Effect of Electrolyte, Applied Voltage, and DNA Concentration on the Translocation Dynamics of a Single-Stranded DNA...

Solid-state nanopores enable label-free detection of biomolecules by monitoring ionic current modulations during molecular translocation. However, reliable measurements are often limited by rapid, poorly controlled translocation dynamics producing short, low-resolution signals. Applied voltage, buffer composition, and analyte concentration influence molecular transport, yet their combined effect in ionic environments, including Tris-based buffers, remains insufficiently understood. Here, we investigated the effects of applied voltage, DNA concentration, and buffer composition on translocation kinetics of a circular 7560-nucleotide single-stranded DNA and the current blockage characteristics. We compared two electrolyte environments: lithium chloride in HEPES (LiCl/HEPES) vs Tris-borate-EDTA (TBE) supplemented with K⁺ and Mg²⁺ cations. The two electrolyte environments differed in ion composition and ionic strength, leading to distinct translocation behaviors. With increasing absolute applied voltage, dwell times decreased and current blockages increased for all DNA concentrations. LiCl/HEPES buffer resulted in slower translocation with broad dwell time distributions, whereas TBE with K⁺ and Mg²⁺ cations produced faster events with narrower dwell time populations and lower current blockage amplitudes. We conclude that the electrolyte environment is a key factor for optimizing solid-state nanopore performance and improving the reliability of single-molecule sensing.