Real-Time Label-Free Kinetics Monitoring of Trypsin-Catalyzed Ester Hydrolysis by a Nanopore Sensor

Trypsin is an important proteolytic enzyme in the digestive system and its activity is a major indicator for evaluating diseases such as chronic pancreatitis. Here, we present a novel label-free method to detect trypsin kinetics using a nanopore technique. A mutant α-hemolysin (M113R)₇ protein nanopore equipped with a polyamine decorated β-cyclodextrin (am₇β-CD) was employed as a sensing platform for the real-time monitoring of the process of trypsin enzymatic cleavage of a substrate Nα-benzoyl-l-arginine ethyl ester (BAEE) at the single molecule level. Significantly, this sensor can exclusively respond to the current modulation caused by the product and prevent interference from the substrate, thus improving detection sensitivity, and it provides a new scheme to detect enzyme activity for cleaving small molecules.