Analytical and Bioanalytical Chemistry

, Volume 411, Issue 25, pp 6637–6644 | Cite as

A competitive thrombin-linked aptamer assay for small molecule: aflatoxin B1

  • Chao Wang
  • Qiang ZhaoEmail author
Research Paper


We described a competitive thrombin-linked aptamer assay for small molecule, using aflatoxin B1 (AFB1) as a model, taking advantage of aptamer affinity binding and enzymatic activity of thrombin. We designed a dual functional DNA probe that contained the aptamer sequence for thrombin and the aptamer sequence for AFB1. Thrombin was labeled on the DNA probe by affinity binding between thrombin and anti-thrombin aptamer. This thrombin-labeled DNA probe was attached on AFB1-bovine serum albumin conjugate (BSA-AFB1) coated on a microplate through the affinity interaction between AFB1 and anti-AFB1 aptamer. The thrombin attached on the microplate catalyzed the cleavage of peptide substrate into detectable product, generating signal for detection. In the presence of AFB1, free AFB1 competed with BSA-AFB1 in the binding to the limited amount of DNA probe, leading to signal decrease. Detection of AFB1 was achieved by measuring the signal change. Under optimized conditions, AFB1 was successfully detected in the range from 0.5 nM to 1 μM when fluorogenic peptide substrate of thrombin and fluorescence analysis were applied. The use of chromogenic peptide substrate in the assay allowed the detection of AFB1 ranging from 0.5 to 125 nM by simple absorbance analysis. The thrombin-linked aptamer assay showed good selectivity towards AFB1, and enabled the detection of AFB1 spiked in diluted beer and corn flour. This TLAA strategy extends the analytical application of thrombin and aptamers in detection of small molecules.

Graphical abstract


Aptamer Thrombin Aflatoxin B1 Enzyme Fluorescence Absorbance 


Funding information

We acknowledged the financial support from the National Natural Science Foundation of China (Grant No. 21575153, 21874146, 21435008) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030200).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2019_2037_MOESM1_ESM.pdf (185 kb)
ESM 1 (PDF 184 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental SciencesChinese Academy of ScienceBeijingChina
  2. 2.University of Chinese Academy of ScienceBeijingChina

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