Constant Pressure-Regulated Microdroplet Polymerase Chain Reaction in Microfluid Chips: A Methodological Study.

ABSTRACT

Digital polymerase chain reaction (PCR) technology in microfluidic systems often results in bubble formation post-amplification, leading to microdroplet fragmentation and compromised detection accuracy. To solve this issue, this study introduces a method based on the constant pressure regulation of microdroplets during PCR within microfluidic chips. An ideal pressure reference value for continuous pressure control was produced by examining air solubility in water at various pressures and temperatures as well as modeling air saturation solubility against pressure for various temperature scenarios. Employing a high-efficiency constant pressure device facilitates precise modulation of the microfluidic chip's inlet and outlet pressure. This ensures that air solubility remains unsaturated during PCR amplification, preventing bubble precipitation and maintaining microdroplet integrity. The device and chip were subsequently utilized for quantitative analysis of the human epidermal growth factor receptor (EGFR) exon 18 gene, with results indicating a strong linear relationship between detection signal and DNA concentration within a range of 101-105 copies/µL (R2 = 0.999). By thwarting bubble generation during PCR process, the constant pressure methodology enhances microdroplet stability and PCR efficiency, underscoring its significant potential for nucleic acid quantification and trace detection.