Fusing MEMS technology with lab-on-chip: nanoliter-scale silicon microcavity arrays for digital DNA quantification and multiplex testing

We report on the development of a microfluidic multiplexing technology for highly parallelized sample analysis via quantitative polymerase chain reaction (PCR) in an array of 96 nanoliter-scale microcavities made from silicon. This PCR array technology features fully automatable aliquoting microfluidics, a robust sample compartmentalization up to temperatures of 95 degrees C, and an application-specific prestorage of reagents within the 25nl microcavities. The here presented hybrid silicon-polymer microfluidic chip allows both a rapid thermal cycling of the liquid compartments and a real-time fluorescence read-out for a tracking of the individual amplification reactions taking place inside the microcavities. We demonstrate that the technology provides very low reagent carryover of prestored reagents < 6x10(-2) and a cross talk rate < 1x10(-3) per PCR cycle, which facilitate a multi-targeted sample analysis via geometric multiplexing. Furthermore, we apply this PCR array technology to introduce a novel digital PCR-based DNA quantification method: by taking the assay-specific amplification characteristics like the limit of detection into account, the method allows for an absolute gene target quantification by means of a statistical analysis of the amplification results. Amplifying the scale of DNA amplificationAn integrated microfluidic device allows researchers to simultaneously detect and quantify multiple target DNA sequences in a single sample. A technique called the polymerase chain reaction (PCR) efficiently generates many copies of a given DNA sequence, even from tiny amounts of source material. Daniel Podbiel and colleagues at Robert Bosch GmbH in Germany have now developed a sophisticated device that can simultaneously perform many PCR reactions from one sample. Within their chip, the DNA is partitioned into multiple reaction wells, each of which can contain reagents needed to PCR amplify a different gene. These reactions are then performed on the chip, generating a fluorescence signal that can be detected and measured to reveal the amount of amplified DNA. Such a device could ultimately prove useful for conducting multi-gene analysis in medical diagnostics or other applications.