Exploring the Single-Molecule Transient Interactions with Nanopore Frequency Spectrum

Interactions at the single-molecule interface play vital roles in biology and nanotechnology. However, nanopore technology hardly describes the transient interaction kinetics of small molecules due to fast translocation events and weak ionic current fluctuations. Here, we employed an improved frequency spectrum method for analyzing the ion current fluctuations induced by small molecules entering the nanopore. This frequency spectrum analysis method allows for the analysis of transient current changes beyond the instrumental temporal resolution. The parameter (peak amplitude, a_m) from the frequency spectrum is used for analyzing the transient interaction kinetics on poly(dA)₂ with the aerolysin nanopore. A nearly consistent linear relationship of a_m and interaction rate (1/τ_on) reveals the kinetics of the interaction by the a_m value. Moreover, we analyzed the ionic current generated by ethylenediaminetetraacetic acid (EDTA) molecules through the aerolysin nanopore. The results demonstrate that kinetics hidden in the open-pore current noise is accessible by this frequency spectrum analysis method, thereby providing insights into elucidating the molecular transient interactions.