The electrochemical characteristics of blue copper protein monolayers on gold L. Andolfi a , D. Bruce b , S. Cannistraro a , G.W. Canters c , J.J. Davis b, * , H.A.O. Hill b , J. Crozier b , M.Ph. Verbeet c , C.L. Wrathmell b , Y. Astier b a Unita INFM Dipartmento di Scienze Ambientali, Universita della Tuscia, I-01100 Viterbo, Italy b Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, UK c Leiden Institute of Chemistry, Gorlaeus Laboratoria, Leiden University, Leiden, The Netherlands Received 31 January 2003; received in revised form 22 August 2003; accepted 14 September 2003 Abstract Site-specifically engineered disulphide or surface cysteine residues have been introduced into two blue copper proteins, Pseu- domonas aeruginosa azurin and Populus nigra plastocyanin, in order to facilitate protein chemisorption on gold electrodes. The subsequently formed well-defined protein monolayers gave rise to robust electrochemical responses and electron transfer rates comparable to those observed at modified electrode surfaces. Proximal probe characterisation confirms the presence, at high coverage, of well-ordered protein adlayers. Additionally, gold-metalloprotein affinity is such that molecular-level tunnelling and topographic analyses can be carried out under aqueous solution. The approaches outlined in this work can, in principal, be extended to the generation of arrays of any redox-active biomolecule. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Plastocyanin; Azurin; Protein monolayer; Self-assembly; Copper metalloprotein; Protein tunnelling 1. Introduction Metalloprotein electrochemical studies have led, not only to an enhanced understanding of the important role these molecules play in biological energy transduc- tion processes (including structure/function correlations) but also to significant biosensing developments and, more recently, the birth of bioelectronics. Much pro- gress has been made during the past 20 years in attaining reproducible electrochemical responses from metallo- proteins and enzymes [1]. Particularly beneficial in this context is the level of control achievable by confining voltammetric investigations to the electrode surface (and thereby removing complicating diffusion-based contri- butions to voltammetry) [2,3]. The process of electron exchange between planar electrode surfaces and immo- bilised metalloproteins has now advanced to the point where detailed kinetic and thermodynamic investiga- tions, including quantitative electronic coupling (such as structure and distance dependence) experiments and reorganisation can be pursued [2,4]. Controlling the electrochemical coupling between biomolecules and man-made circuitry is, further, of considerable impor- tance in the development of improved bio- and immuno- sensing devices. Reliable (that is, reproducible) heterogeneous elec- tron transfer of metalloproteins at man-made electrode surfaces is achievable only through careful consideration of protein and electrode surface chemistry [5–7]. Spe- cifically, much work has centered on ‘‘modelling’’ the electrode surface such that its physiochemical interac- tions with the protein reflect, to variant levels of sim- plification, the natural interfacial interactions of the protein in its native environment. Indeed, the force balance imposed on a metalloprotein confined to an electrode surface differs from that in solution and this commonly leads to a loss of native conformation unless specific steps are taken to create a ‘‘biocompatible Journal of Electroanalytical Chemistry 565 (2004) 21–28 www.elsevier.com/locate/jelechem Journal of Electroanalytical Chemistry * Corresponding author. Tel.: +44-1865-275-914; fax: +44-1865-275- 914. E-mail address: jason.davis@chem.ox.ac.uk (J.J. Davis). 0022-0728/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jelechem.2003.09.038