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Determination of the activity of alkaline phosphatase based on aggregation-induced quenching of the fluorescence of copper nanoclusters 

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Abstract

A rapid method is described for synthesis of copper nanoclusters (CuNCs) by utilizing L-histidine as the stabilizer and ascorbic acid (AA) as the reductant. The CuNCs display blue-green fluorescence with excitation/emission peaks at 390/485 nm. A sensitive fluorometric assay was worked out for determination of alkaline phosphatase (ALP) activity. If the ALP substrate p-nitrophenylphosphate (PNPP) is enzymatically hydrolyzed, it forms p-nitrophenol (PNP) which reduces the fluorescence of CuNCs because its absorption band at 410 nm overlaps the excitation peak of CuNCs at 390 nm. In addition, the amino groups and imidazole groups on the surface of CuNCs possibly form a complex with the phenol groups of PNP. This induces aggregation-induced quenching of the fluorescence of CuNCs. The fluorescent probe has a linear analytical range that extends from 0.5 mU·mL−1 to 40 mU·mL−1 and a detection limit of 45 μU·mL−1.

Schematic illustration of a fluorometric assay for alkaline phosphatase (ALP) activity that uses L-histidine protected copper nanoclusters (CuNCs), aggregation-induced quenching, and the inner filter effect between PNP and CuNCs.

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Acknowledgements

This work was financially supported by Supported by National Natural Science Foundation of China (21707030) and Wuhan Youth Science and technology plan (2016070204010133).

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Authors and Affiliations

  1. Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry-of-Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules, College of Chemistry and Chemical Engineering, Hubei University, Wuhan, 430062, China

    Yanling Hu, Yu He, Yaxue Han, Yili Ge & Gongwu Song

  2. Hubei Province Key Laboratory of Regional Development and Environment Response, Wuhan, 430062, China

    Yu He & Jiangang Zhou

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  1. Yanling Hu
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  2. Yu He
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  3. Yaxue Han
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  4. Yili Ge
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Correspondence to Yu He.

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Hu, Y., He, Y., Han, Y. et al. Determination of the activity of alkaline phosphatase based on aggregation-induced quenching of the fluorescence of copper nanoclusters . Microchim Acta 186, 5 (2019). https://doi.org/10.1007/s00604-018-3122-x

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