Publikationsliste Dr. Silvia Calabrese

• Originalarbeiten in wissenschaftlichen Fachzeitschriften
• Konferenzbeiträge

Originalarbeiten in wissenschaftlichen Fachzeitschriften

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2023

• 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, Band: 23, Seiten: 2623 - 2632
• 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, Band: 148, Seiten: 5243 - 5254

2022

• 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, Band: 12, Nummer: 4, Seite: 237

2020

• 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, Band: 92, Nummer: 19, Seiten: 12833 - 12841
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  Kurzfassung

  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.
• 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, Band: 20, Seiten: 2549 - 2561
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  Kurzfassung

  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.
• 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, Band: 25, Nummer: 8, Seite: 1914
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  Kurzfassung

  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.

Konferenzbeiträge

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2024

• M. Neugebauer, S. Calabrese, A. Markl, L. Schumacher, T. Wolper, T-T. Truong, P. Juelg, N. Borst, T. Hutzenlaub, F. Scherer, F. von Stetten, M. Lehnert
  Increasing digital PCR performance in liquid biopsy using reporter emission multiplexing
  2024 13th CNAPS International Symposium on Circulating Nucleic Acids in Plasma and Serum, Graz/Austria, 4-6 March 2024
• S. Calabrese, A. Markl, M. Neugebauer, L. Schumacher, N. Borst, F. Scherer, F. von Stetten, M. Lehnert
  Reporter emission multiplexing (REM) Increases detection capacities of digital PCR devices leading to sensitive, precise and specific liquid biopsy assays
  2024 EUDIP 2024 - European Digital PCR Symposium 2024, Gent/Belgium, 6-8 February 2024

2023

• S. Murad, M. Heyer, F. Lickert, J. Menges, S. Calabrese, T. Hutzenlaub, N. Paust, P. Juelg
  Fast and bubble-free filling of nano-imprinted high-density picoliter well arrays for digital assays enabled by centrifugal microfluidics
  2023 MicroTAS 2023, Katovice/Poland, 15.-19.10.2023

2021

• Y.-T. Kao, S. Calabrese, N. Borst, M. Lehnert, Y.-K. Lai, F. Schlenker, R. Zengerle, P. Garstecki, F. von Stetten
  One-step amplification-free bacteria detection by optimizes LNA/DNA molecular beacons in droplets
  2021 MicroTAS 2021, Palm Springs/USA, 10.-14.10.2021, online

2020

• J. F. Hess, M. Kotrová, S. Calabrese, T. Hutzenlaub, R. Zengerle, M. Brüggemann, N. Paust
  Automated library preparation for next generation sequencing of immunoglobulin gene rearrangements by centrifugal microfluidics
  2020 MicroTAS 2020, 04.-09.10.2020, virtual

2018

• M. Schulz, N. Borst, M. Specht, S. Calabrese, F. von Stetten, R. Zengerle, N. Paust
  From nasal swab to digital answer: unit operations for antibiotic resistance screening on a single cell level
  2018 MicroTAS 2018, 11. -15. November 2018, Kaohsiung / Taiwan