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A turn-on fluorescent method for determination of the activity of alkaline phosphatase based on dsDNA-templated copper nanoparticles and exonuclease based amplification

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Abstract

The authors describe a method for the determination of the activity of alkaline phosphatase (ALP) that utilizes dsDNA-templated copper nanoparticles (CuNPs) coupled to enzymatic amplification via λ exonuclease. A hybrid of a DNA modified with a phosphate moiety at the 5′-end (P-DNA) and a P-DNA complementary sequence (cP-DNA) is employed as the dsDNA substrate for ALP. In the absence of ALP, the dsDNA is cleaved by the λ exonuclease, which hinders the formation of CuNPs which display fluorescence with excitation/emission peaks at 340/565 nm. However, ALP-mediated hydrolysis of the 5′-phosphoryl end impedes the cleavage of dsDNA by the λ exonuclease, and this promotes the formation of fluorescent dsDNA-templated CuNPs via ascorbate-mediated reduction. Under the optimized experimental conditions, this method exhibits a high specificity to ALP and has a 0.1 U⋅L−1 limit of detection. The strategy also provides the basis for a screening platform for inhibitors of ALP.

Schematic presentation of a fluorescence assay for the detection of alkaline phosphatase based on dsDNA-templated copper nanoparticles and exonuclease based amplification.

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Acknowledgements

This work was supported by National Natural Science Foundation of China (No. 21205142, 31370104), The Research Innovation Program for Graduates of Central South University (2016zzts580).

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Authors and Affiliations

  1. State Key Laboratory of Medical Genetics & School of Life Sciences, Central South University, Changsha, 410013, China

    Haisheng Liu, Changbei Ma, Jun Wang & Kefeng Wu

  2. State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, 410081, China

    Kemin Wang

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  1. Haisheng Liu
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Correspondence to Changbei Ma.

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Liu, H., Ma, C., Wang, J. et al. A turn-on fluorescent method for determination of the activity of alkaline phosphatase based on dsDNA-templated copper nanoparticles and exonuclease based amplification. Microchim Acta 184, 2483–2488 (2017). https://doi.org/10.1007/s00604-017-2256-6

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  • DOI: https://doi.org/10.1007/s00604-017-2256-6

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