High-Density Microfluidic Chip with Vertical Structure for Digital PCR

Digital PCR, as a nucleic acid absolute quantification method at the single-molecule level, has been widely applied in early cancer screening, single-cell analysis, and other biomedical fields. However, existing digital PCR methods still suffer from high costs, complex operations, and low detection dynamic range, which limit their applications. In the study, we developed a microfluidic chip-based digital PCR with a high-density vertical structure using PDMS (polydimethylsiloxane) flexible material. The chip features a three-layer structure of glass–PDMS–glass, with the PDMS structural layer containing 30,000 reaction chambers, each with a volume of 0.713 nL. This vertical-structured chip can increase the total volume and the total number of chambers by 50% without changing the chip area and chamber volume, thereby significantly enhancing dynamic range and sensitivity of the chip detection. This chip is theoretically capable of achieving a nucleic acid detection dynamic range close to 105. Moreover, the digital PCR quantitative detection results of five different concentrations of serially diluted KRAS plasmid DNA templates using this chip also validated the accuracy and reliability of the nucleic acid quantitative detection results. The vertical-structured digital PCR chip, with its simple manufacturing process, uniform and stable sample partitioning, wide detection dynamic range, and low cost, will promote the widespread application of digital PCR.