Proteolytic reaction-based versatile electrochemical biosensor chip for simple and quantitative point-of-care testing

Electrochemical protease biosensors are well-suited for simple and quantitative point-of-care testing. However, there has been limited development of versatile chip formats similar to lateral-flow and glucose test strips for electrochemical proteases biosensors. This paper presents a versatile electrochemical biosensor chip for the wash-free and sensitive detection of proteases. The chip comprises (i) a reaction well, (ii) micropatterned electrodes on a glass substrate, and (iii) target protease-dependent filter paper. All (bio)chemicals required for the proteolytic reaction and signal amplification were predried on filter paper, which was then placed inside the reaction well. Upon injecting the sample solution into the reaction well, most of the dried (bio)chemicals dissolved rapidly. Wash-free and sensitive protease detection was achieved using electrochemical-enzymatic (EN) redox cycling of a redox-active species (4-amino-1-naphthol, AN) liberated from a peptide substrate through a rapid proteolytic reaction. The EN redox cycling involves a working electrode, AN, glucose dehydrogenase, and glucose. To enhance dissolvability and stability, (bio)chemicals were dried using a surfactant and stabilizer and stored at 4 °C with vacuum sealing. To minimize the effect of background variation among the different samples, a high concentration of glucose (200mM) was used, and the difference between the two charge values obtained before and after the incubation period was measured. The biosensor chips successfully detected three proteases: thrombin, leucine aminopeptidase (LAP), and 3CL protease from coronavirus SARS-CoV-2 (Mpro). The detection limits of thrombin in artificial serum, Mpro in artificial saliva, and LAP in human serum were approximately 100, 100pg/mL, and 8U/L, respectively.