Collaborative Research: Ultrasensitive Nucleic Acid Sensing Tools Based on Cas Assays and Solid-State Nanopores

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The urgent need for rapid, inexpensive, and convenient methods to detect viruses has been clearly evidenced by the onset of Covid-19, which caused the death of over 2 million prople worldwide from mid November 2019 to mid January 2021, and continues to take its toll on human life. The 2020 Nobel Prize winning CRISPR/Cas technology, which can be used to rapidly detect DNA sequences in any living organism, offers a promising approach. This approach has been pursued by many companies, but none to date has been able to match the sensitivity of the “gold standard” test (real-time polymerase chain reaction (RT-PCR)), which requires 4-6 hours for completion and costs ~$100 per test. Thus the goal of this project is to develop a method for SARS-CoV-2 (the virus responsible for the COVID-19) detection that is faster, cheaper, more sensitive, and more convenient than the methods presently used for SARS-CoV-2 detection. The project’s goals will be achieved by integrating CRISPR/Cas assays with cutting-edge technologies. Limitations of existing systems will be addressed using a number of advanced analysis tools, advanced devices, artifical inteligence, and novel nanomaterial probes to design an integrated nanopore-microfluidic device for use in point-of-care (POC) settings that is ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end users). Succesful development of this sensor platform will offer a wide range of other uses, as the principles behind it may be applied to other applications that are not related to SARS-CoV-2. The project creates excellent opportunities for interdisciplinary research, as it combines biochemistry, nanoengineering, photonics, and medicine. Outreach programs related to this exciting project will be offered to K-12 schools, attracting young minds and inspiring them to pursue science, technology, engineering and mathematics (STEM) degrees. The goal of this project is to develop a highly sensitive and reliable nucleic acid sensing tool based on CRISPR/Cas assays for SARS-CoV-2 detection. The research will reveal the cleavage activities of Cas enzymes on a variety of composite nanomaterial reporter designs. Solid-state nanopores will be optimized for reading the cleavage patterns of nanomaterial reporters in the Cas assays using a deep neural network to classify the cleavage signatures. Solid-state nanopore readout provides single-molecule quantification and also identifies molecular signatures within the translocating molecules, which has significant advantages over the standard readout methods of today (fluorescence, paper-strip, colorimetric, and electrochemical readout). Once the cleavage activities are understood, a variety of reporters whose cleavage patterns correspond to specific target sequences will be designed. Identification of the cleavage products will enable the development of an integrated nanopore-microfluidic device for use in POC settings that will demonstrate simultaneous nanopore and fluorescence readings of cleavage products in multiplexed CRISPR/Cas assays.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.