Dual-Role Peptide with Capping and Cleavage Site Motifs in Nanoparticle-Based One-Pot Colorimetric and Electrochemical Protease Assay

[Image: see text] A new method for enzyme substrate assembly and its use in proteolytic enzyme assays with colorimetric and electrochemical detection is presented. The novelty of the method is the use of dual-function synthetic peptide containing both gold clustering and protease-sensitive moieties, which not only induces the simple formation of the peptide-decorated gold nanoparticle test substrates but also allows for the detection of proteolysis in the same batch. Protease-treated nanoparticles with a destabilized peptide shell became more prone to electroactivity, and thus, the model enzyme plasmin activity could be quantified with stripping square wave voltammetry analysis as well, giving an alternative method to conduct aggregation-based assays. Spectrophotometric and electrochemical calibration data proved to be linear within the 40–100 nM active enzyme concentration range, with possible extensions of the dynamic range by varying substrate concentration. The simple initial components and the ease of synthesis make the assay substrate preparation economic and easy to implement. The possibility of cross-check analytical results with two independent measurement techniques in the same batch greatly increases the applicability of the proposed system.