www.elsevier.nl/locate/jelechem Journal of Electroanalytical Chemistry 495 (2000) 36 – 41 Electron transfer processes of redox proteins at inherently modified microelectrode array devices Michael Kudera a , Anthony Aitken a , Li Jiang a , Susumu Kaneko a , H. Allen O. Hill a, *, Peter J. Dobson b , Peter A. Leigh b , William S. McIntire c,d a Inorganic Chemistry Laboratory, Uniersity of Oxford, South Parks Road, Oxford OX13QR, UK b Department of Engineering Science, Uniersity of Oxford, Parks Road, Oxford OX13PJ, UK c Molecular Biology Diision, Department of Veterans Affairs Medical Center, San Francisco, CA 94121, USA d Department of Biochemistry and Biophysics and Department of Anesthesia, Uniersity of California, San Francisco, CA 94143, USA Received 21 August 2000; accepted 27 August 2000 Abstract Microelectrode array (MEA) devices were employed to investigate the direct electrochemistry of three redox proteins, namely: horse heart cytochrome c, amicyanin from Thiobacillus ersutus and amicyanin from Paracoccus denitrificans. Cyclic voltammetric experiments were carried out with arrays of 15 ×15 square-shaped gold microdisks of 5 m in dimension. The gold microelec- trodes were defined by a photoresist or silicon nitride coating as the insulating layer. The occurrence of well-behaved, steady-state responses of the redox proteins only at MEAs fabricated with photoresist may be explained by a model which assumes that one component of the photoresist acts as a facilitator enabling electron transfer between the microelectrodes and the protein molecules. It could be shown that the photoresist facilitates the electrochemistry of positively and negatively charged proteins. The formal redox potentials of both types of amicyanin were studied over a range of solution pH. In agreement with previous studies, the formal redox potentials increased with decreasing pH because of the redox inactivity of amicyanin in the Cu I state at low pH. These experiments demonstrated that photoresist-coated MEAs can be employed as versatile tools to gain insight into the properties of redox biomacromolecules. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Amicyanin; Cytochrome c; Direct protein electrochemistry; Electron transfer facilitator; Microelectrode array 1. Introduction During the past two decades the electrochemistry of redox biomacromolecules, i.e. redox proteins and en- zymes, has been investigated extensively [1]. Intra- and inter-molecular electron transfer processes of these molecules are of great interest because they have impor- tant physiological implications. Since the first observation of direct electron transfer of cytochrome c by Yeh and Kuwana [2] and Eddowes and Hill [3] considerable progress has been achieved in the exhibition of direct electrochemistry of various biomacromolecules. A key for this was the functionali- sation of the electrode surfaces [4]. The functionalisa- tion with so-called facilitators simulates the physiological environment of the protein, which en- courages the protein molecules to approach and pre- vents denaturation of the proteins at the electrode surface. Complementary hydrogen bonds, salt-bridges, hydrophobic contacts or electrostatic interactions might be provided by the electroinactive facilitator molecules in order to fulfil this task. The studies presented here are concerned with the application of microelectrode array (MEA) devices in bioelectrochemical experiments (for a review on mi- croelectrodes see Ref. [5]). Photolithography and plasma-enhanced chemical vapour deposition (PECVD) were used to manufacture the MEAs, which were previ- ously characterised by employing simple electroactive species [6]. The redox proteins investigated at these MEAs were the haem protein cytochrome c and the type I blue copper protein amicyanin from the bacteria Thiobacillus ersutus and Paracoccus denitrificans. * Corresponding author. Tel./fax: +44-1865-275900. E-mail address: allen.hill@chemistry.oxford.ac.uk (H.A.O. Hill). 0022-0728/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII:S0022-0728(00)00384-3