Chromogenic and FRET peptide substrates, quantum-dot peptide conjugates and electrochemical peptide interfaces: A 25-year journey from combinatorial chemistry to clinical readouts-A viewpoint.

This Viewpoint synthesises 25 years of work from a single laboratory on activity-based protease sensing and offers a critical appraisal of where each of the three resulting platforms stands today. The activity of proteolytic enzymes-rather than their abundance-determines the outcome of inflammation, tumour progression, viral entry and barrier dysfunction. Measuring it in clinical samples is demanding: closely related proteases share substrate preferences, and biological matrices are crowded with endogenous inhibitors and competing hydrolases. Over the past 25 years, our group at the University of Gdansk has developed a chemical toolkit along three complementary axes, assembled here, as a probe ladder running from solution chemistry to solid-state interface. First, internally quenched chromogenic and Förster resonance energy transfer (FRET) peptide substrates, designed by iterative deconvolution of combinatorial libraries spanning non-primed and primed subsites, provide selective readouts for serine, cysteine, metallo- and viral proteases. Second, peptide-quantum dot conjugates built on non-toxic ZnO nanocrystals improved detection limits 20-30-fold over the parent solution substrate in two published demonstrators. Third, peptide-modified gold electrodes translate protease cleavage and molecular recognition into a label-free electrochemical signal-exemplified by an electrode that reads 20S proteasome activity and by an antimicrobial-peptide interface for bacterial endotoxin. We applied the resulting probes in exploratory studies of bladder cancer, type-2 diabetes and diabetic nephropathy, and positioned them within the broader landscape of activity-based protease sensing-the activity-based probes of the Bogyo and Cravatt laboratories, the PS-SCL and HyCoSuL combinatorial approaches, and the Weissleder near-infrared imaging program.