An electrochemical biosensor was developed for the determination of sarcoma (Src)-related protein kinase-catalyzed phosphorylation reactions in the presence of adenosine 5'-gamma-ferrocenoyl triphosphate (Fc-ATP). The sensing platform is based on a highly specific amino acid sequence Glu-Gly-Ile-Tyr-Asp-Val-Pro (EGIYDVP), to which a Fc-PO2 moiety can be transferred from Fc-ATP by the action of the Src kinase. The enzyme kinetics and kinase inhibition were investigated by square wave voltammetry (SWV). The kinetic parameters K-m and V-max were determined for Src protein kinase with respect to Fc-ATP co-substrate and were found to be 200 mu M and 115 mu A cm(-2) min, for phosphorylation of the EGIYDVP peptide substrate. Furthermore, the Src-catalyzed phosphorylation of Tyr was investigated in the presence of the small molecule inhibitors PP1, PP2, SU6656, and roscoyitine. PP3 does not inhibit Src activity and was used as a control. The percent inhibition at half concentration, IC50, values were determined for all inhibitors under the study and were estimated to be in the 5-30 nM range. The electrochemical study suggests that the increase in inhibition efficiency was in the order PP3 < SU6656 < roscovitine < PP2 < PP1. (C) 2011 Elsevier Ltd. All rights reserved.