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Ultrasensitive amperometric aptasensor for the epithelial cell adhesion molecule by using target-driven toehold-mediated DNA recycling amplification

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Abstract

An amperometric aptasensor is reported for the electrochemical determination of the epithelial cell adhesion molecule (EpCAM). It is based on a combination of EpCAM-driven toehold-mediated DNA recycling amplification, the specific recognition of EpCAM aptamer, and its binding to EpCAM. Hairpin probe 1 (Hp1) with a toehold region was modified with a 5′-thiol group (5’-SH) and self-assembled onto the surface of a gold electrode. Upon addition of EpCAM, the probe A (a 15-mer) is liberated from the aptamer/probe A complex and then hybridizes with the toehold domain of Hp1. This results in the exposure of another toehold for further hybridizing with hairpin probe 2 (Hp2) to displace probe A in the presence of Hp2 that was labeled with the electrochemical probe Methylene Blue (MB). Subsequently, liberated probe A is hybridized again with another Hp1 to start the next round of DNA recycling amplification by reusing probe A. This leads to the formation of plenty of MB-labeled DNA strands on the electrode surface and generates an amplified current. This 1:N probe-response amplification results in ultrasensitive and specific detection of EpCAM, with a 20 pg·mL−1 detection limit. The electrode is highly stable and regenerable. It was successfully applied to the determination of EpCAM in spiked human serum, urine and saliva, and thus provides a promising tool for early clinical diagnosis.

Schematic illustration of the electrochemical detection for EpCAM. The method is based on aptamer-based recognition and EpCAM-driven toehold-mediated DNA recycling amplification. Hp1: Hairpin probe 1; Hp2: Hairpin probe 2; MB: Methylene blue; MCH: 6-Mercapto-1-hexanol; EpCAM: Epithelial cell adhesion molecule.

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Acknowledgements

These works were supported by the Natural Science Foundation of Hubei Provincial Department of Education (Nos.D20172101), the Foundationfor Innovative Research Team of Hubei University of Medicine (2014CXG04), the Science and Technology Key Program of Shiyan (Nos. 16Y70, 17 K74 and 17Y49), the School Foundation for Hubei University of Medicine (No.FDFR201614) and the Key Discipline Project of Hubei University of Medicine.

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  1. Qinhua Chen and Wanbao Hu contributed equally to this work.

Authors and Affiliations

  1. Affiliated Dongfeng Hospital, Hubei University of Medicine, Hubei Shiyan, 442008, China

    Qinhua Chen, Bing Shang, Jian Wei, Long Chen, Xiaojun Guo, Fengying Ran, Wei Chen & Xueru Ding

  2. Sinopharm Dongfeng Huaguo Hospital, Hubei Shiyan, 442008, China

    Wanbao Hu, Ying Xu & Yinhua Wu

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  1. Qinhua Chen
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Correspondence to Qinhua Chen.

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Chen, Q., Hu, W., Shang, B. et al. Ultrasensitive amperometric aptasensor for the epithelial cell adhesion molecule by using target-driven toehold-mediated DNA recycling amplification. Microchim Acta 185, 202 (2018). https://doi.org/10.1007/s00604-018-2739-0

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