Electroosmotic Trap Against the Electrophoretic Force Near a Protein Nanopore Reveals Peptide Dynamics during Capture and Translocation

We report on the ability to control the dynamics of a single peptide capture and passage across a voltage-biased, alpha-hemolysin nanopore (alpha-HL), under conditions that the electroosmotic force exerted on the analyte dominates the electrophoretic transport. We demonstrate that by extending outside the nanopore, the electroosmotic force is able to capture a peptide at either the lumen or vestibule entry of the nanopore, and transiently traps it inside the nanopore, against the electrophoretic force. Statistical analysis of the resolvable dwell-times of a metastable trapped peptide, as it occupies either the beta-barrel or vestibule domain of the alpha-HL nanopore, reveals rich kinetic details regarding the direction and rates of stochastic movement of a peptide inside the nanopore. The presented approach demonstrates the ability to shuttle and study molecules along the passage pathway inside the nanopore, allows to identify the mesoscopic trajectory of a peptide exiting the nanopore through either the vestibule or beta-barrel moiety, thus providing convincing proof of a molecule translocating the pore. The kinetic analysis of a peptide fluctuating between various microstates inside the nanopore, enabled a detailed picture of the free energy description of its interaction with the alpha-HL nanopore. When studied at the limit of vanishingly low transmembrane potentials, this provided a thermodynamic description of peptide reversible binding to and within the alpha-HL nanopore, under equilibrium conditions devoid of electric and electroosmotic contributions.