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.
Kerman
Kagan
K
Department of Chemistry, The University of Western Ontario, 1151 Richmond Street, London, Ont., Canada N6A 5B7.
Kraatz
Heinz-Bernhard
HB
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002384:Catalysis; D004563:Electrochemistry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry*; D008839:Microelectrodes; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019311:Staurosporine / Q000737:chemistry*
D006046:Gold; D011505:Protein-Tyrosine Kinases; D019311:Staurosporine