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 manufactured finished PWA chip was 20.8 mm × 16 mm, with 27,000 closely packed microwells. The height and diameter of the completed wells were 40 μm and 100 μm, respectively, with edge-to-edge spacing of 20 μm and volumes of 314 pL. A scanning electron microscope (SEM) image of the silicon picoliter well array is shown in Fig 2b. Native silicon is an inhibitor of PCR [70] ; accordingly, the chip surface was coated with a compact 200-nm-thick silicon dioxide (SiO 2 ) layer by thermal oxidation. However, the chip surface still absorbed polymerase protein and other reaction reagents, primarily owing to the great increase in the surface-to-volume ratio in the micro-scale environment, thus decreasing the DNA amplification efficiency. The conventional strategy involves treating the reaction chambers with 0.2% bovine serum albumin (BSA) solution at 80°C for 30 min before loading the PCR reaction mix [71] or adding 1 μL of 50 mg mL −1 BSA solution to 20 μL of PCR master mixture as a blocking protein to occupy the adsorption sites and reduce the loss of polymerase [9] .