Sensing Single Molecule Penetration into Nanopores: Pushing the Time Resolution to the Diffusion Limit.

To quantify small molecule penetration into and eventually permeation through nanopores, we applied an improved excess-noise analysis of the ion current fluctuation caused by entering molecules. The kinetic parameters of substrate entry and exit are derived from a two-state Markov model, analyzing the substrate concentration dependence of the average ion current and its variance. Including filter corrections allows one to detect the transition rates beyond the cutoff frequency, f, of the instrumental ion-current filter. As an application of the method, we performed an analysis of the single-channel ion current of Meropenem, an antibiotic of the carbapenem family, interacting with OmpF, the major general outer membrane channel of Escherichia coli bacteria. At 40 °C we detected the residence time of Meropenem inside OmpF of about 500 ns-more than 2 orders of magnitude smaller than f and close to the diffusion limit of few hundred nanoseconds. We also have established theoretical limit conditions under which the substrate-induced channel blockages can be detected and suggest that submicrosecond-scale gating kinetic parameters are accessible with existing experimental equipment.