Spacer Matters: All-Peptide-Based Ligand for Promoting Interfacial Proteolysis and Plasmonic Coupling

Plasmonic coupling via nanoparticle assembly is a popular signal-generation method in bioanalytical sensors. Here, we customized an all-peptide-based ligand that carries an anchoring group, polyproline spacer, biomolecular recognition, and zwitterionic domains for functionalizing gold nanoparticles (AuNPs) as a colorimetric enzyme sensor. Our results underscore the importance of the polyproline module, which enables the SARS-CoV-2 main protease (M^pro) to recognize the peptidic ligand on nanosurfaces for subsequent plasmonic coupling via Coulombic interactions. AuNP aggregation is favored by the lowered surface potential due to enzymatic unveiling of the zwitterionic module. Therefore, this system provides a naked-eye measure for M^pro. No proteolysis occurs on AuNPs modified with a control ligand lacking a spacer domain. Overall, this all-peptide-based ligand does not require complex molecular conjugations and hence offers a simple and promising route for plasmonic sensing other proteases.