Microchimica Acta

, Volume 184, Issue 8, pp 2893–2899 | Cite as

A competitive fluorescent aptasensor for okadaic acid detection assisted by rolling circle amplification

  • Huajie Gu
  • Liling Hao
  • Nuo Duan
  • Shijia Wu
  • Yu Xia
  • Xiaoyuan Ma
  • Zhouping Wang
Original Paper
First Online:
Received:
Accepted:

Abstract

Okadaic acid (OA) is a low molecular weight marine toxin from shellfish which causes diarrheic shellfish poisoning (DSP). Due to its frequent occurrence, OA has become a serious threat to human health and seafood industry. The authors describe a competitive fluorophore-linked aptamer assay for OA that is based on rolling circle amplification (RCA). It consists of the following steps: (a) The wells of a microplate are modified by fixing the OA aptamer on their surface; (b) The aptamer is hybridized with an aptamer-complementary sequence-primer complex; (c) the RCA reaction is performed; (d) the FAM labelled signal probe is added. OA competes with the detection probe for the immobilized aptamer. After the competitive reaction has occurred, the supernatants containing released detection probes are removed and then read with a microplate reader. This method, unlike in competitive assays where the signals negatively correlate with OA concentrations, has a positive correlation between fluorescence intensity and OA concentration. The optimized assay has a lower detection limit (1 pg·mL−1) and a wider linear range (from 1 pg·mL−1 to 100 ng·mL−1) owning to signal amplification via RCA. It also is highly specific, repeatable, has good recovery and can be used to detect OA in seafood.

Graphical abstract

Schematic of a microplate assay for okadaic acid assisted by rolling circle amplification (RCA) and using a fluorophore-linked aptamer. The signal intensities are directly proportional to the concentrations of OA. The assay without RCA was also performed and compared to.

Keywords

Diarrheic shellfish poisoning Aptamer Microplate assay Fluorophore-linked aptamer assay Isothermal amplification Signal enhancement 
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Notes

Acknowledgements

This work was partly supported by the National Science and Technology Support Program of China (2015BAD17B02), the Key Research and Development Program of Jiangsu Province (BE2016306), the National Natural Science Foundation of China (31401576, 31401575), the Technology R&D Program of Suzhou (SYN201513), the National Grain Special Public-Funded Program of China (201513006), and Synergetic Innovation Center of Food Safety and Quality control of Jiangsu Province.

Compliance with ethical standards

The authors declare that they have no conflict of interest.

Supplementary material

604_2017_2293_MOESM1_ESM.pdf (1.4 mb)
Supplementary Material (PDF 1.36 mb)

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Copyright information

© Springer-Verlag Wien 2017

Authors and Affiliations

  • Huajie Gu
    • 1
    • 2
  • Liling Hao
    • 1
  • Nuo Duan
    • 1
  • Shijia Wu
    • 1
  • Yu Xia
    • 1
  • Xiaoyuan Ma
    • 1
  • Zhouping Wang
    • 1
    • 3
  1. 1.State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Synergetic Innovation Center of Food Safety and NutritionJiangnan UniversityWuxiChina
  2. 2.School of Chemical Biology and Materials EngineeringSuzhou University of Science and TechnologySuzhouChina
  3. 3.School of Food Science and Technology, National Engineering Research Center of SeafoodDalian Polytechnic UniversityDalianChina

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