Integrated antigenic and nucleic acid detection in single virions and virion-infected host-derived extracellular vesicles

The coronavirus disease of 2019 (COVID-19) led to the rapid development of novel assays to improve sensitivities for detecting the virion responsible for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite that, there have been over 767 million reported cases and over 6.9 million deaths worldwide. Therefore, tunable, sensitive, and high-throughput assays are warranted to control future outbreaks. Herein, we developed a tunable in situ assay to selectively sort virions and infected host-derived extracellular vesicles (IHD-EVs) and simultaneously detect their antigens and nucleic acid cargo at a single-particle resolution. The Biochip Antigen and RNA Assay (BARATM) integrates positive immunoselection and infection dynamics to sort particles, immunofluorescence to detect antigens, and fluorescence in situ hybridization to detect nucleic acids. BARATM enhanced sensitivities by detecting biomolecular signatures at the single-particle level, enabling the detection of virions in asymptomatic patients, and genetic mutations in single SARS-CoV-2 virions. Furthermore, BARATM revealed the continued long-term expression of virion-RNA in the IHD-EVs of post-acute sequelae SARS-CoV-2 infection (PASC) patients. BARATM was validated on saliva (30 healthy donors and 33 symptomatic and 20 asymptomatic patients) and nasopharyngeal swabs (19 healthy donors and 40 patients), revealing a highly accurate diagnosis by simultaneously detecting the spike glycoprotein and nucleocapsid-encoding RNA on single SARS-CoV-2 virions with sensitivities of 100 % and 95 %, respectively, and specificities of 100 % for both biofluids. Altogether, the single-particle detection of antigens and virion-RNA provides a tunable framework for the diagnosis, monitoring, and mutation screening of current and future outbreaks. Significance Statement Viral outbreaks are imminent and rapidly translatable assays are necessary to keep contagion at bay. The Biochip Antigen and RNA Assay (BARATM) enables the simultaneous detection of antigens and nucleic acid cargo in virions and extracellular vesicles (EVs) from infected host cells at a single-particle resolution. Detecting single particles enhances sensitivities and specificities, enables long-term disease, and latent state monitoring, and provides a unique perspective into genetic mutations and virion lineages. Our work provides evidence for the utility of single-particle technologies as multifaceted diagnostic assays, providing more comprehensive information than traditional diagnostics. Classification Biological Sciences (Applied Biological Sciences) ### Competing Interest Statement E.R. and K.T.N. have filed a patent application for the BARATM technology. ### Funding Statement This work was supported by the U.S. National Institutes of Health (NIH) grants UG3/UH3TR002884 (E.R.) and U18TR003807 (E.R., L.J.L., P.P., K.W., & I.L.). Additional support for E.R. was provided by the William G. Lowrie Department of Chemical and Biomolecular Engineering and the James Comprehensive Cancer Center at The Ohio State University. Additional supports include Kaplan Cancer Research Fund (H.G.K.), Wilke Family Foundation (J.R.H.), the Murdock Trust (J.R.H.), the Parker Institute for Cancer Immunotherapy (J.R.H.), Merck and the Biomedical Advanced Research and Development Authority under Contract HHSO10201600031C (J.R.H.), the Swedish Medical Center Foundation (J.D.G.), DOD W911NF-17-2-0086 (K.W. & I.L.). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study relied on patient samples and was approved by the Biomedical Sciences Committee at The Ohio State University (Institutional Review Board protocol 2021H0246). Any patient identifiers were not known to anyone outside the research group to ensure patient privacy. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.