Microsyst Nanoeng. 2020 Oct 5;6:82. doi: 10.1038/s41378-020-00187-1. eCollection 2020.
ABSTRACT
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 °C, and an application-specific prestorage of reagents within the 25 nl 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 < 6 × 10-2 and a cross talk rate < 1 × 10-3 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.
PMID:34567692 | PMC:PMC8433415 | DOI:10.1038/s41378-020-00187-1
