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 offers a simple one-step detection strategy and does not require off-chip incubation. Traditional nucleic acid based detection requires stringent multi-probe hybridization and washing thus is slow and complicated. 33, 34 In this work, the CRISPR complex is quickly mixed with the target sample and then the CRISPR/crRNA/target DNA complex is injected into the channel for on-chip detection. The chip sits on a hotplate set at 37°C and does not need thermal cycling with temperature control like PCR does. 35, 36 Thus, the detection system we developed is much simpler and more compact, ideal for POC applications. 37,38 To extend the detection limit, our system can be integrated with isothermal amplification methods such as recombinase polymerase amplification for sensitive "one pot" target amplification and CRISPR reaction, without the background issues seen in traditional "one-pot" methods. 17, 39 An advantage of the IMPACT chip compared to other detection apparatuses such as the SHERLOCK test strip is that it is a fully enclosed system without extra packaging need. 40,41 This is advantageous as the treated micropillars are sealed before molecular diagnostics. The reporter probes are immobilized within the channel, avoiding degradation issues from outside contaminants. This is particularly important when dealing with RNA target as it is susceptible to degradation by the presence of RNase. 42 Our fully integrated chip without special packaging needs is able to avoid RNase exposed in air, dust, and human hands in POC settings.