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 IMPACT chip (Fig. 1b) consists of 6 cm long channels patterned with Polydimethylsiloxane (PDMS) micropillars. Fig. 1c shows the SEM image of the channel, revealing periodic curved nature of the channel and even spacing of the micropillars (diameter: 100 µm; height: 120 µm). After channel fabrication and sealing, surface treatment with APTES (3-Aminopropyl)triethoxysilane) and glutaraldehyde was performed for streptavidin immobilization. 20 After streptavidin coating, the static water contact angle decreases from ~105 degree to ~90 degree for a flat PDMS surface and ~138 degree to 110 degree for micropillar surface, respectively (Fig. 1d ). Fig. 1e shows the entire sample preparation and detection protocol. DNA reporter probes with a biotin label were conjugated on the streptavidin coated micropillars. CRISPR Cas12a complex was then introduced into the channel. With the presence of the ASFV target DNA, it cleaves the reporters from the micropillars for detection. The sequence of the DNA target and the crRNA are listed in Fig. 1f . The target was selected based on the PAM region in the ASFV-SY18 genome. 21 It has been shown that auto-fluorescence occurs from the chemical treatment of APTES and glutaraldehyde. 22 In our case, we were able to confirm the phenomenon was specific to glutaraldehyde and APTES ( Fig. SI 1a) , and use it to show that it only occurs on PDMS in the presence of these chemicals (Fig. SI 1b) . Fig. 1g shows the uniformity of the chemical treatment on the surface, as the intensity of the auto-fluorescence (emission: 525 nm) is uniform throughout. We then incubated the reporter probes (emission: 668 nm) in the channel for 3 hrs and showed uniform coating in the channel (Fig. 1h ).