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 widespread impact of the current coronavirus (COVID-19) is a striking indicator of the fact that the global community is struggling to battle infectious diseases. Failure to contain the virus early has resulted in an outbreak that has infected over 120,000 people with over 3,000 deaths. 1, 2 The USA CDC predicts that a nationwide epidemic is unavoidable. 3 The current situation highlights a urgent need for access to real-time detection that is accurate and effective to identify those infected so they can be properly quarantined and treated. 4, 5 An effective vaccine could be the best solution to contain epidemics. However, vaccines take a very long time to develop, as evidenced with the African swine fever virus (ASFV), which was initially discovered in 2018 but only had a preliminary vaccine announced in January of this year. 6 It took over an additional month to show its effectiveness in laboratory tests, and it still needs to be proven effective in the field. 7 Currently, there are no proven treatments for either COVID-19 or ASFV. Even if one were to be developed soon, it can take years to prove its effectiveness in clinical trials and mass produce the vaccine, not to mention distribute and administer it in affected areas. 8 To contain and prevent the spread of these contagious outbreaks, a rapid point-of-care (POC) testing device is essential. Current methods can take up to two days for tittering in a centralized laboratory in order to diagnose whether a sample contains the disease, and is not viable and inefficient when trying to isolate those infected and prevent them from spreading the disease to others. 9,10 In addition, the collected patient samples need to be sent to a centralized laboratory, leading to very long turnaround times and greatly limiting the number of people who could be tested and confirmed. 11 Strides have been made to develop POC detection methods which can be deployed in the field, and currently lab-on-chip (LOC) devices or simple test kits which involve real-time polymerase chain reaction (PCR) have emerged as one of the leading choices for meeting the desired criteria. 12 PCR is particularly sought after due to its ability to amplify the viral RNA/DNA from a few copies to billions. 13 It has also been shown to work with clinical samples in LOC devices with low volumes of both reagents and patient samples. 14 However, there have been many issues with implementing PCR test kits, such as a shortages in test kits and trained professional to use them, as well as potential inaccurate results from the kits. 15, 16 Clustered regularly interspaced short palindromic repeats (CRISPR) provides an alternative to PCR amplification techniques for the detection of viral RNA/DNA. Certain Cas proteins, such as CRISPR-Cas12a, have been shown to be powerful in biological detection due to their ability to indiscriminately cut single-stranded DNA (ssDNA) after they are activated by a target DNA. 17 This is extremely useful when paired with "reporter probes" (ssDNA strands with a fluorescent dye and quencher attached to them), as the CRISPR complex can cleave the reporter probe and release the dye for fluorescence quantification. However, one of the issues with utilizing the CRISPR complex is the high fluorescence background signal associated in the sample as the quencher cannot fully quench the fluorescent dye.