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Synthesis of a highly Zn2+-selective cyanine-based probe and its use for tracing endogenous zinc ions in cells and organisms

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Abstract

The zinc ion has a key role in a variety of physiological and pathological processes. As a consequence, the development of sensitive and reliable methods to monitor the presence of zinc ions in cells and organisms is of great importance to biological research and biomedical applications. This protocol describes detailed procedures for the five-stage synthesis of a zinc ion–selective, cyanine-based fluorescent probe, CTMPA, from 2,6-bis(hydroxymethyl)pyridine. In addition, we describe its applications in the detection of Zn2+ released during apoptosis in cells and endogenous Zn2+ in living zebrafish. Notably, the use of CTMPA enabled our research group to monitor for the first time the presence of zinc ions in neuromasts of zebrafish via fluorescence. The approximate time frame for the synthesis of CTMPA is 4–5 d, and for its use in bioimaging is 8–10 h for cells and 2 h for zebrafish.

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Figure 1: Zn2+-chelating agent CTMPA.
Figure 2: Emission spectra of CTMPA (5.0 μM) in titrations with Zn2+ (0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0 and 5.0 equiv.) in HEPES buffer (10 mM, pH = 7.4) containing 10% CH3CN (λex = 560 nm).
Figure 3: Fluorescence images collected in order to detect the presence of zinc ions released during apoptosis.
Figure 4: Fluorescence detection of endogenous zinc ions in 96-h-old zebrafish incubated with CTMPA for 1 h.

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Acknowledgements

This work was supported by the National Creative Research Initiative program (grant no. 2010-0018272 to I.S. and 2012R1A3A2048814 to J.Y.). Z.G. acknowledges National 973 Program (2013CB733700), National Natural Science Foundation of China (61177034) and Shanghai Pujiang Program (13PJD010).

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Author notes

  1. Zhiqian Guo and Gun-Hee Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Nano Science, Global Top 5 Research Program, Ewha Womans University, Seoul, Korea

    Zhiqian Guo & Juyoung Yoon

  2. Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai, People's Republic of China

    Zhiqian Guo

  3. Department of Chemistry, Center for Biofunctional Molecules, Yonsei University, Seoul, Korea

    Gun-Hee Kim & Injae Shin

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  1. Zhiqian Guo
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  2. Gun-Hee Kim
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  3. Juyoung Yoon
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  4. Injae Shin
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Contributions

Z.G. and G.-H.K. conducted experiments; Z.G., G.-H.K., J.Y. and I.S. designed experiments and wrote the paper.

Corresponding authors

Correspondence to Juyoung Yoon or Injae Shin.

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The authors declare no competing financial interests.

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Guo, Z., Kim, GH., Yoon, J. et al. Synthesis of a highly Zn2+-selective cyanine-based probe and its use for tracing endogenous zinc ions in cells and organisms. Nat Protoc 9, 1245–1254 (2014). https://doi.org/10.1038/nprot.2014.086

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