Enhanced Analytes Capture by Engineering Electrostatics at the Entry of Aerolysin Nanopore

Abstract

Nanopore technology is a powerful single-molecule platform for detecting and sequencing a wide range of biomolecules. Among nanopores, aerolysin has emerged as a particularly promising candidate for peptide sensing. However, its ability to capture long biopolymers is limited due to its lack of a vestibule structure. In this study, we engineered electrostatics at the entry of the aerolysin pore and observed an increase in event frequency – up to 2 times higher for DNA and for the peptide compared to wild-type aerolysin. Importantly, this modification did not affect the pore's current-voltage characteristics. When tested with DNA and α-synuclein peptides, the engineered pore (D209R) exhibited comparable dwell times and current blockages to the wild-type pore, while ion selectivity and electroosmotic flux show an increase. These findings highlight that fine-tuning the electrostatic properties at the pore entry can significantly enhance event frequency without compromising key transport properties such as current blockage or dwell time. This improvement expands the utility of aerolysin nanopores for sensing and sequencing applications and paves the way for more effective diagnostic tools and analytical methods in the field of proteomics and biomarker discovery.