title: Digital droplet PCR on disk.
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.
authors.affiliation: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany and Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Friedrich.Schuler@imtek.uni-freiburg.de.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Schuler
authors.affiliation: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Trotter
authors.affiliation: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Geltman
authors.affiliation: Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Friedrich.Schuler@imtek.uni-freiburg.de.
authors.forename: Frank
authors.initials: F
authors.lastname: Schwemmer
authors.affiliation: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany and Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Friedrich.Schuler@imtek.uni-freiburg.de.
authors.forename: Simon
authors.initials: S
authors.lastname: Wadle
authors.affiliation: Molecular Diagnostics Laboratory, Rioja Salud-Foundation, La Rioja, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Domínguez-Garrido
authors.affiliation: Molecular Diagnostics Laboratory, Rioja Salud-Foundation, La Rioja, Spain.
authors.forename: María
authors.initials: M
authors.lastname: López
authors.affiliation: Molecular Diagnostics Laboratory, Rioja Salud-Foundation, La Rioja, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Cervera-Acedo
authors.affiliation: Molecular Diagnostics Laboratory, Rioja Salud-Foundation, La Rioja, Spain.
authors.forename: Paula
authors.initials: P
authors.lastname: Santibáñez
authors.affiliation: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany and Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Friedrich.Schuler@imtek.uni-freiburg.de.
authors.forename: Felix
authors.initials: F
authors.lastname: von Stetten
authors.affiliation: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany and Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Friedrich.Schuler@imtek.uni-freiburg.de.
authors.forename: Roland
authors.initials: R
authors.lastname: Zengerle
authors.affiliation: Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany and Laboratory for MEMS Applications, IMTEK - Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Friedrich.Schuler@imtek.uni-freiburg.de.
authors.forename: Nils
authors.initials: N
authors.lastname: Paust
mesh_terms: D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000295:instrumentation*; D004655:Emulsions / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D010316:Particle Size; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D013499:Surface Properties
chemical_list: D004655:Emulsions; D004247:DNA