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One-step electrochemical immunosensing for simultaneous detection of two biomarkers using thionine and ferrocene as distinguishable signal tags

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Abstract

We report on a new kind of electrochemical immunosensors for simultaneous determination of the biomarkers carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Thionine and ferrocene were applied as distinguishable electrochemical tags (and mediators) which were covalently conjugated on anti-AFP and anti-CEA antibodies, respectively, via carboxy groups. The resulting conjugates were co-immobilized on a glassy carbon electrode functionalized with gold nanoparticles. Finally, horseradish peroxidase (HRP) was immobilized onto the modified electrode. Labeled thionine and ferrocene, respectively, act as distinguishable tags for simultaneous determination of AFP and CEA due to the difference in the location of their voltammetric peaks. With a one-step immunoassay format, the analytes in the sample produced transparent immunoaffinity reaction with the corresponding antibodies on the electrode. Once the immunocomplex is formed, it partially inhibits the active center of the immobilized HRP, and this decreased the activity of HRP in terms of reduction of hydrogen peroxide. This immunosensor enables the simultaneous determination of AFP and CEA in a single run and within the same dynamic range (0.01–50 ng mL−1) and the same lower detection limit (0.01 ng mL−1). The reproducibility and stability of the immunosensors are acceptable. The dual immunosensor was applied to evaluate several specimens, and the assay results are in acceptable agreement with clinical data.

This contribution devises a novel multiplexed electrochemical immunoassay for simultaneous detection of alpha-fetoprotein and carcinoembryonic antigen by using thionine and ferrocene as distinguishable signal tags on a one-spot immunosensor. The assay was performed by using one-step immunoreaction between the immobilized antibodies and the analytes. Although the linear range is relatively narrow, it completely meets the requirement of clinical diagnosis.

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Acknowledgements

Support by the Research Fund for the Doctoral Program of Higher Education of China (no. 20103514120003), the National Science Foundation of Fujian Province (no. 2011J06003), the National Natural Science Foundation of China (nos. 21075019 and 41176079), the “973” National Basic Research Program of China (no. 2010CB732403), and the Program for Changjiang Scholars and Innovative Research Team in University (no. IRT1116) is gratefully acknowledged.

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Authors and Affiliations

  1. Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, 350108, People’s Republic of China

    Wenqiang Lai, Junyang Zhuang, Juan Tang, Guonan Chen & Dianping Tang

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  1. Wenqiang Lai
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  2. Junyang Zhuang
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  3. Juan Tang
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  5. Dianping Tang
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Correspondence to Dianping Tang.

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Lai, W., Zhuang, J., Tang, J. et al. One-step electrochemical immunosensing for simultaneous detection of two biomarkers using thionine and ferrocene as distinguishable signal tags. Microchim Acta 178, 357–365 (2012). https://doi.org/10.1007/s00604-012-0839-9

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  • DOI: https://doi.org/10.1007/s00604-012-0839-9

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