A fast, ultrasensitive SERS immunoassay to detect SARS-CoV-2 in saliva

The COVID-19 pandemic has emphasized the need for accurate, rapid, point-of-care diagnostics to control disease transmission. We have developed a simple, ultrasensitive single-particle surface-enhanced Raman spectroscopy (SERS) immunoassay to detect the SARS-CoV-2 spike protein in saliva. This assay relies on the use of single chain Fv (scFv) recombinant antibody expressed in E. coli to bind the SARS-CoV-2 spike protein. Recombinant scFv labeled with a SERS-active dye in solution is mixed with unlabeled scFv conjugated to gold-coated magnetic nanoparticles and a sample to be tested. In the presence of the SARS-CoV-2 spike protein, immunocomplexes form and concentrate the labeled scFv close to the gold surface of the nanoparticles, causing an increased SERS signal. The assay detects inactivated SARS-CoV-2 virus and spike protein in saliva at concentrations of 1.94 × 103 genomes mL−1 and 4.7 fg mL−1, respectively, making this direct detection antigen test only 2–3 times less sensitive than some qRT-PCR tests. All tested SARS-CoV-2 spike proteins, including those from alpha, beta, gamma, delta, and omicron variants, were detected without recognition of the closely related SARS and MERS spike proteins. This 30 min, no-wash assay requires only mixing, a magnetic separation step, and signal measurements using a hand-held, battery-powered Raman spectrometer, making this assay ideal for ultrasensitive detection of the SARS-CoV-2 virus at the point-of-care. [Display omitted] •We have developed an ultrasensitive SERS assay to detect the SARS-CoV-2 spike protein.•The assay detects virus at 1.94 × 103 genomes/ml or 4.7 fg mL−1 spike protein in saliva.•SARS-CoV-2 variants of concern, including delta and omicron, are detected.•Cross-reactivity is not detected with SARS-CoV and MERS-CoV spike protein.•The single particle assay is stable for at least two weeks in a standard refrigerator.