The fabrication of electrochemical biosensor consists of the following successive steps: formation of thiol derivative of iminodiacetic acid (IDA-like/N-heterocyclic donor) and N-acetylcysteamine (NAC) self-assembled monolayer on the Au electrode, complexation of Cu(II) by N(IDA-like) attached to the surface of the Au electrode and immobilization of kinase protein Rio1 through N(IDA-like)-Cu(II)-histidine-tag covalent bond formation. Each step of modification was controlled by cyclic voltammetry, electrochemical impedance spectrometry and atomic force microscopy. The interactions between rHis(6)-Rio1 attached to the surface of the electrode and tyrphostin inhibitor (2E)-N-Benzyl-2-cyano-3-(3,4-dihydroxyphenyl)-acrylamide (AG-490) and its analogue (2-cyano-N-(4-methoxyphenyl)-3-(pyridin-3-yl)prop-2-enamide) (CPE), present in aqueous solution were monitored with Osteryoung square wave voltammetry. The basis of the biosensor response was the change in the electrochemical properties of Cu(II) redox centres upon formation of the rHis(6)-Rio1 -inhibitor complex. A linear responses with high reproducibility and stability were observed between 0.10 and 0.40 mu M of AG-490 as well as of CPE. The interaction between rHis(6)-Rio1 and AG-490 was stronger than the interaction with its analogue CPE. Cu(II) redox current decrease of 37.9 +/- 1.6% and 23.3 +/- 1.0% were observed in the presence of 0.40 mu M of AG-490 and CPE, respectively. The presented biosensor could be applied as an analytical tool, useful in screening of interactions between rHis(6)-Rio1 and compounds, that could function as potential kinase inhibitors. (C) 2013 Elsevier Ltd. All rights reserved.