Label-free electrochemical measurement of protein tyrosine kinase activity and inhibition based on electro-catalyzed tyrosine signaling.

Yu Yang, Liang-Hong Guo, Na Qu, Ming-Yuan Wei, Li-Xia Zhao, Bin Wan

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, PO Box 2871, Beijing 100085, China.

Biosensors & bioelectronics 2011 Oct 15

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A novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1a>) peptide, as a substrate of EGFR, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy)(3)(2+) (bpy=2,2'-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 1 UmL(-1). Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC(50) of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants K(i) were estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Citation

Yu Yang, Liang-Hong Guo, Na Qu, Ming-Yuan Wei, Li-Xia Zhao, Bin Wan. Label-free electrochemical measurement of protein tyrosine kinase activity and inhibition based on electro-catalyzed tyrosine signaling. Biosensors & bioelectronics. 2011 Oct 15;28(1):284-90