Protease Biosensor by Conversion of a Homogeneous Assay into a Surface-Tethered Electrochemical Analysis Based on Streptavidin–Biotin Interactions
Ning Xia, Zhifang Sun, Fangyuan Ding, Yanan Wang, Wenna Sun, Lin Liu
Abstract
This work proposed a new sensing strategy for protease detection by converting a homogeneous assay into a surface-tethered electrochemical analysis. Streptavidin (SA), a tetramer protein, was used as the sensing unit based on the SA–biotin coupling chemistry. Caspase-3 was used as the model analyte, and a biotinylated peptide with a sequence of biotin–GDEVDGK–biotin was designed as the substrate. Specifically, the peptide substrate could induce an assembly of SA to form (SA–biotin–GDEVDGK–biotin)n aggregates through SA–biotin interactions, which was confirmed by atomic force microscopy (AFM). The peptide substrate-induced assembly of SA was facilely initiated on an electrode–liquid surface by modification of the electrode with SA. The in situ formation of (SA–biotin–GDEVDGK–biotin)n aggregates created an insulating layer, thus limiting the electron transfer of ferricyanide. Once the peptide substrate was cleaved into two shorter fragments (biotin–GDEVD and GK–biotin) by caspase-3, the resulting products would compete with biotin–GDEVDGK–biotin to bind SA proteins immobilized on the electrode surface and distributed in a solution, thus preventing the in situ formation of (SA–biotin–GDEVDGK–biotin)n assemblies. With the simple principle of the substrate-induced assembly of SA, a dual-signal amplification was achieved with improved sensitivity. Taking advantage of high sensitivity, simple principle, and easy operation, this method can be augmented to design various surface-tethered biosensors for practical applications.