplification (LAMP). After mixing, the reaction mix is aliquoted into several reaction chambers for geometric multiplexing. As a measure for mixing efficiency, the mean coefficient of variation (C —— V, n = 4 LabDisks) of the time to positivity (tp) of the LAMP reactions (n = 11 replicates per LabDisk) is taken. The C —— V of the tp is reduced from 18.5% (when using standard shake mode mixing) to 3.3% (when applying TCR actuated bubble mixing). The bubble mixer has been implemented in a monolithic fashion without the need for any additional actuation besides rotation and temperature control, which are needed anyhow for the assay workflow.
• F. Hegge, R. Moroni, P. Trinke, B. Bensmann, R. Hanke-Rauschenbach, S. Thiele, S. Vierrath
  Three-dimensional microstructure analysis of a polymer electrolyte membrane water electrolyzer anode
  2018 J Power Sources, volume: 393, pages: 62 - 66
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  Abstract

  The anode catalyst layer of a PEM water electrolyzer is reconstructed using a combination of FIB-SEM tomography and ionomer modeling. The pore space is infiltrated with silicone, enabling good discrimination between pores and IrRuOx catalyst material, while the ionomer cannot be imaged. The reconstructed volume of 29 μm × 24 μm x 7 μm contains catalyst particles with a median size of 0.5 μm and has a porosity of 55%. By modeling different ionomer contents inside the pore space, the impact on microstructural and transport parameters is investigated. At an ionomer content of 40–50% of the pore volume, all transport parameters are in a reasonable range, confirming experimental results from literature. At an ionomer content of 48% the catalyst layer has a porosity of 29%, a median pore size of 0.94 μm, a permeability of the pore space of and a mean ionomer film thickness of . The tortuosities of the ionomer and the pore space are calculated to 3.5 and 6.7 at the corresponding phase fractions of 26% and 29% respectively. The electrochemically active surface area estimated from the tomography () is considerably lower than literature values, indicating a roughness below FIB-SEM resolution.
• N. P. Kocatürk-Schumacher, J. Madjarov, P. Viwatthanasittiphong, S. Kerzenmacher
  Toward an Energy Efficient Wastewater Treatment: Combining a Microbial Fuel Cell/Electrolysis Cell Anode With an Anaerobic Membrane Bioreactor
  2018 Frontiers in Energy Research, volume: 6, page: 95
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  Abstract

  Recently, it has been shown that combining a bioelectrochemical system (BES) with an anaerobic membrane bioreactor (AnMBR) to produce electricity can reduce the overall energy consumption of wastewater treatment. In this study, we tested the recently proposed concept that integrates a microbial anode into an AnMBR, under application relevant conditions, for the treatment of synthetic brewery wastewater. We developed two system configurations: a filtering anode with stainless steel filter plate; and a hybrid anode, in which a polymeric membrane is combined with stainless steel mesh. As fouling is problematic in AnMBRs, we investigated the effect of two fouling mitigation methods, namely electrochemical cleaning and application of a turbulence promotor, on the permeate fluxes and current densities.We also investigated the effect of cathode (counter electrode) position on the permeate fluxes and current densities in filtering and hybrid anode. Our results revealed that permeate fluxes were influenced by the membrane pore size; and dropped below 5 L m−2 hr−1 on day 3 with filter grade 0.5μm; whereas similar values of permeate flux were observed after 5 days of operation with the membrane with filter grade 0.1μm. COD removal across the membrane reached up to 644mg L−1 indicating improvement in energy efficiency and effluent quality of the AnMBR. The location of cathode did not influence permeate fluxes and current densities, but permeate pH was largely affected. Electrochemical cleaning improved permeate fluxes more than 2-fold (18.9 L m−2 hr−1 after 7 days of operation) compared to the operation of the 0.1μm membrane without a cleaning procedure. Application of a turbulence promotor increased permeate fluxes and current densities in filtering anode. The hybrid anode resulted in similar current densities, but higher permeate fluxes as compared to the filtering anode, which dropped below 20 L m−2 hr−1 only after 8 days of operation. The Kocatürk-Schumacher et al. Microbial Anode Combined With AnMBR hybrid anode configuration is an attractive option that combines high permeate fluxes on conventional non-conductive filters with current generation on an inexpensive conductive material. In summary, our results demonstrate that combining BES with AnMBR is a promising approach toward an energy efficient wastewater treatment.

2017

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• M. Bühler, C. Klose, F. Hegge, T. Lickert, S. Thiele
  A Novel Fabrication Technique for Electrodes of PEM Water Electrolyzers
  2017 ECS Transactions, volume: 80, pages: 1069 - 1075
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  Abstract

  In this work the improvement of material interfaces at electrodes for proton exchange membrane (PEM) water electrolyzers is addressed by a novel fabrication technique. In this approach the electrode and the membrane are directly deposited on the porous transport layers (PTLs), which serve as substrate for the electrodes. The aim is to stabilize and increase the oxygen and hydrogen evolution rate at high current densities leading to a reduction of the noble metal loading of the electrodes – and additionally to develop a cost effective novel fabrication technique applicable for the large scale industrial fabrication of electrodes for PEM water electrolyzers. The innovative manufacturing technique is described in this transaction, and current challenges regarding the coating of porous substrates in terms of parameter control, reliability and homogeneity are pointed out.
• M. Breitwieser, T. Bayer, A. Büchler, R. Zengerle, S. M. Lyth, S. Thiele
  A fully spray-coated fuel cell membrane electrode assembly using Aquivion ionomer with a graphene oxide/cerium oxide interlayer
  2017 J Power Sources, volume: 351, pages: 145 - 150
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  Abstract

  A novel multilayer membrane electrode assembly (MEA) for polymer electrolyte membrane fuel cells (PEMFCs) is fabricated in this work, within a single spray-coating device. For the first time, direct membrane deposition is used to fabricate a PEMFC by spraying the short-side-chain ionomer Aquivion directly onto the gas diffusion electrodes. The fully sprayed MEA, with an Aquivion membrane 10 mm in thickness, achieved a high power density of 1.6 W/cm2 for H2/air operation at 300 kPaabs. This is one of the highest reported values for thin composite membranes operated in H2/air atmosphere. By the means of confocal laser scanning microscopy, individual carbon fibers from the gas diffusion layer are identified to penetrate through the micro porous layer (MPL), likely causing a low electrical cell resistance in the range of 150 U cm2 through the thin sprayed membranes. By spraying a 200 nm graphene oxide/cerium oxide (GO/CeO2) interlayer between two layers of Aquivion ionomer, the impact of the electrical short is eliminated and the hydrogen crossover current density is reduced to about 1 mA/cm2. The peak power density of the interlayer-containing MEA drops only by 10% compared to a pure Aquivion membrane of similar thickness.
• N. Borst, F. Schuler, S. Wadle, M. Schulz, M. Specht, J. Li, L. Becherer, M. Trotter, A. B. Rodríguez-Martínez, N. Paust, R. Zengerle, F. von Stetten
  A technology platform for digital nucleic acid diagnostics at the point of care
  2017 Laboratoriumsmedizin, volume: 41, issue: 5
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  Abstract

  The combination of digital amplification and centrifugal microfluidics can enable quantitative and fast diagnostics at the point of care (PoC). The new unit operation of centrifugal step emulsification allows high throughput droplet generation. Different methods for digital nucleic acid analysis, including PCR, recombinase polymerase amplification (RPA) and loop mediated isothermal amplification (LAMP), have already been demonstrated. Our novel approach of integrated sample-to-answer analysis is introduced, and examples for the detection of HIV and single cell analysis of antibiotic resistant bacteria are presented. Next to these LabDisk based systems, a microfluidic cartridge termed DropChip allows for digital amplification using only commercially available laboratory devices.
• L. Benning, L. Gutzweiler, K. Tröndle, J. Riba, R. Zengerle, P. Koltay, S. Zimmermann, G. B. Stark, G. Finkenzeller
  Assessment of hydrogels for bioprinting of endothelial cells
  2017 J Biomed Mater Res A, pages: 935 - 947
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  Abstract

  In tissue engineering applications, vascularization can be accomplished by co-implantation of tissue forming cells and endothelial cells (ECs), whereby the latter are able to form functional blood vessels. The use of three-dimensional (3D) bioprinting technologies has the potential to improve the classical tissue engineering approach because these will allow the generation of scaffolds with high spatial control of endothelial cell allocation. This study focuses on a side by side comparisons of popular commercially available bioprinting hydrogels (matrigel, fibrin, collagen, gelatin, agarose, Pluronic F-127, alginate and alginate/gelatin) in the context of their physicochemical parameters, their swelling/degradation characteristics, their biological effects on vasculogenesis-related EC parameters and their printability. The aim of this study was to identify the most suitable hydrogel or hydrogel combination for inkjet printing of ECs to build pre-vascularized tissue constructs. Most tested hydrogels displayed physicochemical characteristics suitable for inkjet printing. However, Pluronic F-127 and the alginate/gelatin blend were rapidly degraded when incubated in cell culture medium. Agarose, Pluronic F-127, alginate and alginate/gelatin hydrogels turned out to be unsuitable for bioprinting of ECs because of their non-adherent properties and/or their incapability to support EC proliferation. Gelatin was able to support EC proliferation and viability but was unable to support endothelial cell sprouting. Our experiments revealed fibrin and collagen to be most suitable for bioprinting of ECs, because these hydrogels showed acceptable swelling/degradation characteristics, supported vasculogenesis-related EC parameters and showed good printability. Moreover, ECs in constructs of preformed spheroids survived the printing process and formed capillary-like cords.
• T. Gleichmann, A. Rostas, J. Wörner, E. Schleicher, L. Gutzweiler, B. Hamouda, R. Zengerle, P. Koltay, L. Riegger
  Atmospheric Photopolymerization of Acrylamide Enabled by Aqueous Glycerol Mixtures: Characterization and Application for Surface-Based Microfluidics
  2017 Macromol Mater Eng, page: 1600518
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  Abstract

  Polyacrylamide usually is the material of choice for electrophoretic separation in slab gels, capillaries, and microfluidic devices. So far its polymerization requires anaerobic environments because oxygen impurities inhibit or even terminate the polymerization reaction of acrylamide. Here, it is demonstrated that gel precursor solutions with glycerol contents above 20 vol% enable direct atmospheric photopolymerization of acrylamide with no need for sealing or degassing the solution in advance. The positive effect of glycerol on the polymerization reaction is proven by simulation-validated electron paramagnetic resonance measurements. Nuclear magnetic resonance reveals that glycerol does not interfere with the reaction indicating that the observed enhancement in polymerization is owed to the low oxygen solubility of aqueous glycerol mixtures. Glycerol concentrations of >60 vol% in the gel precursor solution enable complete polymerization of volumes down to 5 nL within less than 5 s. This enables using liquid handling robots to fabricate channel-free open microfluidic structures of solid polyacrylamide hydrogel in a low-cost automated manner in a standard lab environment.
• Y. Zhao, G. Czilwik, V. Klein, K. Mitsakakis, R. Zengerle, N. Paust
  C-reactive protein and Interleukin 6 microfluidic immunoassays with on-chip pre-stored reagents and centrifugo-pneumatic liquid control
  2017 Lab Chip, volume: 17, issue: 9, pages: 1666 - 1677
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  Abstract

  We present a fully automated centrifugal microfluidic method for particle based protein immunoassays. Stick-pack technology is employed for pre-storage and release of liquid reagents. Quantitative layout of centrifugo-pneumatic particle handling, including timed valving, switching and pumping is assisted by network simulations. The automation is exclusively controlled by the spinning frequency and does not require any additional means. New centrifugal microfluidic process chains are developed in order to sequentially supply wash buffer based on frequency dependent stick-pack opening and pneumatic pumping to perform two washing steps from one stored wash buffer; pre-store and re-suspend functionalized microparticles on disk; and switch between the path of the waste fluid and the path of the substrate reaction product with 100% efficiency. The automated immunoassay concept comprises on demand ligand binding, two washing steps, substrate reaction, timed separation of the reaction products, and termination of the substrate reaction. We demonstrated separation of particles from three different liquids with particle loss below 4 % and residual liquid remaining within particles below 3 %. The automated immunoassay concept was demonstrated by means of detecting Creactive protein (CRP) in the range of 1 - 81 ng ml-1 and Interleukin 6 (IL-6) in the range of 64 - 13500 pg ml-1. The limit of detection and quantification were 1.0 ng ml-1 and 2.1 ng ml-1 for CRP; 64 pg ml-1 and 205 pg ml-1 for IL-6, respectively.
• M. Breitwieser, C. Klose, A. Hartmann, A. Büchler, M. Klingele, S. Vierrath, R. Zengerle, S. Thiele
  Cerium Oxide Decorated Polymer Nanofibers as Effective Membrane Reinforcement for Durable, High-Performance Fuel Cells
  2017 Adv Energy Mater, page: 1602100
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  Abstract

  High-power, durable composite fuel cell membranes are fabricated here by direct membrane deposition (DMD). Poly(vinylidene fluoride-co-hexafluo- ropropylene) (PVDF-HFP) nanofibers, decorated with CeO 2 nanoparticles are directly electrospun onto gas diffusion electrodes. The nanofiber mesh is impregnated by inkjet-printed Nafion ionomer dispersion. This results in 12 µm thin multicomponent composite membranes. The nanofibers provide membrane reinforcement, whereas the attached CeO 2 nanoparticles promote improved chemical membrane durability due to their radical scavenging properties. In a 100 h accelerated stress test under hot and dry conditions, the reinforced DMD fuel cell shows a more than three times lower voltage decay rate (0.39 mV h −1 ) compared to a comparably thin Gore membrane (1.36 mV h −1 ). The maximum power density of the DMD fuel cell drops by 9%, compared to 54% measured for the reference. Impedance spectroscopy reveals that ionic and mass transport resistance of the DMD fuel cell are unaffected by the accelerated stress test. This is in contrast to the reference, where a 90% increase of the mass transport resistance is measured. Energy dispersive X-ray spectroscopy reveals that no significant migration of cerium into the catalyst layers occurs during degradation. This proves that the PVDF- HFP backbone provides strong anchoring of CeO 2 in the membrane.
• P. Lettenmeier, S. Kolb, L. Zielke, S. Thiele, A. Fallisch, N. Sata, A. S. Gago, K. A. Friedrich
  Comprehensive investigation of novel pore-graded gas diffusion layers for high-performance and cost-effective proton exchange membrane electrolyzers
  2017 Energy Environ. Sci., volume: 10, pages: 2521 - 2533
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  Abstract

  Hydrogen produced by water electrolysis is a promising storage medium for renewable energy. Reducing the capital cost of proton exchange membrane (PEM) electrolyzers without losing efficiency is one of its most pressing challenges. Gas diffusion layers (GDL), such as felts, foams, meshes and sintered plates, are key stack components, but these are either inefficient or expensive. This study presents a new type of GDL produced via vacuum plasma spraying (VPS), which offers a large potential for cost reduction. With this technology, it is possible to introduce a gradient in the pore-size distribution along the thickness of the GDL by varying the plasma parameters and titanium powder particle sizes. This feature was confirmed by cross-section scanning electron microscopy (SEM). X-ray computed tomography (CT) and mercury intrusion porosimetry allowed determining the porosity, pore radii distribution, and pore entry distribution. Pore radii of ca. 10 μm could be achieved in the layers of the GDL close to the bipolar plate, while those in contact with the electrodes were in the range of 5 μm. The thermally sprayed Ti-GDLs allowed achieving PEM electrolyzer performances comparable to those of the state-of-the-art sintered plates and far superior than those of meshes. Moreover, a numerical model showed that the reduced capillary pressure and tortuosity eliminates mass transport limitations at 2 A cm−2. The results presented herein demonstrate a promising solution to reduce the cost of one of the most expensive components of the stack.
• L. Benning, L. Gutzweiler, K. Tröndle, J. Riba, R. Zengerle, P. Koltay, S. Zimmermann, G.B. Stark, G. Finkenzeller
  Cytocompatibility testing of hydrogels toward bioprinting of mesenchymal stem cells
  2017 J Biomed Mater Res A, volume: 105, pages: 3231 - 3241
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  Abstract

  Mesenchymal stem cells (MSCs) represent a very attractive cell source for tissue engineering applications aiming at the generation of artificial bone substitutes. The use of three-dimensional bioprinting technologies has the potential to improve the classical tissue engineering approach because bioprinting will allow the generation of hydrogel scaffolds with high spatial control of MSC allocation within the bioprinted construct. In this study, we have performed direct comparisons between commercially available hydrogels in the context of their cytocompatibility toward MSCs and their physicochemical parameters with the aim to identify the most suitable hydrogel for drop-on-demand (DoD) printing of MSCs. In this context, we examined matrigel, fibrin, collagen, gelatin, and gelatin/alginate at various hydrogel concentrations. Matrigel, fibrin, collagen, and gelatin were able to support cell viability, but the latter showed a limited potential to promote MSC proliferation. We concentrated our study on fibrin and collagen hydrogels and investigated the effect of hydroxyapatite (HA) inclusion. The inclusion of HA enhanced proliferation and osteogenic differentiation of MSCs and prevented degradation of fibrin in vitro. According to viscosity and storage moduli measurements, HA-blends displayed physicochemical characteristics suitable for DoD printing. In bioprinting experiments, we confirmed that fibrin and collagen and their respective HA-blends represent excellent hydrogels for DoD-based printing as evidenced by high survival rates of printed MSCs.
• C. Klose, M. Breitwieser, S. Vierrath, M. Klingele, H. Cho, A. Büchler, J. Kerres, S. Thiele
  Electrospun sulfonated poly(ether ketone) nanofibers as proton conductive reinforcement for durable Nafion composite membranes
  2017 J Power Sources, volume: 361, pages: 237 - 242
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  Abstract

  We show that the combination of direct membrane deposition with proton conductive nanofiber reinforcement yields highly durable and high power density fuel cells. Sulfonated poly(ether ketone) (SPEK) was directly electrospun onto gas diffusion electrodes and then filled with Nafion by inkjet-printing resulting in a 12 μm thin membrane. The ionic membrane resistance (30 mΩ*cm2) was well below that of a directly deposited membrane reinforced with chemically inert (PVDF-HFP) nanofibers (47 mΩ*cm2) of comparable thickness. The power density of the fuel cell with SPEK reinforced membrane (2.04 W/cm2) is 30% higher than that of the PVDF-HFP reinforced reference sample (1.57 W/cm2). During humidity cycling and open circuit voltage (OCV) hold, the SPEK reinforced Nafion membrane showed no measurable degradation in terms of H2 crossover current density, thus fulfilling the target of 2 mA/cm2 of the DOE after degradation. The chemical accelerated stress test (100 h OCV hold at 90 °C, 30% RH, H2/air, 50/50 kPa) revealed a degradation rate of about 0.8 mV/h for the fuel cell with SPEK reinforced membrane, compared to 1.0 mV/h for the PVDF-HFP reinforced membrane.
• K. Lehmann, O. Yurchenko, A. Heilemann, S. Vierrath, L. Zielke, S. Thiele, A. Fischer, G. Urban
  High surface hierarchical carbon nanowalls synthesized by plasma deposition using an aromatic precursor
  2017 Carbon, volume: 118, pages: 578 - 587
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  Abstract

  Hierarchical carbon nanowalls (CNW) are synthesized by plasma-enhanced chemical vapor deposition using p-xylene as a complex precursor. In contrast to ordinary CNW, synthesized with short-chained carbons, hierarchical CNW show a unique multi-scale pore structure, made up of micro- and mesopores connected by tubular macropores, offering higher surface area and surface accessibility. Their morphology, graphitic structure, surface area and accessibility are verified by transmission and scanning electron microscopy, gas sorption and impedance spectroscopy. Focused ion beam scanning electron microscopy tomography demonstrates the presence of macropores ensuring pore connectivity down to the substrate. Nitrogen/krypton physisorption confirms the micro- and mesoporous structure contributing extensively to the surface area. The impedance spectra are evaluated according to standard RC and transmission line models. The sample deposited for 60 min, with a structure height of 4.75 μm, features a volumetric capacitance of 2.6 F cm−3 and a response time of 25 ms. Hierarchical CNW exhibit a two to six times higher volumetric capacitance than CNW of similar proportions, reported in literature. Hierarchical CNW offer a promising way to realize high power and energy density requirements in electrochemical energy systems, like supercapacitors, due to their good conductivity, high surface area and open pore structure.
• A. Fallisch, L. Schellhase, J. Fresko, M. Zedda, J. Ohlmann, M. Steiner, A. Bösch, L. Zielke, S. Thiele, F. Dimroth, T. Smolinka
  Hydrogen concentrator demonstrator module with 19.8% solar-to-hydrogen conversion efficiency according to the higher heating value
  2017 Int J Hydrogen Energ, pages: 26804 - 26815
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  Abstract

  Renewable hydrogen is a key element to a sustainable energy system of the future. Therefore solar hydrogen generation has been investigated by various research groups in recent years. The patented concept of the Hydrogen Concentrator (HyCon), which combines III-V multi-junction solar cells with polymer electrolyte membrane electrolysis, has been constantly developed over the last years. In this work, a unique weatherproof HyCon module with an area of 8 × 90.7 cm2 was build and characterized in an outdoor measurement for over two month. During this measurement period, the module showed a stable operation regardless of the water volume flow. The module works under natural convection without any circulation pumps at a suitable maximum temperature of 60–70 °C. The HyCon module consists of eight individual units (HyCon cells), each combining a photovoltaic and an electrolysis cell. Some of the HyCon cells reach a solar-to-hydrogen conversion efficiency of 20% according to the higher heating value at high current densities of 0.8 A/cm2. On the module level a maximum efficiency of 19.8% is reached. To the best of our knowledge this is the highest conversion efficiency so far achieved at such high current densities using a dual junction solar cell.
• A. Fallisch, L. Schellhase, J. Fresko, M. Zechmeister, M. Zedda, J. Ohlmann, L. Zielke, N. Paust, T. Smolinka
  Investigation on PEM water electrolysis cell design and components for a HyCon solar hydrogen generator
  2017 Int J Hydrogen Energ, volume: 42, pages: 13544 - 13553
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  Abstract

  Hydrogen as a secondary energy carrier promises a large potential as a long term storage for fluctuating renewable energies. In this sense a highly efficient solar hydrogen generation is of great interest especially in southern countries having high solar irradiation. The patented Hydrogen Concentrator (HyCon) concept yields high efficiencies combining multi-junction solar cells with proton exchange (PEM) membrane water electrolysis. In this work, a special PEM electrolysis cell for the HyCon concept was developed and investigated. It is shown that the purpose-made PEM cell shows a high performance using a titanium hybrid fiber sinter function both as a porous transport layer and flow field. The electrolysis cell shows a high performance with 1.83 V at 1 A/cm2 and 24 °C working under natural convection with a commercially available catalyst coated membrane. A theoretical examination predicts a total efficiency for the HyCon module from sunlight to hydrogen of approximately 19.5% according to the higher heating value.
• L. Gutzweiler, S. Kartmann, K. Troendle, L. Benning, G. Finkenzeller, R. Zengerle, P. Koltay, B. Stark, S. Zimmermann
  Large scale production and controlled deposition of single HUVEC spheroids for bioprinting applications
  2017 Biofabrication, volume: 9 (2), page: 02502
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  Abstract

  We present 1.) a fast and automated method for large scale production of HUVEC spheroids based on the hanging drop method and 2.) a novel method for well-controlled lateral deposition of single spheroids by drop-on-demand printing. Large scale spheroid production is achieved via printing 1536 droplets of HUVEC cell suspension having a volume of 1 µl each within 3 minutes at a pitch of 2.3 mm within an array of 48 x 32 droplets onto a flat substrate. Printing efficiencies between 97.9% and 100% and plating efficiencies between 87.3% and 100% were achieved. Harvested spheroids (consisting of approx. 250 HUVECs each) appear uniform in size and shape. After incubation and harvesting, the spheroids are deposited individually in user-defined patterns onto hydrogels using an automated drop-on-demand dispenser setup. Controlled by an image detection algorithm focusing the dispenser nozzle, droplets containing exactly one spheroid are printed onto a substrate, while all other droplets are discarded. Using this approach an array of 6 x 3 HUVEC spheroids with intermediate distances of 500 µm embedded in fibrin was generated. Successful progress of spheroid sprouting and merging of neighboring sprouts was observed during the first 72 hours of incubation indicating a good viability of the deposited spheroids.
• M. Kokko, F. Bayerköhler, J. Erben, R. Zengerle, P. Kurz, S. Kerzenmacher
  Molybdenum sulphides on carbon supports as electrocatalysts for hydrogen evolution in acidic industrial wastewater
  2017 Appl Energ, volume: 190, pages: 1221 - 1233
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  Abstract

  Microbial electrolysis cells (MECs) are an attractive future alternative technology to generate renewable hydrogen and simultaneously treat wastewaters. The thermodynamics of hydrogen evolution in MECs can be greatly improved by operating the cathode at acidic pH in combination with a neutral pH microbial anode. This can easily be achieved with acidic industrial wastewaters that have to be neutralised before discharge. For the hydrogen evolution reaction (HER) in acidic wastewater, efficient and inexpensive catalysts are required that are compatible with the often complex chemical composition of wastewaters. In this study, molybdenum sulphides (MoSx) on different carbon supports were successfully used for hydrogen evolution in different acidic media. At first, the cathodes were screened by linear sweep voltammetry in sulphuric acid (pH 0) or phosphate buffer (pH 2.2). After this, the overpotentials for H2 production of the best cathodes and their long term performances (>=1 week) were determined in acidic industrial wastewater (pH 2.4) obtained from a plant mainly producing cellulose acetate. For the most promising MoSx cathodes, the overpotentials for HER (at 3 mA cm-2) were only ~40 mV higher than for a platinum electrode. Most importantly, the catalytic efficiency of the MoSx electrodes improved in the wastewater over time (7–17 days), while Pt electrodes were found to be slowly deactivated. Thus, MoSx emerges as an affordable, efficient and especially durable electrocatalyst for HER in real acidic wastewaters and this could be an important contribution to take energy production from wastewaters in the form of hydrogen towards practical applications.
• M. Klingele, R. Moroni, S. Vierrath, S. Thiele
  Multiscale Tomography-Based Analysis of Fuel Cells: Towards a Fully Resolved Fuel Cell Reconstruction
  2017 J Electrochem Energy, volume: 15, page: 014701
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  Abstract

  The microstructure of a fuel cell electrode largely determines the performance of the whole fuel cell system. In this regard, tomographic imaging is a valuable tool for the understanding and control of the electrode morphology. The distribution of pore- and feature-sizes within fuel cell electrodes covers several orders of magnitude, ranging from millimeters in the gas diffusion layer down to few nanometers in the catalyst layer. This obligates the application of various tomographic methods for imaging every aspect of a fuel cell. This perspective evaluates the capabilities, limits and challenges of each of these methods. Further it highlights and suggests efforts towards the integration of multiple tomographic methods into single multiscale datasets, a venture which aims at large scale, and morphologically fully resolved fuel cell reconstructions.
• M. Frei, J. Erben, J. Martin, R. Zengerle, S. Kerzenmacher
  Nanofiber-deposited porous platinum enables glucose fuel cell anodes with high current density in body fluids
  2017 J Power Sources, volume: 362, pages: 168 - 173
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  Abstract

  The poisoning of platinum anodes by body-fluid constituents such as amino acids is currently the main hurdle preventing the application of abiotic glucose fuel cells as battery-independent power supply for medical implants. We present a novel anode material that enables continuous operation of glucose oxidation anodes in horse serum for at least 30 days at a current density of (7.2 ± 1.9) μA cm−2. The fabrication process is based on the electro-deposition of highly porous platinum onto a 3-dimensional carbon nanofiber support, leading to approximately 2-fold increased electrode roughness factors (up to 16500 ± 2300). The material's superior performance is not only related to its high specific surface area, but also to an improved catalytic activity and/or poisoning resistance. Presumably, this results from the micro- and nanostructure of the platinum deposits. This represents a major step forward in the development of implantable glucose fuel cells based on long-term stable platinum electrodes.
• S. Zehnle, M. Rombach, R. Zengerle, F. von Stetten, N. Paust
  Network simulation-based optimization of centrifugo-pneumatic blood plasma separation
  2017 Biomicrofluidics, volume: 11, page: 024114
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  Abstract

  Automated and robust separation of 14 ll of plasma from 40 ll of whole blood at a purity of 99.81%60.11% within 43 s is demonstrated for the hematocrit range of 20%–60% in a centrifugal microfluidic polymer disk. At high rotational frequency, red blood cells (RBCs) within whole blood are concentrated in a radial outer RBC collection chamber. Simultaneously, plasma is concentrated in a radial inner pneumatic chamber, where a defined air volume is enclosed and compressed. Subsequent reduction of the rotational frequency to not lower than 25 Hz enables rapid transfer of supernatant plasma into a plasma collection chamber, with highly suppressed resuspension of red blood cells. Disk design and the rotational protocol are optimized to make the process fast, robust, and insusceptible for undesired cell resuspension. Numerical network simulation with lumped model elements is used to predict and optimize the fluidic characteristics. Lysis of the remaining red blood cells in the purified plasma, followed by measurement of the hemoglobin concentration, was used to determine plasma purity. Due to the pneumatic actuation, no surface treatment of the fluidic cartridge or any additional external means are required, offering the possibility for low-cost mass fabrication technologies, such as injection molding or thermoforming.
• L. Gutzweiler, T. Gleichmann, L. Tanguy, P. Koltay, R. Zengerle, L. Riegger, , ,
  Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale
  2017 Electrophoresis, volume: 38, pages: 1764 - 1770
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  Abstract

  Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on-demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary- & chip-based methods offering low consumable costs (< 0.1 $) circumventing cost intensive microfluidic chip fabrication, short process times (5 minutes per analysis) and high sensitivity (4 ng/µl dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2-4 µl in volume, a semi-contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non-contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR-grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi-circular cross section of adaptable width (~200-600 µm) and height (~30-80 µm) as well as a typical length of 15-55 mm. Layout of such liquid structures is adaptable on-demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label-free DNA ladder (100-1000 bp) at 100 V/cm via in-line staining and laser induced fluorescent end-point detection using an automated prototype.
• J. Madjarov, S.C. Popat, J. Erben, A. Götze, R. Zengerle, S. Kerzenmacher
  Revisiting methods to characterize bioelectrochemical systems: The influence of uncompensated resistance (iRu-drop), double layer capacitance, and junction potential
  2017 J Power Sources, volume: 356, pages: 408 - 418
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  Abstract

  Bioelectrochemical systems (BES) are characterized with methods derived from the electrochemistry field, for e.g. linear sweep voltammetry (LSV), cyclic voltammetry (CV), and chronoamperometry. The limitations of electrochemical measurements are well known and described, but there are new challenges when these are applied to biological systems. For instance, the electrolyte conditions are predefined by the application involving the use of low conductivities, leading to an increase of two error sources: the iRu-drop and junction potential. Furthermore, the use of electrodes with high surface areas and thus high double layer capacitance lead to capacitive currents that superimpose the biocatalytic current of interest. Even though these problems have often been mentioned in the bioelectrochemistry field, they are seldom considered and reported in publications. The scope of this work is to present and discuss methods to quantify the Ru and double layer capacitance, and to demonstrate their significant influence on the recording of polarization curves. In a typical BES setup, it is exemplarily shown that due to iRu-drop measured potentials can deviate by more than 200 mV from the actual potential. Similarly, more than 40% of a recorded electrode current can originate from the electrode material's double layer capacitance.
• M. Keller, G. Czilwik, J. Schott, I. Schwarz, K. Dormanns, F. von Stetten, R. Zengerle, N. Paust
  Robust temperature change rate actuated valving and switching for highly integrated centrifugal microfluidics
  2017 Lab Chip, volume: 17, pages: 864 - 875
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  Abstract

  We present new unit operations for valving and switching in centrifugal microfluidics that are actuated by a temperature change rate (TCR) and controlled by the rotational frequency. Implementation is realized simply by introducing a comparatively large fluidic resistance to an air vent of a fluidic structure downstream of a siphon channel. During temperature decrease at a given TCR, air pressure inside the downstream structure decreases and the fluidic resistance of the air vent slows down air pressure compensation allowing for a thermally induced underpressure to build up temporarily. Thereby the rate of temperature change determines the time course of the underpressure for a given geometry. The thermally induced underpressure pulls liquid against a centrifugal counterpressure above a siphon crest, which triggers the valve or switch. The centrifugal counterpressure (adjusted by rotation) serves as independent control parameter to allow or prevent valving or switching at any TCR. The unit operations are thus compatible to any temperature or centrifugation protocol prior to valving or switching. In contrast to existing methods, this compatibility is achieved at no additional costs: neither additional fabrication steps, nor additional disk space or external means are required besides global temperature control, which is per se needed for the assay. For layout, an analytical model is provided and verified. The TCR actuated unit operations are demonstrated, first, by a stand-alone switch that routes liquid to either one of the two collection chambers (n = 6) and, second, by studying robustness of TCR actuated valving within a microfluidic cartridge for highly integrated nucleic acid testing. Valving could safely be prevented during PCR by compensating the thermally induced underpressure of 3.52 kPa by centrifugal counterpressure at a rotational frequency of 30 Hz with a minimum safety range to valving of 2.03 kPa. Subsequently, a thermally induced underpressure of 2.55 kPa was utilized for robust siphon valving at 3 Hz with a minimum safety range of 2.32 kPa.
• M. Breitwieser, C. Klose, M. Klingele, A. Hartmann, J. Erben, H. Cho, J. Kerres, R. Zengerle, S. Thiele
  Simple fabrication of 12 μm thin nanocomposite fuel cell membranes by direct electrospinning and printing
  2017 J Power Sources, volume: 337, pages: 137 - 144
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  Abstract

  Direct membrane deposition (DMD) was recently introduced as a novel polymer electrolyte membrane fabrication method. Here, this approach is extended to fabricate 12 μm thin nanocomposite fuel cell membranes. Poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) nanofibers are directly electrospun onto gas diffusion electrodes. By inkjet-printing Nafion ionomer dispersion into the pore space of PVDF-HFP nanofiber mats, composite membranes of 12 mm thickness were fabricated. At 120 C and 35% relative humidity, stoichiometric 1.5/2.5 H2/air flow and atmospheric pressure, the power density of the DMD fuel cell (0.19Wcm-2), was about 1.7 times higher than that of the reference fuel cell (0.11Wcm-2) with Nafion HP membrane and identical catalyst. A lower ionic resistance and, especially at 120 C, a reduced charge transfer resistance is found compared to the Nafion HP membrane. A 100 h accelerated stress test revealed a voltage decay of below 0.8 mV h-1, which is in the range of literature values for significantly thicker reinforced membranes. Finally, this novel fabrication approach enables new degrees of freedom in the design of complex composite membranes. The presented combination of scalable deposition techniques has the potential to simplify and thus reduce cost of composite membrane fabrication at a larger scale.
• F. Sun, R. Moroni, K. Dong, H. Markötter, D. Zhou, A. Hilger, L. Zielke, R. Zengerle, S. Thiele, J. Banhart, I. Manke
  Study of the Mechanisms of Internal Short Circuit in a Li/Li Cell by Synchrotron X‑ray Phase Contrast Tomography
  2017 ACS Energy Lett., volume: 2, pages: 94 - 104
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  Abstract

  Knowledge about degradation and failure of Li-ion batteries (LIBs) is of paramount importance, especially because failure can be accompanied by severe hazards. To contribute to the understanding of such phenomena, synchrotron in-line phase contrast X-ray tomography was employed to investigate internal cell deformation and degradation caused by an internal short circuit (ISC). The tomographic images taken from an uncycled Li/Li cell and a short-circuited Li/Li cell reveal how lithium microstructures (LmSs) develop during electrochemical stripping and plating during discharge and charge and how the three-layer separator used is damaged by growing LmSs and delaminates and melts as a consequence of an ISC. Previously unknown insights into the internal cell degradation and deformation mechanisms caused by an ISC are obtained and provide hints of how the properties of the separator could be modified to improve the reliability and safety of current- and next-generation LIBs.
• M. Klingele, C. Pham, K. Rao Vuyyurud, B. Britton, S. Holdcroft, A. Fischer, S. Thiele
  Sulfur doped reduced graphene oxide as metal-free catalyst for the oxygen reduction reaction in anion and proton exchange fuel cells
  2017 Electrochem Commun, volume: 77, pages: 71 - 75
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  Abstract

  Sulfur doped reduced graphene oxide (S-rGO) is investigated for catalytic activity towards the oxygen reduction reaction (ORR) in acidic and alkaline electrolytes. X-ray photoelectron spectroscopy shows that sulfur in S-rGO is predominantly integrated as thiophene motifs within graphene sheets. The overall sulfur content is determined to be approximately 2.2 at.% (elemental analysis). The catalytic activity of S-rGO towards the ORR is investigated by both rotating disc electrode (RDE) and polymer electrolyte fuel cell (PEFC) measurements. RDE measurements reveal onset potentials of 0.3 V and 0.74 V (vs. RHE) in acidic and alkaline electrolyte, respectively. In a solid electrolyte fuel cell with S-rGO as cathode material, this is reflected in an open circuit voltage of 0.37 V and 0.78 V and a maximum power density of 1.19 mW/cm2 and 2.38 mW/cm2 in acidic and alkaline polymer electrolyte, respectively. This is the first report investigating the catalytic activity of a sulfur doped carbon material in both acidic and alkaline liquid electrolyte, as well as in both proton and anion exchange polymer electrolyte fuel cells.
• M. Breitwieser, M. Klingele, S. Vierrath, R. Zengerle, S. Thiele
  Tailoring the membrane-electrode interface in PEM fuel cells: A review and perspective on novel engineering approaches
  2017 Adv Energy Mater, page: 1701257
• C. Van Pham, M. Klingele, B. Britton, K. R. Vuyyuru, T. Unmuessig, S. Holdcroft, A. Fischer,, S. Thiele
  Tridoped Reduced Graphene Oxide as a Metal-Free Catalyst for Oxygen Reduction Reaction Demonstrated in Acidic and Alkaline Polymer Electrolyte Fuel Cells
  2017 Advanced Sustainable Systems, volume: 1, page: 1600038
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  Abstract

  Recently, it has been demonstrated that doping of graphene by elements such as N, S, or F creates active sites for the oxygen reduction reaction (ORR). This results from bond polarization caused by the difference in electron- egativity between heteroatom dopants and carbon, and/or the presence of defects within the graphene lattice. In this work, fluorine, nitrogen, and sulfur tridoped reduced graphene oxide (F,N,S-rGO) is designed to combine these catalytically active sites. F,N,S-rGO can be inexpensively synthesized by a facile and scalable route involving pyrolysis at 600 °C of sulfur-doped rGO in the presence of Nafion and dimethyl formamide (DMF). The pyrolysis of Nafion and DMF provides F • and N • radicals which serve as doping agents. Rotating disk electrode investigations reveal the ORR catalytic activities of F,N,S-rGO in both acidic and alkaline media, which are consistent with the real performances of the respective polymer electrolyte fuel cells (PEFCs). Maximum power densities of 14 and 46 mW cm −2 are obtained for the acidic and alkaline PEFCs, respectively, using F,N,S-rGO as ORR catalysts. To the best of knowledge, this is the first report on the synthesis of F,N,S tridoped rGO and on its ORR activity in both acidic and alkaline PEFCs.

2016

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• M. Hagner, J. M. Fritz, P. Alknes, C. Scheuerlein, L. Zielke, S. Vierrath, S. Thiele, B. Bordini, A. Ballarino
  3D Analysis of the Porosity in MgB2 Wires Using FIB Nanotomography
  2016 Ieee T Appl Supercon, volume: 26, page: 6200305
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  Abstract

  Porosity is one of several current limiting mechanisms in MgB2 wires. We have compared the microstructural homogeneity and the porosity distribution in different ex situ and in situ MgB2 Powder-in-Tube (PIT) wires. The sub-micrometer structure was determined using Focused Ion Beam (FIB) nanotomography. The ex situ wires exhibit an isotropic microstructure, which has been quantified in terms of an identical tortuosity in transverse and longitudinal filament direction. The very homogenous microstructure in the new ex situ wire generation is probably one reason for its strongly improved critical current density. The in situ wire has an anisotropic microstructure with a lower tortuosity in the axial direction. The microstructural inhomogeneity of the in situ filaments makes microstructural characterization and the comparison between materials and superconducting properties particularly challenging.
• M. Klingele, B. Britton, M. Breitwieser, S. Vierrath, R. Zengerle, S. Holdcroft, S. Thiele
  A Completely Spray-Coated Membrane Electrode Assembly
  2016 Electrochem Commun, volume: 70, pages: 65 - 68
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  Abstract

  We present a proton exchange membrane fuel cell (PEMFC) manufacturing route, in which a thin layer of polymer electrolyte solution is spray-coated on top of gas diffusion electrodes (GDEs) to work as a proton exchange membrane. Without the need for a pre-made membrane foil, this allows inexpensive, fast, large-scale fabrication of membrane-electrode assemblies (MEAs), with a spray-coater comprising the sole manufacturing device. In this work, a catalyst layer and a membrane layer are consecutively sprayed onto a fibrous gas diffusion layer with applied microporous layer as substrate. A fuel cell is then assembled by stacking anode and cathode half-cells with the membrane layers facing each other. The resultant fuel cell with a low catalyst loading of 0.1 mg Pt/cm2 on each anode and cathode side is tested with pure H2 and O2 supply at 80 °C cell temperature and 92% relative humidity at atmospheric pressure. The obtained peak power density is 1.29 W/cm2 at a current density of 3.25 A/cm2. By comparison, a lower peak power density of 0.93 W/cm2 at 2.2 A/cm2 is found for a Nafion NR211 catalyst coated membrane (CCM) reference, although equally thick membrane layers (approx. 25 μm), and identical catalyst layers and gas diffusion media were used. The superior performance of the fuel cell with spray-coated membrane can be explained by a decreased low frequency (mass transport) resistance, especially at high current densities, as determined by electrochemical impedance spectroscopy.
• W.E. Kaman, G. Elshout, P.J.E. Bindels, K. Mitsakakis, J.P. Hays
  Current problems associated with the microbiological point-of-care testing of respiratory tract infections in primary care
  2016 Future Microbiol, volume: 11, pages: 607 - 610
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  Abstract

  Currently, the world is facing a potential ‘antibiotic apocalypse’, whereby clinically relevant bacteria are becoming increasingly resistant to all currently available antibiotics [1]. Several factors are contributing to this global increase in antibiotic resistance, including the unnecessary prescription of antibiotics for nonbacterial (viral and fungal) infections [2]. General practitioners (GPs) sit on the front line of antibiotic prescribing practice, having regular and broad-based contact with members of society that present to their offices with a range of disease states. One of the major reasons for visiting a GP is when a patient is apparently suffering from a respiratory tract infection [3]. The accurate diagnosis of the cause (i.e., the causative pathogen) of this infection can be difficult for the GP to make when only based on clinical symptoms. In the absence of sufficient diagnostic or clinical evidence, the GP may decide to prescribe antibiotics, or alternatively may withhold antibiotic therapy (suspecting a viral infection) when antibiotic therapy is actually required. On the one hand, the ‘overprescribing’ of antibiotics for nonbacterial infections may not only lead to the acquisition of antibiotic resistance in the resident flora of the patient, but also result in unnecessary healthcare costs for the patient and society [4]. It will also expose the patients to unwanted side effects from the antibiotic [5]. On the other hand, not prescribing antibiotics in case of a bacterial pneumonia, may result in extra morbidity for the patient and subsequent healthcare costs [6]. Access to point-of-care (POC) tests may reduce the prescription of antibiotics in primary care [7], although current POC testing performed by GPs for diagnosing respiratory tract infections tends to rely on the use of nonspecific biomarkers, such as CRP [8]. These biomarkers only provide information on the likelihood of a complicated (lower) respiratory tract infection being present. They do not identify pathogens or any antibiotic resistance that may be associated with a pathogen. For this reason, more advanced POC testing devices are currently being developed for specific use in the primary care environment. Such devices could potentially accurately diagnose the causative pathogen associated with a respiratory tract infection and any accompanying antibiotic resistance genes (if present), thereby potentially helping the GP to personalize their antibiotic prescribing practices to the needs of each patient. Unfortunately, however, many hurdles currently exist to the successful implementation of infectious disease POC diagnostics into primary care, the most important of which are described below.
• F. Schuler, M. Trotter, M. Geltman, F. Schwemmer, S. Wadle, E. Domínguez-Garrido, M. López, C. Cervera-Acedo, P. Santibáñez, F. von Stetten, R. Zengerle, N. Paust
  Digital Droplet PCR on Disk
  2016 Lab Chip, volume: 16, pages: 208 - 216
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  Abstract

  Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis.
• F. Schuler, C. Siber, S. Hin, S. Wadle, N. Paust, R. Zengerle, F. von Stetten
  Digital droplet LAMP as microfluidic App on standard laboratory devices
  2016 Anal Methods-uk, volume: 8, pages: 2750 - 2755
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  Abstract

  Digital nucleic acid amplification methods are a growing research field that allows for absolute quantification of DNA making the need of standard curves redundant. However, most existing digital amplification systems require specialized laboratory devices and costly investments. The required disposable cartridges are device specific and not interchangeable. Here, we present digital droplet loopmediated isothermal amplification (ddLAMP) as a microfluidic App on standard laboratory devices. ddLAMP is implemented on a disposable polymer chip (DropChip) of the format of a standard microscope slide. After DNA denaturation off-chip the reaction mix is emulsified in the DropChip in a mini-centrifuge in 6 minutes. The DropChip is transferred to an in situ thermal cycler for 1 hour of incubation. Afterwards, a fluorescence scan in a microarray scanner is performed. The DropChip allows for absolute quantification with a dynamic range of 15-1500 DNA copies μl-1. Assay conditions were optimized for ddLAMP and comparison of ddPCR and ddLAMP for genomic E. coli DNA reveals very good concordance.
• N. Wehkamp, M. Breitwieser, A. Büchler, M. Klingele, R. Zengerle, S. Thiele
  Directly deposited Nafion/TiO2 composite membranes for high power medium temperature fuel cells
  2016 Rsc Adv, volume: 6, pages: 24261 - 24266
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  Abstract

  This work presents a simple production method for TiO2 reinforced Nafion® membranes which are stable up to a 120 °C operation temperature. The novel TiO2 reinforced membranes yield a maximum power density of 2.02 W cm−2 at 120 °C; H2/O2; 0.5/0.5 L min−1; 90% RH, 300/300 kPaabs. This is 2.8 times higher than the highest power density for TiO2 reinforced membranes so far published in literature. The described membranes even exceed the maximum power density of a commercial Nafion® HP membrane in an identical measurement setup at 100 °C and 120 °C. Compared to the commercial Nafion® HP membrane the maximum power density was increased by 27% and 9% at 100 °C and 120 °C, respectively. The membrane is manufactured by drop-casting a dispersion of Nafion® and TiO2 nanoparticles onto both the anode and cathode gas diffusion electrodes. Furthermore pure Nafion® membranes manufactured by the same method had higher membrane resistances at temperatures >100 °C than TiO2-reinforced Nafion® membranes.
• X. Wu, N. Schneider, A. Platen, I. Mitra, R. Zengerle, R. Schüle, M. Meier
  In situ characterization of the mTORC1 during adipogenesis of human adult stem cells on chip
  2016 PNAS, volume: 113, pages: 4143 - 4150
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  Abstract

  Mammalian target of rapamycin (mTOR) is a central kinase in- tegrating nutrient, energy, and metabolite signals. The kinase forms two distinct complexes: mTORC1 and mTORC2. mTORC1 plays an essential but undefined regulatory function for regeneration of adipose tissue. Analysis of mTOR in general is hampered by the complexity of regulatory mechanisms, including protein interac- tions and/or phosphorylation, in an ever-changing cellular microen- vironment. Here, we developed a microfluidic large-scale integration chip platform for culturing and differentiating human adipose- derived stem cells (hASCs) in 128 separated microchambers under standardized nutrient conditions over 3 wk. The progression of the stem cell differentiation was measured by determining the lipid accumulation rates in hASC cultures. For in situ protein analytics, we developed a multiplex in situ proximity ligation assay (mPLA) that can detect mTOR in its two complexes selectively in single cells and implemented it on the same chip. With this combined technology, it was possible to reveal that the mTORC1 is regulated in its abundance, phosphorylation state, and localization in coordination with lyso- somes during adipogenesis. High-content image analysis and param- eterization of the in situ PLA signals in over 1 million cells cultured on four individual chips showed that mTORC1 and lysosomes are temporally and spatially coordinated but not in its composition during adipogenesis.
• M. Zeiger, S. Fleischmann, B. Krüner, A. Tolosa, S. Bechtel, M. Baltes, A. Schreiber, R. Moroni, S. Vierrath, S. Thiele, V. Presser
  Influence of carbon substrate on the electrochemical performance of carbon/manganese oxide hybrids in aqueous and organic electrolytes
  2016 Rsc Adv, volume: 6, pages: 107163 - 107
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  Abstract

  Manganese oxide presents very promising electrochemical properties as an electrode material in supercapacitors, but there remain important open questions to guide further development of the complex manganese oxide/carbon/electrolyte system. Our work addresses specifically the influence of carbon ordering and the difference between outer and inner porosity of carbon particles for the application in aqueous 1 M Na2SO4 and 1 M LiClO4 in acetonitrile. Birnessite-type manganese oxide was hydrothermally hybridized on two kinds of carbon onions with only outer surface area and different electrical conductivity, and conventional activated carbon with a high inner porosity. Carbon onions with a high degree of carbon ordering, high conductivity, and high outer surface area were identified as the most promising material, yielding 179 F g−1. Pore blocking in activated carbon yields unfavorable electrochemical performances. The highest specific energy of 16.4 W h kg−1 was measured for a symmetric full-cell arrangement of manganese oxide coated high temperature carbon onions in the organic electrolyte. High stability during 10 000 cycles was achieved for asymmetric full-cells, which proved as a facile way to enhance the electrochemical performance stability.
• F. Schwemmer, C. E. Blanchet, A. Spilotros, D. Kosse, S. Zehnle, H. D. T. Mertens, M. A. Graewert, M. Rössle, N. Paust, D. I. Svergun, F. von Stetten, R. Zengerle, D. Mark
  LabDisk for SAXS: a centrifugal microfluidic sample preparation platform for small-angle X-ray scattering
  2016 Lab Chip, volume: 16, pages: 1161 - 1170
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  Abstract

  We present a centrifugal microfluidic LabDisk for protein structure analysis via small-angle X-ray scattering (SAXS) on synchrotron beamlines. One LabDisk prepares 120 different measurement conditions, grouped into six dilution matrices. Each dilution matrix: (1) features automatic generation of 20 different measurement conditions from three input liquids and (2) requires only 2.5 μl of protein solution, which corresponds to a tenfold reduction in sample volume in comparison to the state of the art. Total hands on time for preparation of 120 different measurement conditions is less than 5 min. Read-out is performed on disk within the synchrotron beamline P12 at EMBL Hamburg (PETRA III, DESY). We demonstrate: (1) aliquoting of 40 nl aliquots for five different liquids typically used in SAXS and (2) confirm fluidic performance of aliquoting, merging, mixing and read-out from SAXS experiments (2.7–4.4% CV of protein concentration). We apply the LabDisk for SAXS for basic analysis methods, such as measurement of the radius of gyration, and advanced analysis methods, such as the ab initio calculation of 3D models. The suitability of the LabDisk for SAXS for protein structure analysis under different environmental conditions is demonstrated for glucose isomerase under varying protein and NaCl concentrations. We show that the apparent radius of gyration of the negatively charged glucose isomerase decreases with increasing protein concentration at low salt concentration. At high salt concentration the radius of gyration (Rg) does not change with protein concentrations. Such experiments can be performed by a non-expert, since the LabDisk for SAXS does not require attachment of tubings or pumps and can be filled with regular pipettes. The new platform has the potential to introduce routine high-throughput SAXS screening of protein structures with minimal input volumes to the regular operation of synchrotron beamlines.
• F. Stumpf, F. Schwemmer, T. Hutzenlaub, D. Baumann, O. Strohmeier, G. Dingemanns, G. Simons, C. Sager, L. Plobner, F. von Stetten, R. Zengerle, D. Mark
  LabDisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza A H3N2 virus
  2016 Lab Chip, volume: 16, pages: 199 - 207
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  Abstract

  Portable point-of-care devices for pathogen detection require easy, minimal and user-friendly handling steps and need to have the same diagnostic performance compared to centralized laboratories. In this work we present a fully automated sample-to-answer detection of influenza A H3N2 virus in a centrifugal LabDisk with complete prestorage of reagents. Thus, the initial supply of the sample remains the only manual handling step. The self-contained LabDisk automates by centrifugal microfluidics all necessary process chains for PCR-based pathogen detection: pathogen lysis, magnetic bead based nucleic acid extraction, aliquoting of the eluate into 8 reaction cavities, and real-time reverse transcription polymerase chain reaction (RT-PCR). Prestored reagents comprise air dried specific primers and fluorescence probes, lyophilized RT-PCR mastermix and stick-packaged liquid reagents for nucleic acid extraction. Employing two different release frequencies for the stick-packaged liquid reagents enables on-demand release of highly wetting extraction buffers, such as sequential release of lysis and binding buffer. Microfluidic process-flow was successful in 54 out of 55 tested LabDisks. We demonstrate successful detection of the respiratory pathogen influenza A H3N2 virus in a total of 18 LabDisks with sample concentrations down to 2.39 × 104 viral RNA copies per ml, which is in the range of clinical relevance. Furthermore, we detected RNA bacteriophage MS2 acting as internal control in 3 LabDisks with a sample concentration down to 75 plaque forming units (pfu) per ml. All experiments were applied in a 2 kg portable, laptop controlled point-of-care device. The turnaround time of the complete analysis from sample-to-answer was less than 3.5 hours.
• J. Riba, T. Gleichmann, S. Zimmermann, R. Zengerle, P. Koltay
  Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing
  2016 Scientific Reports, volume: 6, page: 32837
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  Abstract

  The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.
• K. G. Kraiczek, J. Mannion, S. Post, A. Tsupryk, V. Raghunathan, R. Brennen, R. Zengerle
  Micromachined Fused Silica Liquid Core Waveguide Capillary Flow Cell
  2016 Anal Chem, volume: 88, pages: 1100 - 1105
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  Abstract

  A planar, chip-based flow cell for UV−vis absorbance detection in HPLC is presented. The device features a microfabricated free-standing liquid core waveguide (LCW) capillary detection tube of long path length that is based on total internal reflection. We report on the linearity and calibration slope characteristics of lithographically produced LCWs with different interior/exterior geometries. 3D ray tracing was indispensable in modeling behavior in the more demanding geometries: multipath behavior may be intrinsic to these waveguides with consequent nonlinearity. Fortunately, nonlinearity in lithographically easy-to-produce waveguide geometries (such as with a flat, concave exterior and a round interior) is not as detrimental as might be initially expected. Experimental performance is predictably affected by the attainable surface quality of the LCW and efficient and reproducible coupling of the input light into the LCW.
• J. Riba, N. Renz, C. Niemöller, S. Bleul, D. Pfeifer, J. M. Stosch, K. H. Metzeler, B. Hackanson, M. Lübbert, J. Duyster, P. Koltay, R. Zengerle, R. Claus, S. Zimmermann, H. Becker
  Molecular Genetic Characterization of Individual Cancer Cells Isolated via Single-Cell Printing
  2016 Plos One, volume: 6, page: 32837
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  Abstract

  Intratumoral genetic heterogeneity may impact disease outcome. Gold standard for dissecting clonal heterogeneity are single-cell analyses. Here, we present an efficient workflow based on an advanced Single-Cell Printer (SCP) device for the study of gene variants in single cancer cells. To allow for precise cell deposition into microwells the SCP was equipped with an automatic dispenser offset compensation, and the 384-microwell plates were electrostatically neutralized. The ejection efficiency was 99.7% for fluorescent beads (n = 2304) and 98.7% for human cells (U-2 OS or Kasumi-1 cancer cell line, acute myeloid leukemia [AML] patient; n = 150). Per fluorescence microscopy, 98.8% of beads were correctly delivered into the wells. A subset of single cells (n = 81) was subjected to whole genome amplification (WGA), which was successful in all cells. On empty droplets, a PCR on LINE1 retrotransposons yielded no product after WGA, verifying the absence of free-floating DNA in SCP-generated droplets. Representative gene variants identified in bulk specimens were sequenced in single-cell WGA DNA. In U-2 OS, 22 of 25 cells yielded results for both an SLC34A2 and TET2 mutation site, including cells harboring the SLC34A2 but not the TET2 mutation. In one cell, the TET2 mutation analysis was inconclusive due to allelic dropout, as assessed via polymorphisms located close to the mutation. Of Kasumi-1, 23 of 33 cells with data on both the KIT and TP53 mutation site harbored both mutations. In the AML patient, 21 of 23 cells were informative for a TP53 polymorphism; the identified alleles matched the loss of chromosome arm 17p. The advanced SCP allows efficient, precise and gentle isolation of individual cells for subsequent WGA and routine PCR/sequencing-based analyses of gene variants. This makes single-cell information readily accessible to a wide range of applications and can provide insights into clonal heterogeneity that were indeterminable solely by analyses of bulk specimens.
• F. Schuler, M. Trotter, R. Zengerle, F. von Stetten
  Monochrome Multiplexing in Polymerase Chain Reaction by Photobleaching of Fluorogenic Hydrolysis Probes
  2016 Anal Chem, volume: 88, issue: 5, pages: 2590 - 2595
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  Abstract

  Multiplexing in polymerase chain reaction (PCR) is a technique widely used to save cost and sample material and to increase sensitivity compared to distributing a sample to several singleplex reactions. One of the most common methods to detect the different amplification products is the use of fluorogenic probes that emit at different wavelengths (colors). To reduce the number of detection channels, several methods for monochrome multiplexing have been suggested. However, they pose restrictions to the amplifiable target length, the sequence, or the melting temperature. To circumvent these limitations, we suggest a novel approach that uses different fluorophores with the same emission maximum. Discrimination is achieved by their different fluorescence stability during photobleaching. Atto488 (emitting at the same wavelength as 6-carboxyfluorescein, FAM) and Atto467N (emitting at the same wavelength as cyanine 5, Cy5) were found to bleach significantly less than FAM and Cy5; i.e., the final fluorescence of Atto dyes was more than tripled compared to FAM and Cy5. We successfully applied this method by performing a 4-plex PCR targeting antibiotic resistance genes in S. aureus using only 2 color channels. Confidence of discrimination between the targets was >99.9% at high copy initial copy numbers of 100 000 copies. Cases where both targets were present could be discriminated with equal confidence for Cy5 channel and reduced levels of confidence (>68%) for FAM channel. Moreover, a 2- plex digital PCR reaction in 1 color channel was shown. In the future, the degree of multiplexing may be increased by adding fluorogenic probe pairs with other emission wavelengths. The method may also be applied to other probe and assay formats, such as Förster resonance energy transfer (FRET) probes and immunoassays.
• F. Sun, L. Zielke, H. Markötter, A. Hilger, D. Zhou, R. Moroni, R. Zengerle, S. Thiele, J. Banhart, I. Manke
  Morphological Evolution of Electrochemically Plated/Stripped Lithium Microstructures Investigated by Synchrotron X‑ray Phase Contrast Tomography
  2016 Acs Nano, volume: 10, pages: 7990 - 7997
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  Abstract

  Due to its low redox potential and high theoretical specific capacity, Li metal has drawn worldwide research attention because of its potential use in nextgeneration battery technologies such as Li−S and Li−O2. Unfortunately, uncontrollable growth of Li microstructures (LmSs, e.g., dendrites, fibers) during electrochemical Li stripping/plating has prevented their practical commercialization. Despite various strategies proposed to mitigate LmS nucleation and/or block its growth, a fundamental understanding of the underlying evolution mechanisms remains elusive. Herein, synchrotron in-line phase contrast X-ray tomography was employed to investigate the morphological evolution of electrochemically deposited/dissolved LmSs nondestructively. We present a 3D characterization of electrochemically stripped Li electrodes with regard to electrochemically plated LmSs. We clarify fundamentally the origin of the porous lithium interface growing into Li electrodes. Moreover, cleavage of the separator caused by growing LmS was experimentally observed and visualized in 3D. Our systematic investigation provides fundamental insights into LmS evolution and enables us to understand the evolution mechanisms in Li electrodes more profoundly.
• R. Moroni, M. Börner, L. Zielke, M. Schroeder, S. Nowak, M. Winter, I. Manke, R. Zengerle, S. Thiele
  Multi-Scale Correlative Tomography of a Li-Ion Battery Composite Cathode
  2016 Sci Rep-uk, volume: 6, page: 30109
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  Abstract

  Focused ion beam/scanning electron microscopy tomography (FIB/SEMt) and synchrotron X-ray tomography (Xt) are used to investigate the same lithium manganese oxide composite cathode at the same specific spot. This correlative approach allows the investigation of three central issues in the tomographic analysis of composite battery electrodes: (i) Validation of state-of-the-art binary active material (AM) segmentation: Although threshold segmentation by standard algorithms leads to very good segmentation results, limited Xt resolution results in an AM underestimation of 6 vol% and severe overestimation of AM connectivity. (ii) Carbon binder domain (CBD) segmentation in Xt data: While threshold segmentation cannot be applied for this purpose, a suitable classification method is introduced. Based on correlative tomography, it allows for reliable ternary segmentation of Xt data into the pore space, CBD, and AM. (iii) Pore space analysis in the micrometer regime: This segmentation technique is applied to an Xt reconstruction with several hundred microns edge length, thus validating the segmentation of pores within the micrometer regime for the first time. The analyzed cathode volume exhibits a bimodal pore size distribution in the ranges between 0–1 μm and 1–12 μm. These ranges can be attributed to different pore formation mechanisms.
• S. Wadle, M. Lehnert, S. Rubenwolf, R. Zengerle, F. von Stetten
  Real-time PCR probe optimization using design of experiments approach
  2016 Biomolecular Detection and Quantification, volume: 7, pages: 1 - 8
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  Abstract

  Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3–14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7–11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times.
• S. Burger, M. Schulz, F. von Stetten, R. Zengerle, N. Paust
  Rigorous buoyancy driven bubble mixing for centrifugal microfluidics
  2016 Lab Chip, pages: 261 - 268
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  Abstract

  We present batch-mode mixing for centrifugal microfluidics operated at fixed rotational frequency. Gas is generated by the disk integrated decomposition of hydrogen peroxide (H2O2) to liquid water (H2O) and gaseous oxygen (O2) and inserted into a mixing chamber. There, bubbles are formed that ascent through the liquid in the artificial gravity field and lead to drag flow. Additionaly, strong buoyancy causes deformation and rupture of the gas bubbles and induces strong mixing flows in the liquids. Buoyancy driven bubble mixing is quantitatively compared to shake mode mixing, mixing by reciprocation and vortex mixing. To determine mixing efficiencies in a meaningful way, the different mixers are employed for mixing of a lysis reagent and human whole blood. Subsequently, DNA is extracted from the lysate and the amount of DNA recovered is taken as a measure for mixing efficiency. Relative to standard vortex mixing, DNA extraction based on buoyancy driven bubble mixing resulted in yields of 92 ± 8% (100 s mixing time) and 100 ± 8% (600 s) at 130g centrifugal acceleration. Shake mode mixing yields 96 ± 11% and is thus equal to buoyancy driven bubble mixing. An advantage of buoyancy driven bubble mixing is that it can be operated at fixed rotational frequency, however. The additional costs of implementing buoyancy driven bubble mixing are low since both the activation liquid and the catalyst are very low cost and no external means are required in the processing device. Furthermore, buoyancy driven bubble mixing can easily be integrated in a monolithic manner and is compatible to scalable manufacturing technologies such as injection moulding or thermoforming. We consider buoyancy driven bubble mixing an excellent alternative to shake mode mixing, in particular if the processing device is not capable of providing fast changes of rotational frequency or if the low average rotational frequency is challenging for the other integrated fluidic operations.
• L. Gutzweiler, F. Stumpf, L. Tanguy, G. Roth, P. Koltay, R. Zengerle, L. Riegger
  Semi-contact-writing of polymer molds for prototyping PDMS chips with low surface roughness, sharp edges and locally varying channel heights
  2016 J Micromech Microeng, volume: 26, issue: 4, pages: 45018 - 45027
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  Abstract

  Microfluidic systems fabricated in polydimethylsiloxane (PDMS) enable a broad variety of applications and are widespread in the field of Lab-on-a-Chip. Here we demonstrate semi-contact-writing, a novel method for fabrication of polymer based molds for casting microfluidic PDMS chips in a highly flexible, time and cost-efficient manner. The method is related to direct-writing of an aqueous polymer solution on a planar glass substrate and substitutes conventional, time- and cost-consuming UV-lithography. This technique facilitates on-demand prototyping in a low-cost manner and is therefore ideally suited for rapid chip layout iterations. No cleanroom facilities and less expertise are required. Fabrication time from scratch to ready-to-use PDMS-chip is less than 5 h. This polymer writing method enables structure widths down to 140 μm and controllable structure heights ranging from 5.5 μm for writing single layers up to 98 μm by stacking. As a unique property, freely selectable height variations across a substrate can be achieved by application of local stacking. Furthermore, the molds exhibit low surface roughness (R a   =  24 nm, R RMS  =  28 nm) and high fidelity edge sharpness. We validated the method by fabrication of molds to cast PDMS chips for droplet based flow-through PCR with single-cell sensitivity.
• S. Wadle, M. Lehnert, F. Schuler, R. Köppel, A. Serr, R. Zengerle, F. von Stetten
  Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters
  2016 BioTechniques, volume: 61, pages: 123 - 128
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  Abstract

  Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2–5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength.
• S. Kartmann, F. Koch, P. Koltay, R. Zengerle, A. Ernst
  Single-use capacitive pressure sensor employing radial expansion of a silicone tube
  2016 Sensor Actuat A-phys, volume: 247
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  Abstract

  This paper reports on a single-use pressure sensor for medical applications. The sensor principle makes use of the radial expansion of a silicone tube which occurs when there is a pressure difference between the inside and the outside of the tube. The change in outer diameter is detected by a capacitive measurement method, whereas the amplification electronics can be separated from the tube being in contact with the fluid. This enables a cross-contamination-free measurement system for patient near application as e.g. within infusion systems. The sensor includes a novel highly sensitive and fast analog amplification circuit in combination with gold electrodes fabricated in PCB technology. This circuit enables to amplify even highly dynamic pressure changes, in the range of a few milliseconds causing capacitive changes on the electrodes in the fF range to a readable voltage level. A functional model is developed based on flex board technology which allows for studying the influence of the electrode geometry as well as the material properties of the silicone tube with respect to the sensitivity of the sensor. The best performing electrode geometry determined by experiments features a sensitivity of 0.195 fF/kPa at a mean coefficient of variation (CV) of 6.4%, considering three individual sensor assemblies. A sensor prototype is designed and fabricated based on the results of the investigated functional model. The performance of the prototype is investigated experimentally in a pressure range from 0 to 40 kPa. As a result a sensitivity of 0.135 V/kPa for DI water as measurement liquid could be achieved and a good linearity of the signal (R² = 0.996) was observed up to 35 kPa.
• L. Zielke, F. Sun, H. Markötter, A. Hilger, R. Moroni, R. Zengerle, S. Thiele, J. Banhart, I. Manke
  Synchrotron X-ray tomographic study of a silicon electrode before and after discharge and the effect of cavities on particle fracturing
  2016 Chemelectrochem, volume: 3, pages: 1170 - 1177
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  Abstract

  Silicon (Si) has been proposed as one of the most promising anode materials for next-generation lithium ion batter- ies, but unsatisfactory discharge capacity and inevitable perfor- mance deterioration prevent their commercialization. We apply in situ synchrotron X-ray tomography to a Si-composite elec- trode in its pristine and firstly discharged state and quantitatively investigate degradation of the electron- and/or ion- conducting network as well as degradation of Si particles. Thus this study is complementary to previous X-ray tomographic studies focusing on the Si particles only. We found that on the electrode level the Si particles located in the central part of the electrode primarily experience crack formation; on the particle level, lithiation be- haviour is heterogeneous and cavities are formed during elec- trode preparation and battery operation. The correlation between the electrochemical activities of Si particles and their individual contact with the conducting network is investigated and quanti- fied: Si particles will experience lithiation only under the condi- tion that at least 40% of their surface is electrically and ionically connected.
• I. Schwarz, S. Zehnle, T. Hutzenlaub, R. Zengerle, N. Paust
  System-level network simulation for robust centrifugal-microfluidic lab-on-a-chip systems
  2016 Lab Chip, volume: 16, pages: 1873 - 1885
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  Abstract

  Centrifugal microfluidics shows a clear trend towards a higher degree of integration and parallelization1. This trend leads to an increase in number and density of integrated microfluidic unit operations. The fact that all unit operations are processed by the same common spin protocol turns higher integration into higher complexity. To allow for efficient development anyhow, we introduce advanced lumped models for network simulations in centrifugal microfluidics. These models consider the interplay of centrifugal and Euler pressures, viscous dissipation, capillary pressures and pneumatic pressures. The simulations are fast and simple to set up and allow for the precise prediction of flow rates as well as switching and valving events. During development, channel and chamber geometry variations due to manufacturing tolerances can be taken into account as well as pipetting errors, variations of contact angles, compliant chamber walls and temperature variations in the processing device. As an example for considering these parameters during development, we demonstrate simulation based robustness analysis for pneumatic siphon valving in centrifugal microfluidics. Subsequently the influence of liquid properties on pumping and valving is studied for four liquids relevant for biochemical analysis, namely: water (large surface tension), blood plasma (large contact angle hysteresis), ethanol/water (highly wetting) and glycerine/water (highly viscous). In a second example, we derive a spin protocol to attain a constant flow rate under varying pressure conditions. Both examples show excellent agreement to experimental validations.
• J. Madjarov, A. Prokhorova, T. Messinger, J. Gescher, S. Kerzenmacher
  The performance of microbial anodes in municipal wastewater: Pre-grown multispecies biofilm vs. natural inocula
  2016 Bioresour Technol., volume: 221, pages: 165 - 171
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  Abstract

  In this study, different inoculation strategies for continuously operated microbial anodes are analyzed and compared. After 20 days of operation with municipal wastewater anodes pre-incubated with a biofilm of the exoelectrogenic species Geobacter and Shewanella showed current densities of (65 ± 8) μA/cm2. This is comparable to the current densities of non-inoculated anodes and anodes inoculated with sewage sludge. Analysis of the barcoded pre-grown multispecies biofilms reveal that 99% of the original biofilm was detached after 20 days of operation with municipal wastewater. This is in contrast to previous experiments where a pre-grown biofilm of exoelectrogens was operated in batch mode. To implement pre-grown biofilms in continuous systems it will thus be necessary to reveal a window of process parameters in which typical exoelectrogenic microorganisms including model organisms can be kept and/or enriched on anodes.
• S. Vierrath, M. Breitwieser, M. Klingele, B. Britton, S. Holdcroft, R. Zengerle, S. Thiele
  The reasons for the high power density of fuel cells fabricated with directly deposited membranes
  2016 J. of Power Sources, volume: 326, pages: 170 - 175
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  Abstract

  In a previous study, we reported that polymer electrolyte fuel cells prepared by direct membrane deposition (DMD) produced power densities in excess of 4 W/cm2. In this study, the underlying origins that give rise to these high power densities are investigated and reported. The membranes of high power, DMD-fabricated fuel cells are relatively thin (12 mm) compared to typical benchmark, commercially available membranes. Electrochemical impedance spectroscopy, at high current densities (2.2 A/cm2) reveals that mass transport resistance was half that of reference, catalyst-coated-membranes (CCM). This is attributed to an improved oxygen supply in the cathode catalyst layer by way of a reduced propensity of flooding, and which is facilitated by an enhancement in the back diffusion of water from cathode to anode through the thin directly deposited membrane. DMD-fabricated membrane-electrode-assemblies possess 50% reduction in ionic resistance (15 mUcm2) compared to conventional CCMs, with contributions of 9 mUcm2 for the membrane resistance and 6 mUcm2 for the contact resistance of the membrane and catalyst layer ionomer. The improved mass transport is responsible for 90% of the increase in power density of the DMD fuel cell, while the reduced ionic resistance accounts for a 10% of the improvement.
• L. Zielke, S. Vierrath, R. Moroni, A. Mondon, R. Zengerle, S. Thiele
  Three-dimensional Morphology of the Interface between Micro Porous Layer and Catalyst Layer in a Polymer Electrolyte Membrane Fuel Cell
  2016 Rsc Advances, volume: 6, pages: 80700 - 80705
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  Abstract

  Interfaces between the different layers in proton exchange membrane fuel cells are expected to influence transport properties and therefore cell performance. So far the interface between micro porous layer (MPL) and catalyst layer (CL) was difficult to investigate due to its nanometer scale morphology. We apply focused ion beam scanning electron microscopy tomography with pore contrasting via atomic layer deposition to reconstruct a representative volume of 5.1 μm x 1.5 μm x 4.5 μm containing CL, MPL and their interface. We find that platinum in the CL results in brighter SEM image intensities, compared to the MPL. This allows i) estimating the extension of the interfacial region (530 nm), ii) evaluating Pt-content homogeneity in the CL and iii) calculating the individual roughnesses for the CL (102 nm) and for the MPL (129 nm). We further calculate porosity, pore sizes, and oxygen diffusivities. Thus, we find that the values of the parameters of the interfacial region are between those of the CL and the MPL, meaning that on the investigated scale, the interface is a homogeneous transitional region. A representativeness analysis shows that our reconstructed volume is sufficiently large concerning all calculated parameters.
• M. Breitwieser, R. Moroni, J. Schock, M. Schulz, B. Schillinger, F. Pfeiffer, R. Zengerle, S. Thiele
  Water management in novel direct membrane deposition fuel cells under low humidification
  2016 Int J Hydrog Energ, volume: 41, pages: 11412 - 11417
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  Abstract

  Polymer electrolyte membrane fuel cells (PEMFCs) fabricated by direct membrane deposition (DMD) were shown to work even at dry conditions without significant deterioration of the membrane resistance. Here, in situ neutron radiography is used to investigate the water management in those fuel cells to uncover the phenomena that lead to the robust operation under low humidification. A constant level of humidification within the membrane electrode assembly (MEA) of a DMD fuel cell is observed even under dry anode operation and 15% relative humidity on the cathode side. This proves a pronounced back diffusion of generated water from the cathode side to the anode side through the thin deposited membrane layer. Over the entire range of polarization curves a very high similarity of the water evolution in anode and cathode flow fields is found in spite of different humidification levels. It is shown that the power density of directly deposited membranes in contrast to a 50 mm thick N-112 membrane is only marginally affected by dry operation conditions. Water profiles in through-plane direction of the MEA reveal that the water content in the DMD fuel cell remains steady even at high current densities. This is in contrast to the N-112 reference fuel cell which shows a strong increase in membrane resistance and a reduced MEA water content with raising current densities. Thus this new MEA fabrication technique has a promising perspective, since dry operation conditions are highly requested in order to reduce fuel cell system costs.

2015

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• S. Kartmann, P. Koltay, R. Zengerle, A. Ernst
  A Disposable Dispensing Valve for Non-Contact Microliter Applications in a 96-Well Plate Format
  2015 Micromachines, volume: 6, pages: 423 - 436
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  Abstract

  We present a miniaturized, disposable, normally-closed electromagnetic dispensing valve for the microliter range to process 96-well plates. The novel injection-molded valve is designed to fit into a 9 mm grid to realize an eight channel dispensing head, enabling the serial processing of well plates row-by-row. The presented dispensing valve design originates from a miniaturization study of a previously developed functional model. The outer diameter of the valve, including all actuating components, was reduced by 8 mm to an overall diameter of 8.5 mm without compromising the performance. Additionally, the actuation current of the valve could be reduced to 5 A. The valve is characterized for a volume range between 840 nL and 5.3 μL. The performance of the injection molded valve is competitive to commercially available dispensing valves, featuring the advantages of low fabrication costs, disposability, low mounting size, easy handling, and super silent actuation.
• D. Czurratis, Y. Beyl, S. Zinober, F. Lärmer, R. Zengerle
  A novel concept for long-term pre-storage and release of liquids for pressure-driven lab-on-a-chip devices
  2015 J Micromech Microeng, volume: 25, page: 045002
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  Abstract

  On-chip storage of liquids is one of the major challenges of polymer-based lab-on-a-chip (LoC) devices. To ensure long-term storage of even highly volatile reagents in polymer disposal LoC cartridges, robust reagent storage concepts are necessary. Tubular bags, so-called stick packs, are widely used in the packaging industry. They offer sufficient vapor barrier properties for liquid storage. Here we present a polymer multilayer LoC-stack with integrated stick packs for the long-term storage of liquid reagents required for diagnostic applications. The storage concept fulfils two main requirements: firstly, the long-term storage of reagents in stick packs without significant losses or interaction with the surroundings and secondly, the on-demand release of liquids, which is realized by the delamination of a stick pack’s peel seam through pneumatic pressure. Furthermore, effects on the opening behavior of stick packs through accelerated aging were investigated after different storage conditions to proof repeatability. This concept enables on-chip storage of liquid reagents at room temperature and allows the implementation in different pressure driven LoC devices or similar applications. Since liquid storage in stick packs is well-established, emerging fields such as lab-on-achip combined with novel reagent release mechanisms should be of great interest for the commercialization of life science products.
• S.K. Vashist, T. van Oordt, E. M. Schneider, R. Zengerle, F. von Stetten, J.H.T. Luong
  A smartphone-based colorimetric reader for bioanalytical applications using the screen-based bottom illumination provided by gadgets
  2015 Biosens Bioelectron, volume: 67, pages: 248 - 255
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  Abstract

  A smartphone-based colorimetric reader (SBCR) was developed using a Samsung Galaxy SIII mini, a gadget (iPAD mini, iPAD4 or iPhone 5s), integrated with a custom-made dark hood and base holder assembly. The smartphone equipped with a back camera (5 megapixels resolution) was used for colorimetric imaging via the hood and base-holder assembly. A 96- or 24-well microtiter plate (MTP) was positioned on the gadget's screensaver that provides white light-based bottom illumination only in the specific regions corresponding to the bottom of MTP's wells. The pixel intensity of the captured images was determined by an image processing algorithm. The developed SBCR was evaluated and compared with a commercial MTP reader (MTPR) for three model assays: our recently developed human C-reactive protein sandwich enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase direct ELISA, and bicinchoninic acid protein estimation assay. SBCR had the same precision, dynamic range, detection limit and sensitivity as MTPR for all three assays. With advanced microfabrication and data processing, SBCR will become more compact, lighter, inexpensive and enriched with more features. Therefore, SBCR with a remarkable computing power could be an ideal point-of-care (POC) colorimetric detection device for the next-generation of cost-effective POC diagnostics, immunoassays and diversified bioanalytical applications.
• M. Blazek, T. Silva Santisteban, R. Zengerle, M. Meier
  Analysis of fast protein phosphorylation kinetics in single cells on a microfluidic chip
  2015 Lab Chip, volume: 15, pages: 726 - 734
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  Abstract

  In the present study, we developed a microfluidic large-scale integration (mLSI) platform for the temporal and chemical control of cell cultures to study fast kinetics of protein phosphorylation. For in situ protein analysis the mLSI chip integrates the Proximity Ligation Assay (PLA). To investigate cell-signaling events with a time resolution of a few seconds we first engineered and optimized the fluidic layout of the chip with 128 individual addressable cell culture chambers. The functionality of the cell culture operations and PLA is demonstrated by the determination of the minimum cell sample size for obtaining robust quantitative PLA signals at the single-cell level. We show that at least 350 cells per assay condition are required to statistically evaluate single cell PLA data. In the following we used the PLA chip with over 500 hundred cells per condition to record sequential phosphorylation reactions of the canonical protein kinase within the Akt pathway, which is activated in various human cancer types. This was achieved by stimulating mouse fibroblast cell cultures with either the platelet-derived growth factor (PDGF) or insulin-like growth factor (IGF-1). Fluidic cell stimulation pulses of 5 seconds were followed by precisely time shifted cell fixation pulses to obtain a temporal resolution of 10 seconds. PLA was then performed on all fixed arrays of cell cultures to extract the characteristic phosphorylation times at the single cell level for either the PDGF, or IGF-1 receptor and the Akt and GSK3 beta kinases. Characteristic phosphorylation times for the receptors were between 13 and 35 seconds, whereas for downstream kinases between 25 and 200 seconds. Thus we could reveal a molecular order of the phosphorylation reactions during the signal transduction through the Akt pathway. In dependence of the stimulus we found a temporal difference for the characteristic phosphorylation time of 20 and 150 seconds for the Ser-473 and Thr-308 residues on the Akt kinase, respectively. Temporal alteration of sequential phosphorylation reactions on Akt has been proposed as molecular mechanism to differentiate between stimuli and biophysically determined in the present study.
• M. Keller, J. Naue, R. Zengerle, F. von Stetten, U. Schmidt
  Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment
  2015 Plos One, volume: 10, issue: 7, page: e0131845
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  Abstract

  Nested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.
• O. Strohmeier, S. Keil, B. Kanat, P. Patel, M. Niedrig, M. Weidmann, F. Hufert, J. Drexler, R. Zengerle, F. von Stetten
  Automated nucleic acid extraction from whole blood, B . subtilis, E. coli, and Rift Valley fever virus on a centrifugal microfluidic LabDisk
  2015 Rsc Adv, volume: 5, page: 32144
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  Abstract

  We present total nucleic acid extraction from whole blood, Gram‐positive Bacillus subtilis, Gram‐negative Escherichia coli, and Rift Valley fever RNA virus on a low‐cost, centrifugal microfluidic LabDisk cartridge processed in a light‐weight (< 2 kg) and portable processing device. Compared to earlier work in disk based centrifugal microfluidics, this includes the following advances: combined lysis and nucleic acid purification on one cartridge and handling of sample volumes as large as 200 μL. The presented system has been validated for logarithmic dilutions of aforementioned bacteria and viruses from various sample matrices including blood plasma and culture media and extraction of human DNA from whole blood. Recovered DNA and RNA concentrations in the eluate were characterized by quantitative PCR to: 58.2 % ‐ 98.5 %, 45.3 % ‐ 102.1 % and 29.5 % ‐ 34.2 % versus manual reference for Bacillus subtilis, Escherichia coli and Rift Valley fever virus, respectively. For extraction of human DNA from whole blood, similar results for on‐disk ((10.1 ± 7.6) x 104 DNA copies) and manual reference extraction ((10.2 ± 6.3) x 104 DNA copies)) could be achieved. Eluates from on‐disk extraction show slightly increased ethanol concentrations of 4.1 ± 0.3 % to 5.5 ± 0.2 % compared to manual reference (2.0 ± 0.5 % to 3.6 ± 0.6 %). The complete process chain for sample preparation is automatically performed within ~30 minutes, including ~15 minutes lysis time. It is amenable to concatenation with downstream modules for multiplex nucleic acid amplification as recently demonstrated for panel testing of various pathogens at the point of care.
• F. Schuler, N. Paust, R. Zengerle, F. von Stetten
  Centrifugal Step Emulsification can Produce Water in Oil Emulsions with Extremely High Internal Volume Fractions
  2015 Micromachines, volume: 6, issue: 8, pages: 1180 - 1188
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  Abstract

  The high throughput preparation of emulsions with high internal volume fractions is important for many different applications, e.g., drug delivery. However, most emulsification techniques reach only low internal volume fractions and need stable flow rates that are often difficult to control. Here, we present a centrifugal high throughput step emulsification disk for the fast and easy production of emulsions with high internal volume fractions above 95%. The disk produces droplets at generation rates of up to 3700 droplets/s and, for the first time, enables the generation of emulsions with internal volume fractions of >97%. The coefficient of variation between droplet sizes is very good (4%). We apply our system to show the in situ generation of gel emulsion. In the future, the recently introduced unit operation of centrifugal step emulsification may be used for the high throughput production of droplets as reaction compartments for clinical diagnostics or as starting material for micromaterial synthesis.
• F. Schuler, F. Schwemmer, M. Trotter, S. Wadle, R. Zengerle, F. von Stetten, N. Paust
  Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA
  2015 Lab Chip, volume: 15, pages: 2759 - 2766
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  Abstract

  Aqueous microdroplets provide miniaturized reaction compartments for numerous chemical, biochemical or pharmaceutical applications. We introduce centrifugal step emulsification for the fast and easy production of monodispers droplets. Homogenous droplets with preselectable diameters in a range from 120 μm to 170 μm were generated with coefficients of variation of 2-4% and zero run-in time or dead volume. The droplet diameter depends on the nozzle geometry (depth, width, and step size) and interfacial tensions, only. Droplet size is demonstrated to be independent of the dispersed phase flow rate between 0.01-1 μl/s, proving the robustness of the centrifugal approach. Centrifugal step emulsification can easily be combined with existing centrifugal microfluidic unit operations, is compatible to scalable manufacturing technologies such as thermoforming or injection moulding and enables fast emulsification (> 500 droplets per second and nozzle) with minimal handling effort (2-3 pipetting steps). The centrifugal microfluidic droplet generation was used to perform the first digital droplet recombinase polymerase amplification (ddRPA). It was used for absolute quantification of Listerias monocytogenes DNA concentration standards with a total analysis time below 30 min. Compared to digital droplet polymerase chain reaction (ddPCR), with processing times of about 2 hours, the overall processing time of digital analysis was reduced by more than a factor of 4.
• F. Schwemmer, T. Hutzenlaub, D. Buselmeier, N. Paust, F. von Stetten, D. Mark, R. Zengerle, D. Kosse
  Centrifugo-pneumatic multi-liquid aliquoting – parallel aliquoting and combination of multiple liquids in centrifugal microfluidics
  2015 Lab Chip, volume: 15, pages: 3250 - 3258
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  Abstract

  The generation of mixtures with precisely metered volumes is essential for reproducible automation of laboratory workflows. Splitting a given liquid into well-defined metered sub-volumes, the so-called aliquoting, has been frequently demonstrated on centrifugal microfluidics. However, so far no solution exists for assays that require simultaneous aliquoting of multiple, different liquids and the subsequent pairwise combination of aliquots with full fluidic separation before combination. Here, we introduce the centrifugo-pneumatic multi-liquid aliquoting designed for parallel aliquoting and pairwise combination of multiple liquids. All pumping and aliquoting steps are based on a combination of centrifugal forces and pneumatic forces. The pneumatic forces are thereby provided intrinsically by centrifugal transport of the assay liquids into dead end chambers to compress the enclosed air. As an example, we demonstrate simultaneous aliquoting of 1.) a common assay reagent into twenty 5 µl aliquots and 2.) five different sample liquids, each into four aliquots of 5 µl. Subsequently, the reagent and sample aliquots are simultaneously transported and combined into twenty collection chambers. All coefficients of variation for metered volumes were between 0.4% - 1.0% for intra-run variations and 0.5% - 1.2% for inter-run variations. The aliquoting structure is compatible to common assay reagents with a wide range of liquid and material properties, demonstrated here for contact angles between 20° and 60°, densities between 789 and 1855 kg / m3 and viscosities between 0.89 and 4.1 mPa s. The centrifugo-pneumatic multi-liquid aliquoting is implemented as a passive fluidic structure into a single fluidic layer. Fabrication is compatible to scalable fabrication technologies such as injection molding or thermoforming and does not require any additional fabrication steps such as hydrophilic or hydrophobic coatings or integration of active valves.
• Y. Zhao, F. Schwemmer, S. Zehnle, F. von Stetten, R. Zengerle, N. Paust
  Centrifugo-pneumatic sedimentation, re-suspension and transport of microparticles
  2015 Lab Chip, pages: 4133 - 4137
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  Abstract

  Microparticles are widely used as solid phase for affinity based separation. Here, we introduce a new method for automated handling of microparticles in centrifugal microfluidics that is not restricted by the particle size and requires neither auxiliary means such as magnets nor coating of microfluidic structures. All steps are initiated and controlled by the speed of rotation, only. It is based on storage and “on demand” release of pneumatic energy within tuneable time frames: A slow release of the pneumatic energy triggers a first fluidic path through which the supernatant above the sedimented particles is removed. An abrupt release triggers a second path which allows for liquid routing and transport of the re-suspended particles. Re-suspension of particles is thereby achieved by quickly changing the speed of rotation. We demonstrate the exchange of the particle carrier medium with supernatant removal efficiency of more than 99.5% and particle loss below 4%. Re-suspension and subsequent transport of suspended particles shows particle loss below 7%. The method targets for the automation of particle based assays e.g. DNA extractions and immunoassays. It is compatible to monolithic integration and suitable for mass production technologies e.g. thermoforming or injection moulding.
• M. Keller, S. Wadle, N. Paust, L. Dreesen, C. Nuese, O. Strohmeier, R. Zengerle, F. von Stetten
  Centrifugo-thermopneumatic fluid control for valving and aliquoting applied to multiplex real-time PCR on off-the-shelf centrifugal thermocycler
  2015 Rsc Adv, volume: 5, pages: 89603 - 89611
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  Abstract

  We introduce microfluidic automation of geometrically multiplexed real-time PCR to off-the-shelf Rotor-Gene Q thermocyclers (RGQ, QIAGEN GmbH, Hilden, Germany). For centrifugal fluid control the RGQ provides low and constant rotation of 400 rpm, only. Compatibility to this very limited flexibility of centrifugal actuation is achieved by using thermal gas compression and expansion for valving and aliquoting. In contrast to existing thermo-pneumatic actuation, centrifugo-thermopneumatic (CTP) fluid control employs the induced change of partial vapor pressure by global temperature control as actuation parameter for two new unit operations: CTP siphon valving and CTP two-stage aliquoting. CTP siphon valving was demonstrated to reliably transfer sample liquid in all cases (n = 35) and CTP two-step aliquoting transfers metered aliquots of 18.2 ± 1.2 μl (CV 6.7%, n = 8) into reaction cavities within 5 s (n = 24). Thermal characteristics of CTP two-stage aliquoting were found to be in good agreement with an introduced analytical model (R2 = 0.9876, n = 3). A microfluidic disk segment comprising both new unit operations was used for automation of real-time PCR amplification of Escherichia coli DNA. Required primers and probes were pre-stored in the reaction cavities and a comparison with reference reactions in conventional PCR tubes yielded the same PCR efficiency, repeatability, and reproducibility.
• L. Zielke, C. Barchasz, S. Waluś, F. Alloin, J.-C. Leprêtre, A. Spettl, V. Schmidt, A. Hilger, I. Manke, J. Banhart, R. Zengerle, S. Thiele
  Degradation of Li/S Battery Electrodes On 3D Current Collectors Studied Using X-ray Phase Contrast Tomography
  2015 Sci Rep-uk, volume: 5, page: 10921
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  Abstract

  Lithium/sulphur batteries are promising candidates for future energy storage systems, mainly due to their high potential capacity. However low sulphur utilization and capacity fading hinder practical realizations. In order to improve understanding of the system, we investigate Li/S electrode morphology changes for different ageing steps, using X-ray phase contrast tomography. Thereby we find a strong decrease of sulphur loading after the first cycle, and a constant loading of about 15% of the initial loading afterwards. While cycling, the mean sulphur particle diameters decrease in a qualitatively similar fashion as the discharge capacity fades. The particles spread, migrate into the current collector and accumulate in the upper part again. Simultaneously sulphur particles lose contact area with the conducting network but regain it after ten cycles because their decreasing size results in higher surface areas. Since the capacity still decreases, this regain could be associated with effects such as surface area passivation and increasing charge transfer resistance.
• M. Klingele, M. Breitwieser, R. Zengerle, S. Thiele
  Direct Deposition of Proton Exchange Membranes Enabling High Performance Hydrogen Fuel Cells
  2015 J. Mat. Chem. A, volume: 3, pages: 11239 - 11245
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  Abstract

  We apply drop-on-demand inkjet printing to fabricate proton exchange membranes for polymer electrolyte fuel cells. This completely substitutes the commonly used membrane foil. A Nafion® dispersion is deposited directly onto the catalyst layers of anode and cathode gas diffusion electrodes, and the two electrodes are pressed together with membrane layers facing each other. Fuel cells constructed utilizing this method reveal a thin overall membrane thickness of 8-25 µm and a good adhesion of membrane and catalyst layers. This results in a membrane ionic resistance of only 12.7 mΩ*cm² without compromising hydrogen crossover, which was determined to be less than 2 mA/cm². We achieve a cell power density exceeding 4 W/cm² with pure oxygen as cathode fuel, which, to our knowledge, is the highest reported power density with a Nafion® membrane hydrogen fuel cell. The membrane shows a stable performance over the entire range of reactant gas humidification from 0 to 100 % relative humidity. Power densities exceeding 1.0 W/cm² are achieved under dry operation with air as cathode fuel. A 576 hour combined mechanical and chemical accelerated stress test reveals no significant degradation of hydrogen crossover, indicating a promising lifetime of the membrane.
• L. Drechsel, M. Schulz, F. von Stetten, C. Moldovanc, R. Zengerle, N. Paust
  Electrochemical pesticide detection with AutoDip – a portable platform for automation of crude sample analyses
  2015 Lab Chip, volume: 15, pages: 704 - 710
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  Abstract

  Lab-on-a-chip devices hold promise for automation of complex workflows from sample to answer with minimal consumption of reagents in portable devices. However, complex, inhomogeneous samples as they occur in environmental or food analysis may block microchannels and thus often cause malfunction of the system. Here we present the novel AutoDip platform which is based on the movement of a solid phase through the reagents and sample instead of transporting a sequence of reagents through a fixed solid phase. A ball-pen mechanism operated by an external actuator automates unit operations such as incubation and washing by consecutively dipping the solid phase into the corresponding liquids. The platform is applied to electrochemical detection of organophosphorus pesticides in real food samples using an acetylcholinesterase (AChE) biosensor. Minimal sample preparation and an integrated reagent pre-storage module hold promise for easy handling of the assay. Detection of the pesticide chlorpyrifos-oxon (CPO) spiked into apple samples at concentrations of 10−7 M has been demonstrated. This concentration is below the maximum residue level for chlorpyrifos in apples defined by the European Commission.
• I. Leibacher, J. Schoendube, J. Dual, R. Zengerle, P. Koltay
  Enhanced single-cell printing by acoustophoretic cell focusing
  2015 Biomicrofluidics, volume: 9, page: 024109
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  Abstract

  Recent years have witnessed a strong trend towards analysis of single-cells. To access and handle single-cells, many new tools are needed and have partly been developed. Here, we present an improved version of a single-cell printer which is able to deliver individual single cells and beads encapsulated in free-flying picoliter droplets at a single-bead efficiency of 96% and with a throughput of more than 10 beads per minute. By integration of acoustophoretic focusing, the cells could be focused in x and y direction. This way, the cells were lined-up in front of a 40 µm nozzle, where they were analyzed individually by an optical system prior to printing. In agreement with acoustic simulations, the focusing of 10 µm beads and Raji cells has been achieved with an efficiency of 99% (beads) and 86% (Raji cells) to a 40 µm wide center region in the 1mm wide microfluidic channel. This enabled improved optical analysis and reduced bead losses. The loss of beads that ended up in the waste (because printing them as single beads arrangements could not be ensured) was reduced from 52 ± 6% to 28 ± 1%. The piezoelectric transducer employed for cell focusing could be positioned on an outer part of the device, which proves the acoustophoretic focusing to be versatile and adaptable.
• S. Vierrath, F. Güder, A. Menzel, M. Hagner, R. Zengerle, M. Zacharias, S. Thiele
  Enhancing the Quality of the Tomography of Nanoporous Materials for Better Understanding of Polymer Electrolyte Fuel Cell Materials
  2015 J Power Sources, pages: 413 - 417
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  Abstract

  To investigate the nanostructure of polymer electrolyte fuel cell catalyst layers, focused ion beam - scanning electron microscopy (FIB-SEM) tomography is a common technique. However, as FIB-SEM tomography lacks of image contrast between the catalyst layer and its pores, state-of-the-art reconstruction methods by threshold cannot accurately distinguish between these two phases. We show that this inability leads to an underestimation of the porosity by a factor of nearly two, a reconstruction with channel-like artifacts and that these artifacts make it impossible to calculate reliable diffusivities. To overcome this problem, we fill the pores of the catalyst layer with ZnO via atomic layer deposition prior to tomography. By using atomic layer deposition, even smallest pores can be filled with ZnO, which exhibits a good contrast to the catalyst layer in SEM images. As a result, we present the porosity of the catalyst layer (65%) and its three-dimensional representation without typical reconstruction artifacts. Calculated O2 diffusivities (23.05 - 25.40 x 10-7 m2s-1) inside the catalyst layer are in good agreement with experimental values from the literature. Furthermore, filling with ZnO permits the identification of large Pt clusters inside the catalyst layer, which were estimated to reduce the catalyst surface area by 9%.
• A.G. Venkatesh, S. K. Vashist, E. M. Schneider, R. Zengerle, F. von Stetten, J. H.T. Luong
  Graphene-based C-reactive protein immunoassay with smartphone readout
  2015 Protocol Exchange
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  Abstract

  A highly-sensitive immunoassay (IA) procedure with minimal process steps has been developed for the detection of human C-reactive protein (CRP) in less than 30 min. Graphene nanoplatelets (GNP) was admixed with 3-aminopropyltriethoxysilane (APTES) and EDC-activated anti-human CRP antibody (Ab) to form a stable complex that was then covalently attached to a KOH-pretreated polystyrene microtiter plate (MTP). The developed one-step kinetics-based IA involves the formation of a sandwich immune complex followed by two washings and an enzyme-based colorimetric reaction that was read by a smartphone-based colorimetric reader (SBCR). The detection range of CRP was 0.03-81 ng mL-1 with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.07 ng mL-1 and 0.9 ng mL-1, respectively. Moreover, the developed IA enabled the detection of CRP spiked in diluted human whole blood and plasma as well as CRP present in clinical plasma samples with high analytical precision, thereby demonstrating its immense utility for biomedical diagnostics.
• M. Breitwieser, M. Klingele, B. Britton, S. Holdcroft, R. Zengerle, S. Thiele
  Improved Pt-utilization efficiency of low Pt-loading PEM fuel cell electrodes using direct membrane deposition
  2015 Electrochem Commun, volume: 60, pages: 168 - 171
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  Abstract

  Direct membrane deposition was used to produce record platinum catalyst utilization efficiency polymer electrolyte membrane fuel cells. The novel membrane fabrication technique was applied to gas diffusion electrodes with low Pt-loadings of 0.102 and 0.029 mg/cm2. Under oxygen atmosphere and 300 kPaabs total pressure, 88 kW/gPt cathodic catalyst utilization efficiency with a symmetrical Pt-loading of 0.029 mg/cm2 on the anode and cathode side was achieved. This is 2.3 times higher than the Pt-utilization efficiency of a reference fuel cell prepared using a commercial Nafion N-211 membrane and identical catalyst layers, emphasizing that the improvement is purely attributable to the novel membrane fabrication technique. This value represents the highest Pt-utilization efficiency reported in literature. The results strongly motivate the application of employing direct membrane deposition techniques to prepare low resistance polymer electrolyte thin films in order to compensate for kinetic losses introduced when using low catalyst loadings.
• D. Czurratis, Y. Beyl, A. Grimm, T. Brettschneider, S. Zinober, F. Lärmer, R. Zengerle
  Liquids on-chip: direct storage and release employing micro-perforated vapor barrier films
  2015 Lab Chip, volume: 15, pages: 2887 - 2895
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  Abstract

  Liquids on-chip describes a reagent storage concept for disposable pressure driven Lab-on-Chip (LoC) devices, which enables liquid storage in reservoirs without additional packaging. On-chip storage of liquids can be considered as one of the major challenges for the commercial break through of polymer-based LoC devices. Especially the ability for long-term storage and reagent release on demand are the most important aspects for a fully developed technology. On-chip storage not only replaces manual pipetting, it creates numerous advantages: fully automated processing, ease of use, reduction of contamination and transportation risks. Previous concepts for on-chip storage are based on liquid packaging solutions (e.g. stick packs, blisters, glass ampoules), which implicate manufacturing complexity and additional pick and place processes. That is why we prefer on-chip storage of liquids directly in reservoirs. The liquids are collected in reservoirs, which are made of high barrier polymers or coated by selected barrier layers. Therefore, commonly used polymers for LoC applications as cyclic olefin polymer (COP) and polycarbonate (PC) were investigated in the context of novel polymer composites. To ensure long-term stability the reservoirs are sealed with a commercially available barrier film by hot embossing. The barrier film is structured by pulsed laser ablation, which installs rated break points without affecting the barrier properties. A flexible membrane is actuated through pneumatic pressure for reagent release on demand. The membrane deflection breaks the barrier film and leads to efficient cleaning of the reservoirs in order to provide the liquids for further processing.
• G.Czilwik, S.K.Vashist, V.Klein, G.Roth, A.Buderer, F. von Stetten, R. Zengerle, D.Mark
  Magnetic chemiluminescent immunoassay for human C-reactive protein on the centrifugal microfluidics platform
  2015 Rsc Adv, volume: 5, pages: 61906 - 61912
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  Abstract

  Human C-reactive protein (CRP) has been reported as an inflammatory biomarker with the highest reference for use in clinical practice. However, the existing analytical techniques are lacking automation and simplicity, as desired for a prospective immunoassay format for point-of-care (PoC) analysis. We have developed an automated magnetic chemiluminescent immunoassay (MCIA) on a mobile analyser for rapid PoC determination of CRP. The MCIA is fully automated after the initial loading of sample and immunoreagents at the inlet ports. The automated protocol involves the transportation of magnetic capture microparticles between adjacent reaction compartments using a set of stationary magnets, a microfluidic polymer disposable and a specific centrifugal protocol. The developed MCIA has a sample-to-answer time of 25 min and hands-on time of approximately 5 min. It detects the entire pathophysiological range of CRP, as desired for clinically-relevant high sensitivity CRP immunoassay format, i.e. 3–81 ng mL−1 in diluted human serum with a limit of detection (LOD) and limit of quantification (LOQ) of 1.5 ng mL−1 and 1.8 ng mL−1, respectively.
• F. Schwemmer, S. Zehnle, D. Mark, F. von Stetten, R. Zengerle, N. Paust
  Microfluidic timer for timed valving and pumping in centrifugal microfluidics
  2015 Lab Chip, volume: 15, pages: 1545 - 1553
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  Abstract

  Accurate timing of microfluidic operations is essential for the automation of complex laboratory workflows, in particular for the supply of sample and reagents. Here we present a new unit operation for timed valving and pumping in centrifugal microfluidics. It is based on temporary storage of pneumatic energy and time delayed sudden release of said energy. The timer is loaded at a relatively higher spinning frequency. The countdown is started by reducing to a relatively lower release frequency, at which the timer releases after a pre-defined delay time. We demonstrate timing for 1.) the sequential release of 4 liquids at times of 2.7 s ± 0.2 s, 14.0 s ± 0.5 s, 43.4 s ± 1.0 s and 133.8 s ± 2.3 s, 2.) timed valving of typical assay reagents (contact angles 36° - 78°, viscosities 0.9 mPa s - 5.6 mPa s) and 3.) “on demand” valving of liquids from 4 inlet chambers in any user defined sequence controlled by the spinning protocol. The microfluidic timer is compatible to all wetting properties and viscosities of common assay reagents and does neither require assistive equipment, nor coatings. It can be monolithically integrated into a microfluidic test carrier and is compatible to scalable fabrication technologies such as thermoforming or injection molding.
• G. Czilwik, I. Schwarz, M. Keller, S. Wadle, S. Zehnle, F. von Stetten, D. Mark, R. Zengerle, N. Paust
  Microfluidic vapor-diffusion barrier for pressure reduction in fully closed PCR modules
  2015 Lab Chip, volume: 15, pages: 1084 - 1091
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  Abstract

  Microfluidic systems for polymerase chain reaction (PCR) should be fully closed to avoid vapor loss and to exclude the risk of contaminating the test environment. In closed systems however, the high temperatures of up to 95°C associated with PCR cause high overpressures up to 100 kPa, dominated by the increase of vapor partial pressure upon evaporation. Such high overpressures pose challenges to the mechanical stability of microfluidic chips as well as to the liquid handling in integrated sample-to-answer systems. In this work, we drastically reduce the pressure increase in fully closed PCR systems by integrating a microchannel that serves as a vapor-diffusion barrier (VDB), separating the liquid-filled PCR chamber from an auxiliary air chamber. In such configurations, propagation of vapor from the PCR chamber into the auxiliary air chamber and as a consequence the increase of pressure is limited by the slow diffusion process of vapor through the VDB. At temperature increase from 23°C to 95°C, we demonstrate the reduction of overpressure from more than 80 kPa without the VDB to only 35 kPa with the VDB. We further demonstrate proper function of VDB and its easy integration with downstream processes for PCR based nucleic acid amplification within centrifugal microfluidics. Without integration of the VDB, malfunction due to pressure-induced delamination of the microfluidic chip occurred.
• G. Czilwik, D. Baumann, D. Mark, R. Zengerle, F. von Stetten
  Mikrofluidische Automatisierung in der Molekulardiagnostik
  2015 BIOspektrum, volume: 21, pages: 622 - 624
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  Abstract

  Easy-to-use point-of-care (POC) tests provide rapid diagnostic results near the patient, enabling fast decision-making in patient management. However, the implementation of automated molecular diagnostic POC tests still faces technical challenges. We demonstrate a highly sensitive POC platform for automated molecular diagnostics of neonatal sepsis that uses a mobile analyzer and unit-use test carriers.
• S. Vierrath, L. Zielke, R. Moroni, A. Mondon, D. R. Wheeler, R. Zengerle, S. Thiele
  Morphology of nanoporous carbon-binder domains in Li-ion batteries—A FIB-SEM study
  2015 Electrochem Commun, volume: 60, pages: 176 - 179
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  Abstract

  FIB-SEM tomography is used to reconstruct the carbon-binder domain (CBD) of a LiCoO2 battery cathode (3.9 × 5 × 2.3 μm3) with contrast enhancement by ZnO infiltration via atomic layer deposition. We calculate the porosity inside the CBD (57.6%), the cluster-size distribution with a peak at 54 nm, and the pore-size distribution with a peak at 64 nm. The tortuosities of the pore space (1.6–2.0) and the CBD (2.3–3.5) show a mild anisotropy, which is attributed to the fabrication process. A comparison to a modeled homogenous CBD reveals that clustering in the CBD decreases its electronic conductivity while increasing the ionic diffusivity. To account for the higher calculated diffusivity compared to experimental values from literature, a simple binder swelling model is implemented, suggesting a swelling of 75 vol%. The prevention of both clustering and swelling could increase the volume available for active material and therefore the energy density.
• S. Zehnle, F. Schwemmer, R. Bergmann, F. von Stetten, R. Zengerle, N. Paust
  Pneumatic siphon valving and switching in centrifugal microfluidics controlled by rotational frequency or rotational acceleration
  2015 Microfluid Nanofluid, volume: 19, pages: 1259 - 1269
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  Abstract

  Air-pressure-mediated, pneumatic siphon valves employ temporary storage and subsequent release of pneumatic energy, exclusively controlled by rotation of the disk. Implementation is easy, and robust valves can be integrated in a monolithic way at minimum additional costs. However, so far, pneumatic siphon valving requires deceleration from high to low rotational frequencies. Valve opening is performed always when the rotation of the disk drops below a critical rotational frequency. To overcome this limitation, we introduce new concepts for pneumatic siphon valving which enable operation of the disk at any rotational frequency without unwanted bursts of the siphon valves. Thus, the design space for pneumatic siphon valves in centrifugal microfluidics is significantly extended. Three types of pneumatic siphon valves are presented with release control at (1) rotational frequencies between 25 and 48 Hz, (2) positive rotational accelerations between 1 and 22 Hz s−1, and (3) negative rotational accelerations between 5 and 20 Hz s−1. Finally, we combine two valve types to realize robust switching into two fluidic paths with flow rate ratios of 94/6 and 0/100.
• C. Köhler, L. Bleck, M. Frei, R. Zengerle, S. Kerzenmacher
  Poisoning of Highly Porous Platinum Electrodes by Amino Acids and Tissue Fluid Constituents
  2015 ChemElectroChem, volume: 2, pages: 1785 - 1793
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  Abstract

  The first comprehensive study of the poisoning behavior of physiological amino acids and important tissue fluid constituents on porous platinum electrodes was performed. On the basis of chronoamperometry under physiological conditions, the results are highly relevant for the development of enzyme-free implantable glucose fuel cells and possibly also glucose sensors. The strongest poisoning was exhibited by positively charged alkaline amino acids and by some of the neutral amino acids, as well as by uric acid and creatinine. In contrast to previous work, sulfur-containing amino acids showed no poisoning effect. This was explained by the use of highly porous platinum electrodes in the current study and experimental methods that were more relevant for practical applications. Furthermore, comprehensive analysis regarding the degree of recovery in the absence of poisoning species and the extent of electrode regeneration by cyclic voltammetry was performed for each of the amino acids and physiological substances.
• M. Klingele, R. Zengerle, S. Thiele
  Quantification of artifacts in scanning electron microscopy tomography: Improving the reliability of calculated transport parameters in energy applications such as fuel cell and battery electrodes
  2015 J Power Sources, volume: 275, pages: 852 - 859
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  Abstract

  Focused ion beam and scanning electron microscopy tomography (FIB-SEMt) is commonly used to extract reactant transport relevant parameters from nano-porous materials in energy applications, such as fuel cells or batteries. Here we present an approach to virtually model the errors in FIB-SEMt which are caused by the FIB cutting distance. The errors are evaluated in terms of connectivity, solid volume fraction (SVF), conductivity, diffusivity, as well as mean grain and pore sizes. For state-of-the-art FIB-SEMt experiments, where a hydrogen fuel cell catalyst layer with 60 nm mean grain size and 40% SVF is sectioned with a cutting distance of 15 nm, the error in our simulation ranges up to 51% (conductivity), whereas other parameters remain largely unaffected (Laplace diffusivity, 4%). We further present a method, employing virtual coarsening and back interpolation, to reduce FIB cutting distance errors in all investigated parameters. Both error evaluation and correction are applicable to sphere based porous materials with relevance for the energy conversion and storage sector such as polymer electrolyte membrane fuel cell catalyst layer (PEMFC CL), battery carbon binder domain (CBD) or supercapacitor electrodes.
• G. Czilwik, T. Messinger, O. Strohmeier, S. Wadle, F. von Stetten, N. Paust, G. Roth, R. Zengerle, P. Saarinen, J. Niittymäki, K. McAllister, O. Sheils, J. O'Leary, D. Mark
  Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation
  2015 Lab Chip, volume: 15, pages: 3749 - 3759
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  Abstract

  Diagnosis of infectious diseases suffers from long turnaround times for gold standard culture-based identification of bacterial pathogens, therefore impeding timely specific antimicrobial treatment based on laboratory evidence. Rapid molecular diagnostics-based technologies enable detection of microorganisms within hours however cumbersome workflows and complex equipment still prevent their widespread use in the routine clinical microbiology setting. We developed a centrifugal-microfluidic “LabDisk” system for rapid and highly-sensitive pathogen detection on a point-of-care analyser. The unit-use LabDisk with pre-stored reagents features fully automated and integrated DNA extraction, consensus multiplex PCR pre-amplification and geometrically-multiplexed species-specific real-time PCR. Processing merely requires loading of the sample and DNA extraction reagents with minimal hands-on time of approximately 5 min. We demonstrate detection of as few as 3 colony-forming-units (cfu) of Staphylococcus warneri, 200 cfu of Streptococcus agalactiae, 5 cfu of Escherichia coli and 2 cfu of Haemophilus influenzae in a 200 μL serum sample. The turnaround time of the complete analysis from “sample-to-result” was 3 h and 45 min. The LabDisk consequently provides an easy-to-use molecular diagnostic platform for rapid and highly-sensitive detection of bacterial pathogens without requiring major hands-on time and complex laboratory instrumentation.
• F. Stumpf, J. Schoendube, A. Gross, C. Rath, S. Niekrawietz, P. Koltay, G. Roth
  Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation
  2015 Biosens Bioelectron, volume: 69, pages: 301 - 306
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  Abstract

  Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cellline) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10 µm diameter) resulting in a single bead delivery in 27 ou tof 28 cases and relative positional precision of ± 350 µm at a printing distance of 6 mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3 ± 2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing.
• J. Schoendube, D. Wright, R. Zengerle, P. Koltay
  Single-cell printing based on impedance detection
  2015 Biomicrofluidics, volume: 9, page: 014117
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  Abstract

  Label-free isolation of single cells is essential for the growing field of single-cell analysis. Here, we present a device which prints single living cells encapsulated in free-flying picoliter droplets. It combines inkjet printing and impedance flow cytometry. Droplet volume can be controlled in the range of 500 pl–800 pl by piezo actuator displacement. Two sets of parallel facing electrodes in a 50 µm x 55 µm channel are applied to measure the presence and velocity of a single cell in real-time. Polystyrene beads with <5% variation in diameter generated signal variations of 12%–17% coefficients of variation. Single bead efficiency (i.e., printing events with single beads vs. total number of printing events) was 73%611% at a throughput of approximately 9 events/min. Viability of printed HeLa cells and human primary fibroblasts was demonstrated by culturing cells for at least eight days.
• C. E. Blanchet, A. Spilotros, F. Schwemmer, M. A. Graewert, A. Kikhney, C. M. Jeffries, D. Franke, D. Mark, R. Zengerle, F. Cipriani, S. Fiedler, M. Roessle, D. I. Sverguna
  Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY)
  2015 J Appl Crystallogr, volume: 48, pages: 431 - 443
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  Abstract

  A high-brilliance synchrotron P12 beamline of the EMBL located at the PETRA III storage ring (DESY, Hamburg) is dedicated to biological smallangle X-ray scattering (SAXS) and has been designed and optimized for scattering experiments on macromolecular solutions. Scatterless slits reduce the parasitic scattering, a custom-designed miniature active beamstop ensures accurate data normalization and the photon-counting PILATUS 2M detector enables the background-free detection of weak scattering signals. The high flux and small beam size allow for rapid experiments with exposure time down to 30– 50 ms covering the resolution range from about 300 to 0.5 nm. P12 possesses a versatile and flexible sample environment system that caters for the diverse experimental needs required to study macromolecular solutions. These include an in-vacuum capillary mode for standard batch sample analyses with robotic sample delivery and for continuous-flow in-line sample purification and characterization, as well as an in-air capillary time-resolved stopped-flow setup. A novel microfluidic centrifugal mixing device (SAXS disc) is developed for a high-throughput screening mode using sub-microlitre sample volumes. Automation is a key feature of P12; it is controlled by a beamline meta server, which coordinates and schedules experiments from either standard or nonstandard operational setups. The integrated SASFLOWpipeline automatically checks for consistency, and processes and analyses the data, providing near real-time assessments of overall parameters and the generation of low-resolution models within minutes of data collection. These advances, combined with a remote access option, allow for rapid high-throughput analysis, as well as time-resolved and screening experiments for novice and expert biological SAXS users.
• J. Schoendube, A. Yusof, K. Kalkandjiev, R. Zengerle, P. Koltay
  Wafer level fabrication of single cell dispenser chips with integrated electrodes for particle detection
  2015 J Micromech Microeng, volume: 25, page: 025008
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  Abstract

  This work presents the microfabrication and experimental evaluation of a dispenser chip, designed for isolation and printing of single cells by combining impedance sensing and drop-on-demand dispensing. The dispenser chip features 50 × 55 μm (width × height) microchannels, a droplet generator and microelectrodes for impedance measurements. The chip is fabricated by sandwiching a dry film photopolymer (TMMF) between a silicon and a Pyrex wafer. TMMF has been used to define microfluidic channels, to serve as low temperature (75 °C) bonding adhesive and as etch mask during 300 μm deep HF etching of the Pyrex wafer. Due to the novel fabrication technology involving the dry film resist, it became possible to fabricate facing electrodes at the top and bottom of the channel and to apply electrical impedance sensing for particle detection with improved performance. The presented microchip is capable of dispensing liquid and detecting microparticles via impedance measurement. Single polystyrene particles of 10 μm size could be detected with a mean signal amplitude of 0.39 ± 0.13 V (n = 439) at particle velocities of up to 9.6 mm s−1 inside the chip.

2014

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• L. Zielke, T. Hutzenlaub, D. R. Wheeler, I. Manke, T. Arlt, N. Paust, R. Zengerle, S. Thiele
  A Combination of X-Ray Tomography and Carbon Binder Modeling: Reconstructing the Three Phases of LiCoO2 Li-Ion Battery Cathodes
  2014 Adv Energy Mater, volume: 4, page: 1301617
  » show abstract« hide abstract
  Abstract

  X-ray tomography allows the active-material domain (LiCoO2) of Li-ion battery cathodes to be imaged, but it is unable to resolve the carbon-binder domain (CBD). Here, a new method for creating a complete 3D representation (virtual design) of all three phases of a cathode is provided; this includes the active-material domain, the CBD, and the electrolyte-filled pore space. It combines X-ray tomographic data of active material with a statistically modeled CBD. Two different statistical CBD morphology models are compared as examples: i) a random cluster model representing a standard mixture of carbon black and polyvenylidene fluoride (PVDF) and ii) a fiber model. The transport parameters are compared in a charged and a discharged cathode. The results demonstrate that the CBD content and morphology changes the ionic and electronic transport parameters dramatically and thus cannot be neglected. Calculations yield that the fiber model shows up to three times higher electrical conductivity at the same CBD content (discharged case) and better ionic diffusion conditions for all CBD contents. In the charged case, the morphology impact on electrical conduction is small. This effective method to generate transport parameters for different CBDs can be transferred to other CBD morphologies and electrodes.
• S.K. Vashist, G. Czilwik, T. van Oordt, F. von Stetten, R. Zengerle,, E.M. Schneider, J.H.T. Luong
  A rapid one-step kinetics-based immunoassay procedure for the highly-sensitive detection of C-reactive protein
  2014 Protocol Exchange
• M. Hoehl, E. Schulte Bocholt, A. Kloke, N. Paust, F. von Stetten, R. Zengerle, J. Steigert, A. Slocum
  A versatile-deployable bacterial detection system for food and environmental safety based on LabTube-automated DNA purification, LabReader-integrated amplification, readout and analysis
  2014 Analyst, volume: 139, pages: 2788 - 2798
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  Abstract

  Contamination of foods is a public health hazard that episodically causes thousands of deaths and sickens millions worldwide. To ensure food safety and quality, rapid, low-cost and easy-to-use detection methods 10 are desirable. Here, the LabSystem is introduced for integrated, automated DNA purification, amplification and detection. It consists of a disposable, centrifugally-driven DNA purification platform (LabTube) and the subsequent amplification and detection in a low-cost UV/vis-reader (LabReader). For demonstration of the LabSystem in the context of food safety, purification of Escherichia coli (nonpathogenic E. coli and pathogenic verotoxin-producing E. coli (VTEC)) in water and milk, and the 15 product-spoiler Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice was integrated and optimized in the LabTube. Inside the LabReader, the purified DNA was amplified, readout and analyzed using both qualitative isothermal loop-mediated DNA amplification (LAMP) and quantitative real-time PCR. For the LAMP-LabSystem, the combined detection limits for purification and amplification of externally lysed VTEC and A. acidoterrestris is 102-103 cell-equivalents. In the PCR-LabSystem for E. coli cells, the quantification limit is 102 20 cell-equivalents including LabTube-integrated lysis. The demonstrated LabSystem only requires a laboratory centrifuge (to operate the disposable, fully closed LabTube) and the low-cost LabReader for DNA amplification, readout and analysis. Compared with commercial DNA amplification devices, the LabReader improves sensitivity and specificity by the simultaneous readout of four wavelengths and the continuous readout during temperature cycling. The 25 use of a detachable eluate tube as an interface affords semi-automation of the LabSystem, which does not require specialized training. It reduces hands-on time from about 50 to 3 min with only two handling steps: sample input and transfer of the detachable detection tube.
• S. K. Vashist, O. Mudanyali, E. M. Schneider, R. Zengerle, A. Ozcan
  Cellphone-based devices for bioanalytical sciences
  2014 Anal Bioanal Chem, volume: 406, issue: 14, pages: 3263 - 3277
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  Abstract

  During the last decade, there has been a rapidly growing trend toward the use of cellphone-based devices (CBDs) in bioanalytical sciences. For example, they have been used for digital microscopy, cytometry, read-out of immunoassays and lateral flow tests, electrochemical and surface plasmon resonance based bio-sensing, colorimetric detection and healthcare monitoring, among others. Cellphone can be considered as one of the most prospective devices for the development of next-generation point-of-care (POC) diagnostics platforms, enabling mobile healthcare delivery and personalized medicine. With more than 6.5 billion cellphone subscribers worldwide and approximately 1.6 billion new devices being sold each year, cellphone technology is also creating new business and research opportunities. Many cellphone-based devices, such as those targeted for diabetic management, weight management, monitoring of blood pressure and pulse rate, have already become commerciallyavailable in recent years. In addition to such monitoring platforms, several other CBDs are also being introduced, targeting e.g., microscopic imaging and sensing applications for medical diagnostics using novel computational algorithms and components already embedded on cellphones. This report aims to review these recent developments in CBDs for bioanalytical sciences along with some of the challenges involved and the future opportunities.
• M. Hoehl, M. Weissert, A. Dannenberg, T. Nesch, N. Paust, F. von Stetten, R. Zengerle, A. Slocum, J. Steigert
  Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating-system
  2014 Biomed Microdevices, volume: 16, issue: 3, pages: 375 - 385
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  Abstract

  This paper introduces a disposable battery-driven heating system for loop-mediated isothermal DNA amplification (LAMP) inside a centrifugally-driven DNA purification platform (LabTube). We demonstrate LabTube-based fully automated DNA purification of as low as 100 cell-equivalents of verotoxin-producing Escherichia coli (VTEC) in water, milk and apple juice in a laboratory centrifuge, followed by integrated and automated LAMP amplification with a reduction of hands-on time from 45 to 1 min. The heating system consists of two parallel SMD thick film resistors and a NTC as heating and temperature sensing elements. They are driven by a 3 V battery and controlled by a microcontroller. The LAMP reagents are stored in the elution chamber and the amplification starts immediately after the eluate is purged into the chamber. The LabTube, including a microcontroller-based heating system, demonstrates contamination-free and automated sample-to-answer nucleic acid testing within a laboratory centrifuge. The heating system can be easily parallelized within one LabTube and it is deployable for a variety of heating and electrical applications.
• K. Dolch, J. Danzer, T. Kabbeck, B. Bierer, J. Erben, A. H. Förster, J. Maisch, P. Nick, S. Kerzenmacher, J. Gescher
  Characterization of microbial current production as a function of microbe-electrode-interaction
  2014 Bioresource Technol, volume: 157, pages: 284 - 292
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  Abstract

  Microbe–electrode-interactions are keys for microbial fuel cell technology. Nevertheless, standard measurement routines to analyze the interplay of microbial physiology and material characteristics have not been introduced yet. In this study, graphite anodes with varying surface properties were evaluated using pure cultures of Shewanella oneidensis and Geobacter sulfurreducens, as well as defined and undefined mixed cultures. The evaluation routine consisted of a galvanostatic period, a current sweep and an evaluation of population density. The results show that surface area correlates only to a certain extent with population density and anode performance. Furthermore, the study highlights a strain-specific microbe–electrode-interaction, which is affected by the introduction of another microorganism. Moreover, evidence is provided for the possibility of translating results from pure culture to undefined mixed species experiments. This is the first study on microbe–electrode-interaction that systematically integrates and compares electrochemical and biological data.
• S. Sané, C. Heilemann, P. Salvei, S. Rubenwolf, C. Jolivalt, C. Madzak, R. Zengerle, P. J. Nielsen, S. Kerzenmacher
  Enzymatic fuel cells solely supplied with unpurified cellobiose dehydrogenaseand laccase in microorganism´s culture supernatans
  2014 ChemElectroChem, volume: 1, pages: 1886 - 1894
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  Abstract

  Enzymatic electrodes have great potential for catalysing the direct conversion of chemical compounds into electricity with the use of redox enzymes. However, expensive and time-consuming enzyme purification and the frequent need to add mediators are considered as drawbacks of enzymatic electrodes. We report a biofuel cell at pH 5, supplied with the unpurified enzymes laccase and cellobiose dehydrogenase (CDH) in crude culture supernatant, without the further addition of mediators. A maximum power output of 6.2±1.2 μW cm−2 was achieved by using supernatants containing laccase from Trametes versicolor (2.84 UmL−1) and CDH (0.90 UmL−1) from a recombinant yeast Yarrowia lipolytica YPC4. In comparison, the supply of purified enzymes (laccase: 2.40 UmL−1, CDH: 0.15 UmL−1) in a buffer solution yielded only about a twofold higher power density. Our results demonstrate the feasibility of using unpurified laccase and CDH in an enzymatic biofuel cell, which can simplify its construction and operation.
• S. K. Vashist, E.M. Schneider, R. Zengerle, F. von Stetten, J.H.T. Luong
  Graphene-based rapid and highly-sensitive immunoassay for C-re active protein using a smartphone-based colorimetric reader
  2014 Biosens Bioelectron, volume: 66, pages: 169 - 176
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  Abstract

  A novel immunoassay (IA) has been developed for human C-reactive protein (CRP), an important biomarker and tissue preserving factor for infection and inflammation. Graphene nanoplatelets (GNP) and 3-aminopropyltriethoxysilane (APTES) were admixed and covalently attached to a polystyrene based-microtiter plate (MTP), pretreated with KOH. The resulting surface served as a stable layer for the covalent attachment of the anti-human CRP antibody. The IA procedure was based on the one-step kinetics-based sandwich IA employing a minimum number of process steps, whereas the enzymatic reaction solution was monitored by a smartphone-based colorimetric reader. With a limit of detection and a limit of quantification of 0.07 ng mL−1 and 0.9 ng mL−1, it precisely detected CRP spiked in diluted human whole blood and plasma as well as the CRP levels in clinical plasma samples. The results obtained for “real-world” patient samples agreed well with those of the conventional immunosorbent assay and the clinically-accredited analyzer-based IA. The antibody-bound GNP-functionalized MTPs retained its original activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C.
• K.G. Kraiczek, R. Bonjour, Y. Salvadé, R. Zengerle
  Highly Flexible UV-Vis Radiation Sources and Novel Detection Schemes for Spectrophotometric HPLC Detection
  2014 Anal Chem, volume: 86, issue: 2, pages: 1146 - 1152
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  Abstract

  The concept and performance of the first multiwavelength deep UV light-emitting-diode-based high-performance liquid chromatography (HPLC) absorbance detector are presented. In single-wavelength mode and with optical reference, the limit of detection (LOD) is comparable to conventional state-of-theart HPLC absorbance detectors. In multiwavelength modeat present up to eight wavelengths without optical referencethe LOD is about 10 times higher than in single-wavelength mode. Multiplexing and demultiplexing methods are used to separate chromatographic signals in multiwavelength mode and keeps the detector configuration simple and yet flexible. Depending on the operation mode, stray light is either totally negligible or controlled electronically and digitally.
• E. Kipf, R. Zengerle, J. Gescher, S. Kerzenmacher
  How Does the Choice of Anode Material Influence Electrical Performance? A Comparison of Two Microbial Fuel Cell Model Organisms
  2014 ChemElectroChem, volume: 1, pages: 1849 - 1853
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  Abstract

  The influence of the anode material on the electrical performance of the two microbial fuel cell model organisms Geobacter sulfurreducens and Shewanella oneidensis is investigated. High-surface-area activated carbon and low-surface-area graphite felt are compared in terms of polarization curves under quasi-steady-state conditions. Unexpectedly, G. sulfurreducens exhibits similar current densities up to 700 mAcm_2 independent of the anode material. This is ~50% higher than steady-state values reported previously. The negligible influence of electrode material on the electrical performance of G. sulfurreducens is attributed to the fact that it performs only direct electron transfer, but forms thick biofilms. In contrast, S. oneidensis, relying mainly on mediated electron transfer, apparently makes better use of high-surface-area activated carbon and achieves higher current densities compared to graphite felt. This underlines the importance of tailoring anode materials according to the used organisms.
• O. Fokina, J. Eipper, L. Winandy, S. Kerzenmacher, R. Fischer
  Improving the performance of a biofuel cell cathode with laccase from Pycnoporus sanguineus
  2014 Bioresource Technol, volume: 175C, pages: 445 - 453
• T. van Oordt, Y. Barb, R. Zengerle, F. von Stetten
  Lamination of Polyethylene Composite Films by Ultrasonic Welding: Investigation of Peel Behavior and Identification of Optimum Welding Parameters
  2014 J Appl Polym Sci, volume: 131, pages: 40291 - 40298
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  Abstract

  The lamination of different polyethylene (PE) composite films by ultrasonic welding to fabricate peelable seals that open at defined burst pressures is investigated. The sealing time, pressure, and amplitude were varied within the range of 100–400 ms, 50– 250 kPa, and 12–24 mm, respectively. T-peel tests and electron micrographs revealed four different peel regimes, depending on the parameter combination: (I) Interlaminar peeling at low-peel strength with uniform peeling along a weakly bonded PE lamination layer; (II) transition tearing at intermediate peel strength showing areas of interlaminar peeling and translaminar tearing; (III) translaminar tearing at high-peel strength showing tears through the entire film; and, (IV) undefined tearing at varying tear strength occurring when vibration effects during welding lead to insufficient contact of the films or high pressures lead to a displacement of PE. This study will allow the systematic adjustment of ultrasonic welding parameters for PE films.
• D. Liang, J. Zhang, M.T. Govindaiah, L. Tanguy, A. Ernst, R. Zengerle, P. Koltay
  Liquid volume measurement method for the picoliter to nanoliter volume range based on quartz crystal microbalance technology
  2014 Meas Sci Technol, volume: 25, page: 095302
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  Abstract

  In this article, a quantitative liquid volume measurement method for the sub-nanoliter range using a quartz crystal microbalance (QCM) is described and experimentally analyzed. The primary measurement device to determine the volume of small liquid droplets is a QCM sensor coated with a surface-attached hydrogel to improve the mechanical coupling of the liquid to the sensor surface. An experimental evaluation of measured volumes in the range of 3 nl to 15 nl in normal room conditions has been performed with three identical sensors prepared with a PDMAA-1%MaBP hydrogel coating with a thickness of 1.5 µm ± 0.12 µm. A linearity of R2 more than 0.87, an average coefficient of variation (CV) within one experimental run of 5.7%, a mean absolute relative bias of 5.5%, and a sensor-to-sensor variation of 6.3% have been experimentally determined. The feasibility of this method has also been experimentally proven for the picoliter volume range down to 200 pl, with an average CV of 5.3% and a mean absolute relative bias of 6.5%. Furthermore, a stability evaluation consisting of 10 experimental series with approximately 150 measurements over the course of one week has been performed. This evaluation showed that the experimental setup, although exhibiting highly consistent performance within one measurement run, is not yet reproducible enough for long-term and repeated use because of undefined swelling and crack formation in the hydrogel layer. The low reproducibility implies a relatively high expanded uncertainty, with k = 2 according to the JCGM 'Evaluation of Measurement Data—Guide to the Expression of Uncertainty in Measurement' (GUM) for the total measurement method of approximately 3.82 nl when measuring a 10 nl liquid droplet. Nevertheless, the QCM method as described here contributes to significant progress beyond the state-of-the-art that might allow new opportunities for precise measurement of sub-nanoliter liquid volumes.
• K. Mutschler, S. Dwivedi, S. Kartmann, S. Bammesberger, P. Koltay, R. Zengerle, L. Tanguy
  Multi physics network simulation of a solenoid dispensing valve
  2014 Mechatronics, volume: 24, pages: 209 - 221
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  Abstract

  significant effects in a complete technical system. In a solenoid dispensing valve the involved physical effects are electro-magnetic (coil and magnet interaction), fluid flow and fluid structure interaction. It is challenging and time consuming to establish a full model description of these different effects even by using the most up-to-date Computational Fluid Dynamics (CFDs) software tools. This article therefore presents an alternative approach using network simulation methods for modelling of a dispensing valve using the simulation software SABER (Synopsys). To create the model, the different parts of the valve and the relevant physical effects occurring therein are described by partial differential equations and implemented as lumped elements. The lumped elements are then linked together to form a complete model of the dispensing valve including electrical, mechanical and fluid dynamic properties. A comparison with experimental data obtained from a real valve is presented at the end of the paper to discuss and validate the model. In particular the correct prediction of the dispensed liquid volume in dependence of the main parameters like pressure and opening time are considered. Using ab initio simulation deviations of the predicted dispensed liquid volume from the experimental results in the range 0.65–7.4% was found for different actuating pressures at valve opening times larger than 20 ms.
• M. Salzer, S. Thiele, R. Zengerle, V. Schmidt
  On the importance of FIB-SEM specific segmentation algorithms for porous media
  2014 Mater Charact, volume: 95, pages: 36 - 43
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  Abstract

  A new algorithmic approach to segmentation of highly porous three dimensional image data gained by focused ion beam tomography is described which extends the key-principle of local threshold backpropagation described in [1]. The technique of focused ion beam tomography has shown to be capable of imaging the microstructure of functional materials. In order to perform a quantitative analysis on the corresponding microstructure a segmentation task needs to be performed. However, algorithmic segmentation of images obtained with focused ion beam tomography is a challenging problem for highly porous materials if filling the pore phase, e.g. with epoxy resin, is difficult. The grey intensities of individual voxels are not sufficient to determine the phase represented by them and usual thresholding methods are not applicable. We thus propose a new approach to segmentation, that pays respect to the specifics of the imaging process of focused ion beam tomography. As an application of our approach, the segmentation of three dimensional images for a cathode material used in polymer electrolyte membrane fuel cells is discussed. We show that our approach preserves significantly more of the original nanostructure than an thresholding approach.
• S.K. Vashist, G. Czilwik, T. van Oordt, F. von Stetten, R. Zengerle, E.M. Schneider, J.H.T. Luong
  One-step kinetics-based immunoassay for the highly-sensitive detection of C-reactive protein in less than 30 minutes
  2014 Anal Biochem, volume: 456, pages: 32 - 37
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  Abstract

  The article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly-sensitive detection of human C-reactive protein (CRP) in less than 30 min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimum process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads® in 15 min followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3-81 ng mL-1 with a limit of detection and analytical sensitivity of 0.4 ng mL-1 and 0.7 ng mL-1, respectively. It detects CRP spiked in diluted human whole blood and serum with high analytical precision as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid (EDTA) plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.
• M. Pospischil, T. Fellmeth, A. Brand, S. Nold, M. Kuchler, M. Klawitter, H. Gentischer, M. König, M. Hörteis, L. Wende, O. Doll, R. Zengerle, F. Clement, D. Biro
  Optimizing fine line dispensed contact grids
  2014 Energy Procedia, volume: 55, pages: 693 - 701
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  Abstract

  Previous studies on dispensing as an alternative front side metallization process in crystalline silicon photovoltaics demonstrated, how an adaption of paste rheology allows for a precise adjustment of contact finger geometry in a wide range. In order to demonstrate the benefit of this advantage, the analytical simulation tool Gridmaster was extended to observe the effect of various geometrical parameters on solar cell results and manufacturing costs. In addition, respective geometrical parameters of thick film printed contact fingers were determined using a special in house developed tool based on MATLAB. As a result, contact geometries as achievable by means of ultrafine line dispensing are ideally suited for contacting silicon solar cells. Compared with standard fine line single screen printed finger geometries, an efficiency increase of up to Δ = 0.4%abs. as well as a reduction of manufacturing costs of 1 €ct./Wp on module level can be achieved using dispensing technology. In order to obtain suitable data, simulation results were compared with solar cell results on industrial pre-processed Cz-Si p-type wafer material applying the novel ten nozzle parallel fine line dispensing unit. Therefore, a new dispensing paste was developed combining optimum optical finger aspect ratios of ARo = 0.7 with excellent contacting behavior. A successful first test of applicability already led to a maximum cell efficiency of  = 18.7%, demonstrated on an industrial emitter with a sheet resistance of around Rsh = 90Ω/sq.
• C. Köhler, M. Frei, R. Zengerle, S. Kerzenmacher
  Performance Loss of a Pt-Based Implantable Glucose Fuel Cell in Simulated Tissue and Cerebrospinal Fluids
  2014 ChemElectroChem, volume: 1, pages: 1895 - 1900
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  Abstract

  We present an electrode-resolved analysis of the performance of platinum-based abiotically catalyzed glucose fuel cells in realistically simulated tissue fluid (STF) and cerebrospinal fluid (SCF). The presence of amino acids and small organic molecules at physiological concentration leads to a drastic breakdown in fuel cell voltage, resulting in a maximum lifetime of 19 h in STF or 37 h in SCF. The performance loss mainly originates from catalyst poisoning at the anode, which causes 92% or 80% of the overall fuel cell voltage loss in STF or SCF, respectively. The results underline the necessity to further investigate and improve catalyst poisoning at the anode under realistic body conditions in order to improve the lifetime of platinum- based implantable glucose fuel cells.
• C. Escadafal, O. Faye, A. A. Sall, O. Faye, M. Weidmann, O. Strohmeier, F. von Stetten, J. Drexler, M. Eberhard, M. Niedrig, P. Patel
  Rapid molecular assys for the detection of yellow fever virus in low-resource settings
  2014 Plos Neglect Trop D, volume: 8, issue: 3, page: e2730
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  Abstract

  Despite the use of a safe and effective vaccine, yellow fever virus is still causing hundreds of thousands of infections and tens of thousands of deaths every year. The disease is widespread in South America and Africa where several outbreaks have occurred in the past years. As the disease is difficult to distinguish from other illnesses during its early stage, it is necessary to develop reliable, rapid and simple diagnostic methods to confirm YF cases to be able to respond effectively to outbreaks through vaccination and vector control. In this study, we describe the development a diagnostic method for YFV, using an isothermal technology called recombinase polymerase amplification which allows detection of the virus within 20 minutes, using a portable and easy-to-use device. The YFV RPA assay proved to be a specific and sensitive detection method during testing in the laboratory and under field conditions in Senegal.
• O. Strohmeier, N. Marquart, D. Mark, G. Roth, R. Zengerle, F. von Stetten
  Real-time PCR based detection of a panel of foodborne pathogens on a centrifugal microfluidic “LabDisk” with on-disk quality controls and standards for quantification
  2014 Anal Methods-uk, volume: 6, pages: 2038 - 2046
• M. Rombach, D. Kosse, B. Faltin, S. Wadle, G. Roth, R. Zengerle, F. von Stetten
  Real-time stability testing of air-dried primers and fluorogenic hydrolysis probes stabilized by trehalose and xanthan
  2014 Biotechniques, volume: 57, issue: 3, pages: 151 - 155
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  Abstract

  A method for conserving primers and differently labeled fluorogenic hydrolysis (i.e., TaqMan) probes at ambient conditions is presented. Primers and hydrolysis probes with four different fluorophore-quencher combinations (6- FAM–BHQ1, HEX–BHQ1, ROX–BHQ650, and Cy5–BHQ2) were mixed with trehalose and xanthan at final concentrations of 56 mM and 2.78 mM, respectively. Mixtures were air-dried at 23°C for 30 min on strips composed of cyclo olefin polymer (COP), a material widely used in the manufacturing of in vitro diagnostic (IVD) test carriers. After one year of storage, the functionality of the primers and fluorophore-quencher combinations was validated by real-time polymerase chain reaction (real-time PCR), confirming their stability when stored in the presence of stabilizers, with the best results achieved using trehalose. This approach could be of great benefit for manufacturing IVD systems, for example, for genotyping applications based on multiplexing using different fluorescent dyes.
• S. K. Vashist, E.M. Schneider, J.H.T. Luong
  Surface plasmon resonance-based immunoassay for human fetuin A
  2014 Analyst, volume: 139, pages: 2237 - 2242
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  Abstract

  This article describes a highly-sensitive surface plasmon resonance (SPR)-based immunoassay (IA) for human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The assay is based on a novel immobilization procedure that simply involves the dilution of an anti-HFA capture antibody (Ab) in 1% (v/v) 3-aminopropyltriethoxysilane (APTES), followed by its dispensing on a KOH-treated gold (Au)-coated SPR chip and incubation for 30 min. The developed SPR IA detected 0.3-20 ng mL(-1) of HFA with a limit of detection and sensitivity of 0.7 ng mL(-1) and 1 ng mL(-1), respectively. The highly-simplified Ab immobilization procedure is also 5-fold more rapid than conventional procedures. It leads to the leach-proof binding of the capture Ab, which means that the developed SPR IA is highly cost-effective, as the Ab-bound SPR chip could be reused for many repeated HFA IAs after regeneration with 10 mM glycine-HCl, pH 2.0. The Ab-bound SPR chip, stored at 4 °C, lost only 18% of its original activity after 4 months. For the detection of HFA spiked in diluted human whole blood and plasma, the results obtained by the developed SPR IA agreed well with the commercial HFA sandwich ELISA.
• A. Kloke, A. R. Fiebach, S. Zhang, L. Drechsel, S. Niekrawietz, M. Hoehl, R. Kneusel, K. Panthel, J. Steigert, F. von Stetten, R. Zengerle, N. Paust
  The LabTube – A novel microfluidic platform for assay automation in laboratory centrifuges
  2014 Lab Chip, volume: 14, pages: 1527 - 1537
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  Abstract

  Assay automation is the key for successful transformation of modern biotechnology into routine workflows. Yet, it requires considerable investment in processing devices and auxiliary infrastructure, which is not cost-efficient for laboratories with low or medium sample throughput or point-of-care testing. To close this gap, we present the LabTube platform, which is based on assay specific disposable cartridges for processing in laboratory centrifuges. LabTube cartridges comprise interfaces for sample loading and downstream applications and fluidic unit operations for release of prestored reagents, mixing, and solid phase extraction. Process control is achieved by a centrifugally-actuated ballpen mechanism. To demonstrate the workflow and functionality of the LabTube platform, we show two LabTube automated sample preparation assays from laboratory routine: DNA extractions from whole blood and purification of His-tagged proteins. Equal DNA and protein yield were observed compared to manual reference runs, while LabTube automation could significantly reduce the hands-ontime to one minute per extraction.
• L. Zielke, T. Hutzenlaub, D. R. Wheeler, C.-W. Chao, I. Manke, A. Hilger, N. Paust, R. Zengerle, S. Thiele
  Three-Phase Multiscale Modeling of a LiCoO 2 Cathode: Combining the Advantages of FIB–SEM Imaging and X-Ray Tomography
  2014 Adv Energy Mater, volume: 5, page: 1401612
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  Abstract

  LiCoO 2 electrodes contain three phases, or domains, each having specific-intended functions: ion-conducting pore space, lithium-ion-reacting active material, and electron conducting carbon-binder domain (CBD). Transport processes take place in all domains on different characteristic length scales: from the micrometer scale in the active material grains through to the nanopores in the carbon-binder phase. Consequently, more than one imaging approach must be utilized to obtain a hierarchical geometric representation of the electrode. An approach incorporating information from the micro- and nanoscale to calculate 3D transport-relevant properties in a largereconstructed active domain is presented. Advantages of focused ion beam/ scanning electron microscopy imaging and X-ray tomography combined by a spatial stochastic model, validated with an artifi cially produced reference structure are used. This novel approach leads to signifi cantly different transport relevant properties compared with previous tomographic approaches: nanoporosity of the CBD leads to up to 42% additional contact area between active material and pore space and increases ionic conduction by a factor of up to 3.6. The results show that nanoporosity within the CBD cannot be neglected.
• T. Hutzenlaub, S. Thiele, N. Paust, R. Spotnitz, R. Zengerle, C. Walchshofer
  Three-dimensional electrochemical Li-ion battery modelling featuring a focused ion-beam/scanning electron microscopy based three-phase reconstruction of a LiCoO2 cathode
  2014 Electrochim Acta, volume: 115, pages: 131 - 139
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  Abstract

  We combine a three-phase, three-dimensional reconstruction of a LiCoO2battery cathode based onfocused ion-beam/scanning electron microscopy (FIB/SEM) imaging with an electrochemical model. Themodel considers the electric potential and lithium/salt concentration distribution in both the liquid elec-trolyte and the solid active-material phases. In contrast to previously presented models, we spatiallyresolve the carbon-binder phase to provide a more realistic description of the electric potential. Weobserve that carbon-binder coverage of the solid electrolyte interface (SEI) impedes local surface reac-tions and thus affects lithium redistribution. For the considered cathode, the total surface to volumeratio of the SEI is reduced from 11.2 × 105to 6.5 × 105m2m−3when the carbon-binder phase is modelledexplicitly. This leads to increased inhomogeneity of the lithium concentration in active-material grainsduring charging.Additionally, we study lithium/salt concentration in the electrolyte, revealing gradients between 0.9and 1.5 kmol m−3depending on the distance to the separator. This is significant because the lithium/saltconcentration directly affects the ion transport properties of the electrolyte.
• T. S. Santisteban, R. Zengerle, M. Meier
  Through-holes, cavities and perforations in polydimethylsiloxane (PDMS) chips
  2014 Rsc Adv, volume: 4, pages: 48012 - 48016
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  Abstract

  We present a method to fabricate through-holes between 10 to 180 µm between polydimethylsiloxane (PDMS) layers of microfluidic large-scale integration platforms. Therefore we employed standard PDMS spin-coating processes onto silicon molds with microstructures formed from SU-8 and AZ photoresists. Our approach is based on the modification of the surface polarity of the PDMS prototyping molds by a 250 nm thick layer of octafluorocyclobutane (C4F8), which resulted in a contact angle of 125 ± 3 degrees for water. This super hydrophobic surface repelled PDMS from microstructures protruding out of the spin coated PDMS layer. Subsequently, we applied and characterized the C4F8 coating for the robust fabrication of interlayer connectors between PDMS membranes of 40 µm thickness. To enable embedding of through-holes, perforations and/or cavities in very thin layers of PDMS (<20 µm) we mixed PDMS with inert silicone oil to reduce its viscosity. In difference to previous attempts to lower the viscosity of PDMS using organic solvents, the silicone oil cross-linked to PDMS and was thus, unable to freely diffuse into the polymerized PDMS. This reduces the risk for bleeding of hazardous components in biological applications. Finally, we manufactured a three layer mLSI chip with integrated cavities for catching fluorescently labeled beads and cells. The presented process parameters can easily be adapted to specific needs in fabrication of multi-layer PDMS arrangements by following the systematic parameter screening.
• L. Zielke, A. Fallisch, N. Paust, R. Zengerle, S. Thiele
  Tomography based screening of flow field / current collector combinations for PEM water electrolysis
  2014 Rsc Adv, volume: 4, pages: 58888 - 58894
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  Abstract

  Current collectors in PEM water electrolysis perform several functions that influence overall performance. These include conducting heat and electrons, as well as transporting water and gas. X-ray tomography, standardized reconstruction, and calculation methods are used to compare the morphological and transport parameters of eight different current collectors functioning as flow fields for PEM water electrolysis. We find simple exponential relations between through-plane thermal conductivity l and porosity p (l20 C(p) ¼ 1749p 1.306 4.420), as well as between through-plane electrical conductivity s and water permeability K (s(K) ¼ 10 6 K 0.6376). In addition, we use both local and global concepts to investigate the pore space of current collectors. We hereby investigate homogeneity and characteristic sizes, like mean pore diameter or mean distance between solid parts at the catalytic interface. Moreover, we find that the local concept of mean chord lengths can be used to explain electrical and thermal conductivity anisotropies. These chords can be used to predict the direction of the largest conductivity for fibrous current collectors.
• S. Sané, K. Richter, S. Rubenwolf, N. J. Matschke, C. Jolivalt, C. Madzak, R. Zengerle, J. Gescher, S. Kerzenmacher
  Using planktonic microorganisms to supply the unpurified multi-copper oxidases laccase and copper efflux oxidases at a biofuel cell cathode
  2014 Bioresource Technol, volume: 158, pages: 231 - 238
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  Abstract

  The feasibility to apply crude culture supernatants that contain the multicopper oxidases laccase or copper efflux oxidase (CueO) as oxygen reducing catalysts in a biofuel cell cathode is shown. As enzyme-secreting recombinant planktonic microorganisms, the yeast Yarrowia lipolytica and the bacterium Escherichia coli were investigated. The cultivation and operation conditions (choice of medium, pH) had distinct effects on the electro-catalytic performance. The highest current density of 119 ± 23 μAcm-2 at 0.400 V vs. NHE was obtained with the crude culture supernatant of E. coli cells overexpressing CueO and tested at pH 5.0. In comparison, at pH 7.4 the electrode potential at 100 μAcm-2 is 0.25 V lower. Laccase-containing supernatants of Y. lipolytica yielded a maximum current density of 6.7 ± 0.4 μAcm-2 at 0.644 V vs. NHE. These results open future possibilities to circumvent elaborate enzyme purification procedures and realize cost effective and easy-to-operate enzymatic biofuel cells.

2013

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• S. Bammesberger, S. Kartmann, L. Tanguy, D. Liang, K. Mutschler, A. Ernst, R. Zengerle, P. Koltay
  A Low-Cost, Normally Closed, Solenoid Valve for Non-Contact Dispensing in the Sub-μL Range
  2013 Micromachines, volume: 4, pages: 9 - 21
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  Abstract

  We present a disposable, normally closed, non-contact dispensing valve for the sub-μL range. The miniaturized solenoid valve (diameter: 8 mm, height: 27.25 mm) is compatible to standard Luer-Lock interfaces. A highly dynamic actuation principle enables opening times down to 1 ms. The dispensing performance was evaluated for water (η = 1.03 mPas) and a 66% (w/w) glycerol/water solution (η = 16.98 mPas), at pressures varying from 200 to 800 mbar. The experimentally determined minimal dispensing volume was 163 nL (CV 1.6%) for water and 123 nL (CV 4.5%) for 66% (w/w) glycerol/water. The low-cost polymer valve enables high precision dispensing of liquid volumes down to the lower end of the sub-μL range comparable to high-end non-disposable micro-dispensing valves.
• S. Bammesberger, I. Malki, A. Ernst, R. Zengerle, P. Koltay
  A calibration-free, noncontact, disposable liquid dispensing cartridge featuring an online process control
  2013 Journal of Laboratory Automation, volume: 27, pages: 394 - 402
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  Abstract

  We present a noncontact liquid dispenser that uses a disposable cartridge for the calibration-free dosage of diverse biochemical reagents from the nanoliter to the microliter range. The dispensing system combines the advantages of a positive displacement syringe pump (responsible for defining the aliquot’s volume with high accuracy) with a highly dynamic noncontact dispenser (providing kinetic energy to detach the liquid from the tip). The disposable, noncontact dispensing cartridge system renders elaborate washing procedures of tips obsolete. A noncontact sensor monitors the dispensing process to enable an online process control. To further increase confidence and reliability for particularly critical biomedical applications, an optional closed-loop control prevents malfunctions. The dispensing performance was characterized experimentally in the range of 0.25 to 10.0 µL using liquids of different rheological properties (viscosity 1.03–16.98 mPas, surface tension 30.49–70.83 mN/m) without adjusting or calibrating the actuation parameters. The precision ranged between a coefficient of variation of 0.5% and 5.3%, and the accuracy was below ±10%. The presented technology has the potential to contribute significantly to the improvement of biochemical liquid handling for laboratory automation in terms of usability, miniaturization, cost reduction, and safety.
• Y. Abbas, J. Miwa, R. Zengerle, F. von Stetten
  Active Continuous-Flow Micromixer Using an External Braille Pin Actuator Array
  2013 Micromachines, volume: 4, pages: 80 - 89
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  Abstract

  We present a continuous-flow active micromixer based on channel-wall deflection in a polydimethylsiloxane (PDMS) chip for volume flows in the range up to 2 μL s−1 which is intended as a novel unit operation for the microfluidic Braille pin actuated platform. The chip design comprises a main microchannel connected to a series of side channels with dead ends aligned on the Braille pins. Computer-controlled deflection of the side-channel walls induces chaotic advection in the main-channel, which substantially accelerates mixing in low-Reynolds number flow. Sufficient mixing (mixing index MI below 0.1) of volume flows up to 0.5 μL s−1 could be achieved within residence times ~500 ms in the micromixer. As an application, continuous dilution of a yeast cell sample by a ratio down to 1:10 was successfully demonstrated. The mixer is intended to serve as a component of bio-analytical devices or as a unit operation in the microfluidic Braille pin actuated platform.
• S. Spieth, A. Schumacher, F. Trenkle, O. Brett, K. Seidl, S. Herwik, S. Kisban, P. Ruther, O. Paul, A. A. Aarts, H. P. Neves, P. D. Rich, D. E. Theobald, T. Holtzman, J. W. Dalley, B.-E. Verhoef, P. Janssen, R. Zengerle
  Approaches for drug delivery with intracortical probes
  2013 Biomed Eng Online, volume: 59, issue: 4, pages: 291 - 303
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  Abstract

  Intracortical microprobes allow the precise monitoring of electrical and chemical signaling and are widely used in neuroscience. Microelectromechanical system (MEMS) technologies have greatly enhanced the integration of multifunctional probes by facilitating the combination of multiple recording electrodes and drug delivery channels in a single probe. Depending on the neuroscientific application, various assembly strategies are required in addition to the microprobe fabrication itself. This paper summarizes recent advances in the fabrication and assembly of micromachined silicon probes for drug delivery achieved within the EU-funded research project NeuroProbes. The described fabrication process combines a two-wafer silicon bonding process with deep reactive ion etching, wafer grinding, and thin film patterning and offers a maximum in design flexibility. By applying this process, three general comb-like microprobe designs featuring up to four 8-mm-long shafts, cross sections from 150×200 to 250×250 µm², and different electrode and fluidic channel configurations are realized. Furthermore, we discuss the development and application of different probe assemblies for acute, semichronic, and chronic applications, including comb and array assemblies, floating microprobe arrays, as well as the complete drug delivery system NeuroMedicator for small animal research.
• N. Wangler, M. Welsche, M. Blazek, M. Blessing, M. Vervliet-Scheebaum, R. Reski, C. Müller, H. Reinecke, J. Steigert, G. Roth, R. Zengerle, N. Paust
  Bubble Jet agent release cartridge for chemical single cell stimulation
  2013 Biomed Microdevices, volume: 15, pages: 1 - 8
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  Abstract

  We present a new method for the distinct specific chemical stimulation of single cells and small cell clusters within their natural environment. By single-drop release of chemical agents with droplets in size of typical cell diameters (d <30 μm) on-demand micro gradients can be generated for the specific manipulation of single cells. A single channel and a double channel agent release cartridge with integrated fluidic structures and integrated agent reservoirs are shown, tested, and compared in this publication. The single channel setup features a fluidic structure fabricated by anisotropic etching of silicon. To allow for simultaneous release of different agents even though maintaining the same device size, the second type comprises a double channel fluidic structure, fabricated by photolithographic patterning of TMMF. Dispensed droplet volumes are V015 pl and V0 10 pl for the silicon and the TMMF based setups, respectively. Utilizing the agent release cartridges, the application in biological assays was demonstrated by hormone-stimulated premature bud formation in Physcomitrella patens and the individual staining of one single L 929 cell within a confluent grown cell culture.
• O. Strohmeier, A. Emperle, G. Roth, D. Mark, R. Zengerle, F. von Stetten
  Centrifugal gas-phase transition magnetophoresis (GTM) – a generic method for automation of magnetic bead based assays on the centrifugal microfluidic platform and application to DNA purification
  2013 Lab Chip (Lab On A Chip) 2013, volume: 13, pages: 146 - 155
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  Abstract

  Transportation of magnetic beads between different reagents plays a crucial role in many biological assays e.g. for purification of biomolecules or cells where the beads act as a mobile solid support. Therefore, usually a complex set-up either for fluidic processing or for manipulation of magnetic beads is required. To circumvent these drawbacks, we present a facile and automated method for the transportation of magnetic beads between multiple microfluidic chambers on a centrifugal microfluidic cartridge ‘‘LabDisk’’. The method excels by requiring only one stack of stationary permanent magnets, a specific microfluidic layout without actively controlled valves and a predefined frequency protocol for rotation of the LabDisk. Magnetic beads were transported through three fluidically separated chambers with a yield of 82.6% ¡ 3.6%. Bead based DNA purification from a dilution series of a Listeria innocua lysate and from a lambda phage DNA standard was demonstrated where the three chambers were used for binding, washing and elution of DNA. Recovery of L. innocua DNA was up to 68% ¡ 24% and for lambda phage DNA 43% ¡ 10% compared to manual reference purification in test tubes. Complete purification was conducted automatically within 12.5 min. Since all reagents can be preloaded onto the LabDisk prior to purification, no further hands-on steps are required during processing. Due to its modular and generic character, the presented method could also be adapted to other magnetic bead based assays e.g. to immunoassays or protein affinity purification, solely requiring the adjustment of number and volumes of the fluidic chambers.
• D. Mark, F. von Stetten, R. Zengerle
  Chips with everything: microfluidics and diagnostics
  2013 Medical Device Developments
• N. Lass, L. Riegger, R. Zengerle, P. Koltay
  Enhanced Liquid Metal Micro Droplet Generation by Pneumatic Actuation Based on the StarJet Method
  2013 Micromachines, volume: 4, pages: 49 - 66
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  Abstract

  We present a novel pneumatic actuation system for generation of liquid metal droplets according to the so-called StarJet method. In contrast to our previous work, the performance of the device has been significantly improved: the maximum droplet generation frequency in continuous mode has been increased to fmax = 11 kHz (formerly fmax = 4 kHz). In addition, the droplet diameter has been reduced to 60 μm. Therefore, a new fabrication process for the silicon nozzle chips has been developed enabling the production of smaller nozzle chips with higher surface quality. The size of the metal reservoir has been increased to hold up to 22 mL liquid metal and the performance and durability of the actuator has been improved by using stainless steel and a second pneumatic connection to control the sheath flow. Experimental results are presented regarding the characterization of the droplet generation, as well as printed metal structures.
• T. Hutzenlaub, A. Asthana, J. Becker, R. Wheeler, R. Zengerle, S. Thiele
  FIB/SEM-based calculation of tortuosity in a porous LiCoO2 cathode for a Li-ion battery
  2013 Electrochem Commun, volume: 27, pages: 77 - 80
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  Abstract

  We present a new method to quantify tortuosity in the porous, LiCoO2 cathode of a Li-ion battery. The starting point is a previously published 3D reconstruction from FIB/SEM images with three phases, the active material domain, carbon-binder domain and pore space. Based on this geometrical configuration, we compute effective diffusivities, from which we in turn derive tortuosity values for the pore space ranging between 5 and 11.6 for the three spatial directions. In a next step, we compare our approach to an imaging method that employs back-filling material. These methods do not differentiate between the carbon-binder domain and the pore space. Thus we remove the carbon-binder domain from our 3D reconstruction and add its volume to the pore space. As a result of this procedure, the tortuosity is greatly reduced to values between 1.5 and 1.9. Experiments suggest that both results for tortuosity are inaccurate and that the real values lie somewhere between these parameter sets. Hence, based on experimental data, we propose a nanoporous carbon-binder domain and derive intermediate tortuosity values between 4.2 and 6.1. These values are consistent with experimental values for similar Li-ion cathodes reported previously.
• A. Kloke, C. Köhler, A. Dryzga, R. Gerwig, K. Schumann, M. Ade, R. Zengerle, S. Kerzenmacher
  Fabrication of Highly Porous Platinum by Cyclic Electrodeposition of PtCu Alloys: How do Process Parameters Affect Morphology?
  2013 J Electrochem Soc, volume: 160, issue: 2, pages: D111 - D118
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  Abstract

  In this work we analyze the influence of process parameters on the morphology of highly porous platinum electrodes, fabricated by co-deposition of platinum-copper alloy and subsequent selective dissolution of the less noble copper during multiple cyclic voltammetry scans. Thereto the Cu2+ concentration in the deposition electrolyte, the negative scan limit, and the scan rate were varied. Corresponding electrodes were characterized by their surface texture (scanning electron microscopy, X-ray diffractometry), specific surface area, and elemental compositions (X-ray photoelectron spectroscopy and energy dispersive X-ray spectroscopy). Under conditions enforcing precursor depletion a sphere-dominated structure is observed, whereas oppositional configurations lead to smoother, mud-crack pattern like structures. Electrodes of high specific surface area are obtained particularly when high copper ion concentrations (100 mmol l−1 CuSO4, roughness factor 3740 ± 270), high negative scan limits (−0.800 V vs. SCE, roughness factor 3790 ± 370) and low scan speeds (1 mV s−1, roughness factor 3930 ± 370) compared to standard parameters (20 mmol l−1 CuSO4, −0.600 V vs. SCE negative scan limit, 1 mV s−1, roughness factor 3040 ± 300) were applied during fabrication. Since the fabrication of these highly rough electrodes requires the deposition of high amounts of platinum (10–20 mg per cm2 footprint area) it is mainly relevant to applications such as neurostimulation electrodes or implantable glucose fuel cells in which platinum costs are less relevant than electrode properties and functionality.
• C. Köhler, A. Kloke, A. Drzyzga, R. Zengerle, S. Kerzenmacher
  Fabrication of highly porous platinum electrodes for micro-scale applications by pulsed electrodeposition and dealloying
  2013 Journal of Power Sources, volume: 242, pages: 255 - 263
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  Abstract

  We present the implementation and optimization of a novel electrodeposition method for the fabrication of highly porous platinum electrodes. It is based on the co-deposition of platinum and copper and the selective dealloying of copper in a pulsed manner. The new process yields mechanically stable platinum electrodes with roughness factors of up to RF = 6500 ± 700, compared to the state-of-the-art cyclic electrodeposition method this corresponds to an improvement in RF by 111 %. Furthermore the time demand for fabrication is reduced by 59 %, whereas the platinum utilization is increased by 53 %. The method is particularly advantageous for applications such as micro fuel cells since it enables the precise deposition of catalytically active electrodes on micro-structured conductive areas. In this context the novel platinum electrodes show higher current densities for the oxidation of formic acid and glucose than state-of-the-art electrodes. In terms of methanol oxidation their catalytic activity is comparable to commercial direct methanol fuel cell (DMFC) electrodes, fabricated from Pt-Ru nanoparticles dispersed on carbon black.
• T. Hutzenlaub, J. Becker, R. Zengerle, S. Thiele
  How Coarsening the 3D Reconstruction of a Porous Material Influences Diffusivity and Conductivity Values
  2013 ECS Electrochemistry Letters, volume: 2, pages: F14 - F17
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  Abstract

  Coarsening the resolution of a 3D reconstruction is a common approach to make simulations feasible with regard to computational resources. We coarsen the reconstruction of a PEMFC cathode catalyst layer and investigate how this influences parameters such as diffusivity and conductivity. This is also an indication of how trustworthy these parameters are in the first place, because imaging itself is a coarsened representation of the real morphology. While diffusivity remains approximately constant due to the opposing behavior of bulk and Knudsen diffusivity, conductivity is strongly affected. The method introduced here is transferable to evaluate 3D reconstructions of other porous materials.
• T. Brettschneider, C. Dorrer, D. Czurratis, R. Zengerle, M. Daub
  Laser micromachining as a metallization tool for microfluidic polymer stacks
  2013 J Micromech Microeng, volume: 23, pages: 035020 - 035030
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  Abstract

  A novel assembly approach for the integration of metal structures into polymeric microfluidic systems is described. The presented production process is completely based on a single solid-state laser source, which is used to incorporate metal foils into a polymeric multi-layer stack by laser bonding and ablation processes. Chemical reagents or glues are not required. The polymer stack contains a flexible membrane which can be used for realizing microfluidic valves and pumps. The metal-to-polymer bond was investigated for different metal foils and plasma treatments, yielding a maximum peel strength of Rps = 1.33N mm−1. A minimum structure size of 10 μm was determined by 3D microscopy of the laser cut line. As an example application, two different metal foils were used in combination to micromachine a standardized type-T thermocouple on a polymer substrate. An additional laser process was developed which allows metal-to-metal welding in close vicinity to the polymer substrate. With this process step, the reliability of the electrical contact could be increased to survive at least 400 PCR temperature cycles at very low contact resistances.
• C. Cupelli, T. Borchardt, T. Steiner, N. Paust, R. Zengerle, M. Santer
  Leukocyte enrichment based on a modified pinched flow fractionation approach
  2013 Microfluid Nanofluid, volume: 14, issue: 3-4, pages: 551 - 563
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  Abstract

  In this paper, a simple and robust design for a passive hydrodynamic cell sorter based on pinched flow-field fractionation is presented and analyzed. Two principal layouts of the sorter are discussed and investigated experimentally as well as numerically based on the dissipative particle dynamics (DPD) method. Experimentally, design 1 approximately sorts 87 % of the erythrocytes to their designated outlet, while 100 % of the leukocytes branch correctly. This also holds for design 2 differing merely in the direction of the outlet for erythrocytes, but here only 69 % of the red blood cells are redirected to the designated outlet. This behavior can be elucidated by employing DPD simulations, where erythrocytes advected with the flow are modeled explicitly. Our results suggest that if a cell sorter is designed to operate at high throughput, its layout may not entirely rely on commonly assumed idealizing conditions, because cells cannot be considered as point-like, isolated objects following definite stream lines. Hydrodynamic forces originating from the cells as extended objects must be taken into account.
• T. van Oordt, Y. Barb, J. Smetana, R. Zengerle, F. von Stetten
  Miniature stick-packaging – an industrial technology for pre-storage and release of reagents in lab-on-a-chip systems
  2013 Lab Chip, volume: 13, issue: 15, pages: 2888 - 2892
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  Abstract

  Stick-packaging of goods in tubular-shaped composite-foil pouches has become a popular technology for food and drug packaging. We miniaturized stick-packaging for use in lab-on-a-chip (LOAC) systems to prestore and on-demand release the liquid and dry reagents in a volume range of 80–500 ml. An integrated frangible seal enables the pressure-controlled release of reagents and simplifies the layout of LOAC systems, thereby making the package a functional microfluidic release unit. The frangible seal is adjusted to defined burst pressures ranging from 20 to 140 kPa. The applied ultrasonic welding process allows the packaging of temperature sensitive reagents. Stick-packs have been successfully tested applying recovery tests (where 99% (STDV = 1%) of 250 ml pre-stored liquid is released), long-term storage tests (where there is loss of only ,0.5% for simulated 2 years) and air transport simulation tests. The developed technology enables the storage of a combination of liquid and dry reagents. It is a scalable technology suitable for rapid prototyping and low-cost mass production.
• D. Trebbels, A. Kern, F. Fellhauer, C. Huebner, R. Zengerle
  Miniaturized FPGA-Based High-Resolution Time-Domain Reflectometer
  2013 Ieee, volume: 62, issue: 7, pages: 2101 - 2113
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  Abstract

  Time-domain reflectometry (TDR) is a well-known measurement principle for evaluating frequency-dependent electric and dielectric properties of various materials and substances. Although TDR is a proven method, the high price for TDR measurement equipment and complex laboratory setups is often a limiting factor for cost-sensitive applications or large-scale field experiments, where a large number of TDR meters is required. This paper reports on the development of a new miniaturized low-cost TDR meter capable of sampling a repetitive rectangular waveform, which is used as an excitation signal. The developed sampling circuit is based on a digital delta modulator (DM) and allows for capturing the waveform of a repetitive measurement signal. A 1-MHz signal can be captured with a virtual sampling resolution of 1 ps within a measurement interval of 1 s. The generated pulses have a rise time of 2 ns and can be captured with an amplitude resolution of approximately 10 bit and an accuracy of approximately 8 bit. The developed digital DM architecture is implemented inside a small field programmable gate array and integrated into a miniaturized low-power TDR meter prototype for battery-powered outdoor applications. The captured measurement data are stored on integrated micro-SD card memory and can be read out either via a Universal Serial Bus, an RS-485 bus system, or a wireless interface. The TDR meter is controlled by an integrated microcontroller and a real-time clock and therefore can operate completely independent from any additional control setup. The TDR meter targets applications within the field of geoscience and agricultural monitoring, where large-scale measurement systems are required.
• N. Losleben, J. Spinke, S. Adler, N. Oranth, R. Zengerle
  Model fluids representing aqueous in-vitro diagnostic reagents for the development of dispensing systems
  2013 Drug Discov Today, pages: 1035 - 1042
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  Abstract

  Analyzers for in-vitro diagnostic (IVD) testing facilitate the determination of medical information from biological samples. To reach a high quality, the detection reagents have to be dispensed with a high degree of precision and accuracy. A technology change from conventional pipetting systems to contactfree dispensers provides the opportunity to reduce carry-over and handle reagents in the microliter range. A great challenge for the development and validation of new systems is the huge variety of the IVD reagents. This work presents the fluidic properties of 646 different aqueous IVD reagents and how they can be represented by a set of easy-to-prepare model fluids, covering the rheological range of the reagents. In addition, based on the model fluids, a standardized approach is presented for the evaluation of dispensers for IVD applications.
• T. Hutzenlaub, J. Becker, R. Zengerle, S. Thiele
  Modelling the water distribution within a hydrophilic and hydrophobic 3D reconstructed cathode catalyst layer of a proton exchange membrane fuel cell
  2013 J Power Sources, volume: 227, pages: 260 - 266
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  Abstract

  We reconstruct a section of the cathode catalyst layer (CCL) of a Gore PEMFC membrane electrode assembly three-dimensionally with nanometre scale resolution. Subsequently, we present a new modelling method to fill the pore space of this matrix stepwise with water, enabling the description of varying saturation conditions of the CCL. The method is based on a 3D pore size distribution and enables to differentiate between a hydrophilic and a hydrophobic CCL. It utilizes a sequence to fill the pores according to their size, going from small to large (hydrophilic) or vice versa (hydrophobic), until a predefined value of water saturation is reached. We compare both cases by calculating an effective diffusivity for oxygen in nitrogen in all spatial directions. Both the hydrophilic and the hydrophobic case display a similar ability to transport oxygen up to approximately 50% water saturation of the pore space. At higher water saturation, we calculate larger diffusivity values for the hydrophobic case. Finally, we calculate the specific reaction surface area that is accessible from the gas diffusion layer via unfilled pores for all water saturation conditions. At 50% saturation, the hydrophobic case displays a twenty times larger reaction surface area than the hydrophilic case.
• O. Strohmeier, S. Laßmann, B. Riedel, D. Mark, G. Roth, M. Werner, R. Zengerle, F. von Stetten
  Multiplex genotyping of KRAS point mutations in tumor cell DNA by allele-specific real-time PCR on a centrifugal microfluidic disk segment
  2013 Microchim Acta, volume: 181, pages: 1681 - 1688
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  Abstract

  Point Mutations on the Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified as an important predictive biomarker for response to cancer therapy targeting the epidermal growth factor receptor. KRAS mutations are prevalent in up to 40 % of all colorectal carcinomas, and routinely conducted KRAS genotyping is becoming mandatory to predict therapy success and to reduce therapy costs. We report a low-cost, disposable and ready-to-use centrifugal microfluidic cartridge (termed GeneSlice) containing preloaded primers and probes. The GeneSlice cartridge enables the parallel detection of the seven most relevant KRAS point mutations by allele-specific real-time PCR. It represents a cost effective alternative to dideoxy-sequencing with a faster time-to-result (~ 2 h versus up to 20 h in case of dd-sequencing). Microfluidic processing of the GeneSlice along with allele-specific amplification and real-time detection are conducted in a slightly modified, commercially available PCR thermocycler. Intra-chip standard deviation of Cq values on the GeneSlices is negligible (GeneSlice 1: Cq,std.dev. = 0.13; GeneSlice 2: Cq,std.dev = 0.26). In 23 of 24 experiments, the data for genotyping 6 cancer cell lines (n = 4 per cell line) agreed with dd-sequencing. Additionally, DNA derived from microdissected formalin-fixed and paraffin embedded colorectal carcinomas of two cases was genotyped correctly and reproducibly (n = 3 per patient; one GeneSlice excluded from evaluation). The GeneSlice therefore clearly demonstrated the potential to become a valuable tool for routine diagnostics of KRAS mutations by reducing costs and hands-on time.
• S. Thiele, T. Fürstenhaupt, D. Banham, T. Hutzenlaub, V. Birss, C. Ziegler, R. Zengerle
  Multiscale tomography of nanoporous carbon-supported noble metal catalyst layers
  2013 J Power Sources, volume: 228, pages: 185 - 192
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  Abstract

  Noble metal catalysts are a scarce, non-renewable resource, and yet are required in a wide range of industrial applications, including in polymer electrolyte membrane fuel cells (PEMFCs). The effectiveness of PEMFCs depends not only on the size, active surface area, and distribution of the Pt catalyst nanoparticles, which affects reaction kinetics, but also on the porous structure of the carbon support, which affects mass transport. Unfortunately, the very different size scales – several nm for the Pt catalyst vs. several μm for the porosity features – cannot be characterized by a single method. Here, we present a novel approach for integrating information from both of these size scales to build a single geometrical model. Focused Ion Beam – Scanning Electron Microscope tomography (SEMt) was carried out on a commercial PEMFC cathode catalyst layer to characterize porosity, connectivity as well as pore-size and grain-size distribution. Transmission Electron Microscopy tomography (TEMt) was used to analyze volume and surface area distributions of nanometer sized platinum catalyst particles. Further, we propose an up-scaling approach to translate the information obtained from TEMt to SEMt. Knowledge of catalyst particle locations within the solid support matrix will be critical in enabling the analysis of limiting transport processes in PEMFC CCLs.
• D. Liang, C. Steinert, S. Bammesberger, L. Tanguy, A. Ernst, R. Zengerle, P. Koltay
  Novel gravimetric measurement technique for quantitative volume calibration in the sub-microliter range
  2013 Meas Sci Technol, volume: 24, pages: 025301 - 025311
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  Abstract

  We present a novel measurement method based on the gravimetric principles adapted from the ASTM E542 and ISO 4787 standards for quantitative volume determination in the sub-microliter range. Such a method is particularly important for the calibration of non-contact micro dispensers as well as other microfluidic devices. The novel method is based on the linear regression analysis of continuously monitored gravimetric results and therefore is referred to as ‘gravimetric regression method (GRM)’. In this context, the regression analysis is necessary to compensate the mass loss due to evaporation that is significant for very small dispensing volumes. A full assessment of the measurement uncertainty of GRM is presented and results in a standard measurement uncertainty around 6 nl for dosage volumes in the range from 40 nl to 1 μl. The GRM has been experimentally benchmarked with a dual-dye ratiometric photometric method (Artel Inc., Westbrook, ME, USA), which can provide traceability of measurement to the International System of Units (SI) through reference standards maintained by NIST. Good precision (max. CV = 2.8%) and consistency (bias around 7 nl in the volume range from 40 to 400 nl) have been observed comparing the two methods. Based on the ASTM and ISO standards on the one hand and the benchmark with the photometric method on the other hand, two different approaches for establishing traceability for the GRM are discussed.
• S. Sané, C. Jolivalt, G. Mittler, P. Nielsen, S. Rubenwolf, R. Zengerle, S. Kerzenmacher
  Overcoming Bottlenecks of Enzymatic Biofuel Cell Cathodes: Crude Fungal Culture Supernatant Can Help to Extend Lifetime and Reduce Cost
  2013 ChemSusChem, volume: 6, issue: 7, pages: 1209 - 1215
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  Abstract

  Enzymatic biofuel cells (BFCs) show great potential for the direct conversion of biochemically stored energy from renewable biomass resources into electricity. However, enzyme purification is time-consuming and expensive. Furthermore, the long-term use of enzymatic BFCs is hindered by enzyme degradation, which limits their lifetime to only a few weeks. We show, for the first time, that crude culture supernatant from enzyme-secreting microorganisms (Trametes versicolor) can be used without further treatment to supply the enzyme laccase to the cathode of a mediatorless BFC. Polarization curves show that there is no significant difference in the cathode performance when using crude supernatant that contains laccase compared to purified laccase in culture medium or buffer solution. Furthermore, we demonstrate that the oxygen reduction activity of this enzymatic cathode can be sustained over a period of at least 120 days by periodic resupply of crude culture supernatant. This is more than five times longer than control cathodes without the resupply of culture supernatant. During the operation period of 120 days, no progressive loss of potential is observed, which suggests that significantly longer lifetimes than shown in this work may be possible. Our results demonstrate the possibility to establish simple, cost efficient, and mediatorless enzymatic BFC cathodes that do not require expensive enzyme purification procedures. Furthermore, they show the feasibility of an enzymatic BFC with an extended lifetime, in which self-replicating microorganisms provide the electrode with catalytically active enzymes in a continuous or periodic manner.
• M. Blazek, C. Betz, M. N. Hall, M. Reth, R. Zengerle, M. Meier
  Proximity Ligation Assay for High Content Profiling of Cell Signaling Pathways on a Microfluidic Chip
  2013 Mol Cell Proteomics, volume: 12, issue: 12, pages: 3898 - 3907
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  Abstract

  Here, we present the full integration of a proximity ligation assay (PLA) on a microfluidic chip for systematic cell signaling studies. PLA is an in situ technology for detection of protein interaction, post-translational modification, concentration, and cellular location with single molecule resolution. Analytical performance advances on chip are achieved, including full automation of the biochemical PLA steps, target multiplexing, and reduction of antibody consumption by two orders of magnitudes compared to standard procedures. In combination with a microfluidic cell-culturing platform, control over 128 cell culture microenvironments is gained. We demonstrate the use of the combined cell-culture and protein analytic assay on chip by characterizing the Akt signaling pathway upon PDGF stimulation. Signal transduction is detected by monitoring the phosphorylation state of Akt, GSK-3β, p70S6K, S6, Erk1/2, mTOR, and the cellular location of FoxO3a in parallel with the PLA. Single-cell PLA results revealed for Akt and direct targets of Akt a maximum activation time of 4–8 min upon PDGF stimulation. Activation times for phosphorylation events downwards in the Akt signaling pathway including phosphorylation of S6, p70S6K, and mTOR are delayed by 8–10 min or exhibit a response time of at least 1 h. Quantitative confirmation of the Akt phosphorylation signal was determined with the help of a mouse embryonic fibroblast cell line (MEF) deficient for rictor. Taken together, the miniaturized PLA chip establishes a biotechnological tool for general cell signaling studies and their dynamics relevant for a broad range of biological inquiry.
• S. Bammesberger, A. Ernst, N. Losleben, L. Tanguy, R. Zengerle, P. Koltay
  Quantitative characterization of non-contact microdispensing technologies for the sub-microliter range
  2013 Drug Discov Today, volume: 18, issue: 9-10, pages: 435 - 446
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  Abstract

  This work describes how to effectively compare non-contact dispensing technologies for automated liquid handling under high-throughput screening (HTS) conditions in the range of 0.05–10 ml. Exemplarily, we characterize five established technologies and categorize them into valve-based and positive displacement-based technologies. Furthermore we introduce dispensing accuracy and precision in an ‘intrarun’, ‘inter-run’ and ‘tip-to-tip’ context as universally applicable performance parameters. A NIST traceable spectrophotometric measurement method is utilized for experimental characterization. It yields an Intra-Run CV (Inter-Run CV) between 0.4% to 7.7% (0.5 to 10.9%) and a Tip-to-Tip CV between 1.4% and 9.9% for target volumes <1 ml. An absolute accuracy of better than 5.0% is generally difficult to achieve in the sub-microliter range.
• T. van Oordt, G. B. Stevens, S. K. Vashist, R. Zengerle, F. von Stetten
  Rapid and highly sensitive luciferase reporter assay for the automated detection of botulinum toxin in the centrifugal microfluidic LabDisk platform
  2013 Rsc Adv, volume: 3, pages: 22046 - 22052
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  Abstract

  We developed a system for the detection of botulinum neurotoxin (BoNT) type A based on a highly sensitive luciferase reporter assay automated by the centrifugal microfluidic LabDisk platform. The assay is based on the detection of BoNT's proteolytic activity and generation of a bioluminescent signal due to the release of firefly luciferase, pre-bound to microbeads via a cleavable peptide linker, in response to BoNT. It detected purified BoNT, BoNT in complex with neurotoxin associated proteins, and the recombinant enzymatic BoNT light chain in buffer and whole milk in the concentration range of 8 pM to 6 nM with an analytical sensitivity and limit of detection of 10–39 pM and 6–14 pM, respectively. The intra-disk, intra-day and inter-day variability were in the range of 1–13%, 1–7% and 10–13%, respectively. The developed assay correlated well with the conventional microwell plate assay. It is superior to the conventional BoNT assays in terms of portability, cost-effectiveness, lesser sample requirement, lesser number of steps and detection of a broad spectrum of BoNT serotypes and subtypes. It takes only 30 minutes, which is ideal for point-of-need BoNT detection in the case of security threats and food monitoring.
• K. G. Kraiczek, G. P. Rozing, R. Zengerle
  Relation between chromatographic resolution and signal-to-noise ratio in spectrophotometric HPLC detection
  2013 Anal Chem, volume: 85, pages: 4829 - 4835
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  Abstract

  Absorption spectrophotometry has been and still is the industry standard for detection in HPLC. Limit of detection (LOD) and linear dynamic range (LDR) are the primary performance requirements and have driven continuous improvement of spectrophotometric HPLC detectors. Recent advances in HPLC column technology have led to low flow-rate HPLC such as capillary HPLC and nanoflow HPLC and put higher demands on optical HPLC signal detection. However, fundamental principles in spectrophotometric HPLC detection have not been reviewed for many years. In particular the relationship between the detector’s signalto- noise ratio (SNR) and band broadening needs to be re-evaluated. In this work, a new quantitative model is presented which allows the calculation of the trade-off made between chromatographic resolution and SNR in spectrophotometric HPLC detection. Modern optics flow cells based on total internal reflection are included and compared to conventional flow cells.
• K. Kliche, G. Kattinger, S. Billat, L. Shen, S. Messner, R. Zengerle
  Sensor for thermal gas analysis based on micromachined silicon-microwires
  2013 Ieee Sens J, volume: 13, issue: 7, pages: 2626 - 2635
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  Abstract

  We present a microelectromechanical systems-based sensor for the thermal detection of changes of gas mixtures such as the CO2 concentration in air that is of interest in air conditioning climate control within buildings. Key properties of the system are low power consumption (<10 mW) and high longterm stability through the absence of moving or consumptive components. The used sensor chip has three silicon-microwires (thermistors) surrounded by the gas mixture to be analyzed. A centered wire (heater) is supplied with sinusoidal heating power. This induces a thermal response via the surrounding gas to measurement wires (detectors) located in different distances from the heater. The phase shift between heating power and induced thermal responses at the detectors is analyzed and depends on the thermal properties of the gas. After calibration, the sensor is able to quantify the concentration of an individual component within a mixture of different but known gas components. This is demonstrated by measuring the CO2 concentration in N2/CO2 mixtures with a resolution of 0.2% at constant pressure and temperature.
• A. Gross, J. Schöndube, S. Niekrawitz, W. Streule, L. Riegger, R. Zengerle, P. Koltay
  Single-Cell Printer: Automated,On Demand, and Label Free
  2013 JALA - J Lab Autom, volume: 18, issue: 6, pages: 504 - 518
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  Abstract

  Within the past years, single-cell analysis has developed into a key topic in cell biology to study cellular functions that are not accessible by investigation of larger cell populations. Engineering approaches aiming to access single cells to extract information about their physiology, phenotype, and genotype at the single-cell level are going manifold ways, meanwhile allowing separation, sorting, culturing, and analysis of individual cells. Based on our earlier research toward inkjet-like printing of single cells, this article presents further characterization results obtained with a fully automated prototype instrument for printing of single living cells in a noncontact inkjet-like manner. The presented technology is based on a transparent microfluidic drop-on-demand dispenser chip coupled with a camera-assisted automatic detection system. Cells inside the chip are detected and classified with this detection system before they are expelled from the nozzle confined in microdroplets, thus enabling a “one cell per droplet” printing mode. To demonstrate the prototype instrument’s suitability for biological and biomedical applications, basic experiments such as printing of single-bead and cell arrays as well as deposition and culture of single cells in microwell plates are presented. Printing efficiencies greater than 80% and viability rates about 90% were achieved.
• E. Kipf, J. Koch, B. Geiger, J. Erben, K. Richter, J. Gescher, R. Zengerle, S. Kerzenmacher
  Systematic screening of carbon-based anode materials for microbial fuel cells with Shewanella oneidensis MR-1
  2013 Bioresource Technol, volume: 146, pages: 386 - 329
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  Abstract

  We present a systematic screening of carbon-based anode materials for microbial fuel cells with Shewanella oneidensis MR-1. Under anoxic conditions nanoporous activated carbon cloth is a superior anode material in terms of current density normalized to the projected anode area and anode volume (24.0 ± 0.3 μA cm−2 and 482 ± 7 μA cm−3 at −0.2 vs. SCE, respectively). The good performance can be attributed to the high specific surface area of the material, which is available for mediated electron transfer through self-secreted flavins. Under aerated conditions no influence of the specific surface area is observed, which we attribute to a shift from primary indirect electron transfer by mediators to direct electron transfer via adherent cells. Furthermore, we show that an aerated initial growth phase enhances the current density under subsequent anoxic conditions fivefold when compared to a similar experiment that was conducted under permanently anoxic conditions.
• T. Brettschneider, C. Dorrer, M. Bründel, R. Zengerle, M. Daub
  Wafer-level packaging and laser bonding as an approach for silicon-into-lab-on-chip integration
  2013 J. Micromech. Microeng., volume: 23, pages: 055005 - 055014
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  Abstract

  A novel approach for the integration of silicon biosensors into microfluidics is presented. Our approach is based on wafer-level packaging of the silicon die and a laser-bonding process of the resulting mold package into a polymer-multilayer stack. The introduction of a flexible and 40 μm thin hot melt foil as an intermediate layer enables laser bonding between materials with different melting temperatures, where standard laser welding processes cannot be employed. All process steps are suitable for mass production, e.g. the approach does not involve any dispensing steps for glue or underfiller. The integration approach was demonstrated and evaluated regarding process technology by wafer-level redistribution of daisy chain silicon dies representing a generic biosensor. Electrical connection was successfully established and laser-bonding tensile strength of 5.7 N mm−2 and burst pressure of 587 kPa at a temperature of 100 °C were achieved for the new material combination. The feasibility of the complete packaging approach was shown by the fabrication of a microfluidic flow cell with embedded mold package.

2012

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• Vosseler M, Clemenz M, Zengerle R
  A flat and cost effective actuator based on superabsorbent polymer driving a skin attachable drug delivery system
  2012 Smart Materials and Structures, volume: 21, pages: 105002 - 105013
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  Abstract

  We present a flat and cost effective volume displacement actuator based on superabsorbent polymer. It offers slow kinetics and is able to work against reasonable back-pressures,e.g. 0.50 ml in 235 min at 140 kPa. It is predestined for low-cost skin attachable drug delivery devices. The actuator consists of a plastic ring filled with superabsorbent polymer granulate. It is sealed with a thermoplastic elastomeric membrane on one side and a stiff filter membrane on the other side. After adding a defined amount (e.g. 2 or 10 ml) of swelling agent the actuator shows a fast initial volume displacement within a few minutes followed by a slow continuous increase of this volume within hours. Minimized initial volume displacement and maximized displaced volume after 4 h cannot be combined in one actuator. A minimized initial displacement can be as low as 0.10 ml +/- 0.03 ml. A maximized displaced volume after 4 h can be 1.71 ml +/- 0.18 ml, not considering the initial effect. The back-pressure dependency of one selected actuator design was studied. At a back-pressure of 100 kPa the displaced volume is reduced by 33%. We investigated various actuator designs with varying surface area, hardness of the elastomeric membrane and superabsorbent polymer. Finally, we demonstrate a skin attachable drug delivery system based on the employment of the superabsorbent polymer actuator.
• M Kunze, J Merz, W-J Hummel, H Glosch, S Messner, R Zengerle
  A micro dew point sensor with a thermal detection principle
  2012 Meas Sci Technol, volume: 23, pages: 014004 - (10pp)
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  Abstract

  We present a dew point temperature sensor with the thermal detection of condensed water on a thin membrane, fabricated by silicon micromachining. The membrane (600 × 600×∼1 μm3) is part of a silicon chip and contains a heating element as well as a thermopile for temperature measurement. By dynamically heating the membrane and simultaneously analyzing the transient increase of its temperature it is detected whether condensed water is on the membrane or not. To cool the membrane down, a peltier cooler is used and electronically controlled in a way that the temperature of the membrane is constantly held at a value where condensation of water begins. This temperature is measured and output as dew point temperature. The sensor system works in a wide range of dew point temperatures between 1 K and down to 44 K below air temperature. In experimental investigations it could be proven that the deviation of the measured dew point temperatures compared to reference values is below ±0.2 K in an air temperature range of 22 to 70 ◦C. At low dew point temperatures of −20 ◦C (air temperature = 22 ◦C) the deviation increases to nearly −1 K.
• S. Spieth, A. Schumacher, T. Holtzman, P. D. Rich, D. E. Theobald, J. W. Dalley, R. Nouna, S. Messner, R. Zengerle
  An intra-cerebral drug delivery system for freely moving animals
  2012 Biomed Microdevices, volume: 14, pages: 799 - 809
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  Abstract

  Microinfusions of drugs directly into the central nervous system of awake animals represent a widely used means of unravelling brain functions related to behaviour. However, current approaches generally use tethered liquid infusion systems and a syringe pump to deliver drugs into the brain, which often interfere with behaviour. We address this shortfall with a miniaturised electronically-controlled drug delivery system (20×17.5×5 mm3) designed to be skull-mounted in rats. The device features a micropump connected to two 8-mm-long silicon microprobes with a cross section of 250×250 μm2 and integrated fluid microchannels. Using an external electronic control unit, the device allows infusion of 16 metered doses (0.25 μL each, 8 per silicon shaft). Each dosage requires 3.375 Ws of electrical power making the device additionally compatible with state-of-the-art wireless headstages. A dosage precision of 0.25±0.01 μL was determined in vitro before in vivo tests were carried out in awake rats. No passive leakage from the loaded devices into the brain could be detected using methylene blue dye. Finally, the device was used to investigate the effects of the NMDA-receptor antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1- phosphonic acid, (R)-CPP, administered directly into the prefrontal cortex of rats during performance on a task to assess visual attention and impulsivity. In agreement with previous findings using conventional tethered infusion systems, acute (R)-CPP administration produced a marked increase in impulsivity.
• S. Zehnle, F. Schwemmer, G. Roth, F. von Stetten, R. Zengerle, N. Paust
  Centrifugo-dynamic inward pumping of liquids on a centrifugal microfluidic platform
  2012 Lab Chip (Lab on a Chip) 2012, volume: 12, pages: 5142 - 5145
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  Abstract

  We present a method to pump liquids in a centrifugal microfluidic spinning disk from a radial outward position to a radial inward position. Centrifugal forces are applied to compress air in a cavity, this way storing pneumatic energy. The cavity is connected to an outlet channel having a lower hydraulic resistance compared to the inlet channel. The stored pneumatic energy is quickly released by fast reduction of rotational frequency. This way liquid is transported mainly through the channel with lower resistance, directing the liquid radially inwards. Pump efficiencies of .75% per pump cycle have been demonstrated for water, ethanol, a highly viscous lysis buffer and whole blood. By employing three pump cycles, water has been pumped radially inwards with an efficiency of .90%. The inward pumping requires centrifugation only, which is intrinsically available on every centrifugal microfluidic platform.
  
  
   
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• A. Tropmann, L. Tanguy, P. Koltay, R. Zengerle, L. Riegger
  Completely superhydrophobic PDMS surfaces for microfluidics
  2012 Langmuir, volume: 28, issue: 22, pages: 8292 - 8295
• G. Lammers, G. Roth, M. Heck, R. Zengerle, G. S. Tjabringa, E. M. Versteeg, T. Hafmans, R. Wismans, D. P. Reinhardt, E. T. P. Verwiel, P. L. J. M. Zeeuwen, J. Schalkwijk, R. Brock, W. F. Daamen, T. H. van Kuppevelt
  Construction of a Microstructured Collagen Membrane Mimicking the Papillary Dermis Architecture and Guiding Keratinocyte Morphology and Gene Expression
  2012 Macromol Biosci, volume: 12, pages: 675 - 691
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  Abstract

  A papillary-structured collagen fibril membrane is created, mimicking the 3D-architecture of the human papillary dermis. Primary human keratinocytes cultured to confluency on papillarstructured films are compared to keratinocytes cultured on flat membranes. Microscopical evaluation reveals the presence of morphologically distinct cells at the base of the papillar structures that are not observed on flat membranes. Gene expression microarrays and RT-qPCR indicate that these cells are in a more proliferative/ migrational state, whereas cells on flat membranes have a more differentiated expression profile. Immunohistochemical stainings confirm these results. In conclusion, specific collagen architecture can direct keratinocyte behavior, and this may be used to further improve skin regeneration.
• Mitsakakis K, Sekula-Neuner S, Lenhert S, Fuchs H, Gizeli E
  Convergence of Dip-pen nanolithography and acoustic biosensors towards a rapid-analysis multi-sample microsystem
  2012 Analyst, volume: 137, pages: 3076 - 3082
• A. Kloke, C. Köhler, R. Gerwig, R. Zengerle, S. Kerzenmacher
  Cyclic Electrodeposition of PtCu Alloy: Facile Fabrication of Highly Porous Platinum Electrodes
  2012 Adv Mater, volume: 24, pages: 2916 - 2921
• B. Faltin, S. Wadle, G. Roth, R. Zengerle, F. von Stetten
  Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters
  2012 Clin Chem, volume: 58, issue: 11, pages: 1546 - 1556
• Daniel Mark, Felix von Stetten, Roland Zengerle
  Microfluidic Apps for off-the-shelf instruments
  2012 Lab Chip, volume: 12, pages: 2464 - 2468
• B. Landenberger, H. Hoefemann, S. Wadle, A. Rohrbacha
  Microfluidic sorting of arbitrary cells with dynamic optical tweezers
  2012 Lab Chip, volume: 12, pages: 3177 - 3183
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  Abstract

  Optical gradient forces generated by fast steerable optical tweezers are highly effective for sorting small populations of cells in a lab-on-a-chip environment. The presented system can sort a broad range of different biological specimens by an automated optimisation of the tweezer path and velocity profile. The optimal grab positions for subsequent trap and cell displacements are estimated from the intensity of the bright field image, which is derived theoretically and proven experimentally. We exhibit rapid displacements of 2 µm small mitochondria, yeast cells, rod-shaped bacteria and 30 mm large protoplasts. Reliable sorting of yeast cells in a microfluidic chamber by both morphological criteria and by fluorescence emission is demonstrated.
• S.K. Vashist, A. G. Venkatesh, K. Mitsakakis, G. Czilwik, G. Roth, F. von Stetten, R. Zengerle
  Nanotechnology-Based Biosensors and Diagnostics: Technology Push versus Industrial/Healthcare Requirements
  2012 BioNanoSci., volume: 2, pages: 115 - 126
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  Abstract

  There have been considerable advances in the field of nanotechnology-based biosensors and diagnostics (NBBD) during the last two decades. These include the production of nanomaterials (NMs), employing them for new biosensing and diagnostic applications, their extensive characterization for in vitro and in vivo applications, and toxicity analysis. All these developments have led to tremendous technology push and successful demonstrations of several promising NBBD. However, there has been a significant lag in their commercialization, especially due to the lack of international regulatory guidelines for evaluating the safety of NMs and the growing public concerns about their toxicity. Despite these numerous advances and the recent regulatory approval of several NMs, it still remains to be seen if NBBD are superior to conventional ones (not based on NMs), reliable, reproducible, cost effective, and robust enough to meet the requirements of industries and healthcare. This manuscript provides a critical review of NBBD, the technology push, and the industrial/healthcare requirements.
• A. Ernst, K. Mutschler, L. Tanguy, N. Paust, R. Zengerle, P. Koltay
  Numerical Investigations on Electric Field Characteristics with Respect to Capacitive Detection of Free-Flying Droplets
  2012 sensors, volume: 12, pages: 10550 - 10565
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  Abstract

  In this paper a multi-disciplinary simulation of a capacitive droplet sensor based on an open plate capacitor as transducing element is presented. The numerical simulations are based on the finite volume method (FVM), including calculations of an electric field which changes according to the presence of a liquid droplet. The volume of fluid (VOF) method is applied for the simulation of the ejection process of a liquid droplet out of a dispenser nozzle. The simulations were realised using the computational fluid dynamic (CFD) software CFD ACE+. The investigated capacitive sensing principle enables to determine the volume of a micro droplet passing the sensor capacitor due to the induced change in capacity. It could be found that single droplets in the considered volume range of 5 nL < Vdrop < 100 nL lead to a linear change of the capacity up to ΔQ < 30 fC. The sensitivity of the focused capacitor geometry was evaluated to be Si = 0.3 fC/nL. The simulation results are validated by experiments which exhibit good agreement.
• Dennis Trebbels, Felix Fellhauer, Michael Jugl, Gerd Haimerl, Mart Min, Roland Zengerle
  Online Tissue Discrimination for Transcutaneous Needle Guidance Applications Using Broadband Impedance Spectroscopy
  2012 Ieee T Bio-med Eng, volume: 59, issue: 2, pages: 494 - 503
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  Abstract

  This paper reports on a novel system architecture for measuring impedance spectra of a biological tissue close to the tip of a hollow needle. The measurement is performed online using fast broadband chirp signals. The time domain measurement raw data are transformed into the transfer function of the tissue in frequency domain. Correlation technique is used to analyze the characteristic shape of the derived tissue transfer function with respect to known “library functions” for different types of tissue derived in earlier experiments. Based on the resulting correlation coefficients the exact type of tissue is determined. A bipolar coaxial needle is constructed, simulated by finite element method and tested during various in vitro and in vivo experiments. The results show a good spatial resolution of approximately 1.0mm for a needle with a diameter of 2.0 mm. The correlation coefficients for the three tested tissue types muscle, fat, and blood allow for a clear tissue classification. Best results have been obtained using the characteristic phase diagrams for each tissue. Correlated to the corresponding library transfer function the coefficients are in the range of+0.96 to +0.99 for the matching tissue. In return, the resulting coefficients for correlation with nonmatching tissues are in the range of −0.93 to +0.81.
• J. Hoffmann, L. Riegger, F. Bundgaard, D. Mark, R. Zengerle, J. Ducrée
  Optical non-contact localization of liquid-gas interfaces on disk during rotation for measuring flow rates and viscosities
  2012 Lab Chip (Lab on a Chip) 2012, volume: 12, pages: 5231 - 5236
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  Abstract

  We present a novel technique for the spatio-temporally resolved localization of liquid-gas interfaces on centrifugal microfluidic platforms based on total internal reflection (TIR) at the channel wall. The simple setup consists of a line laser and a linear image sensor array mounted in a stationary instrument. Apart from identifying the presence of usually unwanted gas bubbles, the here described online meniscus detection allows to measure liquid volumes with a high precision of 1.9%. Additionally, flow rates and viscosities (range: 1–12 mPa s, precision of 4.3%) can be sensed even during rotation at frequencies up to 30 Hz.
  
  
   
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• S.Herrlich, S.Spieth, S.Messner, R.Zengerle
  Osmotic micropumps for drug delivery
  2012 Adv Drug Deliver Rev, volume: 64, pages: 1617 - 1627
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  Abstract

  This paper reviews miniaturized drug delivery systems applying osmotic principles for pumping. Osmotic micropumps require no electrical energy and consequently enable drug delivery systems of smallest size for a broad field of new applications. In contrast to common tablets, these pumps provide constant (zeroorder) drug release rates. This facilitates systems for long term use not limited by gastrointestinal transit time and first-pass metabolism. The review focuses on parenteral routes of administration targeting drug delivery either in a site-specific or systemic way. Osmotic pumps consist of three building blocks: osmotic agent, solvent, and drug. This is used to categorize pumps into (i) single compartment systems using water from body fluids as solvent and the drug itself as the osmotic agent, (ii) two compartment systems employing a separate osmotic agent, and (iii) multi-compartment architectures employing solvent, drug and osmotic agent separately. In parallel to the micropumps, relevant applications and therapies are discussed.
• A. Tropmann, N. Lass, N. Paust, T. Metz, C. Ziegler, R. Zengerle, P. Koltay
  Pneumatic Dispensing of Nano- to Picoliter Droplets of Liquid Metal with the StarJet Method for Rapid Prototyping of Metal Microstructures
  2012 Microfluid Nanofluid, volume: 12, pages: 75 - 84
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  Abstract

  This work presents a new, simple and robust, pneumatically actuated method for the generation of liquid metal micro droplets in the nano- to picoliter range. The so called StarJet dispenser utilizes a star-shaped nozzle geometry that stabilizes liquid plugs in its center by means of capillary forces. Single droplets of liquid metal can be pneumatically generated by interaction of the sheathing gas flow in the outer grooves of the nozzle and the liquid metal. For experimental validation, a print head was build consisting of silicon chips with a star-shaped nozzle geometry and a heated actuator (up to 280 °C). The silicon chips are fabricated by Deep Reactive Ion Etching (DRIE). Chip designs with different star-shaped geometries were able to generate droplets with diameters in the range of the corresponding nozzle diameters. The StarJet can be operated in two modes: Either continuous droplet dispensing mode or drop on demand (DoD) mode. The continuous droplet generation mode for a nozzle with 188 µm diameter shows tear-off frequencies between 25 Hz and 120 Hz, while droplet diameters remain constant at 210 µm for each pressure level. Metal columns were printed with a thickness of 0.5 to 1.0 mm and 30 mm height (aspect ratio >30) to demonstrate the directional stability of droplet ejection and its potential as a suitable tool for direct prototyping of metal microstructures.
• Arne Kloke, Christian Köhler, Roland Zengerle, Sven Kerzenmacher
  Porous Platinum Electrodes Fabricated by Cyclic Electrodeposition of PtCu Alloy: Application to Implantable Glucose Fuel Cells
  2012 J Phys Chem C, volume: 116, pages: 19689 - 19698
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  Abstract

  We demonstrate the application of cyclic electrodeposition of PtCu alloy to fabricate porous platinum electrodes for implantable glucose fuel cells. Depending on the number of deposition cycles, electrodes with controllable specific surface area can be fabricated, their roughness factors ranging from 20 (slightly roughened) to 3100 (highly porous). Adjusting the specific surface area of the electrode from low to high determines whether it functions as cathode or anode, respectively. Compared to state of the art, this process is beneficial due to shorter fabrication times, lower temperatures, and the requirement of only one process for the fabrication of both electrodes. Correspondingly fabricated glucose fuel cells showed a power density of 5.1 μW cm−2 under close to physiological conditions, which is an improvement by 16% compared to earlier designs. During continuous operation over 90 days the fuel cell showed a mean continuous decay of about 0.8% per day, which is related to catalyst poisoning at the anode. Future work will thus have to focus on the improvement of long-term stability instead of power density. ■ INTRODUCTION
• S. Rubenwolf, S. Sané, L. Hussein, J. Kestel, F. von Stetten, G. Urban, M. Krueger, R. Zengerle, S. Kerzenmacher
  Prolongation of electrode lifetime in biofuel cells by periodic enzyme renewal
  2012 Appl Microbiol Biotechnol, volume: 96, pages: 841 - 849
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  Abstract

  Abstract Enzymatically catalyzed biofuel cells show unique specificity and promise high power densities, but suffer from a limited lifetime due to enzyme deactivation. In the present work, we demonstrate a novel concept to extend the lifetime of a laccase-catalyzed oxygen reduction cathode in which we decouple the electrode lifetime from the limited enzyme lifetime by a regular resupply of fresh enzymes. Thereto, the adsorption behavior of laccase from Trametes versicolor to buckypaper electrode material, as well as its time-dependent deactivation characteristics, has been investigated. Laccase shows a Langmuir-type adsorption to the carbon nanotube-based buckypaper electrodes, with a mean residence time of 2 days per molecule. In a citrate buffer of pH 5, laccase does not show any deactivation at room temperature for 2 days and exhibits a half-life of 9 days. In a long-term experiment, the laccase electrodes were operated at a constant galvanostatic load. The laccase-containing catholyte was periodically exchanged against a freshly prepared one every second day to provide sufficient active enzymes in the catholyte for the replacement of desorbed inactive enzymes. Compared to a corresponding control experiment without catholyte exchange, this procedure resulted in a 2.5 times longer cathode lifetime of 19±9 days in which the electrode showed a potential above 0.744 V vs. normal hydrogen electrode at 110 μAcm−2. This clearly indicates the successful exchange of molecules by desorption and re-adsorption and is a first step toward the realization of a selfregenerating enzymatic biofuel cell in which enzyme-producing microorganisms are integrated into the electrode to continuously resupply fresh enzymes.
• Jochen Rupp, Manuela Schmidt, Susanne Münch, Markus Cavalar, Ulf Steller, Jürgen Steigert, Michael Stumber, Christian Dorrer, Peter Rothacher, Roland Zengerle, Martina Daub
  Rapid microarray processing using a disposable hybridization chamber with an integrated micropump
  2012 Lab Chip, volume: 12, pages: 1384 - 1388
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  Abstract

  We present a disposable microarray hybridization chamber with an integrated micropump to speed up diffusion based reaction kinetics by generating convective flow. The time-to-result for the hybridization reaction was reduced from 60 min (standard protocol) down to 15 min for a commercially available microarray. The integrated displacement micropump is pneumatically actuated. It includes two active microvalves and is designed for low-cost, high volume manufacturing. The setup is made out of two microstructured polymer parts realized in polycarbonate (PC) separated by a 25 mm thermoplastic elastomer (TPE) membrane. Pump rate can be controlled between 0.3 µl/s and 5.7 µl/s at actuation frequencies between 0.2 Hz and 8.0 Hz, respectively.
• Jochen Hoffmann, Martin Trotter, Felix von Stetten, Roland Zengerle, Günter Roth
  Solid-phase PCR in a picowell array for immobilizing and arraying 100,000 PCR products to a microscope slide
  2012 Lab Chip, volume: 12, issue: 17, pages: 3049 - 3054
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  Abstract

  We present a method for performing highly parallel PCR reactions in a picowell array (PWA) simultaneously immobilizing generated PCR products covalent and spatially-resolved onto a microscope slide via solid-phase PCR (SP-PCR). This so called PWA-SP-PCR is performed in picowell arrays featuring 100,000 wells∙cm^-2 of 19 pL reaction volumes with a surface-to-volume-ratio of 0.2 µm-1. Positive signals are obtained in 97.2 % of the 110,000 wells on an area of 110 mm^2. Immobilized DNA is either detected sequence-unspecific via streptavidin-Cy5 or sequence-specific by Cy3 labeled hybridization probes. Amplification and immobilization is demonstrated for template DNA ranging from 100 bp up to 1513 bp length. Compared to widely established emulsion based PCR (emPCR) approaches, leading to PCR products immobilized onto bead surfaces in highly parallel manner, the novel technique results in direct spatial registration of immobilized PCR products in a microarray format. This enables the subsequent use for massively parallel analysis similar to standard microarrays.
• H. Hoefemann, S. Wadle, N. Bakhtina, V. Kondrashov, N. Wangler, R. Zengerle
  Sorting and lysis of single cells by BubbleJet technology
  2012 Sensors and Actuators B, volume: 168, pages: 442 - 445
• S Spieth, A Schumacher, C Kallenbach, S Messner, R Zengerle
  The NeuroMedicator—a micropump integrated with silicon microprobes for drug delivery in neural research
  2012 J Micromech Microeng, volume: 22, page: 065020
• T. Hutzenlaub, S. Thiele, R. Zengerle, C. Ziegler
  Three-Dimensional Reconstruction of a LiCoO2 Li-Ion Battery Cathode
  2012 Electrochem Solid St, volume: 15, issue: 3, pages: A33 - A36
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  Abstract

  In the reported work, we reconstruct a LiCoO2 cathode three-dimensionally, for the first time differentiating between all three constituents: (i) active material, (ii) binder and (iii) pore space for this specific material. We apply a hybrid method of manual and grey-scale threshold segmentation to reconstruct a cuboid with a volume of approximately 4500 µm3. The reconstructed geometry is characterised to support the data basis of homogenized cathode models. We solve numerically for electrical conductivity and derive electrical tortuosity analytically from the result. Pore space connectivity and pore size distribution are also calculated. The segmented images are provided as supplementary electronic material.
• A. Gulliksen, H. Keegan, C. Martin, J. O’Leary, L. A. Solli, I. M. Falang, P. Grønn, A. Karlg°ard, M. M. Mielnik, Ib-R.Johansen, Terje R. Tofteberg, T. Baier, R. Gransee, K. Drese, T. Hansen-Hagge, L. Riegger, P. Koltay, R. Zengerle, F. Karlsen, D. Ausen, , , Liv Furuberg2, 4
  Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7mRNAModel System
  2012 Journal of Oncology, volume: 2012, page: ID 905024
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  Abstract

  The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.
• Jochen Hoffmann, Sebastian Hin, Felix von Stetten, Roland Zengerle, Günter Roth
  Universal protocol for grafting PCR primers onto various lab-on-a-chip substrates for solid-phase PCR
  2012 RSC Advances, volume: 2, issue: 9, pages: 3885 - 3889
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  Abstract

  A universal protocol for grafting PCR primers onto glass, PDMS, COP, COC, and PP is developed and evaluated by solid-phase PCR (SP-PCR). Primers are immobilized in a PCR compatible way featuring spots with high homogeneity and integrity. Furthermore, we show a protocol for binding a PCR product to immobilized PCR primers via solid-phase PCR (SP-PCR). Previously reported ‘‘enhanced SP-PCR’’ (Z. Kahn et al. Anal. Biochem., 2008, 375, 391–393) is improved in terms of factorial signal increase from 9.9 to 86.8 and specificity from 11.7 to 45.9. The presented immobilization- and SP-PCR protocols may enable integration of DNA microarrays directly into microfluidic lab-on-a-chip cartridges of various materials for analysis via SP-PCR. Beside planar microarrays, another interesting application could be to coat the inner surfaces of a chip with PCR primers to recover generated PCR products in digital PCR systems.

2011

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• A. Kloke, B. Biller, U. Kräling, S. Kerzenmacher, R. Zengerle, F. von Stetten
  A Single Layer Glucose Fuel Cell Intended as Power Supplying Coating for Medical Implants
  2011 Fuel Cells, volume: 11, issue: 2, pages: 316 - 326
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  Abstract

  We present a novel type of abiotically catalysed implantable glucose fuel cell with anode and cathode placed side by side,using a Raney-platinum glucose oxidation anode with high tolerance towards oxygen. In contrast to conventional assembly designs used for implantable glucose fuel cells, no permeable cathode mounted in front of the anode to effect oxygen depletion is required. At 2.2 ± 0.1 lW cm–2 the single layer fuel cell exhibits only half the maximum power density of the conventional fuel cell, which solely stems from the doubled total fuel cell area demand. Nevertheless, the novel single layer design is advantageous in terms of simplified fabrication and reduced overall thickness, facilitating implementation of the fuel cell as a power supplying coating directly on the surface of medical implants. Furthermore,the single layer design offers an attractive possibility to diminish the reduction of power density by limited oxygen mass transfer to the cathode by increasing the cathode to anode area proportion. With doubled cathode area proportion a by 36% higher power density can be reached. To calculate the optimum cathode to anode area proportions,we introduced an analytical model based on the experimentally determined polarisation resistances of the individual electrodes. Keywords: Biomedical Devices, Energy Harvesting, Implantable Glucose Fuel Cell, Oxygen Tolerant Platinum Electrodes, Power Supply
• S Spieth, O Brett, K Seidl, A A A Aarts, M A Erismis, S Herwik, F Trenkle, S Tätzner, J Auber, M Daub, H P Neves, R Puers, O Paul, P Ruther, R Zengerle
  A floating 3D silicon microprobe array for neural drug delivery compatible with electrical recording
  2011 J Micromech Microeng, volume: 21, pages: 125001 - (16pp)
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  Abstract

  This paper reports on the design, fabrication, assembly, and characterization of a three-dimensional (3D) silicon-based floating microprobe array for localized drug delivery to be applied in neuroscience research. The microprobe array is composed of a silicon platform into which up to four silicon probe combs with needle-like probe shafts can be inserted. Two dedicated positions in the array allow the integration of combs for drug delivery. The implemented comb variants feature 8-mm-long probe shafts with two individually addressable microchannels incorporated in one single shaft or distributed to two shafts. Liquid supply to the array is realized by a highly flexible 250-µm-thick multi-lumen microfluidic cable made from polydimethylsiloxane (PDMS). The specific design concept of the slim-base platform enables floating implantation of the array in the small space between brain and skull. In turn, the flexible cable mechanically decouples the array from any microfluidic interface rigidly fixed to the skull. After assembly of the array, full functionality is demonstrated and characterized at infusion rates from 1 to 5 µL/min. Further, the effect of a parylene-C coating on the water vapour and osmotic liquid water transport through the PDMS cable walls is experimentally evaluated by determining the respective transmission rates including the water vapour permeability of the used PDMS type.
• L. Hussein, S. Rubenwolf, F. von Stetten, G. Urban, R. Zengerle, M. Krueger, S. Kerzenmacher
  A highly efficient buckypaper-based electrode material for mediatorless laccase-catalyzed dioxygen reduction
  2011 Biosens Bioelectron, volume: 26, pages: 4133 - 4138
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  Abstract

  The redox enzyme laccase from Trametes versicolor efficiently catalyzes the oxygen reduction reaction (ORR) in mediatorless biofuel cell cathodes when adsorbed onto multi-walled carbon nanotubes (MWCNTs). In this work we demonstrate that the fabrication of MWCNTs in form of buckypaper (BP) results in an excellent electrode material for laccase-catalyzed cathodes. BPs are mechanically stable, self-entangling mats with high dispersion of MWCNTs resulting in easy to handle homogeneous layers with highly mesoporous structures and excellent electrical conductivities. All biocathodes have been electrochemically investigated in oxygen-saturated buffer at pH 5 by galvanostatic polarization and potentiodynamic linear sweep voltammetry. Both methods confirm an efficient direct interaction of laccase with BP with a high open circuit potential of 0.882V vs. normal hydrogen electrode (NHE). The high oxygen reduction performance leads to high current densities of 422±71 µAcm−2 at a typical cathode potential of 0.744V vs. NHE. When the current density is normalized to the mass of the electrode material (mass activity), the BPbased film electrodes exhibit a 68-fold higher current density at 0.744Vvs.NHEthan electrodes fabricated from the same MWCNTs in a non-dispersed agglomerated form as packed electrodes. This clearly shows that MWCNTs can act more efficiently as cathode when prepared in form of BP. This can be attributed to reduced diffusional mass transfer limitations and enhanced electrical conductivity. BP is thus a very promising material for the construction of mediatorless laccase cathodes for ORR in biofuel cells. In addition we demonstrated that these electrodes exhibit a high tolerance towards glucose, the most common bioanode fuel.
• S. Kerzenmacher, U. Kräling, T. Metz, R. Zengerle, F. von Stetten
  A potentially implantable glucose fuel cell with Raney-platinum film electrodes for improved hydrolytic and oxidative stability
  2011 J Power Sources, volume: 196, pages: 1264 - 1272
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  Abstract

  We present an improved abiotically catalyzed glucose fuel cell, intended as energy harvesting tissue implantable power supply for medical implants. The fuel cell is constructed from a Raney-platinum film cathode deposited on a silicon substrate with micro-machined feedholes for glucose permeability, arranged in front of a Raney-platinum film anode. A novelty is the application of platinum for both electrodes and the complete abdication of hydrogel binders. This overcomes the limited stability against hydrolytic and oxidative attack encountered with previous glucose fuel cells fabricated from activated carbon particles dispersed in a hydrogel matrix. During performance characterization in phosphate buffered saline under physiological concentrations of glucose and oxygen the diffusion resistance to be expected from tissue capsule formation was taken into account. Despite the resulting limited oxygen supply, the Raney-platinum fuel cells exhibit a power density of up to (4.4±0.2)µWcm−2 at 7.0% oxygen saturation. This exceeds the performance of our previous carbon-based prototypes, and can be attributed to the higher catalytic activity of platinum cathodes and in particular the increased oxygen tolerance of the Raney-platinum film anodes.
• Michael Vosseler, Michael Jugl, Roland Zengerle
  A smart interface for reliable intradermal injection and infusion of high and low viscosity solutions
  2011 Pharm Res-dordr, volume: 28, pages: 647 - 661
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  Abstract

  Purpose: We present a smart intradermal interface suitable for skin attached drug delivery devices. Our solution enables injections or infusions that are less invasive compared to subcutaneous injections and is leakage-free at the location of penetration. Methods: The intradermal interface is based on a 31 gauge cannula embedded in a slider, movable relative to a carrier plate that can easily be fixed onto the skin. By simply pushing the slider the cannula is inserted into the dermis. Results: We performed injections and infusions with stained water and polyethylene glycol (PEG) solution in ex vivo pig skin. The sizes of coloured spots in the skin range from 3.5 mm² to 15.4 mm² for stained water depending on the infused volume. Infusing stained PEG solution resulted in stained tissue areas about one order of magnitude larger. One of three investigated leakage modes is unacceptable but can be reliably avoided by proper site selection. At low flow rates and at the beginning of an infusion an initial back pressure overshoot was identified. This effect was identified as the limiting parameter for the design of small programmable or intelligent devices based on micro actuators. Conclusions: With the proposed easy-to-use interface, intradermal injections and infusions can be performed reliably. Therefore, it is supposed to be an ideal and clinically relevant solution for self-administration of parenteral drugs in home care applications.
• Daniel Mark, Patrick Weber, Sascha Lutz, Maximilian Focke, Roland Zengerle, Felix von Stetten
  Aliquoting on the centrifugal microfluidic platform based on centrifugo-pneumatic valves
  2011 Microfluid Nanofluid, volume: 10, pages: 1279 - 1288
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  Abstract

  We present a new method for aliquoting liquids on the centrifugal microfluidic platform. Aliquoting is an essential unit operation to perform multiple parallel assays (‘‘geometric multiplexing’’) from one individual sample, such as genotyping by real-time polymerase chain reactions (PCR), or homogeneous immunoassay panels. Our method is a two-stage process with an initial metering phase and a subsequent transport phase initiated by switching a centrifugo- pneumatic valve. The method enables aliquoting liquids into completely separated reaction cavities. It includes precise metering that is independent on the volume of prestored reagents in the receiving cavities. It further excludes any cross-contamination between the receiving cavities. We characterized the performance for prototypes fabricated by three different technologies: micro-milling, thermoforming of foils, and injection molding. An initial volume of*90 ll was split into 8 aliquots of 10 ll volume each plus a waste reservoir on a thermoformed foil disk resulting in a coefficient of variation (CV) of the metered volumes of 3.6%. A similar volume of*105 ll was split into 16 aliquots of 6 ll volume each on micro-milled and injection-molded disks and the corresponding CVs were 2.8 and 2.2%, respectively. Thus, the compatibility of the novel aliquoting structure to the aforementioned prototyping and production technologies is demonstrated. Additionally, the important question of achievable volume precision of the aliquoting structure with respect to the production tolerances inherent to each of these production technologies is addressed experimentally and theoretically. The new method is amenable to low cost mass production, since it does not require any post-replication surface modifications like hydrophobic patches.
• O Frey, P D van derWal, S Spieth, O Brett, K Seidl, O Paul, P Ruther, R Zengerle, N F de Rooij
  Biosensor microprobes with integrated microfluidic channels for bi-directional neurochemical interaction
  2011 J Neural Eng, volume: 8, page: 066001
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  Abstract

  This paper reports on silicon-based microprobes, 8 mm long and 250 μm × 250 μm cross-section, comprising four recessed biosensor microelectrodes (50 μm × 150 μm) per probe shank coated with an enzymatic layer for the selective detection of choline at multiple sites in brain tissue. Integrated in the same probe shank are up to two microfluidic channels for controlled local liquid delivery at a defined distance from the biosensor microelectrodes. State-of-the-art silicon micromachining processing was applied for reproducible fabrication of these experiment-tailored multi-functional probe arrays. Reliable electric and fluidic interconnections to the microprobes are guaranteed by a custom-made holder. The reversible packaging method implemented in this holder significantly reduces cost and assembly time and simplifies storage of the biosensor probes between consecutive experiments. The functionalization of the electrodes is carried out using electrochemically aided adsorption. This spatially controlled deposition technique enables a parallel deposition of membranes and is especially useful when working with microelectrode arrays. The achieved biosensors show adequate characteristics to detect choline in physiologically relevant concentrations at sufficient temporal and spatial resolution for brain research. Sensitivity to choline better than 10 pA μM−1, detection limit below 1 μM and response time of 2 s were obtained. The proposed combination of biosensors and microfluidic injectors on the same microprobe allows simultaneous chemical stimulation and recording as demonstrated in an agarose gel-based brain phantom.
• Marc Karle, Johannes Wöhrle, Junichi Miwa, Nils Paust, Günter Roth, Roland Zengerle, Felix von Stetten
  Controlled counter-flow motion of magnetic bead chains rolling along microchannels
  2011 Microfluid Nanofluid, volume: 10, issue: 4, pages: 935 - 939
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  Abstract

  We demonstrate controlled transport of superparamagnetic beads in the opposite direction of a laminar flow. A permanent magnet assembles 200 nm magnetic particles into about 200 lm long bead chains that are aligned in parallel to the magnetic field lines. Due to a magnetic field gradient, the bead chains are attracted towards the wall of a microfluidic channel. A rotation of the permanent magnet results in a rotation of the bead chains in the opposite direction to the magnet. Due to friction on the surface, the bead chains roll along the channel wall, even in counter-flow direction, up to at a maximum counter-flow velocity of 8 mm s(-1). Based on this approach, magnetic beads can be accurately manoeuvred within microfluidic channels. This counter-flow motion can be efficiently be used in Lab-on-a-Chip systems, e.g. for implementing washing steps in DNA purification.
• Mitsakakis K, Gizeli E
  Detection of multiple cardiac markers with an integrated acoustic platform for cardiovascular risk assessment
  2011 Anal Chim Acta, volume: 699, pages: 1 - 5
• Christoph Ziegler, Simon Thiele, Roland Zengerle
  Direct three-dimensional reconstruction of a nanoporous catalyst layer for a polymer electrolyte fuel cell
  2011 J Power Sources, volume: 196, issue: 4, pages: 2094 - 2097
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  Abstract

  The direct three-dimensional reconstruction of a polymer electrolyte fuel cell cathode catalyst layer from focused ion beam/scanning electron microscope (FIB/SEM) images is presented. The carbon and pore distribution is shown and quantitatively analysed. A new catalyst layer sample (Fumapem-F950/HiSpec13100) is sliced with FIB and a series of SEM images is taken. The images are registered, segmented and a three-dimensional stack is reconstructed. The three-dimensional carbon and pore distribution is shown. Based on the reconstruction the pore size distribution is evaluated. The total porosity and the unconnected pores space is analysed. The fully segmented 2D images are provided as supplemental material to this paper for future analysis and modeling work. Keywords Polymer electrolyte fuel cell; Porous catalyst layer; Three-dimensional reconstruction
• N Wangler, L Gutzweiler, K Kalkandjiev, C Müller, F Mayenfels, H Reinecke, R Zengerle, N Paust
  High-resolution permanent photoresist laminate TMMF for sealed microfluidic structures in biological applications
  2011 J Micromech Microeng, volume: 21, page: 095009
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  Abstract

  We demonstrate the use of photosensitive epoxy laminate TMMF S2045 for the fabrication and sealing of tapered microfluidic channels. The 45 μm thick resist enables the fabrication of shallow sealed cavities featuring extreme aspect ratios of less than 1:40 (h = 45 μm, w = 2000 μm). It also provides high resolution and enables minimum feature sizes of 10 μm. For the fabrication of free-standing structures, an aspect ratio of up to 7:1 was achieved. The dry-film photoresist can be applied easily by lamination onto structured substrates. The total thickness variation of the resist across a 100 mm wafer was determined to be less than ±0.6 μm. Process parameters for the fabrication and sealing of various micro-channels are discussed and optimized in this paper. The main focus was to minimize thermal impact during lamination, soft-bake, exposure and post–exposure bake, which could lead to lid sagging or channel clogging due to liquefaction of uncured resist. We tested TMMF according to ISO 10995-5 and found it to be non-cytotoxic, enabling its use for biological applications. Swelling of less than 5% for incubation of the dry-film resist in several biologically relevant solvents, buffers and cleaning solutions was observed.
• Azmi Yusof, Helen Keegan, Cathy D. Spillane, Orla M. Sheils, Cara M. Martin, John J. O’Leary, Roland Zengerle, Peter Koltay
  Inkjet-like printing of single-cells
  2011 Lab Chip, volume: 11, pages: 2447 - 2454
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  Abstract

  Cell sorting and separation techniques are essential tools for cell biology research and for many diagnostic and therapeutic applications. For many of these applications, it is imperative that heterogeneous populations of cells are segregated according to their cell type and that individual cells can be isolated and analysed. We present a novel technique to isolate single cells encapsulated in a picolitre sized droplet that are then deposited by inkjet-like printing at defined locations for downstream genomic analysis. The single-cell-manipulator (SCM) developed for this purpose consists of a dispenser chip to print cells contained in a free flying droplet, a computer vision system to detect single-cells inside the dispenser chip prior to printing, and appropriate automation equipment to print single-cells onto defined locations on a substrate. This technique is spatially dynamic, enabling cell printing on a wide range of commonly used substrates such as microscope slides, membranes and microtiter plates. Demonstration experiments performed using the SCM resulted in a printing efficiency of 87% for polystyrene microbeads of 10 mm size. When the SCM was applied to a cervical cancer cell line (HeLa), a printing efficiency of 87% was observed and a post-SCM cell viability rate of 75% was achieved.
• M. Pospischil, K. Zengerle, J. Specht, G. Birkle, P. Koltay, R. Zengerle, A. Henning, M. Neidert, C. Mohr, F. Clement, D. Biro
  Investigations on Thick-Film-Paste Rheology for Dispensing Applications
  2011 Energy Procedia, volume: 8, pages: 449 - 454
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  Abstract

  In order to establish dispensing as a promising metallization process in silicon photovoltaics, equipment and metal pastes require further optimization. By conducting several rheological experiments based on rotational tests, shear thinning as well as thixotropic behavior of thick-film pastes were investigated. Both are crucial parameters for continuous dispensing of 60 μm fingers with high aspect ratios. Flow rate fluctuations during dispensing though imply stress peaks that may disturb a continuous paste flow. Thus, a comparison of the flow rate of two pastes was conducted. A comparison of dispensed cells with screen-printed reference cells, on multi-crystalline wafer-material, showed an efficiency increase of 0.3%abs. on average. This is mainly caused by reduced finger widths and higher aspect ratios of dispensed fingers.
• K Kalkandjiev, L Riegger, D Kosse, M Welsche, L Gutzweiler, R Zengerle, P Koltay
  Microfluidics in silicon/polymer technology as a cost-efficient alternative to silicon/glas
  2011 J Micromech Microeng, volume: 21, page: 025008 (8p
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  Abstract

  We investigate TMMF photopolymer as a cost efficient alternative to glass for the liquid tight sealing of high density silicon microchannels. TMMF enables low temperature sealing and access to structures underneath via lamination and standard UV lithography instead of costly glass machining and anodic bonding. TMMF is highly transparent and has a low autofluorescence for wavelengths larger than 400 nm. As the photopolymer is too thin for implementing bulky World-to-Chip-interfaces, we propose adhesive bonding of COC modules. All materials were tested according ISO 10993-5 and showed no cytotoxic effects on the proliferation of L929 cells. To quantify the cost efficiency of the proposed techniques, we used an established Si/Pyrex nanoliter dispenser as a reference and replaced structured Pyrex wafers by TMMF laminates and COC modules. Thus, consumable costs and time effort were reduced by 90 % respectively 35 % for the sealing and 80 % respectively 75 % for implementing the World-to-Chip interface. Liquid tightness was proved by applying a pressure of 0,2 MPa for 5 h without delamination or cross talk between neighbouring microchannels separated from each other by 100 µm. In contrast to anodic bonding, the proposed techniques are tolerant to surface inhomogenities. They enable manufacturing of silicon/polymer microfluidics at significantly lower costs and without compromising the performance compared to corresponding silicon/glass devices.
• M. Focke, D. Kosse, D. Al-Bamerni, S. Lutz, C. Mueller, H. Reinecke, R. Zengerle, F. von Stetten
  Microthermoforming of microfluidic substrates by soft lithography (µTSL): optimization using design of experiments
  2011 J Micromech Microeng, volume: 21, issue: 11, pages: 115001 - 115012
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  Abstract

  We present a detailed analysis of microthermoforming by soft lithography (µTSL) for replication of foil-based microfluidic substrates. The process was systematically optimized by design of experiments (DOE) enabling fabrication of defect-free lab-on-a-chip devices. After the assessment of typical error patterns we optimized the process toward the minimum deviation between mold and thermoformed foil substrates. The following process parameters have most significant impact on the dimensional responses (p < 0.05): critical temperature before start of evacuation, molding temperature, pressure of pre-stretching and duration of pre-stretching as well as duration of molding pressure. The most relevant parameter is molding temperature with >40% relative impact. The DOE results in an empirical process model with a maximum deviation between the prediction and experimental proof of 2% for the optimum parameter set. Finally, process optimization is validated by the fabrication and testing of a microfluidic structure for blood plasma separation from human whole blood. The optimized process enabled metering of a nominal volume of 4.0 mu l of blood plasma with an accuracy deviation of 3% and a metering precision of +/- 7.0%. The µTSL process takes about 30 min and easily enables the replication of 300 mu m wide microchannels having vertical sidewalls without any draft angles in a well-controllable way. It proves to be suitable for multiple applications in the field of microfluidic devices.
• Mitsakakis K, Gizeli E
  Multi-sample acoustic biosensing microsystem for protein interaction analysis
  2011 Biosens Bioelectron, volume: 26, pages: 4579 - 4584
• Simon Thiele, Roland Zengerle, Christoph Ziegler
  Nano-Morphology of a Polymer Electrolyte Fuel Cell Catalyst Layer—Imaging, Reconstruction and Analysis
  2011 Nano Res, volume: 4, issue: 9, pages: 849 - 860
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  Abstract

  The oxygen reduction reaction (ORR) in the cathode catalyst layer (CCL) of polymer electrolyte fuel cells (PEFC) is one of the major causes of performance loss during operation. In addition, the CCL is the most expensive component due to the use of a Pt catalyst. Apart from the ORR itself, the species transport to and from the reactive sites determines the performance of the PEFC. The effective transport properties of the species in the CCL depend on its nanostructure. Therefore a three-dimensional reconstruction of the CCL is required. A series of two-dimensional images was obtained from focused ion beam - scanning electron microscope (FIB-SEM) imaging and a segmentation method for the two-dimensional images has been developed. The pore size distribution (PSD) was calculated for the three-dimensional geometry. The influence of the alignment and the anisotropic pixel size on the PSD has been investigated. Pores were found in the range between 5 nm and 205 nm. Evaluation of the Knudsen number showed that gas transport in the CCL is governed by the transition flow regime. The liquid water transport can be described within continuum hydrodynamics by including suitable slip flow boundary conditions.
• Ernst, Andreas, Ju, Lin, Vondenbusch, Bernhard, Zengerle, Roland, Koltay, Peter
  Noncontact Determination of Velocity and Volume of Nanoliter Droplets on the Fly
  2011 Ieee Sens J, volume: 11, issue: 8, pages: 1736 - 1742
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  Abstract

  We present a sensor for measuring volume and velocity of dispensed nanoliter droplets in a noncontact manner on the fly. The sensor-setup has a total thickness of 3.2 mm and can easily be mounted underneath any given nanoliter dispenser for continuous online monitoring of its dispensing performance. The principle is based on the interaction of dispensed single droplets of sample liquid passing the electric field of an open plate capacitor. The effect depends on droplet parameters like volume, velocity, and dielectric constant and is discussed in the paper. The presented data analysis enables a velocity independent volume determination of water droplets in the range from 26 to 82 nl with an accuracy of 3 nl. The sensor signal is sensitive to the alignment of the flight path of the nanoliter droplets within the 1.2-mm wide open capacitor and can lead to systematic volume errors of up to Delta V approx. 12 nl. The impact of different dielectric constants can only be differentiated for very high variations; thus, the sensor needs to be calibrated to the different types of liquids.
• F. Trenkle, S. Haeberle, R. Zengerle
  Normally-closed peristaltic micropump with re-usable actuator and disposable fluidic chip
  2011 Sensor Actuat B-chem, volume: 154, issue: 2, pages: 137 - 141
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  Abstract

  We present a new peristaltic micropump offering three key features: (i) a disposable pump body and a reuseable actuator unit, (ii) an intrinsic normally-closed mechanism blocking unintended liquid flows up to a pressure of 100 kPa and (iii) a backpressure independent pump performance up to 40 kPa. The modular concept basing on a re-usable actuator unit and a low-cost disposable microfluidic chip enables an easy and cost-efficient exchange of all contaminated parts after use, which addresses especially the needs in the health care sector. The intrinsic normally-closed feature blocks liquid flow in both directions up to a pressure difference of 100 kPa when the electric power is off. The micropump is actuated in a peristaltic manner by three piezostack actuators. Up to a frequency of 15 Hz the pump rate increases linearly with operation frequency leading to a pump rate of 120 µL/min. This was proved for an operation voltage of 140V by pumping water. In addition the pump rate is independent on backpressure up to 40 kPa and shows a linear decrease for higher pressure differences. The maximum achievable backpressure at zero flow rate was extrapolated to be 180 kPa. As for all peristaltic micropumps, the pump is bidirectional, e.g. the pump direction can be changed forward to reverse mode.
• Stefanie Rubenwolf, Sven Kerzenmacher, Roland Zengerle, Felix von Stetten
  Strategies to extend the lifetime of bioelectrochemical enzyme electrodes for biosensing and biofuel cell applications
  2011 Appl Microbiol Biot, volume: 89, issue: 5, pages: 1315 - 1322
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  Abstract

  Enzymes are powerful catalysts for biosensor and biofuel cell electrodes due to their unique substrate specificity. This specificity is defined by the amino acid chain's complex three-dimensional structure based on noncovalent forces, being also responsible for the very limited enzyme lifetime of days to weeks. Many electrochemical applications, however, would benefit from lifetimes over months to years. This mini-review provides a critical overview of strategies and ideas dealing with the problem of short enzyme lifetime, which limits the overall lifetime of bioelectrochemical electrodes. The most common approaches aim to stabilize the enzyme itself. Various immobilization techniques have been used to reduce flexibility of the amino acid chain by introducing covalent or non-covalent binding forces to external molecules. The enzyme can also be stabilized using genetic engineering methods to increase the binding forces within the protein or by optimizing the environment in order to reduce destabilizing interactions. In contrast, renewing the inactivated catalyst decouples overall system lifetime from the limited enzyme lifetime and thereby promises theoretically unlimited electrode lifetimes. Active catalyst can be supplied by exchanging the electrolyte repeatedly. Alternatively, integrated microorganisms can display the enzymes on their surface or secrete them to the electrolyte, allowing unattended power supply for long-term applications. Keywords Enzyme inactivation . Biofuel cell . Biosensor . Amino acid replacement . Immobilization . Self-regeneration
• G. Grossmann, Woei-Jiun Guo, D. W. Ehrhardt, W. B. Frommer, R. V. Sit, S. R. Quake, M. Meier
  The RootChip: An Integrated Microfluidic Chip for Plant Science
  2011 The Plant Cell, volume: 23, pages: 4234 - 4240
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  Abstract

  Studying development and physiology of growing roots is challenging due to limitations regarding cellular and subcellular analysis under controlled environmental conditions. We describe a microfluidic chip platform, called RootChip, that integrates live-cell imaging of growth and metabolism of Arabidopsis thaliana roots with rapid modulation of environmental conditions. The RootChip has separate chambers for individual regulation of the microenvironment of multiple roots from multiple seedlings in parallel. We demonstrate the utility of The RootChip by monitoring time-resolved growth and cytosolic sugar levels at subcellular resolution in plants by a genetically encoded fluorescence sensor for glucose and galactose. The RootChip can be modified for use with roots from other plant species by adapting the chamber geometry and facilitates the systematic analysis of root growth and metabolism from multiple seedlings, paving the way for large-scale phenotyping of root metabolism and signaling.
• T. Hutzenlaub, N. Paust, R. Zengerle, C. Ziegler
  The effect of wetting properties on bubble dynamics and fuel distribution in the flow field of direct methanol fuel cells
  2011 J Power Sources, volume: 196, pages: 8048 - 8056
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  Abstract

  We investigate CO2 bubble dynamics on the anode side of a direct methanol fuel cell (DMFC). In contrast to previous studies, we analyse the effect of both channel wall and diffusion layer wettability by observing two-phase flow from the side at different mean velocities of the fuel supply. Hydrophobic and hydrophilic flow channel surfaces are compared experimentally. The hydrophilic flow channel leads to a minimum pressure drop along the channel. Bubbles show virtually no pinning and consequently travel at approximately the mean fuel velocity inside the channel. In contrast to this, we observe bubble pinning in the hydrophobic flow channels. The critical fuel velocities necessary for detachment of the bubbles mainly depends on bubble length. We identify and describe a new bubble bypass configuration where fuel bypass channels are solely generated in a favourable position underneath a blocking bubble along the diffusion layer. This enforces fuel to bypass the CO2 bubble at a large relative velocity close to the diffusion layer, thus enhancing mass transfer. Our experimental findings are in excellent agreement with a CFD/analytical model. This model allows for quantitative prediction of average bypass flow velocity.

2010

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• A. Kloke, S. Rubenwolf, C. Bücking, J. Gescher, S. Kerzenmacher, R. Zengerle, F. von Stetten
  A versatile miniature bioreactor and its application to bioelectrochemistry studies
  2010 Biosens Bioelectron, volume: 25, pages: 2559 - 2565
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  Abstract

  Often, reproducible investigations on bio-microsystems essentially require a flexible but well-defined experimental setup, which in its features corresponds to a bioreactor. We therefore developed a miniature bioreactor with a volume in the range of a few millilitre that is assembled by alternate stacking of individual polycarbonate elements and silicone gaskets. All the necessary supply pipes are incorporated as bore holes or cavities within the individual elements. Their combination allows for a bioreactor assembly that is easily adaptable in size and functionality to experimental demands. It allows for controlling oxygen transfer as well as the monitoring of dissolved oxygen concentration and pH-value. The system provides access for media exchange or sterile sampling. A mass transfer coefficient for oxygen (kLa) of 4.3×10−3 s−1 at aflowrate of only 15 ml min−1 and a mixing time of 1.5 s at aflowrate of 11 ml min−1 were observed for the modular bioreactor. Single reactor chambers can be interconnected via ion-conductive membranes to form a two-chamber test setup for investigations on electrochemical systems such as fuel cells or sensors. The versatile applicability of this modular and flexible bioreactor was demonstrated by recording a growth curve of Escherichia coli (including monitoring of pH and oxygen) saturation, and also as by two bioelectrochemical experiments. In the first electrochemical experiment the use of the bioreactor enabled a direct comparison of electrode materials for a laccase-catalyzed oxygen reduction electrode. In a second experiment, the bioreactor was utilized to characterize the influence of outer membrane cytochromes on the performance of Shewanella oneidensis in a microbial fuel cell.
• Lutz Riegger, Oliver Strohmeier, Bernd Faltin, Roland Zengerle, Peter Koltay
  Adhesive bonding of microfluidic chips: influence of process parameters
  2010 J Micromech Microeng, volume: 20, pages: 087003 - (5pp)
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  Abstract

  In this note, the influence of process parameters for adhesive bonding as a versatile approach for the sealing of polymer microfluidic chips is investigated. Specifically, a process chain comprising pre-processing, adhesive transfer as well as post-processing is presented and parameter recommendations are provided. As a device for adhesive transfer, a modified laminator is utilized which transfers thin layers of adhesive onto the chip surface, only via a silicone roll. Using this device and a high temperature (Tg > 100 ◦C) epoxy adhesive, adhesive layers in the range of 2–4 μm can be reproducibly transferred (CV < 4%). For best bonding results, it is recommended to provide 2.5 μm thin layers of adhesive in combination with a subsequent evacuation step at 10 mbar for 3 h. Further, it is proposed to integrate capture channels near large, featureless areas to compensate for variations in processing and thus prevent clogging of channels.
• K. Bürk, M. Wick, G. Roth, P. Decker, R. Voltz
  Antineuronal antibodies in sporadic late-onset cerebellar ataxia
  2010 J Neurol, volume: 257, pages: 59 - 62
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  Abstract

  Sporadic late-onset cerebellar ataxia of unknown cause is considered a neurodegenerative disorder whose underlying mechanisms are still unknown. To identify antineuronal autoantibodies, immunohistochemical and immunoblotting techniques were performed in 67 patients with sporadic cerebellar degeneration of unknown cause. Elevated P/Q-type voltage-gated calcium channel (VGCC)-specific antibodies were found in eight patients (11.9%). There was no hint of a paraneoplastic disorder in any of the patients. The present findings suggest an autoimmune contribution to the pathophysiology of a subgroup of sporadic late-onset cerebellar ataxia.
• Tobias Metz, Nils Paust, Roland Zengerle, Peter Koltay
  Capillary driven movement of gas bubbles in tapered structures
  2010 Microfluid Nanofluid, volume: 9, pages: 341 - 355
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  Abstract

  This article presents a study on the capillary driven movement of gas bubbles confined in tapered channel configurations. These configurations can be used to transport growing gas bubbles in micro fluidic systems in a passive way, i.e. without external actuation. A typical application is the passive degassing of CO2 in micro direct methanol fuel cells (lDMFC). Here, a one-dimensional model for the bubble movement in wide tapered channels is derived and calibrated by experimental observations. The movement of gas bubbles is modelled on straight trajectories based on a balance of forces. The bubble geometry is considered as three dimensional. In the development of the model, the effects of surface tension, inertia, viscosity, dynamic contact angle and thin film deposition are considered. It is found that in addition to viscous dissipation, the dynamics related to the contact line—dynamic contact angle and thin film deposition—are essential to describe the gas bubble’s movement. Nevertheless, it was also found that both of these effects, as modelled within this work, have similar impact and are hard to distinguish. The model was calibrated against experiments in a parameter range relevant for the application of travelling gas bubbles in passive degassing structures for lDMFCs.
• Stefanie Rubenwolf, Oliver Strohmeier, Arne Kloke, Sven Kerzenmacher, Roland Zengerle, Felix von Stetten
  Carbon electrodes for direct electron transfer type laccase cathodes investigated by current density–cathode potential behaviour
  2010 Biosens Bioelectron, volume: 26, pages: 841 - 845
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  Abstract

  Direct electron transfer from carbon electrodes to adsorbed laccase (EC 1.10.3.2) from Trametes versicolor is widely used to enable mediatorless enzymatic biofuel cell cathodes. However, data published so far are poorly comparable in terms of oxygen reduction performance. We thus present a comparative characterization of carbon-based electrode materials as cathode in half-cell configuration, employing adsorbed laccase as oxygen reduction catalyst. Open circuit potentials and performances were significantly increased by laccase adsorption, indicating the occurrence of direct electron transfer. At a potential of 0.5V vs. SCE volume-normalized current densities of approximately 10, 37, 40, 70, and 77 µAcm−3 were measured for cathodes nanotubes, carbon nanofibers and multi-walled carbon nanotubes, respectively. In addition, we could show that both, carbon nanotubes and porous carbon tubes exhibit dramatically lower current densities compared to graphite felt and carbon nanofibers when normalized to BET surface instead of electrode volume. Further work will be required to clarify whether this stems from material-dependent interaction of enzyme and electrode surface or constricted enzyme adsorption due to agglomeration of the nanotubes. In case of the latter, an improved dispersion of the nanotubes upon electrode fabrication may greatly enhance their performance.
• Maximilian Focke, Fabian Stumpf, Günter Roth, Roland Zengerle, Felix von Stetten
  Centrifugal microfluidic system für primary amplification and secondary real-time PCR
  2010 Lab Chip, volume: 10, pages: 3210 - 3212
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  Abstract

  Pre-amplification is a basis for numerous polymerase chain reaction (PCR) protocols but bears severe contamination risks due to handling of high-copy DNA samples. Therefore we developed a selfcontained centrifugal microfluidic system comprising pre-stored reagents; it enables pre-amplification of specific DNA sequences prior to automated aliquoting and real-time PCR in a modified commercial thermocycler.
• Marc Karle, Junichi Miwa, Gregor Czilwik, Volker Auwärter, Günter Roth, Roland Zengerle, Felix von Stetten
  Continuous microfluidic DNA extraction using phase-transfer magnetophoresis
  2010 Lab Chip, volume: 10, pages: 3284 - 3290
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  Abstract

  This paper reports a novel microfluidic-chip based platform using "phase-transfer magnetophoresis" enabling continuous biomolecule processing. As an example we demonstrate for the first time continuous DNA extraction from cell lysate on a microfluidic chip. After mixing bacterial Escherichia coli culture with superparamagnetic bead suspension, lysis and binding buffers, DNA is released from cells and captured by the beads. These DNA carrying beads are continuously transported across the interfaces between co-flowing laminar streams of sample mixture, washing and elution buffer. Bead actuation is achieved by applying a time-varying magnetic field generated by a rotating permanent magnet. Flagella-like chains of magnetic beads are formed and transported along the microfluidic channels by an interplay of fluid drag and periodic magnetic entrapment. The turnover time for DNA extraction was approximately 2 minutes with a sample flow rate of 0.75 µl/s and an eluate flow rate of 0.35 µl/s. DNA recovery was 147% (on average) compared to bead based batch-wise extraction in reference tubes within a dilution series experiment over 7 orders of magnitude. The novel platform is suggested for automation of various magnetic bead based applications that require continuous sample processing, e.g. continuous DNA extraction for flow-through PCR, capture and analysis of cells and continuous immunoassays. Potential applications are seen in the field of biological safety monitoring, bioprocess control, environmental monitoring, or epidemiological studies such as monitoring the load of antibiotic resistant bacteria in waste water from hospitals.
• L. Riegger, M.M. Mielnik, A. Gulliksen, D. Mark, J. Steigert, S. Lutz, M. Clad, R. Zengerle, P. Koltay, J. Hoffmann
  Dye-based coatings for hydrophobic valves and their application to polymer labs-on-a-chip
  2010 J Micromech Microeng, volume: 20, page: 045021
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  Abstract

  We provide a method for the selective surface patterning of microfluidic chips with hydrophobic fluoropolymers which is demonstrated by the fabrication of hydrophobic valves via dispensing. It enables efficient optical quality control for the surface patterning thus permitting the low-cost production of highly reproducible hydrophobic valves. Specifically, different dyes for fluoropolymers enabling visual quality control (QC) are investigated, and two fluoropolymer-solvent-dye solutions based on fluorescent quantum dots (QD) and carbon black (CB) are presented in detail. The latter creates superhydrophobic surfaces on arbitrary substrates, e.g. chips made from cyclic olefin copolymer (COC, water contact angle = 157.9◦), provides good visibility for the visual QC in polymer labs-on-a-chip and increases the burst pressures of the hydrophobic valves. Finally, an application is presented which aims at the on-chip amplification of mRNA based on defined flow control by hydrophobic valves is presented. Here, the optimization based on QC in combination with the Teflon-CB coating improves the burst pressure reproducibility from 14.5% down to 6.1% compared to Teflon-coated valves.
• K Seidl, S Spieth, S Herwik, J Steigert, R Zengerle, O Paul, P Ruther
  In-plane silicon probes for simultaneous neural recording and drug delivery
  2010 J Micromech Microeng, volume: 20, pages: 105006 - (11pp)
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  Abstract

  This paper reports on the design, fabrication and characterization of silicon-based microprobes for simultaneous neural recording and drug delivery. The fabrication technology is based on two-stage deep reactive ion etching combined with silicon wafer bonding and grinding to realize channel structures integrated in needle-like probe shafts. Liquids can be supplied to microfluidic devices via in-plane and out-of-plane ports. The liquid is dispensed at circular out-of-plane ports with a diameter of 25 μm and rectangular in-plane ports with dimensions of 50 × 50 μm2. Two-shaft probes with a pitch between shafts of 1.0 and 1.5 mm were realized. The probe shafts have a length of 8 mm and rectangular cross-sections of w x h (w = 250 μm and h = 200 or 250 μm). Each shaft contains one or two fluidic channels with a cross-section of 50 x 50 μm2. In addition, each probe shaft comprises four recording sites with diameters of 20 μm close to the outlet ports. Mechanical and fluidic characterization demonstrated the functionality of the probes. Typical infusion rates of 1.5 μL min−1 are achieved at a differential pressure of 1 kPa. The Pt-gray electrodes have an average electrode impedance of 260 ± 59 k Ohm at 1 kHz.
• C. Bücking, F. Popp, S. Kerzenmacher, J. Gescher
  Involvement and specificity of Shewanella oneidensis outer membrane cytochromes in the reduction of soluble and solid-phase terminal electronacceptors
  2010 Fems Microbiol Lett, volume: 306, pages: 144 - 151
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  Abstract

  The formation of outer membrane (OM) cytochromes seems to be a key step in the evolution of dissimilatory iron-reducing bacteria. They are believed to be the endpoints of an extended respiratory chain to the surface of the cell that establishes the connection to insoluble electron acceptors such as iron or manganese oxides. The gammaproteobacterium Shewanella oneidensis MR-1 contains the genetic information for five putative OM cytochromes. In this study, the role and specificity of these proteins were investigated. All experiments were conducted using a markerless deletion mutant in all five OM cytochromes that was complemented via the expression of single, plasmid-encoded genes. MtrC and MtrF were shown to be potent reductases of chelated ferric iron, birnessite, and a carbon anode in a microbial fuel cell. OmcA-producing cells were unable to catalyze iron and electrode reduction, although the protein was correctly produced and oriented. However, OmcA production resulted in a higher birnessite reduction rate compared with the mutant. The presence of the decaheme cytochrome SO_2931 as well as the diheme cytochrome SO_1659 did not rescue the phenotype of the deletion mutant.
• Sascha Lutz, Patrick Weber, Max Focke, Bernd Faltin, Jochen Hoffmann, Claas Müller, Daniel Mark, Günter Roth, Peter Munday, Niall Armes, Olaf Piepenburg, Roland Zengerle, Felix von Stetten
  Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA)
  2010 Lab Chip, volume: 10, pages: 887 - 893
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  Abstract

  For the first time we demonstrate a self-sufficient lab-on-a-foil system for the fully automated analysis of nucleic acids which is based on the recently available isothermal recombinase polymerase amplification (RPA). The system consists of a novel, foil-based centrifugal microfluidic cartridge including prestored liquid and dry reagents, and a commercially available centrifugal analyzer for incubation at 37 C and real-time fluorescence detection. The system was characterized with an assay for the detection of the antibiotic resistance gene mecA of Staphylococcus aureus. The limit of detection was <10 copies and time-to-result was <20 min. Microfluidic unit operations comprise storage and release of liquid reagents, reconstitution of lyophilized reagents, aliquoting the sample into #30 independent reaction cavities, and mixing of reagents with the DNA samples. The foil-based cartridge was produced by blow-molding and sealed with a self-adhesive tape. The demonstrated system excels existing PCR based lab-on-a-chip platforms in terms of energy efficiency and time-to-result. Applications are suggested in the field of mobile point-of-care analysis, B-detection, or in combination with continuous monitoring systems.
• M. Focke, F. Stumpf, B. Faltin, P. Reith, D. Bamarni, S. Wadle, C. Müller, H. Reinecke, J. Schrenzel, P. Francois, D. Mark, G. Roth, R. Zengerle, F. von Stetten
  Microstructuring of polymer films for sensitive genotyping by real-time PCR on a centrifugal microfluidic platform
  2010 Lab Chip, volume: 10, pages: 2519 - 2526
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  Abstract

  We present a novel process flow enabling prototyping of microfluidic cartridges made out of polymer films. Its high performance is proven by implementation of a microfluidic genotyping assay testing 22 DNA samples including clinical isolates from patients infected by methicilin-resistant Staphylococcus aureus (MRSA). The microfluidic cartridges (disks) are fabricated by a novel process called microthermoforming by soft lithography (mTSL). Positive moulds are applied allowing for higher moulding precision and very easy demoulding when compared to conventional microthermoforming. High replication accuracies with geometric disk-to-disk variations of less than 1% are typical. We describe and characterise fabrication and application of microfluidic cartridges with wall thicknesses <188 mm thus enabling efficient thermocycling during real-time polymerase chain reaction (PCR). The microfluidic cartridges are designed for operation in a slightly modified commercial thermocycling instrument. This approach demonstrates new opportunities for both microfluidic developments and well-established laboratory instruments. The microfluidic protocol is controlled by centrifugal forces and divides the liquid sample parallely into independent aliquots of 9.8 ml (CV 3.4%, N ¼ 32 wells). The genotyping assays are performed with pre-stored primers and probes for real-time PCR showing a limit of detection well below 10 copies of DNA per reaction well (N ¼ 24 wells in 3 independent disks). The system was evaluated by 44 genotyping assays comprising 22 DNA samples plus duplicates in a total of 11 disks. The samples contained clinical samples of seven different genotypes of MRSA as well as positive and negative controls. The results are in excellent agreement with the reference in microtubes.
• J. Tröndle, A. Ernst, W. Streule, R. Zengerle, P. Koltay
  Non-contact optical sensor to detect free flying droplets in the nanolitre range
  2010 Sensor Actuat A-phys, volume: 158, pages: 254 - 262
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  Abstract

  This paper reports on a non-contact optical sensor for the detection of single droplets in flight. The sensor allows for online process control of non-contact dispensing systems delivering droplets in the nanolitre range. A dispensed liquid droplet, which passes through the optical transducer, leads to a change of the light intensity caused by absorption, reflection and diffraction. The change in light intensity measured by a photo transistor provides information about, e.g. droplet size, velocity and shape. This information is encoded in the time dependent signal shape that yields a characteristic “fingerprint”- signal for each droplet. The fabrication of the sensor is achieved by standard printing circuit board (PCB) technology. Therefore, an easy adaption of the sensor to different kinds of contactless dispensing system and furthermore a very cost efficient production is granted.
• A. Hoff, A.-C. Bagu, T. André, G. Roth, K.-H. Wiesmüller, B. Gückel, R. Brock
  Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells
  2010 Cancer Immunol Immunother, volume: 59, pages: 1379 - 1387
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  Abstract

  The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T cells. Microarrays have enabled an unprecedented miniaturization and parallelization in biological assays. Here, we developed peptide microarrays for the detection of CTL activity. MHC class I-binding peptide epitopes were pipetted onto polymer-coated glass slides. Target cells, loaded with the cell-impermeant dye calcein, were incubated on these arrays, followed by incubation with antigen-expanded CTLs. Cytotoxic activity was detected by release of calcein and detachment of target cells. With only 200,000 cells per microarray, CTLs could be detected at a frequency of 0.5% corresponding to 1,000 antigen-specific T cells. Target cells and CTLs only settled on peptide spots enabling a clear separation of individual epitopes. Even though no physical boundaries were present between the individual spots, peptide loading only occurred locally and cytolytic activity was confined to the spots carrying the specific epitope. The peptide microarrays provide a robust platform that implements the whole process from antigen presentation to the detection of CTL activity in a miniaturized format. The method surpasses all established methods in the minimum numbers of cells required. With antigen uptake occurring on the microarray, further applications are foreseen in the testing of antigen precursors that require uptake and processing prior to presentation.
• Jochen Hoffmann, Daniel Mark, Sascha Lutz, Roland Zengerle, Felix von Stetten
  Pre-storage of liquid reagents in glass ampoules for DNA extraction on a fully integrated lab-on-a-chip cartridge
  2010 Lab Chip, volume: 10, pages: 1480 - 1484
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  Abstract

  Self-containing, ready-to-use cartridges are essential for mobile Lab-on-a-Chip (LoaC) systems intended for Point-of-Care (POC) use. Up to now, a common weak point in many LoaC developments is the need to dispense liquid reagents into the test cartridge before or during processing of the assay. To address this issue we have developed an efficient method for fusing liquid reagents into glass ampoules, which are then sealed into a centrifugally operated cartridge. For on-demand reagent release, the ampoules are disrupted through the flexible lid of the cartridge. Upon centrifugation, 98.7 mL out of 100 mL (CV ¼ 2.5%) of the pre-stored contents are released into the microfluidic system. No liquid loss is observed for ethanol and H2O stored for 300 days at room temperature. Frozen storage is possible without damage to the ampoules. Applicability of this concept is demonstrated by performing a LoaC integrated DNA extraction after 140 days of reagent pre-storage. DNA yield from 32 mL of whole blood was up to 199 ng, which is 77% of an off-chip reference extraction. The presented approach allows the improvement of existing LoaC cartridges where pre-storage of liquid reagents was not implemented yet.
• S. Kerzenmacher, U. Kräling, M. Schroeder, R. Brämer, R. Zengerle, F. von Stetten
  Raney-platinum film electrodes for potentially implantable glucose fuel cells. Part 1: Nickel-free glucose oxidation anodes
  2010 J Power Sources, volume: 195, pages: 6516 - 6523
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  Abstract

  We present a novel fabrication route yielding Raney-platinum film electrodes intended as glucose oxidation anodes for potentially implantable fuel cells. Fabrication roots on thermal alloying of an extractable metal with bulk platinum at 200 ◦C for 48 h. In contrast to earlier works using carcinogenic nickel, we employ zinc as potentially biocompatible alloying partner. Microstructure analysis indicates that after removal of extractable zinc the porous Raney-platinum film (roughness factor ∼2700) consists predominantly of the Pt3Zn phase. Release of zinc during electrode operation can be expected to have no significant effect on physiological normal levels in blood and serum, which promises good biocompatibility. In contrast to previous anodes based on hydrogel-bound catalyst particles the novel anodes exhibit excellent resistance against hydrolytic and oxidative attack. Furthermore, they exhibit significantly lower polarization with up to approximately 100mV more negative electrode potentials in the current density range relevant for fuel cell operation. The anodes’ amenability to surface modification with protective polymers is demonstrated by the exemplary application of an approximately 300nm thin Nafion coating. This had only a marginal effect on the anode long-term stability and amino acid tolerance. While in physiological glucose solution after approximately 100 h of operation gradually increasing performance degradation occurs, rapid electrode polarization within 24 h is observed in artificial tissue fluid. Optimization approaches may include catalyst enhancement by adatom surface modification and the application of specifically designed protective polymers with controlled charge and mesh size.
• S. Kerzenmacher, U. Kräling, M. Schroeder, R. Brämer, R. Zengerle, F. von Stetten
  Raney-platinum film electrodes for potentially implantable glucose fuel cells. Part 2: Glucose-tolerant oxygen reduction cathodes
  2010 J Power Sources, volume: 195, pages: 6524 - 6531
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  Abstract

  We report the fabrication and characterization of glucose-tolerant Raney-platinum cathodes for oxygen reduction in potentially implantable glucose fuel. Fabricated by extraction of aluminum from 1µm thin platinum–aluminum bi-layers annealed at 300 °C, the novel cathodes show excellent resistance against hydrolytic and oxidative attack. This renders them superior over previous cathodes fabricated from hydrogel-bound catalyst particles. Annealing times of 60, 120, and 240 min result in approximately 400–550nm thin porous films (roughness factors ∼100–150), which contain platinum and aluminum in a ratio of ∼9:1. Aluminum release during electrode operation can be expected to have no significant effect on physiological normal levels, which promises good biocompatibility. Annealing time has a distinct influence on the density of trenches formed in the cathode. Higher trench densities lead to lower electrode potentials in the presence of glucose. This suggests that glucose sensitivity is governed by mixed potential formation resulting from oxygen depletion within the trenches. During performance characterization the diffusion resistance to be expected from tissue capsule formation upon electrode implantation was taken into account by placing a membrane in front of the cathode. Despite the resulting limited oxygen supply, cathodes prepared by annealing for 60 min show more positive electrode potentials than previous cathodes fabricated from hydrogel-bound activated carbon. Compared to operation in phosphate buffered saline containing 3.0mMglucose, a potential loss of approximately 120mV occurs in artificial tissue fluid. This can be reduced to approximately 90mV with a protective Nafion layer that is easily electro-coated onto the Raney-platinum film.
• J. Leemhuis, E. Bouché, M. Frotscher, F. Henle, L. Hein, J. Herz, D. K. Meyer, M. Pichler, G. Roth, C. Schwan, H. H. Bock
  Reelin Signals through Apolipoprotein E Receptor 2 and Cdc42 to Increase Growth Cone Motility and Filopodia Formation
  2010 The Journal of Neuroscience, volume: 30, issue: 44, pages: 14759 - 14772
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  Abstract

  Lipoprotein receptor signaling regulates the positioning and differentiation of postmitotic neurons during development and modulates neuronal plasticity in the mature brain. Depending on the contextual situation, the lipoprotein receptor ligand Reelin can have opposing effects on cortical neurons. We show that Reelin increases growth cone motility and filopodia formation, and identify the underlying signaling cascade. Reelin activates the Rho GTPase Cdc42, known for its role in neuronal morphogenesis and directed migration, in an apolipoprotein E receptor 2-, Disabled-1-, and phosphatidylinositol 3-kinase-dependent manner. We demonstrate that neuronal vesicle trafficking, a Cdc42-controlled process, is increased after Reelin treatment and further provide evidence that the peptidergic VIP/ PACAP38 system and Reelin can functionally interact to promote axonal branching. In conclusion, Reelin-induced activation of Cdc42 contributes to the regulation of the cytoskeleton of individual responsive neurons and converges with other signaling cascades to orchestrate Rho GTPase activity and promote neuronal development. Our data link the observation that defects in Rho GTPases and Reelin signaling are responsible for developmental defects leading to neurological and psychiatric disorders.
• J. D. Jeyaprakash S. Samuel, T. Brenner, O. Prucker, M. Grumann, J. Ducree, R. Zengerle, J. Rühe
  Tailormade Microfluidic Devices Through Photochemical Surface Modification
  2010 Macromol Chem Phys, volume: 211, pages: 195 - 203
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  Abstract

  A new pathway for the generation of polymer-based microfluidic devices with tailor-made surface chemistry is described. A simple photochemical process is used to covalently bind polymer molecules to the surfaces of microchannels fabricated by hot embossing. The substrates for the embossing process have the format of a compact disk (CD). CDs from polymethylmethacrylate and polyethylene-co-norbornene were chosen due to their good optical properties. Thin films of polymers containing photoactive benzophenone units were deposited onto the surface of the thus generated devices. These films were subsequently irradiated with UV light leading to the surface-attachment of ultrathin polymer networks. In contrast to their unmodified peers, the obtained,modified microfluidic channels coated with hydrophilic, photoattached layers can be filled in a straightforward manner with water by capillary forces. Channels coated by thin films of poly(ethyloxazoline) show complete resistance to non-specific protein binding. Generation of hydrophobic patches inside the modified microfluidic channels using benzophenone-containing fluoropolymers allows the generation of passive microfluidic valves to direct fluid motion in these CD-based devices.
• F. Hedrich, K. Kliche, M. Storz, S. Billat, M. Ashauer, R. Zengerle
  Thermal flow sensors for MEMS spirometric devices
  2010 Sensors and Actuators A, volume: 162, pages: 373 - 378
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  Abstract

  Sleep-specific respiration disturbances and their coprevalences with basic cardiovascular diseases constitute a supplementary risk for the cardiovascular system and require both urgent diagnostic assessment as well as consistent therapeutic measures. Thus, the need for controlling the breathing of these patients over a long period has become a new focus of interest. Due to its small thermal mass, the micro-machined thermal flow sensor fits the specific requirements in the patient respiration control of high dynamic flow range combined with a fast response time. We present the development of our flow sensor with a special adaptation for spirometric applications. The sensor exhibits a high accuracy, a short response time (<1 ms) and a low power consumption (10mW). Furthermore, heating structures are added onto the chip to prevent condensation. Hence, it represents an attractive solution for the use of portable equipment for preventive exploration of breathing in home-care applications.

2009

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• A. Ernst, W. Streule, N. Schmitt, R. Zengerle, P. Koltay
  A capacitive sensor for non-contact nanoliter droplet detection
  2009 Sensor Actuat A-phys, volume: 153, pages: 57 - 63
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  Abstract

  This paper reports on a sensor for the detection of microdroplets in flight. The presented sensor is based on a capacitive principle, which allows for non-contact monitoring of a complete droplet dispensing process. In the presented experiments the change in capacity caused by liquid droplets in the range of a few nanoliters passing through the electric field of the sensor is studied. From the capacitive change the droplet presence can be deduced with a reliability of 100%, which means that every single droplet dispensed within the experiments caused a significant signal change. In addition, the sensor signal is sensitive to the droplet’s volume V, dielectric constant εr (epsilon) and velocity v. It turns out that every specific droplet exhibits a characteristic “fingerprint” signal dependingonthese parameters. Especially the droplet volume correlates very well with the peak value of the extracted signal. Therefore, the calibrated sensor is able to determine the volume of dispensed droplets in the range from20 to 65 nl with a resolution of less than 2 nl. Furthermore, the printed circuit board (PCB) technology applied for fabrication of the sensor enables a very cost efficient and flexible realisation of the whole sensor unit. The non-contact capacitive principle prevents contamination and loss of media. Therefore, the proposed approach is well suited for high precision droplet presence detection and low cost online monitoring of liquid volumes in microdispensing processes for various applications.
• S. Kerzenmacher, K. Mutschler, U. Kräling, H. Baumer, J. Ducrée, R. Zengerle, F. von Stetten
  A complete testing environment for the automated parallel performance characterization of biofuel cells: design, validation, and application
  2009 J Appl Electrochem, volume: 39, issue: 9, pages: 1477 - 1485
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  Abstract

  We present a complete testing environment for the parallel performance characterization of biofuel cells. Besides rapid-assembly electrode fixtures and an aseptic electrochemical reactor, it comprises a 24-channel electrical testing system that bridges the gap between simple load resistors and costly multi-channel potentiostats. The computer- controlled testing system features active current control to enable the forced operation of half-cell electrodes, whereas galvanic isolation between individual channels ensures interference-free operation of multiple fuel cells immersed in a common testing solution. Implemented into the control software is an automated procedure for the step-wise recording of polarization curves. This way, performance overestimation due to a too fast increase in load current can be circumvented. As an applicational example, three abiotically catalyzed glucose fuel cells are characterized simultaneously in a common testing solution. Complete disclosure of the electrical system (incl. printed circuit board layout, control software, and circuit diagrams) in the online supplementary material accompanying this paper allows researchers to replicate our setup in their lab and can serve as inspiration for the design of similar systems adapted to specific requirements.
• Steigert, Jürgen, Strasser, Monika, Wangler, Nicolai, Brett, Olivia, Streule, Wolfgang, Koltay, Peter, Daub, Martina, Zengerle, Roland
  A modular diffusion barrier based on phase separation for localized delivery of discrete drug volumes in aqueous environments
  2009 Lab Chip, volume: 9, pages: 1801 - 1805
  » show abstract« hide abstract
  Abstract

  We present a new tool for the precisely controlled transfer of individual picoliter (pL) droplets in the range of 150–950 pL at user defined local positions within aqueous liquid environments while avoiding any leakage by diffusion. This is achieved by a low-cost, disposable and biocompatible cap that can be placed on top of any pL-dispenser and generates a phase-gap between dispensing agent and target liquid when the dispenser is dipped into the latter. We developed two different working modes: (i) the standard mode enables an instant injection (<< 1 ms) of the droplet into the liquid environment and (ii) the focus mode further increases the spatial resolution from 100 µm to 50 µm at the cost of slowing down the injection time. For the phase-gap we have proven an excellent long-term stability of more than 30 hours against capillary priming.
• N. Paust, Ch. Litterst, T. Metz, M. Eck, Ch. Ziegler, R. Zengerle, P. Koltay
  Capillary-driven pumping for passive degassing and fuel supply in direct methanol fuel cells
  2009 Microfluid Nanofluid, volume: 7, pages: 531 - 543
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  Abstract

  In this paper we present a new concept of creating and using capillary pressure gradients for passive degassing and passive methanol supply in direct methanol fuel cells (DMFCs). An anode flow field consisting of parallel tapered channels structures is applied to achieve the passive supply mechanism. The flow is propelled by the surface forces of deformed CO2 bubbles, generated as a reaction product during DMFC operation. This work focuses on studying the influence of channel geometry and surface properties on the capillary-induced liquid flow rates at various bubbly gas flow rates. Besides the aspect ratios and opening angles of the tapered channels, the static contact angle as well as the effect of contact angle hysteresis has been identified to significantly influence the liquid flow rates induced by capillary forces at the bubble menisci. Applying the novel concept, we show that the liquid flow rates are up to thirteen times higher than the methanol oxidation reaction on the anode requires. Experimental results are presented that demonstrate the continuous passive operation of a DMFC for more than 15 h.
• Daniel Mark, Tobias Metz, Stefan Haeberle, Sascha Lutz, Jens Ducrée, Roland Zengerle, Felix von Stetten
  Centrifugo-Pneumatic Valve for Metering of Highly Wetting Liquids on Centrifugal Microfluidic Platforms
  2009 Lab Chip, volume: 9, pages: 3599 - 3603
  » show abstract« hide abstract
  Abstract

  We designed and experimentally validated a new type of passive valve for centrifugal microfluidic platforms. A liquid column entering an unvented receiving chamber is stopped by the counter-pressure of compressed air. This valve opens under defined conditions at high centrifugal frequencies at which the interface between liquid and air becomes unstable and enables a phase exchange, forwarding the liquid. Burst frequencies of the valve were determined for liquids typically used in biochemical assays: pure water, water with detergent concentrations between 0.01 % and 10 %, and pure ethanol. Burst frequencies between 8.5 ± 0.6 Hz and 27.9 ± 2.0 Hz were measured for different surface tensions. The burst frequencies can be tuned by simple geometrical changes in the valving structure. The valve does not require ultra-precise structures or local surface modifications and is therefore ideal for low-cost microfluidic polymer disks. Potential applications are in the field of mulitparameter- and panel analysis, such as PCR-genotyping.
• Mark, Daniel, Haeberle, Stefan, Zengerle, Roland, Ducree, Jens, Vladisavljevic, Goran T.
  Manufacture of chitosan microbeads using centrifugally driven flow of gel-forming solutions through a polymeric micronozzle
  2009 J Colloid Interf Sci, volume: 336, pages: 634 - 641
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  Abstract

  A centrifugally driven pulse-free flow was used for the generation of uniform chitosan beads tunable in diameter between 148 and 257 µm. The 2 % chitosan solution was extruded through a polymeric nozzle and the drops were gelated in 10 % tripolyphosphate (TPP) solution at pH 4.0. The coefficient of variation (CV) of the bead diameters issued from different nozzles could be reduced to 15 %.The production process requires a single motor as the sole actively actuated component. The production rate was 45 beads per second with one nozzle spinning at 44 Hz, scaling with the sixth power of the rotational frequency. An analytical model for the bead diameter and production rate is presented and validated by the experimental data. The shrinkage of chitosan drops during gelation was estimated from the observations and the theoretical model. Keywords: microbeads, chitosan, centrifugal, encapsulation, microfluidics
• Katrin Bürk, Marie-Louise Farecki, Georg Lamprecht, Guenter Roth, Patrice Decker, Michael Weller, Hans-Georg Rammensee, Wolfang Oertel
  Neurological Symptoms in Patients with Biopsy Proven Celiac Disease
  2009 Movement Disord, volume: 24, issue: 26, pages: 2358 - 2362
  » show abstract« hide abstract
  Abstract

  In celiac disease (CD), the gut is the typical manifestation site but atypical neurological presentations are thought to occur in 6 to 10% with cerebellar ataxia being the most frequent symptom. Most studies in this field are focused on patients under primary neurological care. To exclude such an observation bias, patients with biopsy proven celiac disease were screened for neurological disease. A total of 72 patients with biopsy proven celiac disease (CD) (mean age 51 6 15 years, mean disease duration 8 6 11 years) were recruited through advertisements. All participants adhered to a gluten-free diet. Patients were interviewed following a standard questionnaire and examined clinically for neurological symptoms. Medical history revealed neurological disorders such as migraine (28%), carpal tunnel syndrome (20%), vestibular dysfunction (8%), seizures (6%), and myelitis (3%). Interestingly, 35% of patients with CD reported of a history of psychiatric disease including depression, personality changes, or even psychosis. Physical examination yielded stance and gait problems in about one third of patients that could be attributed to afferent ataxia in 26%, vestibular dysfunction in 6%, and cerebellar ataxia in 6%. Other motor features such as basal ganglia symptoms, pyramidal tract signs, tics, and myoclonus were infrequent. 35% of patients with CD showed deep sensory loss and reduced ankle reflexes in 14%. Gait disturbances in CD do not only result from cerebellar ataxia but also from proprioceptive or vestibular impairment. Neurological problems may even develop despite strict adherence to a gluten-free diet.
• Paust, N, Krumbholz, S, Munt, S, Müller, C, Koltay, P, Zengerle, R, Ziegler, C
  Self-Regulating Passive Fuel Supply for Small Direct Methanol Fuel Cells Operating in All Orientations
  2009 J Power Sources, volume: 192, pages: 442 - 450
  » show abstract« hide abstract
  Abstract

  A microfluidic fuel supply concept for passive and portable Direct Methanol Fuel Cells (DMFCs) that operate in all spatial orientations is presented. The concept has been proven by fabricating and testing a passive DMFC prototype. Methanol transport at the anode is propelled by the surface energy of deformed carbon dioxide bubbles, generated as a reaction product during DMFC operation. The experimental study reveals that in any orientation, the proposed pumping mechanism transports at least 3.5 times more methanol to the reactive area of the DMFC than the stoichiometry of the methanol oxidation would require to sustain DMFC operation. Additionally, the flow rates closely follow the applied electric load; hence the pumping mechanism is self-regulating. Oxygen is supplied to the cathode by diffusion and the reaction product water is transported out of the fuel cell along a continuous capillary pressure gradient. Results are presented that demonstrate the continuous passive operation for more than 40 hours at ambient temperature with a power output of p = 4 mW cm-2 in the preferred vertical orientation and of p = 3.2 mW cm-2 in the least favourable horizontal orientation with the anode facing downwards. Keywords: passive DMFC; self-regulating fuel supply; capillary-force-driven bubble pump
• Thomas Steiner, Claudio Cupelli, Roland Zengerle, Mark Santer
  Simulation of advanced microfluidic systems with dissipative particle dynamics
  2009 Microfluid Nanofluid, volume: 7, issue: 3, pages: 307 - 323
• Jochen Rupp, Manuela Schmidt, Bettina Günther, Michael Stumber, Sven Zinober, Roland Müller-Fiedler, Bashir Alabsi, Peter Rothacher, Claas Müller, Holger Reinecke, Roland Zengerle, Martina Daub
  The Way to High Volume Fabrication of Lab-on-a-Chip Devices – A Technological Approach for Polymer Based Microfluidic Systems with Integrated Active Valves and Pumps
  2009 J Electron Packaging, volume: 6, pages: 1 - 7
• Steinert C.P., Kalkandjiev, K., Zengerle, R., Koltay, P.
  TopSpot® Vario: a novel microarrayer system for highly flexible and highly parallel picoliter dispensing
  2009 Biomed Microdevices, volume: 11, issue: 4, pages: 755 - 761
  » show abstract« hide abstract
  Abstract

  The standard TopSpot® technology has been successfully used in the recent years for the highly parallel nanoliter dispensing of bio-chemical substances for microarray printing. It is based on a pneumatically actuated printhead which enables non-contact microarray fabrication at a pitch of typically 500 μm. This paper reports on a new and improved way of operating the printheads termed TopSpot® Vario technology, using an incompressible material between the piezo actuator and the dispensing medium. The advantage of the incompressible medium is the direct relation between the displacement amplitude and the ejected liquid volume. Earlier reports stated that the filling of the printheads is a key issue. In this paper we report on the implementation and characterization of a new printhead design including microchannels for bubble free priming of blind channels. Microarray fabrication was successfully conducted with four different types of protein.
• C. Ziegler, D. Gerteisen
  Validity of two-phase polymer electrolyte membrane fuel cell models with respect to the gas diffusion layer
  2009 J Power Sources, volume: 188, issue: 1, pages: 184 - 191
  » show abstract« hide abstract
  Abstract

  A dynamic two-phase model of a proton exchange membrane fuel cell with respect to the gas diffusion layer (GDL) is presented and compared with chronoamperometric experiments. Very good agreement between experiment and simulation is achieved for potential step voltammetry (PSV) and sine wave testing (SWT). Homogenized two-phase models can be categorized in unsaturated flow theory (UFT) and multiphase mixture (M2) models. Both model approaches use the continuum hypothesis as fundamental assumption. Cyclic voltammetry experiments show that there is a deterministic and a stochastic liquid transport mode depending on the fraction of hydrophilic pores of the GDL. ESEM imaging is used to investigate the morphology of the liquid water accumulation in the pores oftwo different media (unteflonated Toray-TGP-H-090 and hydrophobic Freudenberg H2315 I3). The morphology of the liquid water accumulation are related with the cell behavior. The results show that UFT and M2two-phase models are a valid approach for diffusion media with large fraction of hydrophilic pores such as unteflonated Toray-TGP-H paper. However, the use of the homgenized UFT and M2 models appears to be invalid for GDLs with large fraction of hydrophobic pores that corresponds to a high average contact angle of the GDL. Keywords Proton exchange membrane fuel cell; Two-phase model; Validity; GDL

2008

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• Jürgen Steigert, Olivia Brett, Claas Müller, Monika Strasser, Nicolai Wangler, Holger Reinecke, Martina Daub, Roland Zengerle
  A versatile and flexible low-temperature full-wafer bonding process of monolithic 3D microfluidic structures in SU-8
  2008 J Micromech Microeng, volume: 18, issue: 9, pages: 1 - 8
  » show abstract« hide abstract
  Abstract

  We present a versatile fabrication process for the precise fabrication of embedded three-dimensional microfluidic structures in SU-8 photoresist. The full-wafer bond process based on a polyester (PET) handling layer enhances the previous low-temperature bonding technology. We achieved an extremely high bond strength of 45 MPa while requiring only small anchoring structures. Small channel structures with an aspect ratio >2 as well as wide membranes with an aspect ratio <0.02 were successfully bonded to realize precisely defined channel structures. Furthermore, the developed process features high yields (>80%) and enables the integration of microelectronics. The flexibility of the fabrication process is presented in two contrary applications. A completely freestanding and transparent SU-8 foil with a thickness of 225 μm featuring embedded 3D microchannels was fabricated. Also, high quality ink-jet dispensers were successfully fabricated whereas the dispenser quality mainly depends on the channel quality.
• S. Haeberle, L. Naegele, R. Burger, F. von Stetten, R. Zengerle, J. Ducrée
  Alginate bead fabrication and encapsulation of living cells under centrifugally induced artificial gravity conditions
  2008 J Microencapsul, volume: 25, issue: 4, pages: 267 - 274
  » show abstract« hide abstract
  Abstract

  We present a novel method for the direct, centrifugally induced fabrication of small, Ca2+-hardened alginate beads at polymer-tube micronozzles. The bead diameter can arbitrarily be adjusted between 180 µm and 800 µm by the nozzle geometry and spinning frequencies between 5 Hz and 28 Hz. The size distribution of the main peak features a CV of 7 – 16%, only. Up to 600 beads per second and channel are issued from the micronozzle through an air gap towards the curing agent contained in a standard lab tube (“Eppi”). Several tubes can be mounted on a “flying bucket” rotor where they align horizontally under rotation and return to a vertical position as soon as the rotor is at rest. The centrifugally induced, ultra-high artificial gravity conditions (up to 180 g) even allow the micro-encapsulation of alginate solutions displaying viscosities up to 50 Pa s, i.e. about 50,000 times the viscosity of water! With this low cost technology for micro encapsulation, HN25 and PC12 cells have successfully been encapsulated while maintaining vitality.
• Kerzenmacher S, Ducree J, Zengerle R, von Stetten F
  An Abiotically Catalyzed Glucose Fuel Cell for Powering Medical Implants: Reconstructed Manufacturing Protocol and Analysis of Performance
  2008 J Power Sources, volume: 182, issue: 1, pages: 66 - 75
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  Abstract

  Although the first abiotically catalyzed glucose fuel cells have already been developed as sustainable power supply for medical implants in the 1970s, no detailed information concerning the fabrication of these devices has been published so far. Here we present a comprehensive manufacturing protocol for such a fuel cell, together with a detailed analysis of long-term stability performance in neutral buffer containing physiological amounts of glucose and oxygen. In air saturated solution a power density of (3.3 +/- 0.2) µW cm-2 is displayed after 10 days of operation, that gradually decreases to a value of (1.0 +/- 0.05) µW cm-2 in the course of 224 days. A novelty of this work is the characterization of fuel cell performance with individually resolved electrode potentials. Using this technique, we can show that the major part of performance degradation originates from a positive shift of the anode potential, indicating that a more poisoning resistant glucose oxidation catalyst would improve the degradation behavior of the fuel cell. As further factors influencing performance an incomplete reactant separation and a mass transfer governed cathode reaction under the relatively low oxygen partial pressures of body tissue have been identified. Consequently we propose an oxygen depleting electrode interlayer and the application of more effective oxygen reduction catalysts as promising strategies to further improve the fuel cell performance under physiological concentrations of glucose and oxygen.
• M. Daub, R. Zengerle
  Bioprinting on Chip
  2008 Encyclopedia of Microfluidics and Nanofluidics, pages: 124 - 138
• Glatzel T, Cupelli C, Lindemann T, Litterst C, Moosmann C, Niekrawietz R, Streule W, Zengerle R, Koltay P
  Computational fluid dynamics (CFD) software tools for microfluidic applications - A case study
  2008 Comput Fluids, volume: 37, issue: 3, pages: 218 - 235
• Claudio Cupelli, Björn Henrich, Thomas Glatzel, Roland Zengerle, Michael Moseler, Mark Santer
  Dynamic capillary wetting studied with dissipative particle dynamics
  2008 New Journal of Physics, volume: 10, page: 043009
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  Abstract

  We present a study on dynamic capillary wetting in the framework of dissipative particle dynamics (DPD) based on a novel wall model for wetting on solid boundaries. We consider capillary impregnation of a slit pore in two situations: (i) forced (piston-driven) steady state flow and (ii) capillarity driven imbibition out of a finite reservoir. The dynamic contact angle behavior under condition (i) is consistent with the hydrodynamic theories of Cox under partial wetting conditions and Eggers for complete wetting. The flow field near the contact line shows a region of apparent slip flow which provides a natural way of avoiding a stress singularity at the triple line. The dynamics of the capillary imbibition, i.e. condition (ii), is consistently described by the Lucas–Washburn equation augmented by expressions that account for inertia and the influence of the dynamic contact angle.
• Diana Hodgins, Arnaud Bertsch, Nils Post, Manfred Frischholz, Bart Volckaerts, John Spensley, J.M. Wasikiewicz, Henry Higgins, Felix von Stetten, Laurence Kenney
  Healthy Aims: Developing New Medical Implants and Diagnostic Equipment
  2008 Pervasive Computing, volume: 7, issue: 1, pages: 14 - 21
• J. Hermann, C. Ziegler
  Modeling the Dynamic Water Transport in the Porous Layers of PEM Fuel Cells Based on Numerical Upscaling
  2008 J Electrochem Soc, volume: 155, issue: 10, pages: B1066 - B1076
• Billat,S., Kliche,K., Gronmaier,R, Nommensen,P., Auber,J., Hedrich,F., Zengerle,R.
  Monolithic integration of micro-channel on disposable flow sensors for medical applications
  2008 Sensors and Actuators A, volume: 145-146, pages: 66 - 74
• T. Metz, J. Viertel, C. Müller, S. Kerzenmacher, N. Paust, R. Zengerle, P. Koltay
  Passive water management for µfuel-cells using capillary microstructures
  2008 J Micromech Microeng, volume: 18, issue: 10
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  Abstract

  In this work we present a novel system for the passive water management in polymer electrolyte fuel cells (PEMFC) based on capillary effects in microstructures. The system removes abundant water that occurs at low temperatures at a fuel cell cathode and secures the humidity of the electrolyte membrane on higher temperatures. Liquid water is removed by hydrophilic gas supply channels with a tapered cross section as presented previously, and further transported by a system of capillary channels and a layer of nonwoven material. To prevent the membrane from running dry, a storage area in the nonwoven layer is introduced, controlled by a novel passive capillary overflow valve. The valve controls whether water is stored or finally disposed by gravity and evaporation. Experiments in a model system show that the nonwoven material is capable of removing all liquid water that can be produced by the fuel cell. A miniaturized fuel cell utilizing the novel water removal system was fabricated and experiments show that the system can stabilize the performance during changes of electrical load. Clearing the drowned miniaturized fuel cell flow field was proven and required 2 min. To make the capillary effects available for the originally hydrophobic graphite composite materials that were used to fabricate the flow fields, hydrophilic grafting based on photochemistry was applied to the material and contact angles of about 40° could be achieved and preserved for at least three months.
• Tobias Metz, Wolfgang Streule, Roland Zengerle, Peter Koltay
  StarTube: A tube with reduced contact line for minimized gas bubble resistance
  2008 Langmuir, volume: 24, issue: 17, pages: 9204 - 9206
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  Abstract

  In this work we introduce a novel tubing design for multiphase flow that minimizes gas bubble resistance. The design termed “StarTube” has a lamella-like wall structure and was developed to prevent clogging by gas bubbles. This is performed by forcing gas bubbles into the center of the tube by capillary forces, allowing liquid to bypass in the outer grooves. It was found that the mobility of gas bubbles in such a tube is increased more than 1 order of magnitude. The reason is that the contact line perpendicular to the direction of flow is minimized, reducing resistant effects related to the contact linesin particular, contact angle hysteresis.
• C. Litterst, T. Metz, R. Zengerle, P. Koltay
  Static and dynamic behaviour of gas bubbles in T-shaped non-clogging micro-channels
  2008 Microfluid Nanofluid, volume: 5, issue: 6, pages: 775 - 784
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  Abstract

  Preventing micro-channels from clogging is a major issue in most micro and nanofluidic systems. The T-shaped channel first reported by Kohnle et al. prevents micro-channels from clogging by the aid of the equilibrium bubble position in such a geometry. This work is concerned with the static and dynamic behaviour of bubbles in such T-shaped microchannels. The aspect ratio of a rectangle enclosing the Tshaped channel and the contact angle of the walls are the main parameters influencing the static and dynamic bubble behaviour. It is investigated in this article how these parameters relate to the equilibrium bubble shape and how optimum bubble velocities can be achieved inside the channel. An analytical model depending on the contact angle and the channel geometry is presented that allows to determine the bubble configuration inside the channel by minimizing the bubble’s surface energy. A second model is derived to predict the velocity of gas bubbles driven by buoyancy in vertical T-shaped channels. The model is applied to design T-shaped channels with a maximum mobility of gas bubbles. Experiments with MEMS fabricated devices and CFD simulations are used to verify the models. Furthermore design rules for an optimum nonclogging channel geometry which provides the highest gas bubble mobility are given. Keywords Gas bubble, Micro-channel, Clogging, Interfacial energy

2007

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• Björn Henrich, Claudio Cupelli, Michael Moseler, Mark Santer
  An adhesive DPD wall model for dynamic wetting
  2007 Europhys Lett, volume: 80, page: 60004
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  Abstract

  A novel method for the treatment of solid-liquid interfaces in the framework of dissipative particle dynamics (DPD) is presented. The solid is represented by an amorphous, thermally rough ensemble of particles. Since the density of this phase is chosen commensurate to the liquid, artefacts of alternative wall models such as particle layering or spurious variations in temperature are avoided. An adjustable static contact angle permits the convenient study of complex wetting phenomena. We explore the physical behaviour of this boundary model with respect to bulk Poiseuille and Couette flow as well as forced wetting and the behaviour of dynamic contact angles.
• Haeberle S, Zengerle R, Ducrée J
  Centrifugal Generation and Manipulation of Droplet Emulsions
  2007 Microfluidics Nanofluidics, volume: 3, issue: 1, pages: 65 - 75
• Haeberle,S., Schmitt,N., Zengerle,R., Ducrée,J.
  Centrifugo-Magnetic Pump for Gas-to-Liquid Sampling
  2007 Sensors and Actuators A-Physical, volume: 135, issue: 1, pages: 28 - 33
• Hu Min, Lindemann T, Goettsche T, Kohnle J, Zengerle R, Koltay P
  Discrete Chemical Release from a Microfluidic Chip
  2007 J Microelectromech S, volume: 16, issue: 4, pages: 786 - 794
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  Abstract

  We demonstrate a discrete chemical release method, capable of delivering picoliter volumes of chemical solutions with 100 μm of spatial resolution and 20 μs of response time. The releasing mechanism is based on the transfer of pulsed liquid plugs through a hydrophobic air chamber. A microfluidic chip consisting of such a releasing array (2 × 10) is designed and fabricated. Numerical simulation and experimental testing are performed to verify the working principle. Advantages of this release-on-demand technology include leakage-free, fast response and versatile control of release profile. This new method could be a key enabling technology for precisely controlled release of biochemicals for modern pharmacological and biological research. Index Terms: Air gap, discrete chemical stimulation, microfluidic chip, picoliter chemical release, pulsed liquid ejection.
• Steigert,J., Brenner,T., Grumann,M., Riegger,L., Lutz,S., Zengerle,R., Ducree,J.
  Integrated siphon-based metering and sedimentation of whole blood on a hydrophilic lab-on-a-disk
  2007 Biomed.Microdevices, issue: 9, pages: 675 - 679
• Zibek S, Stett A, Koltay P, Hu M, Zengerle R, Nisch W, Stelzle M
  Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine
  2007 Biophys J, volume: 92, issue: 1, pages: L04 - 6L
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  Abstract

  Acetylcholine sensitive TE 671 cells were cultured on nanoporous membranes and chemically stimulated by localized application of i), calcein-AM and ii), acetylcholine, respectively, onto the bottom face of the membrane employing an ink jet print head. Stimulus correlated response of cells was recorded by fluorescence microscopy with temporal and spatial resolution. Calcein fluorescence develops as a result of intracellular enzymatic conversion of calcein-AM, whereas Ca2+ imaging using fluo-4 dye was employed to visualize cellular response to acetylcholine stimulation. Using 25 pl droplets and substance concentration ranging from 10 µM to 1 mM on Nucleopore membranes with pore diameters between 50 nm and 1 µm, a resolution on the order of 50 µm was achieved.
• Haeberle,S., Zengerle,R.
  Microfluidic platforms for lab-on-a-chip applications
  2007 Lab Chip, volume: 7, pages: 1094 - 1110
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  Abstract

  We review microfluidic platforms that enable the miniaturization, integration and automation of biochemical assays. Nowadays nearly an unmanageable variety of alternative approaches exists that can do this in principle. Here we focus on those kinds of platforms only that allow performance of a set of microfluidic functions—defined as microfluidic unit operations—which can be easily combined within a well defined and consistent fabrication technology to implement application specific biochemical assays in an easy, flexible and ideally monolithically way. The microfluidic platforms discussed in the following are capillary test strips, also known as lateral flow assays, the ‘‘microfluidic large scale integration’’ approach, centrifugal microfluidics, the electrokinetic platform, pressure driven droplet based microfluidics, electrowetting based microfluidics, SAW driven microfluidics and, last but not least, ‘‘free scalable non-contact dispensing’’. The microfluidic unit operations discussed within those platforms are fluid transport, metering, mixing, switching, incubation, separation, droplet formation, droplet splitting, nL and pL dispensing, and detection.
• Lindemann,T., Ashauer,H., Duccio,Y.Y., Sassano,S., Zengerle,R., Koltay,P.
  One Inch Thermal Bubble Jet Printhead With Laser Structured Integrated Polyimide Nozzle Plate
  2007 Journal of Microelectromechanical Systems, volume: 16, issue: 2, pages: 420 - 428
• Metz,T., Paust,N., Müller,C., Zengerle,R., Koltay,P.
  Passive water removal in fuel cells by capillary droplet actuation
  2007 Sensors & Actuators: A.Physical, volume: Special Issue MEMS 2007
• Steigert J, Haeberle S, Brenner T, Mueller C, Steinert C P, Koltay P, Gottschlich N, Reinecke H, Ruehe J, Zengerle R, Ducrée J
  Rapid prototyping of microfluidic chips in COC
  2007 J Micromech Microeng, volume: 17, pages: 333 - 341
• L. Riegger, M. Grumann, J. Steigert, S. Lutz, C.P. Steinert, C. Mueller, J. Viertel, O. Prucker, J. Rühe, R. Zengerle, J. Ducree
  Single-step centrifugal hematocrit determination on a 10-$ processing device
  2007 Biomed Microdevices, volume: 9, pages: 795 - 799
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  Abstract

  We present a novel concept to process human blood on a spinning polymer disk for the determination of the hematocrit level by simple visual inspection. The microfluidic disk which is spun by a macroscopic drive unit features an upstream metering structure and a downstream blind channel where the centrifugally enforced sedimentation of the blood is performed. The bubble-free priming of the blind channel is governed by centrifugally assisted capillary filling along the sloped hydrophilic sidewall and the lid as well as the special shape of the dead end of the two-layer channel. The hematocrit is indicated at the sharp phase boundary between the plasma and the segregated cellular pellet on a disk-imprinted calibrated scale. This way, we conduct the hematocrit determination of human blood within 5 min at a high degree of linearity (R2=0.999) and at a high accuracy (CV=4.7%) spanning over the physiological to pathological working range. As all processing steps including the priming, the metering to a defined volume as well as the centrifugation are executed automatically during rotation, the concept is successfully demonstrated in a conventional PC-CDROM drive while delivering the same performance (R2=0.999, CV=4.3%).

2006

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• Messner S, Schaible J, Sandmaier H, Zengerle R
  3-way silicon microvalve for pneumatic applications with electrostatic actuation principle
  2006 Microfluidics and Nanofluidics, issue: 2, pages: 89 - 96
• Haeberle S, Brenner T, Zengerle R, Ducree J
  Centrifugal extraction of plasma from whole blood on a rotating disk
  2006 Lab on A Chip, volume: 6, issue: 6, pages: 776 - 781
• Steigert J, Grumann M, Dube M, Streule W, Riegger L, Brenner T, Koltay P, Mittmann K, Zengerle R, Ducrée J
  Direct Hemoglobin Measurement on a Centrifugal Microfluidic Platform for Point-of-Care Diagnostics
  2006 Sensors and Actuators, pages: 228 - 233
• Steigert J, Grumann M, Brenner T, Riegger L, Harter J, Zengerle R, Ducrée J
  Fully Integrated Whole Blood Testing by Real-Time Absorption Measurement on a Centrifugal Platform
  2006 Lab Chip, volume: 6, issue: 8, pages: 1040 - 1044
• Litterst C, Eccarius S, Hebling C, Zengerle R, Koltay P
  Increasing µDMFC efficiency by passive CO2 bubble removal and discontinuous operation
  2006 Journal of Micromechanics and Microengineering, volume: 16, issue: 9, pages: S248 - S253
• Boettcher M, Jaeger M S, Riegger L, Ducrée J, Zengerle R, Duschl C
  Lab-on-chip-based cell separation by combining dielectrophoresis and centrifugation
  2006 Biophysical Reviews & Letters, volume: 1, issue: 4, pages: 443 - 451
• Ducrée J, Brenner T, Haeberle S, Glatzel T, Zengerle R
  Multilamination of flows in planar networks of rotating microchannels
  2006 Microfluidics and Nanofluidics, volume: 2006, issue: 2, pages: 78 - 84
• Ducrée J, Zengerle R, Grumann M, Brenner T
  One Droplet of Blood – Lab-on-a-Chip System for Rapid Diagnostic
  2006 G.I.T Laboratory Journal, volume: 1, pages: 34 - 36
• Ducrée J, Haeberle S, Brenner T, Glatzel T, Zengerle R
  Patterning of flow and mixing in rotating radial microchannels
  2006 Microfluidics and Nanofluidics, volume: 2, issue: 2, pages: 97 - 105
• Riegger L, Grumann M, Nann T, Riegler J, Ehlert O, Mittenbühler K, Urban G, Pastewka L, Brenner T, Zengerle R, Ducrée J
  Read-out Concepts for Multiplexed Bead-based Fluorescence Immunoassays on Centrifugal Microfluidic Platforms
  2006 Sensors and Actuators A-Physical, volume: 126, pages: 455 - 462
• Grumann M, Steigert J, Riegger L, Moser I, Enderle B, Riebeseel K, Urban G, Zengerle R, Ducree J
  Sensitivity enhancement for colorimetric glucose assays on whole blood by on-chip beam-guidance
  2006 Biomed Microdevices, pages: 209 - 214

2005

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• Grumann M, Geipel A, Riegger L, Zengerle R, Ducrée J
  Batch-mode mixing on centrifugal microfluidic platforms
  2005 Lab on A Chip, volume: 5, issue: 5, pages: 560 - 565
• Haeberle S, Brenner T, Schlosser HP, Zengerle R, Ducrée J
  Centrifugal micromixer
  2005 Chemical Engineering & Technology, volume: 28, issue: 5, pages: 613 - 616
• Gutmann O, Kuehlewein R, Reinbold S, Niekrawietz R, Steinert CP, de Heij B, Zengerle R, Daub M
  Fast and reliable protein microarray production by a new drop-in-drop technique
  2005 Lab on A Chip, volume: 5, issue: 6, pages: 675 - 681
• Brenner T, Glatzel T, Zengerle R, Ducrée J
  Frequency-dependent transversal flow control in centrifugal microfluidics
  2005 Lab on A Chip, volume: 5, issue: 2, pages: 146 - 150
• Steigert J, Grumann M, Brenner T, Mittenbühler K, Nann T, Rühe J, Moser I, Haeberle S, Riegger L, Riegler J, Bessler W, Zengerle R, Ducrée J
  Integrated Sample Preparation, Reaction, and Detection on a High-frequency Centrifugal Microfluidic Platform
  2005 Journal of the Association for Laboratory Automation (JALA), volume: 10, issue: 5, pages: 331 - 341
• Zengerle R
  Micro- and Nano Cluster Freiburg
  2005 mst-news, volume: 6, page: 38
• Jeyaprakash JDS, Steger R, Birkle G, Zengerle R, Koltay P, Ruehe J
  Modification of Micronozzle Surfaces Using Fluorinated Polymeric Nanofilms for Enhanced Dispensing of Polar and Nonpolar Fluids
  2005 Analytical Chemistry, volume: 77, issue: 19, pages: 6469 - 6474
• Kaack R, Dankbar D, Müller-Chorus B, Jung A, Daub M
  Modulare Plattform mit integrierter Nanoliterdosierung
  2005 Laborwelt, volume: 6, issue: 2, pages: 10 - 13
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  Abstract

  Im Bereich der Genom-Forschung sind in den letzten Jahren erhebliche Fortschritte erzielt worden. Möglich wurden diese durch innovative Entwicklungen in der Automatisierung und Mikrofluidik, die als Schlüsselgebiete für die moderne Biotechnologie betrachtet werden können. Ein Beispiel dafür sind die ersten kommerziellen Ansätze zur hochparallelen Expressionsanalyse in miniaturisierten Formaten. Der nächste Schritt, der zwangsläufig folgen muss, sind Weiterent wicklungen hinsichtlich Quantifizierbarkeit (Sensitivität und Selektivität), Schnelligkeit (Parallelität) und Modularität, so dass mit nur einer Geräteplattform eine Vielzahl möglicher Anwendungen, wie z.B. die routinemäßige Diagnostik von SNPs (single nucleotide polymorphisms) oder die Identifikation und Validierung neuer Rezeptoren in der Wirkstoff-Forschung kostengünstig erschlossen werden können.
• Bohl B, Steger R, Zengerle R, Koltay P
  Multi-layer SU-8 Lift-Off Technology For Microfluidic Devices
  2005 Journal of Micromechanics and Microengineering, volume: 15, pages: 1125 - 1130
• Goettsche T, Kohnle J, Willmann M, Ernst H, Spieth S, Tischler R, Messner S, Zengerle R, Sandmaier H
  Novel approaches to particle tolerant valves for use in drug delivery systems
  2005 Sensors and Actuators A-Physical, volume: 118, issue: 1, pages: 70 - 77
• Ducrée J, Zengerle R, Grumann M, Brenner T
  Schnelle Diagnose aus einem Tropfen Blut
  2005 Mikrosystemtechnik, volume: 3
• Daub M
  TopSpot Quality Microarrays
  2005 Labor Medizin & Diagnostik, pages: 25 - 26
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  Abstract

  TopSpot® technology introduces a new concept into the microarray laboratory: highly parallel non-contact dispensing of bioanalytical reagents for the production of low to medium density microarrays. The technology is based on micromachined multi-channel printheads combined with a piezo-driven droplet ejection process. Different automation environments for these print modules allow TopSpot® technology to cover needs from decentralized laboratories to core facilities
• Grumann M, Brenner T, Beer C, Zengerle R, Ducrée J
  Visualization of flow patterning in high-speed centrifugal microfluidics
  2005 Review of Scientific Instruments, volume: 76, issue: 2, pages: 025101 - 025101

2004

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• Gutmann O, Kuehlewein R, Reinbold S, Niekrawietz R, Steinert CP, De HB, Zengerle R, Daub M
  A highly parallel nanoliter dispenser for microarray fabrication
  2004 Biomed Microdevices, volume: 6, issue: 2, pages: 131 - 137
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  Abstract

  We report about the correlation between satellite free droplet release and liquid viscosity in a highly parallel, pressure driven nanoliter dispenser. In extensive studies we found that for liquids of different viscosities the duration of the pressure pulse is the predominant effect compared to pressure amplitude. This result is of essential importance when actuation parameters have to be adopted for different media like oligonucleotide, DNA or protein solutions as it is the case for the non contact high throughput fabrication of microarrays (Ducree et al., 2000). Experiments with oligonucleotides as well as with different proteins showed ascertained carry-over and cross-contamination free printing of DNA and protein microarrays. With it a prime critical point of microarray production is solved, leading to high quality whilst high throughput microarray fabrication. For oligonucleotides printing we found CVs to be better than 1% within one single dispensing channel and 1.5 % within all 24 channels of a 24 channel printhead for each used printing buffer. By optimizing the protein printing buffer the CVs for protein printing were reduced to about 1% within all 24 channels. As a serious practical application test oligonucleotides microarrays were produced using our nanoliter dispenser system. With it a full DNA hybridization experiment was performed. Clear positive signals one hand and no signals in the negative controls on the other hand showed that our system is suited for microarray production.
• Steinert CP, Goutier I, Gutmann O, Sandmaier H, Daub M, de Heij B, Zengerle R
  A highly parallel picoliter dispenser with an integrated, novel capillary channel structure
  2004 Sensors and Actuators A-Physical, volume: 116, issue: 1, pages: 171 - 177
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  Abstract

  We present a cross-contamination free highly-parallel picoliter dispenser based on direct liquid displacement. Such dispensers are essential for the (mass) fabrication of microarrays [J. Ducrée, H. Gruhler, N. Hey, M. Mueller, S. Békési, M. Freygang, H. Sandmaier, R. Zengerle, TopSpot—a new method for the fabrication of microarrays, Tech. Digest, The Thirteenth IEEE Annual International Conference on Micro Electro Mechanical Systems, Mizyazaki, Japan, 23–27 January 2000, pp. 317–322] and are able to dispense up to 384 different reagents at a pitch of 500 µm simultaneously [A. Kuoni, M. Boillat, N.F. de Rooij, A highly parallel piezoelectric printing device for microarray technology, Tech. Digest, The 17th International Conference on Micro Electro Mechanical Systems, Maastricht, The Netherlands, 25–29 January 2004, pp. 466–469]. In contrast to an earlier design [Sens. Actuators A 103 (2003) 88] we investigated different nozzle diameters and a novel capillary channel design.We present a systematic study concerning the relation between nozzle diameter and ejected droplet volume. The change from 35 to 60 µm in nozzle diameter resulted in a doubling of dispensed volume for most used elastomers and irrespective of actuation parameters. Minimum and maximum dispensed volumes have been determined to be 125 and 1700 pl. Those results are based on a new design, which also includes passive microstructures for droplet homogeneity as well as modified microchannels for improved priming and prevention of cross-contamination. Based on this, the coefficient of variation (CV) of droplet velocity could be reduced from 50% down to less than 5%. The CV of droplet volume is clearly below the measurement error (8%). Keywords: Picoliter dispenser; Non-contact printing; Microchannel; Microfluidics; Microarrays; Biochips
• Grumann M, Dobmeier M, Schippers P, Brenner T, Kuhn C, Fritsche M, Zengerle R, Ducrée J
  Aggregation of bead-monolayers in flat microfluidic chambers simulation by the model of porous media
  2004 Lab on A Chip, volume: 4, issue: 3, pages: 209 - 213
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  Abstract

  In this paper, we for the first time simulate the process of hydrodynamic bead aggregation in a flat micro-fluidic chamber by a porous-media model in an iterative routine. This allows us to optimize the chamber design of our recently developed experimental method to form periodical monolayers from the flow of bead suspension. Periodical monolayers are advantageous for parallel assay formats since they enhance the mechanical rigidity of the aggregated pattern. This is important to avoid a spatial rearrangement along various steps of a read-out procedure which would impair the correlation between measurements. Furthermore, the monolayer formation guarantees the individual optical accessibility of all probe beads. By modelling the monolayers with porous media, we can drastically reduce the degrees of freedom in a two-phase, multi-particle problem. This way, we are able to compute stationary hydrodynamic flow patterns in the chamber. In order to simulate the complete filling process from these stationary solutions, we developed an iterative master routine which takes the transient aggregation pattern as the initial condition, then evaluates the placement of the newly introduced beads, and finally converts the points of aggregation into porous media.
• de Heij B, Daub M, Gutmann O, Niekrawietz R, Sandmaier H, Zengerle R
  Highly parallel dispensing of chemical and biological reagents
  2004 Analytical and Bioanalytical Chemistry, volume: 378, issue: 1, pages: 119 - 122
• Gutmann O, Niekrawietz R, Kuehlewein R, Steinert CP, de Heij B, Zengerle R, Daub M
  Impact of medium properties on droplet release in a highly parallel nanoliter dispenser
  2004 Sensors and Actuators A-Physical, volume: 116, issue: 2, pages: 187 - 194
• Gutmann O, Niekrawietz R, Kuehlewein R, Steinert CP, Reinbold S, De HB, Daub M, Zengerle R
  Non-contact production of oligonucleotide microarrays using the highly integrated TopSpot nanoliter dispenser
  2004 Analyst, volume: 129, issue: 9, pages: 835 - 840
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  Abstract

  For the first time we report on the production of oligonucleotide microarrays using a highly parallel and highly integrated, pressure driven TopSpot nanoliter dispenser. The system enables non-contact printing of different media like oligonucleotides, DNA or protein solutions. We optimized the printing buffer needed for oligonucleotides microarrays production with respect to two major aspects: microfluidical optimum for droplet dispensing and biochemical coupling efficiency on different commercially available microarray slides. Coefficient of variations (CVs) of generated spot diameters were measured to be smaller than 1% within one single dispensing nozzle and smaller than 1.5% within all 24 parallel nozzles of the printhead for all printing buffers used. No carry-over and no cross-talk was found, in extensive experiments with oligonucleotides. Optimized printing buffer compositions and concentrations for oligonucleotide microarrays were found, as well as optimized coupling protocols. Furthermore, buffers and protocols were adapted to a host of different microarray slides used. With this system, prime critical points of microarray production are solved, leading to high quality high throughput microarray fabrication.
• Streule W, Lindemann T, Birkle G, Zengerle R, Koltay P
  PipeJet: A Simple Disposable Dispenser for the Nano- and Microliter Range
  2004 Journal of the Association for Laboratory Automation (JALA), volume: 9, issue: 5, pages: 300 - 306
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  Abstract

  This paper reports on a simple, disposable non-contact dispenser for the nano- and microliter range. In contrast to other known dispensers manufactured by silicon micromachining the new device simply consists of an elastic polymer tube with a circular cross section. Actuation is done by a piezostack driven piston, squeezing the tube at a defined position near the open end by a significant fraction of the cross section. In contrast to drop-on-demand devices based on an acoustic actuation principle,5 the squeezing of the tube leads to a significant mechanical displacement of the liquid. Our experiments tested a large number of media in the viscosity range from 1 to 27 mPas. Some of our experiments tested up to approximately 2,000 mPas. Frequency characteristics showed an independent dosage volume for water up to a frequency of 15 Hz for tubes with an inner diameter of approximately 200 lm. Standard deviation within 1,000 shots resulted in an excellent CV (standard deviation/ dosage volume) of less than 2% of the dosage volume. Using tubes with an inner diameter of approximately 1,000 µm and a print frequency of 340 Hz, a flowrate of less than or equal to 143 µL/s could be reached. Beyond the possibility to dispense pure liquids, emulsion paints with particles that have a diameter of approximately 40 µm have also been printed successfully.
• Breisch S, de Heij B, Lohr M, Stelzle M
  Selective chemical surface modification of fluidic microsystems and characterization studies
  2004 Journal of Micromechanics and Microengineering, volume: 14, issue: 4, pages: 497 - 505
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  Abstract

  Control of wetting behaviour of fluidic microsystems was achieved by selective chemical surface modification using either a solution-based procedure or micro-contact printing. The modification procedures were designed in such a way as to obtain optimum wetting in fluid channels, sample reservoirs and nozzles, while at the same time preventing intermixing of fluids from different nozzle exits and thus enabling multiple long-term stable delivery of nano-litre droplets. Analysis included thickness measurements by ellipsometry, contact angle measurements and fluorescence microscopy. Selectively coated TopSpot dosage chips as are used for the fabrication of DNA and protein micro-arrays exhibit superior performance over uncoated dosage chips. Mixed and back-filled silane coatings show enhanced stability versus hydrolysis in basic and acidic solutions, as has been determined from measurements of contact angle as a function of immersion time.
• Steger R, Bohl B, Zengerle R, Koltay P
  The dispensing well plate: a novel device for nanoliter liquid handling in ultra high-throughput screening
  2004 Journal of the Association for Laboratory Automation, volume: 9, issue: 5, pages: 291 - 299
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  Abstract

  This article reports on a novel dispensing system for the massive parallel delivery of liquid volumes in the range of 50 nL. Due to the similarity of the device to conventional micro-well plates used for the storage of liquids, the device has been termed ‘‘dispensing well plate’’ (DWP). In contrast to other known micro dispensers, the DWP can consist of up to 1,536 dispensing units in parallel, all of which hold different reagents. The dispensing units can be arranged very closely at the pitch of conventional micro-well plates (2.25 mm or 4.5 mm). Driven by pneumatic actuation, a fixed volume of different liquids can be dispensed simultaneously and contact-free into micro-well plates or onto flat substrates. Because of this, the liquid handling in many chemical, biochemical, and pharmaceutical applications—especially within highthroughput screening (HTS)—can be sped up by a factor of 10 to 100. In our article, the basic operation principle of the device is presented, and experimental evidence of its extraordinary performance is given: a reproducibility of 2% to 5% and a homogeneity within individual droplet arrays of 1% to 2% has been measured, as well as viscosity independent performance for liquids in the range from 1 to 5 mPas. The applicability of the DWP technology within HTS is demonstrated by performing a miniaturized kinase assay at 1 lL assay volume in a 1536- well plate format.
• Koltay P, Steger R, Bohl B, Zengerle R
  The dispensing well plate: a novel nanodispenser for the multiparallel delivery of liquids (DWP Part I)
  2004 Sensors and Actuators A-Physical, volume: 116, issue: 3, pages: 483 - 491
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  Abstract

  This paper reports on a novel dispensing system for the massive parallel delivery of liquid volumes in the range of 50 nL. Due to the similarity of the device to conventional microwell plates used for storage of liquids, the device has been termed “dispensing well plate” (DWP). In contrast to other known microdispensers the DWP can consist of up to 1536 dispensing units in parallel all holding different reagents. The dispensing units can be arranged very closely at the pitch of conventional microwell plates (2.25 or 4.5 mm). Driven by pneumatic actuation a fixed volume of different liquids can be dispensed simultaneously and contact free into microwell plates or onto flat substrates. By this the liquid-handling in many chemical, biochemical and pharmaceutical applications—especially within high throughput screening (HTS)—can be speed up by a factor 10–100. In this paper the basic operation principle of the device is presented and experimental evidence is given of its extraordinary performance: a reproducibility of 2–5% and a homogeneity within individual droplet arrays of 1–2% has been measured as well as viscosity independent performance for liquids in the range from 1 to 5mPas. The fabrication of DWP prototypes by different micromachining technologies based on silicon dry etching and SU-8 technology is described and various DWP prototypes with different dosage volumes are presented. © 2004 Elsevier B.V. All rights reserved. Keywords: Non-contact dispenser; Jets; High throughput screening; SU-8
• Koltay P, Kalix J, Zengerle R
  Theoretical evaluation of the dispensing well plate method (DWP part II)
  2004 Sensors and Actuators A-Physical, volume: 116, issue: 3, pages: 472 - 482
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  Abstract

  The “dispensing well plate” (DWPTM) method is a technique to deliver fluidic jets or droplets with volumes of several nanoliters contact free to targets likemicro well plates or slides. A cheap and simple pneumatic actuation mechanism allows for the simultaneous delivery of a large number of different liquids. In this paper the dispensing dynamics of a DWP-type nanodispenser is studied theoretically. The device is modelled using computational fluid dynamics (CFD) simulations which are benchmarked to experimental data. Furthermore an analytical discussion is presented providing some insight into the dispensing dynamics. Based on these modelling approaches the dispensing process is studied in detail. Influence of system parameters like driving pressure, nozzle size, channel layout etc. on the jet formation and dispensed volume are quantified and design rules are given to improve the performance. © 2004 Elsevier B.V. All rights reserved. Keywords: Droplet dispensing; Jets; CFD-simulation; Microfluidics

2003

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• de Heij B, Steinert C, Sandmaier H, Zengerle R
  A tuneable and highly-parallel picolitre-dispenser based on direct liquid displacement
  2003 Sensors and Actuators A-Physical, volume: 103, issue: 1-2, pages: 88 - 92
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  Abstract

  We present a new method for the highly-parallel and simultaneous delivery of a multitude of reagents in the picolitre range. This method is based on direct displacement of the liquids using an elastomer-stamp, which simultaneously actuates up to 96 dosing channels, at a pitch of 500 mm. We were able to tune droplet volume from 100 to 700 pl and droplet speed from 0.3 to 4 m/s using printheads with 50 mm diameter nozzles. In contrast to all other inkjet techniques the new direct displacement method enables the precise control of dispensing quantity in the picolitre range regardless of reagent viscosity. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Highly-parallel; Picolitre-dispenser; Bio-molecule; Microfluidics
• Waibel G, Kohnle J, Cernosa R, Storz M, Schmitt M, Ernst H, Sandmaier H, Zengerle R, Strobelt T
  Highly integrated autonomous microdosage system
  2003 Sensors and Actuators A-Physical, volume: 103, issue: 1-2, pages: 225 - 230

2002

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• Ducrée J, Zengerle R
  Microfluidics - Markets and Technologies
  2002 mstnews, volume: 05, issue: 02, pages: 8 - 9
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  Abstract

  The field of microfluidics has become one of the most dynamically emerging disciplines of microtechnology in the recent decade. Microfluidics offers the mere benefits of miniaturization enabling portable and inexpensive devices, mostly for analytical and diagnostic purposes where the costs per test can be greatly reduced. It has also become evident that microfluidics provides a unique access to the nanoworld of biomolecular chemistry thus setting the pace for many leading edge biotechnological innovations. In the course of the “FlowMap” project , a consortium of European partners such as IMTEK (D), HSG-IMIT (D), Cranfield Biotechnology Center (UK), Yole Dévelopement (F) and Wicht Technology Consulting (D) has analyzed the structure of the field in terms of markets, products and technologies. This information will be the basis for an explorative technology roadmap which we develop for microfluidics in the life sciences. A time frame for the development of technological and economic landmarks will be introduced and sequences as well as dependencies will be illustrated. This will allow to plot the complete road between technologies and markets.