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Colorimetric determination of thrombin by exploiting a triple enzyme-mimetic activity and dual-aptamer strategy

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Abstract

The article describes a colorimetric assay for the determination of thrombin. It is based on the application of a triple enzyme-mimetic activity and a dual aptamer binding strategy. The triple signal amplification relies on oxidation of the chromogenic enzyme substrate 3,3,5,5-tetramethylbenzidine (TMB) that is catalyzed by composites consisting of graphene oxide (GO), gold/platinum nanoparticles (AuPtNP), and aptamer (Apt15), a G-quadruplex/hemin conjugate. The dual-aptamer target binding strategy is based on the fact that thrombin has two active sites to be recognized by its aptamers (Apt15 and Apt29). Magnetic beads (MBs) were modified with Apt29 (Apt29-MB) and then are bound by the GO-AuPtNP-Apt15/G-quadruplex/hemin composites. In the presence of thrombin, Apt29-MB and the GO-AuPtNP-Apt15/G-quadruplex/hemin composites form a sandwich-like superstructure. Thus, the absorbance increases due to the formation of TMB oxide produced by catalysis of the composites. Under optimized conditions, the absorbance at 450 nm increases linearly in the 0.30 to 100 nM thrombin concentration range, and the limit of detection is 0.15 nM. The method is simple, rapid, and does not require complicated instrumentation. Bovine serum albumin, human serum albumin and other proteins were found not to interfere.

Schematic presentation of the photometric thrombin assay based on a triple enzyme-mimetic activity of combined nanomaterials (consisting of GO, AuPtNPs and the G-quadruplex/hemin DNAzyme) and two aptamers TMB: 3,3,5,5-tetramethylbenzidine, TMBox: 3,3,5,5-tetramethylbenzidine oxide, AuPtNP: gold/platinum nanoparticles).

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 81573387, 81603072, 81673390), Jiangsu Provincial key research and development program (No. BE2016745), Natural Science Foundation of Jiangsu Province (No. BK20151445) and sponsored by Qing Lan Project.

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Authors and Affiliations

  1. Department of Pharmaceutical Analysis, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, China

    Lei Wang, Wen Yang, Tengfei Li, Duo Li, Zhimin Cui, Yan Wang, Shunli Ji, Qinxin Song, Chang Shu & Li Ding

  2. College of Pharmacy and Chemistry, Dali University, Wanhua Road, Dali, 671000, China

    Yan Wang

  3. Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009, China

    Qinxin Song

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  1. Lei Wang
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  2. Wen Yang
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  10. Li Ding
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Correspondence to Li Ding.

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Institutional Review Board approval was not required because the study is not on animals. Written informed consent was not required for this study because the study is not on human subjects.

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Wang, L., Yang, W., Li, T. et al. Colorimetric determination of thrombin by exploiting a triple enzyme-mimetic activity and dual-aptamer strategy. Microchim Acta 184, 3145–3151 (2017). https://doi.org/10.1007/s00604-017-2327-8

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  • DOI: https://doi.org/10.1007/s00604-017-2327-8

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