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Preparation of an activatable benzothiadiazole-based nanoprobe for multispectral optoacoustic and NIR-II fluorescence dual-mode imaging of liver injury

(2026)Cite this article

Abstract

Light-excited dual-mode imaging that integrates multispectral optoacoustic tomography (MSOT) and near-infrared region II fluorescence (NIR-II FL) imaging allows complementary deep-tissue visualization with high anatomical resolution and molecular sensitivity, thereby enhancing the accuracy of biomedical diagnostics. Activatable probes further advance this approach by producing signals upon recognition of disease-related biomarkers, which reduces background interference and improves imaging specificity. BTPE-NO2@F127, a benzothiadiazole-based nanoprobe, exhibits selective activation of both optoacoustic and NIR-II FL signals in response to hydrogen peroxide (H2O2), a crucial early stage biomarker of liver injury, thus permitting cross-validated detection of hepatic damage in vivo with excellent signal-to-background ratio contrast. Here we present detailed procedures for preparation of the BTPE-NO2@F127 nanoprobe and its applications in MSOT/NIR-II FL dual-mode imaging of trazodone- or ischemia–reperfusion-induced liver injury in mice. Compared to conventional liver injury diagnostic methods, such as invasive tissue biopsy, ex vivo blood analysis and previously reported fluorescent/optoacoustic probes, BTPE-NO2@F127 offers real-time, in situ monitoring with high sensitivity and signal-to-background ratio, as well as mutually corroborating signals for increased reliability. The fabrication of BTPE-NO2@F127, including the chemical synthesis and characterization, requires ~17 d, while the in vitro validation of its H2O2 responsiveness takes ~5 d. Notably, the complete workflow of data acquisition and analysis for MSOT/NIR-II FL dual-mode imaging of liver injury in mice using BTPE-NO2@F127 can be accomplished within 10 h. The protocol is easy to follow and suitable for clinicians and researchers with a basic understanding of chemistry and bioimaging techniques.

Key points

  • This is a protocol for the preparation and characterization of the activatable BTPE-NO2@F127 nanoprobe and its applications in multispectral optoacoustic and near-infrared region II fluorescence dual-mode imaging of liver injury models in mice.

  • Compared to conventional liver injury diagnostic methods, such as invasive tissue biopsy and ex vivo blood analysis, this approach offers real-time, in situ monitoring with high sensitivity and low noise, as well as mutually corroborating signals for increased reliability.

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Fig. 1: A schematic illustration of the preparation of the BTPE-NO2@F127 nanoprobe and its biomedical applications.
Fig. 2: Preparation of the activatable benzothiadiazole-based nanoprobe BTPE-NO2@F127.
Fig. 3: Overview of the procedures for in vitro property validation and in vivo biomedical applications of the activatable benzothiadiazole-based nanoprobe BTPE-NO2@F127.
Fig. 4: Characterizations of BTPE-NO2@F127.
Fig. 5: An assessment of the specific activation behavior of BTPE-NO2@F127 in response to H2O2 in vitro.
Fig. 6: The evaluation of BTPE-NO2@F127 for in vivo detection of liver injury induced by trazodone administration or I–R.

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Data availability

The main data discussed in this protocol are available in the supporting primary research paper41. The raw datasets for Figs. 4 and 5 are provided in the Source Data file, and for Supplementary Figs. 18 and 20–25, they are provided in Supplementary Data 1. All other data are available for research purposes from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

The work was supported by the National Research Foundation Singapore under its Competitive Research Programme (grant no. NRF-CRP26-2021-0002 to Y.Z.). This work was also supported by the National Natural Science Foundation of China (grant no. 52373209 to S.W. and 22274057 to F.Z.) and the Fund of Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates (grant no. 2023B1212060003 to S.W.).

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Author notes

  1. These authors contributed equally: Yinglong Wu, Chaobang Zhang.

Authors and Affiliations

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

    Yinglong Wu & Yanli Zhao

  2. Biomedical Division, State Key Laboratory of Luminescent Materials and Devices, Guangdong Provincial Key Laboratory of Luminescence from Molecular Aggregates, College of Materials Science and Engineering, South China University of Technology, Guangzhou, China

    Chaobang Zhang, Junjie Chen, Fang Zeng & Shuizhu Wu

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Contributions

Y.Z., S.W., F.Z., Y.W. and C.Z. conceived the idea and initiated the project. Y.W., C.Z. and J.C. contributed to the experimental work involved in this protocol. Y.W. and C.Z. wrote the protocol. Y.Z., S.W. and F.Z. supervised the study and the manuscript preparation. All authors contributed to the editing and reviewing of the draft and approved the final manuscript.

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Correspondence to Shuizhu Wu or Yanli Zhao.

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Key references

Wu, Y. et al. Angew. Chem. Int. Ed. 64, e202503850 (2025): https://doi.org/10.1002/anie.202503850

Zhang, C. et al. Angew. Chem. Int. Ed. 63, e202406694 (2024): https://doi.org/10.1002/anie.202406694

Chen, J. et al. Nat. Commun. 12, 6870 (2021): https://doi.org/10.1038/s41467-021-27233-4

Wu, Y. et al. Nat. Commun. 9, 3983 (2018): https://doi.org/10.1038/s41467-018-06499-1

Supplementary information

Supplementary Information (download PDF )

Supplementary Figs. 1–25, Procedure and Tables 1–4.

Reporting Summary (download PDF )

Supplementary Data 1 (download XLSX )

Statistical source data for Supplementary Figs. 18 and 20–25.

Source data

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Wu, Y., Zhang, C., Chen, J. et al. Preparation of an activatable benzothiadiazole-based nanoprobe for multispectral optoacoustic and NIR-II fluorescence dual-mode imaging of liver injury. Nat Protoc (2026). https://doi.org/10.1038/s41596-026-01338-w

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