Electrochemical detection of protein tyrosine kinase-catalysed phosphorylation using gold nanoparticles

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• 作者：Kagan Kerman ; Heinz-Bernhard Kraatz
• 关键词：Electrochemical biosensor ; Colloidal gold nanoparticles ; Phosphorylation ; Protein tyrosine kinase ; Abl-T315I ; Staurosporine
• 刊名：Biosensors and Bioelectronics
• 出版年：2009
• 出版时间：1 January 2009
• 年：2009
• 卷：24
• 期：5
• 页码：1484-1489
• 全文大小：373 K

文摘

Protein tyrosine kinase (PTK)-catalysed phosphorylation reactions of peptides are monitored electrochemically in the presence of Au nanoparticles (NPs) on peptide modified indium tin-oxide (ITO) electrodes. The method is based on the thiophosphorylation using adenosine 5′-[γ-thio] triphosphate (ATP-S) as the co-substrate. Upon thiophosphorylation of the peptides, the Au NPs accumulate on the surface and can be detected electrochemically by monitoring Cl⁻ oxidation at the Au NP surface. The activity of a clinically important PTK, Abl-T315I, which is implicated in the therapy of chronic myelogenous leukaemia (CML), is determined. Abl-T315I was assayed in combination with its highly specific substrate peptide EGIYDVP. The detection limit for the electrochemical detection of Abl-T315I activity was 10 ng/mL. The performance of the biosensor was optimized and control experiment using non-specific peptide-modified electrodes were carried out. The electrochemical current response obtained from the Cl⁻ oxidation chemistry enabled monitoring the inhibition of the thiophosphorylation reactions using staurosporine, a small molecule inhibitor.