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

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'-[gamma-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 10ng/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.