Protease Biosensor by Conversion of a Homogeneous Assay into a Surface-Tethered Electrochemical Analysis Based on Streptavidin–Biotin Interactions

• DOI: 10.1021/acssensors.0c02415
• Publication Date: 2021-01-23T01:41:29Z
• Authors: Ning Xia; Zhifang Sun; Fangyuan Ding; Yanan Wang; Wenna Sun; Lin Liu
• Keywords: Biotin; Biosensing Techniques; Electrochemical Techniques; Streptavidin; 01 natural sciences; Peptide Hydrolases; 0104 chemical sciences

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.