Trypsin Detection Using Electrochemical Reduction-based Redox Cycling

It is more difficult to obtain high signal-to-background ratios in biosensors using electrochemical reduction than using electrochemical oxidation. Here, we present a method for trypsin detection using electrochemical reduction-based redox cycling. Electrochemical-enzymatic (EN) redox cycling and electrochemical-chemical (EC) redox cycling for trypsin detection were tested and compared. Trypsin cleaves a peptide bond in an electrochemically inactive p-aminophenol (AP)-conjugated oligopeptide, and this cleavage results in the release of electrochemically active AP, which is involved in EN and EC redox-cycling reactions. Horseradish peroxidase and cytochrome c (Cyt c) were tested as redox enzymes for EN redox cycling involving a redox enzyme and H2O2. Cyt c was better than horseradish peroxidase, as its use resulted in lower background levels. The trypsin detection based on the EN redox cycling involving Cyt c and H2O2 (similar to 50 ng/mL) exhibited lower detection limits than the detection based on EC redox cycling involving IO3- (similar to 100 ng/mL), because of higher signal levels.

Automated summary

It is more challenging to achieve high signal-to-background ratios in biosensors with electrochemical reduction compared to electrochemical oxidation. This study introduced a method for trypsin detection using electrochemical reduction-based redox cycling, comparing enzymatic and chemical redox cycling. Results showed that using cytochrome c as a redox enzyme in EN redox cycling had lower background levels compared to horseradish peroxidase, leading to more sensitive trypsin detection.