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 sample loading instrument included a scraping liquid blade and a chip carrier. Detailed operation steps are shown in Fig 1. The scraping liquid blade was composed of a hard glass slide and a piece of soft silica gel (thickness, 3 mm), which were pasted together at an end; the chip carrier was composed of another glass slide and a 3M adhesive tape, which prevented the chip from sliding in the process of scraping (Fig 1a) . The prepared 20-μL digital RPA reagents were carefully transferred onto one end of the PWA chip by a pipette. Depressing the pipette to the second stop was unsuitable for reducing the introduction of air bubbles to the chip. The blade was held at a 40-60°angle relative to the chip carrier so that the edge of silica gel was placed at the end of PWA chip. The angle of the blade was adjusted slightly until visual confirmation that RPA reagents were wetting the chip. Then, in one smooth motion, the blade was dragged slowly across the chip, while a slight downward pressure was applied to dispense the reagent ( and 1b). The scraping time was approximately 5 s. The PWA chip sat at room temperature for 20 s for residual liquid evaporation. The chip was then gently overlaid with excess mineral oil (M8410; Sigma-Aldrich, St. Louis, MO, USA) via a disposable pipette until the entire surface was fully covered (Fig 1b) . Mineral oil was used because it is lighter than water (0.82-0.88 g mL −1 ) and its solubility in water is extremely low [9] . It makes the RPA reaction independent of each other. Therefore, the subsequent packaging operation can not affect the accuracy of dRPA results, even at room temperature. The copper chamber was filled with mineral oil prior to transferring and packaging the finished PWA chip after sample loading (Fig 1c and 1d) . Finally, a piece of 2.5-mm-thick quartz glass cover-plate was fixed on the copper chamber by four screws (Fig 1e and 1f) . The whole process avoided air bubbles. To strengthen the air tightness between the glass and the copper chamber, a rubber O-ring was added around the chamber. The bright images of the sample loading and chip packaging process can be found in S2 Fig.