Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids

Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

The "BSA strategy" can similarly ensure the success of RPA reactions in the picoliter wells, but it cannot eliminate cross-contamination that may occur between adjacent microwells owing to the edge-to-edge spacing of only 20 μm. There may be a thin liquid film on the chip surface after scraping PRA reagents into the picoliter wells. Although it will evaporate quickly, i.e., in 10-30 s, before oil sealing, the precise evaporation time is unknown because it is related to environmental humidity and temperature, which vary among experiments. The DNA amplification products enter adjacent microwells by residual liquid passage; thus, many clusters of positive RPA wells appear gradually from 5 min to 10 min with cross-contamination on the non-silanization chip (Fig 3a) . Passivating the PWA chip surface by a silanizing agent may effectively eliminate cross-contamination during dRPA. Methoxy-PEG-silane could produce a compact monomolecular layer on the SiO 2 surface by covalent binding (Fig 2c) , which can protect PCR enzymes from being adsorbed and does not interfere with PCR reactions [72] . Additionally, it is highly compatible with mineral oil for sealing and fixes water molecules by hydrogen bond interactions. Accordingly, even when minor residual liquid remains after evaporation for 20 s, it is unable to flow on the methoxy-PEG-silane surface and connect adjacent microwells. There is no cross-contamination on the silanization chip; positive wells with a single DNA template amplify independently and the fluorescence intensity for adjacent negative wells does not change (Fig 3b) . Moreover, the silanization of the PWA chip is very easy to perform, unlike the complicated modifications required for OpenArray, with a hydrophilic interior surface and hydrophobic exterior surface [72] .