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

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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.

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.

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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.

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu, 214122, China

    Huajie Gu, Liling Hao, Nuo Duan, Shijia Wu, Yu Xia, Xiaoyuan Ma & Zhouping Wang

  2. School of Chemical Biology and Materials Engineering, Suzhou University of Science and Technology, Suzhou, Jiangsu, 215009, China

    Huajie Gu

  3. School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian, 116034, China

    Zhouping Wang

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  1. Huajie Gu
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Correspondence to Zhouping Wang.

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Gu, H., Hao, L., Duan, N. et al. A competitive fluorescent aptasensor for okadaic acid detection assisted by rolling circle amplification. Microchim Acta 184, 2893–2899 (2017). https://doi.org/10.1007/s00604-017-2293-1

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  • DOI: https://doi.org/10.1007/s00604-017-2293-1

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