A simple and general approach to assay protease activity with electrochemical technique.

Proteases are involved in a large number of serious disease processes, while the assay of proteolytic activity can be used for clinical diagnostics. In this paper we report a simple electrochemical method to assay protease activity. This method makes use of an unlabeled peptide that comprises the specific substrate domain of a protease, which can be easily operated and generalized for assay of various kinds of proteases. Specifically, the peptide is immobilized onto a gold electrode surface via the chemical adsorption of the C-terminal cysteine residue, forming a positively charged interface derived from the N-terminal cationic residue. Therefore, the positive electrochemical probes [Ru(NH3)5Cl](2+) cannot get across to the electrode to generate signal. Nevertheless, the proteolytic digestion of the peptide will decrease the number of positive charges on the electrode surface and weaken the blocking effect against the positive electrochemical species, resulting in an increased electrochemical signal. Under optimized conditions, the activity of the model protease, trypsin, can be assayed with a detection limit of 0.026 U/mL. The method may also allow the determination of trypsin activity in serum samples. Moreover, since this approach can be used for the assay of other proteases by simply changing the substrate domain of the peptide, it may have great potential in biomedical applications in the future.