Understanding the Characteristics of Small DNA Detection and Effect of Electrolyte on Translocation Performance in Nanopore Sensors

Nanopore sensing provides a powerful single‐molecule platform for detecting and characterizing nucleic acids. In this study, track‐etched poly(ethylene terephthalate) (PET) nanopores were fabricated and used to investigate the translocation dynamics of ultrashort DNA fragments that is 25‐bp single‐stranded (ss‐DNA) and 25‐bp double‐stranded DNA (ds‐DNA). Experimental ionic current recordings and finite‐element simulations were combined to elucidate the influence of electrolyte type, particularly KCl and LiCl, on translocation kinetics. The results revealed that ds‐DNA exhibited higher translocation frequency and larger current blockades than ss‐DNA, attributed to its stiffer structure, higher charge density, and stronger coupling to the pore's electric field. Switching from KCl to LiCl significantly increased dwell times and reduced current amplitudes, consistent with stronger Li + –DNA interactions that partially neutralize the phosphate backbone and suppress electro‐osmotic flow. Quantitatively, the transition from K + to Li + resulted in approximately a 40%–50% increase in event duration and a 25%–35% reduction in current‐pulse amplitude. Finite‐element simulations reproduced these experimental trends and confirmed the dominant role of electrophoretic and electro‐osmotic forces in governing translocation. The findings provide valuable insight into the design of nanopore‐based biosensors and demonstrate that electrolyte selection can effectively tune DNA translocation dynamics for improved detection and analytical resolution.