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

A wide-field, high-resolution imaging setup was required to adequately resolve all 27,000 picoliter wells. As illustrated in Fig 2a, large field of view images were captured using an Olympus microscope SZX7 (Olympus, Tokyo, Japan) and a monochrome cooled camera MC21 (Microshot Technology, Guangzhou, China) equipped with a 1.4 million pixel high-resolution CCD sensor (SONY, Tokyo, Japan). A resolution of 16 pixels per microwell can be achieved for the whole PWA chip. These were not sufficient to generate greater than 20 mW cm -2 light intensity on the entire chip area, which is necessary to excite the fluorescence signal for detection by the CCD camera. To address this problem, an optical cube was placed between the chip and object lens to shorten the path of excitation light to reach the fluorescent material. This improvement could reduce the loss of light intensity using a conventional microscope. The cube filters (Chroma Technology Corp., Bellows Fall, VT, USA) were composed of a 505-nm dichroscope, a filter set of 480/30 nm for fluorescence excitation and 535/40 nm for emission collection of the FAM dye, and were positioned 10 mm above the chip packaging device. The light source, a high-power 3 W blue LED bead (Micro-shot Technology, Guangzhou, China), was coupled to the cube in a horizontal direction.