Single-molecule sensing with an anthrax nanopore enabled by pH-asymmetric ionic liquids

The anthrax protective antigen self-assembles into a heptameric transmembrane channel under acidic conditions, exhibiting characteristic biological nanopore properties. However, the anthrax channel faces significant challenges in single-molecule nanopore sensing due to inherent instability issues such as sudden current attenuation, frequent channel occlusion and noise interference, primarily mediated by phenylalanine clamps within the channel lumen. To address these limitations on nanopore sensing, we designed four electrolyte systems (KCl, NaCl, LiCl, and 1-butyl-3-methylimidazolium chloride [BMIM]Cl) with asymmetric ionic strength and pH condition to stabilize the anthrax nanopore platform. While traditional alkali metal chlorides (KCl, NaCl, LiCl) showed partial improvement in electrical properties, the [BMIM]Cl system with pH asymmetry enabled a stable nanopore with optimal electrical characteristics, including diminished channel occlusion, enhanced signal-to-noise ratio, steady discrete constant and current strength of open-pore, all indicative of uniform ion transport. Furthermore, the stabilized anthrax nanopore demonstrated exceptional molecular detection capability, resolving single-molecule discrimination of homologous peptides of MAPKK gene families which are key signaling proteins involved in the regulation of various biology processes in eukaryotic organisms. This work establishes a robust nanopore platform for high-resolution small-molecule detection and highlights the unique advantages of ionic liquids in regulating nanoscale molecule transport.