Rapid distribution of a liquid column into a matrix of nanoliter wells for parallel real-time quantitative PCR

A simple and reliable microfluidic method to load and seal a two-dimensional matrix of nanoliter wells, preloaded with primer pairs, for performing PCR without well-to-well cross-contamination was developed. With a vacuum aided microfluidics the distribution of sample mixture into 100 wells was achieved within 0.2 s, and all the wells were fluidically isolated within 0.5 s. The wells were subsequently sealed with poly-(dimethylsiloxane) (PDMS) prepolymer to prevent evaporation of PCR mixture and primer cross-contamination. The performance of the PDMS matrix chip was successfully tested by simultaneous (parallel) real-time quantitative PCR amplification of 10 different gene targets against cDNA template from human hepatocellular carcinoma, in 120 nl volume using EvaGreen fluorescent dye. Negligible cross-contamination between primers in different wells was confirmed with the PCR result, and by investigating the diffusion of the primers. The microfluidic channel design, chip structure, vacuum level, and operation schedule were optimized to achieve successful vacuum aided microfluidics for PCR mixture loading without air trapping. The microfluidics and the well matrix chip platform are suitable for both low- and high-density well applications including PCR related applications as well as immunoassays and cell-based assays.