Figure 4.

Tunning sensitivity and specificity of the sensor. (a-c) The operation window of M^pro sensors based on NR10, OR8, and YR9 peptide, which contains 0, 1, and 4 arginine residues in its aggregating sequence, respectively; see Table 1. (d) The M^pro LoD of sensors based on four peptides of varying number of arginine: no LoD observed for NR10 and OR8 substrate; 27.7 and 3.4 nM is determined for YR9 and ZY7 substrate, respectively. (e) Time progression of ratiometric signal (Abs₆₀₀/Abs₅₂₀) in inhibitor assays. Increasing molar ratio of [inhibitor]/[M^pro] from 0−10 was employed. The control curve (Ct.) designates inhibitor only without M^pro additive. (f) A typical inhibition titration curve fitted with the Morrison equation (Eqn. S2) is shown for the competitive inhibitor, GC376.^[30] Inset shows the chemical structure of GC376 inhibitor. A Henderson equation was applied to resolve the apparent inhibitor dissociation constant, K_{i (app)} =15 nM_, and active enzyme concentration, [E]₀ =60 nM (out of 100 nM)_. The IC₅₀ is 45 nM. (g) Sensor activation by other mammalian proteins (100 nM), including bovine serum albumin (BSA), hemoglobin, α-amylase (100 U/mL), thrombin, and trypsin. Assay with and without M^pro is included as positive and negative control. (h) The response of sensors based on ZY7 and DS12 substrate to M^pro or trypsin in Tris buffer. LoD for M^pro is 27.7 and 114.4 nM for ZY7 and DS12, respectively; LoD for trypsin is 9.7 and 30.3 nM for ZY7 and DS12, respectively.