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Aptamer-based fluorescent solid-phase thrombin assay using a silver-coated glass substrate and signal amplification by glucose oxidase

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Abstract

We describe an aptamer-based solid-state biosensor for the fluorometric determination of thrombin. The surface of silver-coated glass was modified with the thrombin-binding aptamer 1 (TBA 1) of the sequence 5′-HS-TTT TTT TTT TTT TTT GGT TGG TGT GGT TGG-3′. A second (and biotinylated) thrombin -binding aptamer (TBA 2) with the sequence 5′-biotin-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′ was applied as the signaling aptamer. Following binding of thrombin by TBA 1 on the surface, TBA 2 is added and then binds to the thrombin on the surface of the silver-coated glass to form the thrombin-aptamer complex. The biotin groups on TBA 2 are then coated with streptavidin, and biotin-labeled glucose oxidase (biotin-GOx) is added to bind to streptavidin. The quantity of GOx immobilized in this way is directly related to the quantity of thrombin bound on the surface. Following cleavage of the aptamer with DNase I, glucose is added and oxidized by GOx to yield H2O2. Horseradish peroxidase is added and causes the oxidation of 3-p-hydroxyphenylpropanoic acid to yield a fluorescent product. The intensity of the blue fluorescence is directly related to the thrombin concentration in the 300 pM to 6500 pM range, and the detection limit is as low as 82 pM. The assay has good selectivity and practicability.

A fluorescent assay was developed for determination of thrombin by using an aptamer immobilized on the surface of silver-coated glass and by exploiting hydrolysis by DNase I and amplification by glucose oxidase

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Acknowledgments

The work was supported by National Natural Science Foundation of China (No: 21375153), the Fundamental Research Funds for the Central Universities (No: 13lgzd05) and open project of Beijing National Laboratory for Molecular Sciences.

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Authors and Affiliations

  1. School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, 510275, People’s Republic of China

    Yubin Li & Liansheng Ling

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  1. Yubin Li
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  2. Liansheng Ling
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Correspondence to Liansheng Ling.

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Li, Y., Ling, L. Aptamer-based fluorescent solid-phase thrombin assay using a silver-coated glass substrate and signal amplification by glucose oxidase. Microchim Acta 182, 1849–1854 (2015). https://doi.org/10.1007/s00604-015-1515-7

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  • DOI: https://doi.org/10.1007/s00604-015-1515-7

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