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Colorimetric and electrochemical determination of the activity of protein kinase based on retarded particle growth due to binding of phosphorylated peptides to DNA – capped silver nanoclusters

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Abstract

The authors describe a method for highly sensitive and selective determination of the activity of protein kinase (PKA). It is based on the finding that silver nanoclusters (AgNCs) can act as a nucleus to catalyze further deposition of silver nanoparticles. This causes the color of a solution to change from pale yellow to black. In the detection scheme presented here, the substrate peptide is phosphorylated by PKA in the presence of ATP. The resulting phosphopeptides bind to oligonucleotide-stabilized AgNCs in the presence of Zr(IV) ions due to electrostatic interactions between Zr(IV) and the phosphate groups, thereby capping the AgNCs. The silver enhancement process (leading to a color change to black) does not work if the AgNCs are capped. The degree of inhibition is proportional to the activity of the kinase. The color change can be detected visually or photographically in a microplate format by exploiting the changes in the grey values of the digital photos. In addition, the DNA-AgNCs display fluorescence emission at 635 nm when excited at 565 nm. Electrochemical assays were performed (at a working voltage as low as 38 mV vs. Ag/AgCl) by using a glassy carbon electrode modified with a solution containing AgNCs, Zr(IV) ions and the peptide, and immersing it into the silver enhancement solution. The assay is highly sensitive and selective. It was applied to the determination of PKA in lysates of HeLa cells. The detection limits typically are between 32 and 37 U⋅ L-1 based on a signal-to-noise ratio of 3.

A method for colorimetric and electrochemical determination of the activity of protein kinase activity is described that is based on silver nanocluster (AgNC) based signal amplification. AgNCs act as nucleus for further deposition of silver nanoparticles, but protein kinase can inhibit this process.

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Acknowledgments

The authors thank the support of this work by the National Key Basic Research Program of China (2014CB744502), the National Natural Science Foundation of China (No. 21575165) and the Natural Science Foundation of Hunan province (No. 2015JJ1019).

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Authors and Affiliations

  1. College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, China

    Congcong Shen, Kaina Zhang, Nanxing Gao, Shijiong Wei, Guimei Liu, Yuanlin Chai & Minghui Yang

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  1. Congcong Shen
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  2. Kaina Zhang
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  3. Nanxing Gao
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  4. Shijiong Wei
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  5. Guimei Liu
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  6. Yuanlin Chai
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  7. Minghui Yang
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Correspondence to Minghui Yang.

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The author(s) declare that they have no competing interests.

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Congcong Shen and Kaina Zhang contributed equally to this work.

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Shen, C., Zhang, K., Gao, N. et al. Colorimetric and electrochemical determination of the activity of protein kinase based on retarded particle growth due to binding of phosphorylated peptides to DNA – capped silver nanoclusters. Microchim Acta 183, 2933–2939 (2016). https://doi.org/10.1007/s00604-016-1944-y

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  • DOI: https://doi.org/10.1007/s00604-016-1944-y

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