A Prototype Assay Multiplexing SARS-CoV-2 3CL-Protease and Angiotensin-Converting Enzyme 2 for Saliva-Based Diagnostics in COVID-19

Abstract

With the current state of COVID-19 changing from a pandemic to being more endemic, the priorities of diagnostics will likely vary from rapid detection to stratification for the treatment of the most vulnerable patients. Such patient stratification can be facilitated using multiple markers, including SARS-CoV-2-specific viral enzymes, like the 3CL protease, and viral-life-cycle-associated host proteins, such as ACE2. To enable future explorations, we have developed a fluorescent and Raman spectroscopic SARS-CoV-2 3CL protease assay that can be run sequentially with a fluorescent ACE2 activity measurement within the same sample. Our prototype assay functions well in saliva, enabling non-invasive sampling. ACE2 and 3CL protease activity can be run with minimal sample volumes in 30 min. To test the prototype, a small initial cohort of eight clinical samples was used to check if the assay could differentiate COVID-19-positive and -negative samples. Though these small clinical cohort samples did not reach statistical significance, results trended as expected. The high sensitivity of the assay also allowed the detection of a low-activity 3CL protease mutant.

Keywords: COVID-19; enzymatic assays; proteases.

Scheme 1

Design of a selective substrate and its usage in a sequential dual-protease assay allowing measurement of ACE2 and 3CL protease activity in the same sample.

Figure 1

(A). Selectivity of the assay for a panel of additives; soybean trypsin inhibitor used at 1 mg/mL; all other proteins were used at 1 µM; EDTA and DTT were used at 1 mM; saliva matrices were used at 1:3 ratio with Buffer I; (B). 3CL protease concentration dependence of the assay; Y−axes normalized to concentration of product generated in the reaction using a pre-generated standard curve of the substrate dye (Figure S2); (C). fluorescent assays showing enzyme concentration dependence under final conditions in Buffer II (150 mM NaCl, 1 mM EDTA, 1 mg/mL soybean trypsin inhibitor), or 1:3 mixtures of saliva matrices and Buffer II.

Figure 2

(A). Assay schematic in which ACE2 activity is first measured followed by 3CL protease immediately after in the same sample; (B). fluorescence background generated by ACE2 reaction in different matrices; (C). 3CL protease fluorescence readout standardized for product formation in the presence of the background generated in the ACE2 assay prior.

Figure 3

(A). Twenty-minute 3CL protease assays differentiate blank and low−concentration samples even after a pre−measurement freeze–thaw cycle; (B). at 62 nM or above, 3CL protease samples can be differentiated by fluorescence from blank samples after ACE2 measurement; (C). SERS allows more sensitive detection down to 15.6 nM 3CL protease, even after ACE2 reaction; (D). SERS spectra associated with these samples. * p < 0.05, ** p < 0.01, *** p < 0.001 according to two-tailed paired heteroscedastic t-tests between samples containing 3CL protease and ones that do not.

Figure 4

(A). Fluorescent assays show the potential activity of a mutant enzyme (termed monomer here) with a disrupted dimer interface. (B). Quantified SERS activity for the monomer and WT enzyme. SERS further identified similar enzyme activity at a lower concentration of the mutant enzyme. (C). SERS spectra are generated by reactions of the monomer enzyme. (D). MALDI-TOF MS spectra of a reaction of 1 µM wild-type 3CL protease. (E). MALDI-TOF MS spectra of a reaction of 25 µM mutant monomer 3CL protease. Statistical significance was determined using two−tailed paired heteroscedastic t-tests between samples containing enzymes and ones that do not.