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

Abstract

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.

Label‐free Sensing of Main Protease Activity of SARS‐CoV‐2 with an Aerolysin Nanopore

“… [7] Notably, the M pro can act as a natural SARS‐CoV‐2 biomarker, because it is not closely linked to any human protease. [8] While the fluorescent method enables the sensitive measurement of M pro activity and the colorimetric assay enables the visual detection of SARS‐CoV‐2, [9] they both require tedious labeling and cautious probe designs and have weak anti‐interference capabilities. In this context, the accurate, sensitive, and rapid evaluation of M pro activity will offer an efficient tool for the detection of SARS‐CoV‐2.…”

A Self-Immolative Fluorescent Probe for Selective Detection of SARS-CoV-2 Main Protease

“… 14 Another useful approach for detecting M pro is the colorimetric sensor. 14 , 17 , 19 , 20 One representative gold nanoparticle-based colorimetric protease assay was reported by Willis and coauthors to detect the exogenously expressed M pro in HEK 293T cells. 19 Meanwhile, based on the versatility of nanomaterials, 21 − 26 we developed sensitive colorimetric sensors for the detection of M pro .…”

“… 19 Meanwhile, based on the versatility of nanomaterials, 21 − 26 we developed sensitive colorimetric sensors for the detection of M pro . 17 , 20 , 27 However, these colorimetric sensors cannot be used to accurately visualize M pro activity in infected cells. As another approach, genetically encoded biosensors have generated several excellent sensors for M pro .…”

“…Despite that fact that the levels of detection (LoDs) of the electrochemical sensors can be as low as 0.1 pM, these sensors rarely detect M pro in real cell samples due to their low electrochemical stability in cells . Another useful approach for detecting M pro is the colorimetric sensor. ,,, One representative gold nanoparticle-based colorimetric protease assay was reported by Willis and coauthors to detect the exogenously expressed M pro in HEK 293T cells . Meanwhile, based on the versatility of nanomaterials, − we developed sensitive colorimetric sensors for the detection of M pro . ,, However, these colorimetric sensors cannot be used to accurately visualize M pro activity in infected cells.…”

“…Another useful approach for detecting M pro is the colorimetric sensor. ,,, One representative gold nanoparticle-based colorimetric protease assay was reported by Willis and coauthors to detect the exogenously expressed M pro in HEK 293T cells . Meanwhile, based on the versatility of nanomaterials, − we developed sensitive colorimetric sensors for the detection of M pro . ,, However, these colorimetric sensors cannot be used to accurately visualize M pro activity in infected cells. As another approach, genetically encoded biosensors have generated several excellent sensors for M pro . , Although these genetically encoded biosensors provide the selective and noninvasive detection of M pro , they can only be used in gene-edited cells.…”