Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

Kenneth N. Hass; Mengdi Bao; Qian He; Myeongkee Park; Peiwu Qin; Ke Du

Solid-phase detection assays have been developed as one potential solution to overcome this issue presented in the liquid phase. On the solid surface, reporter probes do not require a quencher since they are only measured in the liquid phase after degradation, thus no fluorescent signal will be detected without the target DNA present in the assay. However, an extended surface with a larger surface area is always needed to increase the probe binding capacity. It has been shown that these extended surface can increase the amount of probe binding, lower the detection limit of the target of interest, and extend the detection dynamic range. 18, 19 Here, we present a fully enclosed Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) system for nucleic acid target detection. The reporter probes were firstly immobilized in the enclosed channel and the CRISPR complex was pumped into the system for reaction (Fig. 1a) . Leveraging the high activity of CRISPR-Cas12a enzyme and the ability of micropillars to bind more reporter probes, we successfully detect double-stranded DNA target without background issues. In addition, the IMPACT chip requires low volumes of reagents, operates in a one-step detection fashion, and does not require complicated temperature control, making it ideal for POC applications. To prove the concept of the IMPACT chip for viral DNA detection, the target sequence selected for the crRNA was synthesized after a segment of the ASFV-SY18 genome (B646L). In the future, our device could work with any RNA or DNA based pathogens, such as the emerging COVID-19.