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Amperometric determination of the activity of protein kinase a using a glassy carbon electrode modified with IgG functionalized gold nanoparticles conjugated to horseradish peroxidase

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Abstract

The authors describe the fabrication of an electrochemical immunosensor for the determination of the activity of protein kinase A (PKA). The method involves (a) electrochemical deposition of gold nanoparticles (AuNPs) on a glassy carbon electrode, (b) PKA-induced catalytic phosphorylation of serine, and (c) the use of phosphoserine antibody and horseradish peroxidase conjugated to IgG on gold nanoparticles (HRP-IgG-AuNPs). The use of AuNPs and HRP-IgG-AuNPs results in large amplification so that the method, at a typical working potential as low as 0.08 V (vs. SCE), has a linear range that extends from 0.1 to 50 activity units per mL, and the detection limit is 0.026 units per mL (at an S/N ratio of 3). The assay is selective (not the least due to a rather low working potential) and well reproducible. It may also be applied to screening for PKA inhibitors and to quantify the PKA activity in human cell lysates.

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Acknowledgements

This work was supported by China Postdoctoral Science Foundation (No. 2014 M550369), the National Natural Science Foundation of China (No. 21375079), the Natural Science Foundation of Shandong province, China (No. ZR2014BQ029), and the Project of Development of Science and Technology of Shandong Province, China (No. 2013GZX20109).

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Authors and Affiliations

  1. College of Chemistry and Material Science, Shandong Agricultural University, 271018, Taian, Shandong, People’s Republic of China

    Yunlei Zhou, Minghui Wang, Huanshun Yin & Shiyun Ai

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  1. Yunlei Zhou
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  2. Minghui Wang
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  3. Huanshun Yin
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  4. Shiyun Ai
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Correspondence to Huanshun Yin.

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Zhou, Y., Wang, M., Yin, H. et al. Amperometric determination of the activity of protein kinase a using a glassy carbon electrode modified with IgG functionalized gold nanoparticles conjugated to horseradish peroxidase. Microchim Acta 184, 3301–3308 (2017). https://doi.org/10.1007/s00604-017-2341-x

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