The transmission of SARS-CoV-2 coronavirus has led to the COVID-19 pandemic. Nucleic acid testing while specific has limitations for mass surveillance. One alternative is the main protease (M pro ) due to its functional importance in mediating the viral life cycle. Here, we describe a combination of modular substrate and gold colloids to detect M pro via visual readout. The strategy involves zwitterionic peptide that carries opposite charges at the C-/N-terminus to exploit the specific recognition by M pro . Autolytic cleavage releases a positively charged moiety that assembles the nanoparticles with rapid color changes (t < 10 min). We determine a limit of detection for M pro in breath condensate matrices < 10 nM. We further assayed ten COVID-negative subjects and found no false-positive result. In the light of simplicity, our test for viral protease is not limited to an equipped laboratory, but also is amenable to integrating as portable point-of-care devices including those on face-coverings.

Activatable contrast agents are of ongoing research interest because they offer low background and high specificity to the imaging target. Engineered sensitivity to protease activity is particularly desirable because proteases are critical biomarkers in cancer, infectious disease, inflammatory disorders, and so forth. Herein, we developed and characterized a set of peptide-linked cyanine conjugates for dual-modal detection of protease activity via photoacoustic (PA) and fluorescence imaging. The peptide−dye conjugates were designed to undergo contact quenching via intramolecular dimerization and contained n dyes (n = 2, 3, or 4) with n − 1 cleavable peptide substrates. The absorption peaks of the conjugates were blue-shifted 50 nm relative to the free dye and had quenched fluorescence. This effect was sensitive to solvent polarity and could be reversed by solvent switching from water to dimethyl sulfoxide. Employing trypsin as a model protease, we observed a 2.5-fold recovery of the peak absorbance, 330−4600-fold fluorescent enhancement, and picomolar detection limits following proteolysis. The dimer probe was further characterized for PA activation. Proteolysis released single dye−peptide fragments that produced a 5-fold PA enhancement through the increased absorption at 680 nm with nanomolar sensitivity to trypsin. The peptide substrate could also be tuned for protease selectivity; as a proof-of-concept, we detected the main protease (M pro ) associated with the viral replication in SARS-CoV-2 infection. Last, the activated probe was imaged subcutaneously in mice and signal was linearly correlated to the cleaved probe. Overall, these results demonstrate a tunable scaffold for the PA molecular imaging of protease activity with potential value in areas such as disease monitoring, tumor imaging, intraoperative imaging, in vitro diagnostics, and point-of-care sensing.

The main protease (Mpro) and papain‐like protease (PLpro) play critical roles in SARS‐CoV‐2 replication and are promising targets for antiviral inhibitors. The simultaneous visualization of Mpro and PLpro is extremely valuable for SARS‐CoV‐2 detection and rapid inhibitor screening. However, such a crucial investigation has remained challenging because of the lack of suitable probes. We have now developed a dual‐color probe (3MBP5) for the simultaneous detection of Mpro and PLpro by fluorescence (or Förster) resonance energy transfer (FRET). This probe produces fluorescence from both the Cy3 and Cy5 fluorophores that are cleaved by Mpro and PLpro. 3MBP5‐activatable specificity was demonstrated with recombinant proteins, inhibitors, plasmid‐transfected HEK 293T cells, and SARS‐CoV‐2‐infected TMPRSS2‐Vero cells. Results from the dual‐color probe first verified the simultaneous detection and intracellular distribution of SARS‐CoV‐2 Mpro and PLpro. This is a powerful tool for the simultaneous detection of different proteases with value for the rapid screening of inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to human health and lacks an effective treatment. There is an urgent need for both real-time tracking and precise treatment of the SARS-CoV-2-infected cells to mitigate and ultimately prevent viral transmission. However, selective triggering and tracking of the therapeutic process in the infected cells remains challenging. Here, we report a main protease (M pro )-responsive, mitochondrial-targeting, and modular-peptide-conjugated probe (PSGMR) for selective imaging and inhibition of SARS-CoV-2-infected cells via enzyme-instructed self-assembly and aggregation-induced emission (AIE) effect. The amphiphilic PSGMR was constructed with tunable structure and responsive efficiency and validated with recombinant proteins, cells transfected with M pro plasmid or infected by SARS-CoV-2, and a M pro inhibitor. By rational construction of AIE luminogen (AIEgen) with modular peptides and M pro , we verified that the cleavage of PSGMR yielded gradual aggregation with bright fluorescence and enhanced cytotoxicity to induce mitochondrial interference of the infected cells. This strategy may have value for selective detection and treatment of SARS-CoV-2-infected cells.

We report the reversible aggregation of gold nanoparticles (AuNPs) assemblies via a di-arginine peptide additive and thiolated PEGs (HS-PEGs). The AuNPs were first aggregated by attractive forces between the citrate-capped surface and the arginine side chains. We found that the HS-PEG thiol group has a higher affinity for the AuNP surface, thus leading to redispersion and colloidal stability. In turn, there was a robust and obvious color change due to on/off plasmonic coupling. The assemblies' dissociation was directly related to the HS-PEG structural properties such as their size or charge. As an example, HS-PEGs with a molecular weight below 1 kDa could dissociate 100% of the assemblies and restore the exact optical properties of the initial AuNP suspension (prior to the assembly). Surprisingly, the dissociation capacity of HS-PEGs was not affected by the composition of the operating medium and could be performed in complex matrices such as plasma, saliva, bile, urine, cell lysates, or even seawater. The high affinity of thiols for the gold surface encompasses by far the one of endogenous molecules and is thus favored. Moreover, starting with AuNPs already aggregated ensured the absence of a background signal as the dissociation of the assemblies was far from spontaneous. Remarkably, it was possible to dry the AuNP assemblies and solubilize them back with HS-PEGs, improving the colorimetric signal generation. We used this system for protease sensing in biological fluids. Trypsin was chosen as the model enzyme, and highly positively charged peptides were conjugated to HS-PEG molecules as cleavage substrates. The increase of positive charge of the HS-PEG−peptide conjugate quenched the dissociation capacity of the HS-PEG molecules, which could only be restored by the proteolytic cleavage. Picomolar limit of detection was obtained as well as the detection in saliva or urine.