Enzymatic Deposition of Silver Particles for Detecting Protease Activity

Abstract

A protease assay is reported by using enzymatic deposition of silver particles to report protease activity as optical readouts. In this assay, a biotinylated oligopeptide, which is a protease substrate, is first immobilized on a glass surface. In the absence of trypsin, streptavidin−alkaline phosphatase (SA−ALP) complex can bind to biotin and catalyze enzymatic silver deposition (ESD), which leads to a layer of highly reflective silver particles deposited on the surface. In the presence of trypsin, the immobilized oligopeptide is cleaved and biotin is removed. As a result, SA−ALP does not bind to the surface, and ESD does not happen. The limit of detection (LOD) of this assay is closely related to the surface density of oligopeptide substrate. When the surface density of oligopeptide is 0.12 molecule nm−2, the LOD for trypsin can reach 10 ng mL−1 (400 × 10−12m). It is also demonstrated that this ESD‐based protease assay can be applied to screening of protease inhibitors, such as benzamidine hydrochloride.