High filling rate digital PCR through-hole array chip with double independent S-shaped flow channels

Sample digital technology is a powerful method for absolute quantification of target molecules such as nucleic acids and proteins. The excellent sample stability and mass production capability has enabled the development of microwell array-based sample digitizing methods. However, in current microwell array chips, samples are loaded by the liquid scraping method, which requires complex manual operation and results in a low filling rate and limited hole filling uniformity. Here, we perform sample loading of a through-hole array chip by a microfluidics-driven method and design a double independent S-shaped flow channels sandwiched through-hole array chip. Because of the capillary force and capillary burst pressure, the sample flowing in the channel can be trapped into through-holes, but cannot flow through the other side. Via air flow and displacement of the remaining sample in the channel, the sample can be partitioned consistently, with zero surplus sample residue in the channel. We evaluated the actual performance of the sample-loading process: the chip enables 99.10% filling rate of 18 500 through-holes, with a grayscale coefficient of variation value of 6.03% determined from fluorescence images. In performing digital polymerase chain reaction on chip, the chip demonstrates good performance for the absolute quantification of target DNA. The simple and robust design of our chip, with excellent filling rate and microsample uniformity, indicates potential for use in a variety of sample digitization applications.