Single‐Molecule Interaction of Peptides with a Biological Nanopore for Identification of Protease Activity

Abstract

Abstract The facile and sensitive detection of peptides is essential for drug screening, pathogen detection, and protein sequencing. There are still challenges for the real‐time single‐molecule sensing and detection of peptides due to their versatile shape, structure, and charges brought by amino acids. Nanopore sensing is an emerging technology for sensing of biomolecules including DNA, RNA, and proteins. In this study, the interaction between peptides of different lengths (N6–N10) and charges with an engineered Mycobacterium smegmatis porin A nanopore are systematically studied, and two types blockage events can be identified by quantifying their dwell times and amplitude of blockades. The findings are further applied to the label‐free and real‐time quantification of protease activity of caseinolytic protease P at nanomolar concentration in 14 min. The protease activity with inhibitor can also be monitored real time by nanopore assay. In summary, this nanopore‐based sensing platform shows promising capacity for peptide detection, protease activities assay, and inhibitor screening.