Peptide cleavage induced assembly enables highly sensitive electrochemiluminescence detection of protease activity

Proteases perform essential functions in a multitude of physiological processes and participate in many human diseases. Probing protease activity sensitively and accurately is critical for both basic research and clinical diagnosis. Herein, a novel optical biosensor for high-sensitive detection of protease was developed based on the specific protease cleavage of synthesized peptide substrate and subsequent assembly between exposed cysteine and Ru(bpy)32+-2-cyanobenzothiazole (Ru(bpy)32+-CBT). The assembly complexes can be enriched by streptavidin coated magnetic beads on work electrode for high-sensitive electrochemiluminescence (ECL) analysis. Using trypsase and caspase-3 as the examples, the limit of detection (LOD) of 8.4 × 10−4 U mL−1 and 5 × 10−6 U mL−1 were obtained respectively. This represents one of most sensitive protease assay reported. Current peptide cleavage induced assembly (PCIA) method can be easily extended to detect other proteases by changing peptide substrate sequence, shows its great potential as versatile biosensor in sensing techniques and clinical diagnosis.