Label-free nanopore single-molecule measurement of trypsin activity

Trypsin is the most important digestive enzyme produced in the pancreas, and is a useful biomarker for pancreatitis. In this work, a rapid and sensitive method for the quantitative determination of trypsin activity is developed by using a biological alpha-hemolysin protein nanopore. Due to its much larger molecular diameter than the narrow pore constriction, trypsin itself cannot transport through the alpha-hemolysin channel. Hence, an indirect trypsin detection method is developed by monitoring its proteolytic cleavage of a lysine-containing peptide substrate. Based on the current modulations produced by the translocation of the substrate degradation products in the nanopore, the activity levels of trypsin could be determined. The method is rapid and highly sensitive, with picomolar concentrations of trypsin detected in minutes. In addition, the effects of cation and temperature on the sensor sensitivity, trypsin inhibition, and serum sample analysis are also investigated.