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

In the scraping process, a relatively slow operation is suitable to fill microwells with the liquid reagent; otherwise, the wells may contain air bubbles in the bottom. Once heated, the bubbles rush out, diffuse, and cause reaction failure on the PWA chip. In contrast to previously performed dRPA reactions on the SlipChip, the PWA chip works without precise "slip" control [66] . If the SlipChip reduces the microwell volume and increases the density rapidly for a high-throughput detection like the PWA chip, the complexity and precision of the "slip" operation would increase rapidly, too. In contrast to previously performed dRPA reactions in droplets produced by centrifugal step emulsification, the droplets of the PWA chip are more consistently sized, avoiding the uneven droplets volumes affecting the quantification results, and their volumes are smaller, i.e. picoliter-sized [67] . Reagent consumption for each experiment on the PWA chip was less than 20 μL. A small reaction volume not only contributes to powerful and high-throughput detection, but also generates much less reagent waste and reduces the cost of each reaction.