Ultrasensitive Protease Sensors Using Selective Affinity Binding, Selective Proteolytic Reaction, and Proximity-Dependent Electrochemical Reaction.

The development of a fast and ultrasensitive protease detection method is a challenging task. This paper reports ultrasensitive protease sensors exploiting (i) selective affinity binding, (ii) selective proteolytic reaction, and (iii) proximity-dependent electrochemical reaction. The selective affinity binding to capture IgG increases the concentration of the target protease (trypsin as a model protease) near the electrode, and the selective proteolytic reaction by trypsin increases the concentration of the redox-active species near the electrode. The electrochemical reaction, which is more sensitive to the concentration of the redox-active species near the electrode than to its bulk concentration, provides an increased electrochemical signal, which is further amplified by the electrochemical-chemical redox cycling. An indium-tin oxide electrode modified with reduced graphene oxide, avidin, and biotinylated capture IgG is used as the electrode, and p-aminophenol liberated from an oligopeptide is used as the redox-active species. The new sensor scheme using no washing process is compared with the new sensor scheme using washing process, and with the conventional scheme using only proteolytic reaction. The new scheme provides a higher signal-to-background ratio and a lower detection limit. Moreover, the increased electrochemical signal offers a more selective protease detection. Trypsin can be detected in phosphate-buffered saline and in artificial serum containing l-ascorbic acid with a low detection limit of 0.5 pg/mL, over a wide range of concentrations, and with an incubation period of only 30 min without washing process. The washing-free electrochemical protease sensor is highly promising for simple, fast, ultrasensitive, and selective point-of-care testing of low-abundance proteases.