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 modified a commercial fluorescence microscope to minimize the distance between the optical cube and the PWA chip, which ensures sufficient light intensity to fully excite the fluorescence signal to obtain wide-field, high-resolution images. This method affords several significant advantages over previous approaches: (a) The macro-fluorescence imaging setup is capable of viewing 6-cm 2 areas in a single snapshot at 0.8× magnification, without the need to obtain images by scan shooting and stitching serial small images together [34] . Thus, image acquisition and processing are rapid. (b) This approach allows the excitation light to vertically illuminate the chip through the optical cube and avoids flatfield corrections due to oblique beam illumination [74] . An uneven light field affects the quantification results. (c) The widefield images are captured in situ, enabling real-time fluorescence detection. Real-time fluorescence imaging of RPA amplification is demonstrated in Fig 4 for a 20 -min period at 5-min increments (these images are enlarged sections from the wide-field fluorescence images for enhanced visualization).