Peptidic Sulfhydryl for Interfacing Nanocrystals and Subsequent Sensing of SARS-CoV-2 Protease

There is a need for surveillance of COVID-19 to identify individuals infected with SARS-CoV-2 coronavirus. Although specific, nucleic acid testing has limitations in terms of point-of-care testing. One potential alternative is the nonstructural protease (nsp5, also known as M-pro/3CL(pro)) implicated in SARS-CoV-2 viral replication but not incorporated into virions. Here, we report a divalent substrate with a novel design, (Cys)(2)-(AA)(x)-(Asp)(3), to interface gold colloids in the specific presence of M-pro leading to a rapid and colorimetric readout. Citrate- and tris(2-carboxyethyl)phosphine (TCEP)-AuNPs were identified as the best reporter out of the 17 ligated nanoparticles. Furthermore, we empirically determined the effects of varying cysteine valence and biological media on the sensor specificity and sensitivity. The divalent peptide was specific to M-pro, that is, there was no response when tested with other proteins or enzymes. Furthermore, the M-pro detection limits in Tris buffer and exhaled breath matrices are 12.2 and 18.9 nM, respectively, which are comparable to other reported methods (i.e., at low nanomolar concentrations) yet with a rapid and visual readout. These results from our work would provide informative rationales to design a practical and noninvasive alternative for COVID-19 diagnostic testing.the presence of viral proteases in biofluids is validated.

Automated summary

This study reports a divalent substrate that can interact with M-pro, facilitating the rapid and visual detection of SARS-CoV-2 coronavirus in COVID-19. The method shows comparable sensitivity to other reported methods and exhibits high specificity to M-pro.