Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) has been regarded as one of the ideal targets for the development of antiviral drugs. The currently used methods for the probing of Mpro activity and the screening of its inhibitors require the use of a double-labeled peptide substrate. In this work, we suggested that the label-free peptide substrate could induce the aggregation of AuNPs through the electrostatic interactions, and the cleavage of the peptide by the Mpro inhibited the aggregation of AuNPs. This fact allowed for the visual analysis of Mpro activity by observing the color change of the AuNPs suspension. Furthermore, the co-assembly of AuNPs and peptide was achieved on the peptide-covered electrode surface. Cleavage of the peptide substrate by the Mpro limited the formation of AuNPs/peptide assembles, thus allowing for the development of a simple and sensitive electrochemical method for Mpro detection in serum samples. The change of the electrochemical signal was easily monitored by electrochemical impedance spectroscopy (EIS). The detection limits of the colorimetric and electrochemical methods are 10 and 0.1 pM, respectively. This work should be valuable for the development of effective antiviral drugs and the design of novel optical and electrical biosensors.

Enzymes play a critical role in most complex biochemical processes. Some of them can be regarded as biomarkers for disease diagnosis. Taking advantage of aggregation-induced emission (AIE)-based biosensors, a series of fluorogens with AIE characteristics (AIEgens) have been designed and synthesized for the detection and imaging of enzymes. In this work, we summarized the advances in AIEgens-based probes and sensing platforms for the fluorescent detection of enzymes, including proteases, phosphatases, glycosidases, cholinesterases, telomerase and others. The AIEgens involve organic dyes and metal nanoclusters. This work provides valuable references for the design of novel AIE-based sensing platforms.

Copper ions, as the active centers of natural enzymes, play an important role in many physiological processes. Copper ion-based catalysts which mimic the activity of enzymes have been widely used in the field of industrial catalysis and sensing devices. As an important class of small biological molecules, peptides have the advantages of easy synthesis, excellent biocompatibility, low toxicity, and good water solubility. The peptide–copper complexes exhibit the characteristics of low molecular weight, high tenability, and unique catalytic and photophysical properties. Biosensors with peptide–copper complexes as the signal probes have promising application prospects in environmental monitoring and biomedical analysis and diagnosis. In this review, we discussed the design and application of fluorescent, colorimetric and electrochemical biosensors based on the peptide–copper coordination interaction.

The heterogeneous assays of proteases usually require the immobilization of peptide substrates on the solid surface for enzymatic hydrolysis reactions. However, immobilization of peptides on the solid surface may cause a steric hindrance to prevent the interaction between the substrate and the active center of protease, thus limiting the enzymatic cleavage of the peptide. In this work, we reported a heterogeneous surface plasmon resonance (SPR) method for protease detection by integration of homogeneous reaction. The sensitivity was enhanced by the signal amplification of streptavidin (SA)-conjugated immunoglobulin G (SA-IgG). Caspase-3 (Cas-3) was determined as the model. A peptide labeled with two biotin tags at the N- and C-terminals (bio-GDEVDGK-bio) was used as the substrate. In the absence of Cas-3, the substrate peptide was captured by neutravidin (NA)-covered SPR chip to facilitate the attachment of SA-IgG by the avidin-biotin interaction. However, once the peptide substrate was digested by Cas-3 in the aqueous phase, the products of bio-GDEVD and GK-bio would compete with the substrate to bond NA on the chip surface, thus limiting the attachment of SA-IgG. The method integrated the advantages of both heterogeneous and homogeneous assays and has been used to determine Cas-3 inhibitor and evaluate cell apoptosis with satisfactory results.

This work reported gold nanoparticles (AuNPs)-based colorimetric immunoassay with the Cu-based metal–organic framework (MOF) to load pyrroloquinoline quinone (PQQ) for the catalytic oxidation of cysteine. In this method, both Cu²⁺ and PQQ in the MOF could promote the oxidation of inducer cysteine by redox cycling, thus limiting the cysteine-induced aggregation of AuNPs and achieving dual signal amplification. Specifically, the recombinant carcinoembryonic antigen (CEA) targets were anchored on the MOF through the metal coordination interactions between the hexahistidine (His₆) tag in CEA and the unsaturated Cu²⁺ sites in MOF. The CEA/PQQ-loaded MOF could be captured by the antibody-coated ELISA plate to catalyze the oxidation of cysteine. However, once the target CEA in the samples bound to the antibody immobilized on the plate surface, the attachment of CEA/PQQ-loaded MOF would be limited. Cysteine remaining in the solution would trigger the aggregation of AuNPs and cause a color change from red to blue. The target concentration was positively related to the aggregation and color change of AuNPs. The signal-on competitive plasmonic immunoassay exhibited a low detection limit with a linear range of 0.01–1 ng/mL. Note that most of the proteins in commercial ELISA kits are recombinant with a His₆ tag in the N- or C-terminal, so the work could provide a sensitive plasmonic platform for the detection of biomarkers.

This work reports the development of a fluorescence method for the detection of poly(ADP-ribose) polymerase-1 (PARP1), in which a phenylboronic acid-modified fluorescein isothiocyanate dye (FITC-PBA) was used to recognize the formed poly(ADP-ribose) (PAR) polymer. The detection system was designed by conjugating recombinant streptavidin (rSA) with PARP1-specific double-stranded DNA (dsDNA) through streptavidin–biotin interaction. Capture of PARP1 via rSA–biotin–dsDNA allowed for the poly-ADP-ribosylation (PARylation) of both rSA and PARP1 in a homogeneous solution. The resulting rSA–biotin–dsDNA/PAR conjugates were then captured and separated via the commercialized nitrilotriacetic acid–nickel ion-modified magnetic bead (MB-NTA-Ni) through the interaction between NTA–Ni on MB surface and oligohistidine (His₆) tag in rSA. The PAR polymer could capture the dye of FITC-PBA through the borate ester interaction between the boronic acid moiety in PBA and the cis-diol group in ribose, thus causing a decrease in fluorescence signal. The PARylation of streptavidin and the influence of steric hindrance on PARylation efficiency were confirmed using reasonable detection strategies. The method showed a wide linear range (0.01~20 U) and a low detection limit (0.01 U). This work should be valuable for the development of novel biosensors for the detection of poly(ADP-ribose) polymerases and diol-containing species.