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Preparation and biomedical applications of single-metal atom catalysts

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Abstract

Nanocatalysts, including nanozymes, photocatalysts and sonocatalysts, have been investigated to trigger catalytic reactions in vivo to regulate biological microenvironments and stimulate therapeutic effects. Compared with lower metal atom utilization rate and catalytic activity of conventional nanocatalysts, single-metal atom catalysts (SACs) usually possess higher catalytic activity and selectivity owing to their well-defined structures and maximized atom utilization. Their properties are, however, strongly dependent on their composition and the preparation procedure. Here we describe the design, preparation and functionalization of SACs with single-metal atoms positioned within nitrogen-doped carbon supports. The SACs are prepared by pyrolysis of zeolitic imidazolate framework-8 (ZIF-8) or polydopamine-derived materials. Their properties depend on, for example, the metal chosen and atoms available for coordination; four example procedures are described: Cu–N4 from Cu–ZIF-8, Ir–N5 from Ir@ZIF-8 plus melamine, Co–PN3 from triphenylphosphine@Co-ZIF-8 and Cu–SN3 from ZnS@Cu-polydopamine. These SACs need to be functionalized to, for example, reduce aggregation and in vivo corona formation before they can be used in biological applications. In this Protocol, functionalization with the proteins (that is, cholesterol oxidase and pyruvate oxidase) is used as an example. The Protocol provides advice regarding physicochemical and functional characterization, as well as for performing experiments in tumor-bearing mice. The functional experiments were designed with the aim of identifying nanocatalysts with peroxidase-like activity that generate reactive oxygen species within areas of the tumor microenvironment that have increased levels of hydrogen peroxide. SAC synthesis takes 3–4 days, functional modification requires one extra day and the most basic and essential in vitro and in vivo assays require 2–3 months.

Key points

  • Single-metal atom catalysts exhibit enhanced catalytic activity and selectivity owing to their well-defined structures and maximized atom utilization, making them suitable for biomedical applications, especially in tumor treatment. The properties of single-metal atom catalysts are strongly dependent on their composition and preparation procedure.

  • This Protocol describes the design, preparation methods, functionalization strategies, physicochemical characterization, performance evaluation and biomedical application operations of single-metal atom catalysts based on nitrogen-doped carbon supports.

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Fig. 1: Schematic representation of the structure and synthesis process of SACs.
Fig. 2: Block diagram of the designed protocol for the preparation and biomedical applications of SACs.
Fig. 3: Scheme for the synthesis of SACs.
Fig. 4: TEM images and XRD patterns of SACs.
Fig. 5: XAS characterization of Cu–N4 SACs.
Fig. 6: XAS characterization of Cu–SN3 SACs.
Fig. 7: XAS characterization of Ir–N5 SACs.
Fig. 8: XAS characterization of Co–PN3 SACs.
Fig. 9: Hydrodynamic size, Zeta potentials and POD-like properties of SACs.
Fig. 10: In vitro and in vivo anticancer effects.

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Data availability

The main data discussed in this protocol are available in the supporting primary research papers84,85,96. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Research Foundation Singapore under its Competitive Research Programme (grant no. NRF-CRP26-2021-0002).

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Author notes

  1. These authors contributed equally: Yang Liu, Rui Niu.

Authors and Affiliations

  1. School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore

    Yang Liu & Yanli Zhao

  2. State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, People’s Republic of China

    Rui Niu, Yinghui Wang & Hongjie Zhang

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  1. Yang Liu
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  2. Rui Niu
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  3. Yinghui Wang
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  4. Hongjie Zhang
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  5. Yanli Zhao
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Contributions

Y.L. and R.N. contributed equally to this work. All authors contributed to developing this protocol and writing this paper. Y.W., H.Z. and Y.Z. supervised the project.

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Correspondence to Yanli Zhao.

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Nature Protocols thanks Ilaria Armenia, Stefano Fedeli, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Niu, R. et al. Adv. Mater. 36, 2312124 (2024): https://doi.org/10.1002/adma.202312124

Liu, Y. et al. Adv. Mater. 35, 2208512 (2023): https://doi.org/10.1002/adma.202208512

Liu, Y. et al. Adv. Mater. 36, 2307752 (2024): https://doi.org/10.1002/adma.202307752

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Liu, Y., Niu, R., Wang, Y. et al. Preparation and biomedical applications of single-metal atom catalysts. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01199-9

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