Turn-on chemiluminescent sensing platform for label-free protease detection using streptavidin-modified magnetic beads

We report a label-free streptavidin-modified magnetic beads (SA-MBs)-based sensing platform for turn-on chemiluminescent (CL) detection of protease using trypsin as model analyte. In the assay, a biotinylated peptide containing an arginine and a terminal cysteine was used as the substrate of trypsin. Upon adding the peptide into a basic luminol-NaIO4 solution, the terminal cysteine induced a strong CL signal. Surprisingly a much lower CL was emitted when the peptide was immobilized on the surface of SA-MBs. Based on this phenomenon, we designed a turn-on CL sensing system for protease using trypsin as model and its inhibitors screening. In the absence of trypsin, the peptide was coupled to the SA-MBs surface, resulting in a low CL background. Upon the addition of trypsin, the peptide can be catalytically hydrolyzed at the C-terminus of arginine, resulting in the formation of free cysteine-containing residues and subsequent CL recovery with the addition of luminol and NaIO4. The simple method does not require washing or separating procedures. Trypsin at a concentration as low as 10 pM can be assayed using this new CL sensing system. Additionally, the proposed method can be employed for screening the inhibitors of trypsin. This new sensing strategy could be easily extended to assay other proteases by simply changing the peptide substrate.