Nanopores have become transformative tools in single-molecule chemical analysis, enabling detailed interrogation of molecular interactions and reaction dynamics. These advancements have revolutionized the characterization of chemical kinetics and stereospecificity, broadening nanopore applications. This review evaluates the principles of nanopore single-molecule chemistry, highlighting breakthroughs in chemically reactive nanopore construction via site-specific mutagenesis, semisynthetic engineering, and orthogonal modifications. Notably, we highlight the innovative strategies enabling precise subunit stoichiometry control to ensure single-molecule reactions, and the integration of machine learning for high-fidelity ionic current analysis. These developments position nanopores as versatile tools for intricate molecular detection in fundamental and applied research. Looking forward, nanopore single-molecule chemistry promises an impact on diagnostics, environmental monitoring, and precision medicine. Integration of molecular dynamics simulations, artificial intelligence-driven protein design frameworks, and microsystems technology may expand detectable species, enhancing robustness and lowering detection limits. Such advancements will deepen our understanding of chemical transformations and support meaningful real-world applications of nanopore technologies.
Keywords: biosensor; hetero-nanopore; machine learning; nanopore engineering; single-molecule chemistry.