For the Sensing of Viral DNA: An Integrated Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System

Abstract

Infectious disease outbreaks have become more frequent and extreme in recent years, and as populations continue to grow and the world becomes more interconnected, they show no signs of stopping. The current COVID-19 pandemic affecting the world and grinding economies to a halt was known about months ago but could not be contained. One of the largest issues facing the containment of infectious disease is a lack of real-time, point-of-care detection devices which can accurately and effectively identify those who are infected so they can be treated and quarantined. Here, an Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) system is developed for detection of viral DNA. Single-stranded DNA reporter probes with fluorescent dyes are immobilized within the system, taking advantage of the increased surface area from the micropillar. A CRISPR-Cas12a and crRNA complex is then injected into the system, and if double-stranded target DNA is present, the CRISPR enzyme is activated and indiscriminately cleaves reporter probes, greatly increasing the fluorescent signal. The system can then be washed and the supernatant collected and measured, revealing accurate detection of the viral DNA target down to 0.1 nM concentration with no fluorescence background.