Leveraging Electroosmosis in OmpF for Robust Peptide Sensing

Abstract Peptide sensing and sequencing are essential for elucidating sequence-function relationships and evaluating the peptide drug quality. Nanopore electrochemistry enables label-free characterization of peptide composition, structure, and interactions at the single-molecule level through ionic-current fingerprints. However, efficient capture remains challenging for low-abundance peptides with heterogeneous charge and conformational properties. Herein, we study peptide capture by the asymmetric OmpF nanopore and examine the relative contributions of electroosmotic flow (EOF), electrophoretic force (EPF), peptide conformation, and field-gradient-mediated interactions. The laterally heterogeneous charge distribution within OmpF modulates the salt dependence of ionic screening and electroosmotic transport, supporting an efficient EOF-associated peptide capture under high-salt conditions. Atomistic molecular dynamics simulations reveal salt-dependent changes in ionic conductance, water flux, and counterion accumulation within the constriction zone, suggesting that the local counterion accumulation approaches saturation at high ionic strength. This behavior enables OmpF to combine high signal-to-noise ratio with low-concentration peptide detection across net charges from −2 e to +6 e. This OmpF sensor provides a linear detection range spanning more than four orders of magnitude, nanomolar concentration limits, and femtogram-level sample consumption. Furthermore, by utilizing the stereoselective recognition capability of OmpF’s asymmetric constriction zone, this approach was further applied to the identification of chiral impurities in near-neutral peptide drugs. The OmpF-based nanopore sensor provides a sensitive and label-free platform with a broad dynamic range for single-molecule peptide analysis, paving the way for versatile nanopore applications.