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Determination of the platelet-derived growth factor BB by a competitive thrombin-linked aptamer-based Fluorometric assay

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Abstract

The authors describe a competitive aptamer based assay for detection of the platelet-derived growth factor BB (PDGF-BB; used as a model protein). The assay is making use of thrombin (a serine protease) as an enzyme label for reporting signals. It is taking advantage of a highly selective aptamer and of the fairly specific enzymatic activity of thrombin in terms of cleaving artificial fluorogenic peptide substrates. In a first step, the surface of wells of microplates is coated with PDGF-BB. On addition of a sample containing PDGF-BB, free and bound PDGF-BB compete with each other for binding to a DNA probe that consists of an aptamer sequence for PDGF-BB and a 29-mer aptamer sequence for thrombin. After washing, thrombin is added and will attach to the DNA probe that bound to the PDGF-BB on the microplates. Following addition of a fluorogenic peptide substrate, the bound thrombin will catalyze the cleavage of the substrate to generate a fluorescent product whose fluorescence intensity is measured at excitation/emission wavelengths of 370/440 nm. Fluorescence intensity decreases with increasing PDGF-BB concentration in the sample because less thrombin will bind to the PDGF-BB coated surface of the microplate. Under optimal conditions, PDGF-BB can be quantified in the 0.125 to 3 nM concentration range. This assay was successfully applied to the determination of PDGF-BB in spiked 100-fold diluted human serum.

In a competitive thrombin-linked aptamer assay, free platelet-derived growth factor BB (PDGF-BB) sample competes with the PDGF-BB coated on microplates for binding to a DNA probe containing PDGF-BB-binding aptamer and thrombin-binding aptamer. The labeled thrombin cleaves substrate into product, achieving signal generation.

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Acknowledgments

This work was supported by National Natural Science Foundation of China (Grant No. 21222503, 21435008, 21575153), Outstanding Youth Talents Program of Shanxi Province, and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030200).

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Authors and Affiliations

  1. Institute of Environmental Science, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan, 030006, China

    Limin Guo & Qiang Zhao

  2. School of Chinese Materia Medica, Shanxi University of Traditional Chinese Medicine, Taiyuan, 030024, China

    Limin Guo

  3. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China

    Qiang Zhao

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  1. Limin Guo
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Correspondence to Qiang Zhao.

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Guo, L., Zhao, Q. Determination of the platelet-derived growth factor BB by a competitive thrombin-linked aptamer-based Fluorometric assay. Microchim Acta 183, 3229–3235 (2016). https://doi.org/10.1007/s00604-016-1978-1

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  • DOI: https://doi.org/10.1007/s00604-016-1978-1

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