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  • Review Article
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Understanding single-molecule reactions using nanopore-based techniques

Nature Chemistry volume 17, pages 1450–1461 (2025)Cite this article

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Abstract

The formation and cleavage of chemical bonds are two fundamental processes in chemistry, and the nature of these bonds determines the physical and chemical properties of a molecule. Real-time observation of chemical bonding at the single-molecule level offers insights into transient intermediates that are normally inaccessible via ensemble measurements. Protein nanopores, with their unique geometries, can be tailored into nanoreactors. Molecular bond-making and -cleavage at the reactive site of a protein nanopore’s interior wall can be visualized by monitoring ionic current changes. Therefore, nanopore-based techniques can enhance the understanding of complex binding kinetics and reaction mechanisms. In this Review we summarize recent advances in using biological nanopores as both single-molecule nanoreactors and single-molecule biosensors. The discussion covers the kinetics of single-molecule reactions under nanopore confinement, the strategies for designing biological nanopores and the latest progress in revealing reaction intermediates and pathways at the single-molecule level. Finally, we emphasize unresolved challenges and anticipate future developments in this rapidly evolving field.

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Fig. 1: Biological nanopores for studying single-molecule reactions.
Fig. 2: Designing nanopore confinement for single-molecule reactions.
Fig. 3: Recording single-molecule covalent reactions with designed nanopores.
Fig. 4: Tuning and controlling the reaction equilibrium inside a nanopore using an external electric field and a neighbouring amino acid.
Fig. 5: Concept of a designed nanopore for single-molecule synthesis.

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Acknowledgements

This research was supported by the National Key R&D Program of China (2022YFA1304604), the National Natural Science Foundation of China (2247040607, 22027806 and 22334006) and programs for high-level entrepreneurial and innovative talent introduction of Jiangsu Province.

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  1. Molecular Sensing and Imaging Center, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China

    Yi-Lun Ying, Chao-Nan Yang, Wei Liu, Xue-Yuan Wu & Yi-Tao Long

  2. Chemistry and Biomedicine Innovation Center, Nanjing University, Nanjing, China

    Yi-Lun Ying

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Y.-L.Y. and Y.-T.L. conceived the concept of this Review. Y.-L.Y., C.-N.Y., W.L. and X.-Y.W. wrote the manuscript. C.-N.Y., W.L. and Y.-L.Y. prepared the figures. All authors contributed to the reviewing and editing of the manuscript.

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Ying, YL., Yang, CN., Liu, W. et al. Understanding single-molecule reactions using nanopore-based techniques. Nat. Chem. 17, 1450–1461 (2025). https://doi.org/10.1038/s41557-025-01905-w

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