LAPSE:2022.0004
Published Article
LAPSE:2022.0004
Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations
Franziska Schlenker, Elena Kipf, Nadine Borst, Nils Paust, Roland Zengerle, Felix von Stetten, Peter Juelg, Tobias Hutzenlaub
January 24, 2022
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 four-color 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.
Keywords
centrifugal step emulsification, digital droplet polymerase chain reaction (ddPCR), droplet fluorescence evaluation, droplet stability, microfluidics, multiplexing
Subject
Suggested Citation
Schlenker F, Kipf E, Borst N, Paust N, Zengerle R, von Stetten F, Juelg P, Hutzenlaub T. Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations. (2022). LAPSE:2022.0004
Author Affiliations
Schlenker F: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany
Kipf E: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany
Borst N: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Laboratory for MEMS Applications, IMTEK—Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany
Paust N: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Laboratory for MEMS Applications, IMTEK—Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany
Zengerle R: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Laboratory for MEMS Applications, IMTEK—Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany
von Stetten F: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Laboratory for MEMS Applications, IMTEK—Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany
Juelg P: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany [ORCID]
Hutzenlaub T: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Laboratory for MEMS Applications, IMTEK—Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany
Journal Name
Processes
Volume
9
Issue
1
First Page
pr9010097
Year
2021
Publication Date
2021-01-05
Published Version
ISSN
2227-9717
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Original Submission
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PII: pr9010097, Publication Type: Journal Article
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LAPSE:2022.0004
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doi:10.3390/pr9010097
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Jan 24, 2022
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Jan 24, 2022
 
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Calvin Tsay
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