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One-pot electrochemical detection of foodborne pathogen based on in situ nucleic acid amplification and wash-free assay

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Abstract

A signal amplification electrochemical biosensor chip was developed to integrate loop-mediated isothermal amplification (LAMP) based on in situ nucleic acid amplification and methyl blue (MB) serving as the hybridization redox indicator for sensitive and selective foodborne pathogen detection without a washing step. The electrochemical biosensor chip was designed by a screen-printed carbon electrode modified with gold nanoparticles (Au NPs) and covered with polydimethylsiloxane membrane to form a microcell. The primers of the target were immobilized on the Au NPs by covalent attachment for in situ amplification. The electroactive MB was used as the electrochemical signal reporter and embedded into the double-stranded DNA (dsDNA) amplicons generated by LAMP. Differential pulse voltammetry was introduced to survey the dsDNA hybridization with MB, which differentiates the specifically electrode-unbound and -bound labels without a washing step. Pyrene as the back-filling agent can further improve response signaling by reducing non-specific adsorption. This method is operationally simple, specific, and effective. The biosensor showed a detection linear range of 102–107 CFU mL−1 with the limit of detection of 17.7 CFU mL−1 within 40 min. This method showed promise for on-site testing of foodborne pathogens and could be integrated into an all-in-one device.

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Acknowledgements

This work was supported by the Science and Technology Benefiting the People Demonstration Project of Qingdao (22-2-7-smjk-2-nsh) and the Key Project of Shandong Province Natural Science Foundation (ZR2020KH030). This work is supported by the Taishan Industrial Experts Program.

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  1. ** Ma

  2. Department of Clinical Laboratory, The Affiliated Hospital of Qingdao University, Qingdao, 266071, People’s Republic of China

    Longqiang Xu

  3. Qingdao JianMa Gene Technology Co., Ltd, Qingdao, 266114, People’s Republic of China

    Yanli Ji & Chao Shi

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** Ma: Resources, Funding acquisition. Chao Shi: Conceptualization, Project administration, Funding acquisition, Supervision.

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Li, X., Fan, Y., Gong, H. et al. One-pot electrochemical detection of foodborne pathogen based on in situ nucleic acid amplification and wash-free assay. Microchim Acta 191, 431 (2024). https://doi.org/10.1007/s00604-024-06500-3

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