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

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.17.994137 doi: bioRxiv preprint Figure 3 : Released reporter probe intensity versus incubation time (10 min to 24 hrs). Treated samples received surface modification and streptavidin treatment before photocleavable reporter probe incubation (1x10 6 nmoles). The sample was retrieved via UV photocleavage. Error bars are standard deviation of the mean We combined the DNA probe modified micropillar channel with CRISPR Cas12a assay for solid-phase and background free viral DNA sensing. We first mixed CRISPR Cas12a/crRNA/target DNA in an Eppendorf tube and then injected the complex into the IMPACT chip and allowed it to incubate for 2 hrs. The activated complex diffuses in the microchannel and nonspecifically cleaves the reporter probes from the micropillars. The uncorrected emission curve and the integrated fluorescence signal of the CRISPR experiments are shown in Fig. 5a and 5b, respectively. The measured fluorescence intensity linearly increases with the target concentration ranging from 0.1-10 nM (Pearson's R=0.9653). On the other hand, the supernatant without any ASFV target DNA input does not present a fluorescence signal as the CRISPR Cas12a cannot be activated, demonstrating a fully enclosed and efficient microdevice for background-free viral DNA quantification.