Voltammetric sensing of trypsin activity using gelatin as a substrate

The relevance of proteases in many areas such as the industry, microbiology, and clinical analysis has prompted the development of different methods for protease detection and proteolytic activity determination. Electro-chemical sensors and assays are valuable techniques for their short response time, simple fabrication, high sensitivity and possibility of miniaturization. Here, we describe the determination of the proteolytic activity of trypsin by using a gelatin-embedded electrochemical redox mediator the (4-((4-aminophenyl)imino)-2,6-dime-thoxycyclohexa-2,5-dien-1-one). The peak current (ip) was used as the analytical signal and measured by means of alternating current voltammetry (ACV). Upon exposure to different concentrations of trypsin, a decrease in the ip expressed as the relative change was observed due to the release of the redox mediator in solution. A linear response range (5-80 mu g/mL) was obtained, this trend was further corroborated using standard techniques such as the azo-casein, and the gel diffusion assay. A significant and negative correlation between these assays and the trypsin activity measured as the ip relative change was observed.