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Gold nanocluster-encapsulated glucoamylase as a biolabel for sensitive detection of thrombin with glucometer readout

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Abstract

This article reports on a sensitive aptamer-based assay for thrombin. The assay includes the following steps: (a) a first thrombin-specific aptamer (P1) was immobilized on the surface of the wells of a microtiter plate via biotin-streptavidin interaction; (b) gold nanoclusters (GNCs) were used to cover the enzyme glucoamylase via a reverse micelle method; (c) the GNCs were then coated with the second aptamer (P2) via thiol chemistry; (d) addition of a solution containing thrombin to the well, and (e) subsequent addition of amylopectin. The glucoamylase in the GNC label catalytically hydrolyzes the amylopectin to form glucose which then is quantified with a glucometer. Under optimal conditions, the signal for glucose increases with the concentration of thrombin in range from 0.05 to 100 nM, and the detection limit is as low as 10 pM. The assay has a good repeatability and displays an intermediate precision of down to 11 %. Nonspecific adsorption was not observed in a series of analyses. The method was applied to the determination of thrombin in spiked serum samples, and the recoveries ranged from 94 to 110 %. The method is assumed to have a wide scope in that it may be extended to numerous other analytes for which appropriate aptamers are available.

An aptamer-based sandwich assay for thrombin was developed using aptamer-coated microwell and using glucoamylase coated with a gold nanocluster as the biolabel.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (grant no.: 21405128) and the Research (Initial) Fund for the Doctoral Program of Xinxiang University.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Engineering, Institute of Biotechnology, Xinxiang University, Xinxiang 453000, People’s Republic of China

    Ai-Li Sun, Yan-Fang Zhang & Xuan-Nian Wang

  2. Department of Electrical Engineering, Henan Mechanical and Electrical Engineering College, Xinxiang 453000, People’s Republic of China

    Feng-Chun Jia

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  1. Ai-Li Sun
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  2. Feng-Chun Jia
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  3. Yan-Fang Zhang
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  4. Xuan-Nian Wang
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Correspondence to Ai-Li Sun or Xuan-Nian Wang.

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Sun, AL., Jia, FC., Zhang, YF. et al. Gold nanocluster-encapsulated glucoamylase as a biolabel for sensitive detection of thrombin with glucometer readout. Microchim Acta 182, 1169–1175 (2015). https://doi.org/10.1007/s00604-014-1440-1

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  • DOI: https://doi.org/10.1007/s00604-014-1440-1

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