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

Alternatively, the emerging additive manufacturing technology can also be used to create such ultra-high aspect ratio microstructures as an efficient target capture platform. 27, 28 One of the main advantages of the IMPACT chip compared to traditional CRISPR assays is its ability to limit the background caused by dye-quencher probes, which is typically seen in CRISPR detection in the liquid state and needs to be designed around to lower the detection limit. 29, 30 Our device utilizing solid-phase CRISPR does not need to tether a quencher on the probe. The cleaved CRISPR products are sent to a separate reservoir for detection thus completely avoiding the fluorescence background caused by the tradition "onepot" detection. As shown in Fig. 5 , without the presence of ASFV target DNA, no fluorescence background signal was detected, demonstrating this powerful backgroundfree detection. This is an important improvement for molecular diagnostics, especially in low light settings in which the amount of background fluorescence present can significantly affect the detection limit . 31,32 Leveraging this advantage with increasing the aspect ratio either with PDMS or a silicon substrate will be able to further increase the accuracy and robustness of the device and improve the limit of detection.