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

We developed a PWA chip with 27,000 consistently sized wells (314 pL) to perform isothermal DNA quantification using dRPA at 39°C for 20 min on a homemade isothermal heater. Sample loading using a scraping liquid blade was simple, fast, and consumed minimal reagents (< 20 μL). Passivating the PWA chip surface by a methoxy-PEG-silane agent eliminated crosscontamination among wells. Our optical design enabled wide-field fluorescence imaging in situ, with both end-point and real-time analysis of picoliter wells in 6-cm 2 areas during dRPA. We applied the PWA chip to accurately quantify serial dilutions of a L. monocytogenes gDNA stock solution. The dRPA reaction was robust and free of cross-contamination on the PWA chip, but the specificity of the device and strategies for multiplex detection remain to be examined. The digital PWA chip can be readily applied to other nucleic acid amplification techniques, such as PCR, RCA, ELISA, and LAMP. Fig. (a) Bright-field image of the sample loading instrument containing a chip carrier and a scraping liquid blade, displayed on a ruler to show scale. The scraping liquid blade is composed of a glass slide and a piece of silica gel (thickness, 3 mm), which are pasted together at an end; the chip carrier is composed of another glass slide and a 3M adhesive tape, which prevents the chip from sliding in the process of scraping. (b) After sample loading by scraping the RPA reagents into the picoliter wells array, let the PWA chip sit quietly in room temperature for 20 seconds for the little residual liquid evaporating. Then sealing the chip with excess mineral oil via a disposable pipette until the entire surface was fully covered. (c-e) Transferring the sample loading finished PWA chip from the chip carrier to the copper chamber filled with mineral oil. (f) Fixing the glass cover-plate on the copper chamber with screws. The rubber O-ring is added around the chamber to strengthen the air tightness. The whole process avoids air bubbles. Table. The sequences of the primers and probe for dRPA-on-chip (all in 5 0 ! 3 0 direction). (PDF)