title: Microstructuring of polymer films for sensitive genotyping by real-time PCR on a centrifugal microfluidic platform.
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 (microTSL). 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 microm 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 microl (CV 3.4%, N = 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 = 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.
authors.affiliation: Laboratory for MEMS Applications, Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Koehler-Allee 106, 79110 Freiburg, Germany.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Focke
authors.forename: Fabian
authors.initials: F
authors.lastname: Stumpf
authors.forename: Bernd
authors.initials: B
authors.lastname: Faltin
authors.forename: Patrick
authors.initials: P
authors.lastname: Reith
authors.forename: Dylan
authors.initials: D
authors.lastname: Bamarni
authors.forename: Simon
authors.initials: S
authors.lastname: Wadle
authors.forename: Claas
authors.initials: C
authors.lastname: Müller
authors.forename: Holger
authors.initials: H
authors.lastname: Reinecke
authors.forename: Jacques
authors.initials: J
authors.lastname: Schrenzel
authors.forename: Patrice
authors.initials: P
authors.lastname: Francois
authors.forename: Daniel
authors.initials: D
authors.lastname: Mark
authors.forename: Günter
authors.initials: G
authors.lastname: Roth
authors.forename: Roland
authors.initials: R
authors.lastname: Zengerle
authors.forename: Felix
authors.initials: F
authors.lastname: von Stetten
mesh_terms: D002498:Centrifugation / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005838:Genotype; D008567:Membranes, Artificial; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics* / Q000302:isolation & purification; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D011108:Polymers / Q000138:chemical synthesis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation*; D012680:Sensitivity and Specificity
chemical_list: D008567:Membranes, Artificial; D011108:Polymers