Centrifugal microfluidic system for primary amplification and secondary real-time PCR.

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 self-contained 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.

Lab-on-a-Foil: microfluidics on thin and flexible films.

This critical review is motivated by an increasing interest of the microfluidics community in developing complete Lab-on-a-Chip solutions based on thin and flexible films (Lab-on-a-Foil). Those implementations benefit from a broad range of fabrication methods that are partly adopted from well-established macroscale processes or are completely new and promising. In addition, thin and flexible foils enable various features like low thermal resistance for efficient thermocycling or integration of easily deformable chambers paving the way for new means of on-chip reagent storage or fluid transport. From an economical perspective, Lab-on-a-Foil systems are characterised by low material consumption and often low-cost materials which are attractive for cost-effective high-volume fabrication of self-contained disposable chips. The first part of this review focuses on available materials, fabrication processes and approaches for integration of microfluidic functions including liquid control and transport as well as storage and release of reagents. In the second part, an analysis of the state of Lab-on-a-Foil applications is provided with a special focus on nucleic acid analysis, immunoassays, cell-based assays and home care testing. We conclude that the Lab-on-a-Foil approach is very versatile and significantly expands the toolbox for the development of Lab-on-a-Chip solutions.

Microstructuring of polymer films for sensitive genotyping by real-time PCR on a centrifugal microfluidic platform.

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.