Multi-parametric Integrated Molecular Detection of SARS-CoV-2 from Biofluids by Adapting Single Extracellular Vesicle Characterization Technologies

AbstractThe World Health Organization has recognized a global pandemic of novel coronavirus pneumonia (COVID-19)from exposure to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses (CoVs)are membrane-enveloped positive-sense, single-stranded RNA viruses decorated with membrane proteins. Thespike (S) glycoprotein is implicated in the viral attachment and fusion to host cells via the human angiotensin-converting enzyme 2 (hACE2). There are different assays to test for COVID-19, including nucleic acid, antigen,and serological tests that can be used in hospitals, point-of-care, and large-scale population testing. Nucleic acidtesting is the standard method for the detection of SARS-CoV-2, which consists of the amplification of viral RNAfrom nasopharyngeal swabs (NPS) by quantitative reverse-transcription polymerase chain reaction (qRT-PCR).Furthermore, given the invasive nature of NPS, saliva is being considered an alternative for detection. Methodsthat bypass RNA extraction, as well as isothermal amplification such as loop-mediated isothermal amplification(LAMP), have been developed to improve the speed of viral RNA detection. However, viral protein expressioncannot be detected by qRT-PCR. Serological tests, on the other hand, are based on host antibodies against thevirus (IgG/IgM). Although fast, these tests suffer from significant false negative/positive. Besides, they do notdetect a current infection. Therefore, to relieve the current healthcare crisis, new technologies capable ofsimultaneous viral RNA/protein detection at the single virus level and host antibody response detection from abody fluid in an integrated device would be highly valuable for enhanced COVID-19 diagnosis.Recently, our group, as part of Phase 2 of the Extracellular RNA Communication Consortium (ERCC2), hassuccessfully developed a microfluidics technology capable of capturing individual exosomes from biofluids andthen simultaneously quantify both exosomal surface proteins and RNA cargo. Given the resemblance in sizeand other characteristics between exosomes and coronaviruses, our technology can be adapted for COVID-19diagnosis. Therefore, we propose to develop and validate a safe-to-use version of our microfluidics system fordirect detection of SARS-CoV-2. The integrated system is capable of multi-parametric detection for enhancedCOVID-19 diagnosis. The platform will be engineered to simultaneously quantify both viral protein, viral RNA,and host antibodies (IgG/IgM) in the same sample, enabling diagnosis, disease status, and prognostic assessment. Model systems, including host IgG/IgM from patient serum, standard synthetic vesicles (SVs), andheat-inactivated SARS-CoV-2 viral particles (SVVs), will be designed and spiked in biofluids to validate andcalibrate the system. To demonstrate the clinical utility, our biochip technology will be deployed and tested usingdifferent biofluids from COVID-19 patients at two independent laboratories (Institute of Systems Biology inSeattle and The Ohio State University (OSU) Wexner Medical Center in Columbus). Measurements obtainedfrom the biochips will be compared to standard qRT-PCR and ELISA methods. A transition plan will be preparedfor FDA Emergency Use Authorization (EUA) application of the biochip technology through a COVID-19 clinicaltesting laboratory at OSU Wexner Medical Center. A commercialization plan will also be developed via licensingto a biotech company.We have assembled a multi-disciplinary team with extensive knowledge and experience in nanobiotechnology,microfluidics, micro/nano-fabrication, infectious diseases, and clinical COVID-19 patient sample collection andtesting. The proposed aims and milestones are given as follows:Specific Aim 1: Development of an integrated biochip to simultaneously capture, fix, and characterizesingle SARS-CoV-2 and IgG/IgM proteins. Milestones. (i) Sorting, capture, and quantitative analysis ofselected proteins and viral RNA in single virus in spike experiments with >95% repeatability; (ii) A sensitivity ofsingle virus detection with >90% repeatability and 5-fold better sensitivity than the current qRT-PCR and ELISAmethods. Specific Aim 2: Testing of single SARS-CoV-2 virus and associated IgG/IgM in biofluids fromCOVID-19 patients. Milestones. (i) Quantitative analysis of clinical samples with >95% repeatability; (ii) 95%of concordance for the detection of SARS-CoV-2 between the biochip technology and the lab-based qRT-PCRand ELISA. Specific Aim 3: Biochip technology transition plan. Milestones. (i) Submission of documentationto the FDA Center for Devices and Radiological Health (CDRH) for EUA; (ii) Scale-up commercialization plan forGMP chip production.