Transparent Gold as a Platform for Adsorbed Protein Spectroelectrochemistry: Investigation of Cytochrome c and Azurin Idan Ashur, † Olaf Schulz, ‡ Chelsea L. McIntosh, † Iddo Pinkas, ⊥ Robert Ros, ‡ and Anne K. Jones* ,†,§ † Department of Chemistry and Biochemistry, ‡ Department of Physics, and § Center for Bioenergy and Photosynthesis, Arizona State University, Tempe, Arizona 85287, United States ⊥ Department of Plant Sciences, The Weizmann Institute of Science, Rehovot, Israel * S Supporting Information ABSTRACT: The majority of protein spectroelectrochemical methods utilize a diffusing, chemical mediator to exchange electrons between the electrode and the protein. In such methods, electrochemical potential control is limited by mediator choice and its ability to interact with the protein of interest. We report an approach for unmediated, protein spectroelectrochemistry that overcomes this limitation by adsorbing protein directly to thiol self-assembled monolayer (SAM) modified, thin (10 nm), semitransparent gold. The viability of the method is demonstrated with two diverse and important redox proteins: cytochrome c and azurin. Fast, reversible electrochemical signals comparable to those previously reported for these proteins on ordinary disk gold electrodes were observed. Although the quantity of protein in a submonolayer adsorbed at an electrode is expected to be insufficient for detection of UV-vis absorption bands based on bulk extinction coefficients, excellent spectra were detected for each of the proteins in the adsorbed state. Furthermore, AFM imaging confirmed that only a single layer of protein was adsorbed to the electrode. We hypothesize that interaction of the relatively broad gold surface plasmon with the proteins’ electronic transitions results in surface signal enhancement of the molecular transitions of between 8 and 112 times, allowing detection of the proteins at much lower than expected concentrations. Since many other proteins are known to interact with gold SAMs and the technical requirements for implementation of these experiments are simple, this approach is expected to be very generally applicable to exploring mechanisms of redox proteins and enzymes as well as development of sensors and other redox protein based applications. ■ INTRODUCTION Redox proteins are at the heart of a number of biologically and technologically important processes including respiration and photosynthesis. They currently form the basis for a number of analytic biosensors and are widely investigated for their potential applications in renewable energy technologies. 1,2 Redox proteins consist of protein coordinating one or more redox active cofactors, often prosthetic groups that undergo spectroscopic changes upon oxidation or reduction. Thus, the mechanisms of redox proteins can be explored via both electrochemical and spectroscopic analyses. Although electro- chemistry allows precise investigation of the kinetics and thermodynamics of redox reactions, it can be difficult to chemically identify intermediate or product species via electrochemical experiments alone. On the other hand, spectroscopic analyses can be used nondestructively to characterize the chemical features of unknown species but with little control over solution potential or the corresponding redox state of the analyte. The two techniques provide a complementary view of protein function, but correlating independently obtained data is often nontrivial. Thus, spectroelectrochemical methods, those that simultaneously investigate electrochemistry and spectroscopy, provide excellent opportunities to explore mechanisms of redox proteins. 3,4 Although a number of types of spectroscopies including FTIR, resonance Raman, and EPR have been used to characterize redox proteins, UV-vis is almost certainly the most commonly utilized. Often, the metallocofactors in proteins have strong metal-to-ligand or ligand-to-metal charge transfer transitions in the UV or visible range. Reduction or oxidation of these cofactors results in electronic changes reflected in altered UV-vis spectroscopic features that can be directly correlated to the redox process. Additionally, at a practical level, UV-vis experiments are relatively easy to undertake. The most widely utilized methodology for protein UV-vis spectroelectrochemistry (SEC) employs a thin layer config- uration to optimize diffusion of protein and/or chemical mediator to the working electrode surface, usually a partially transparent Au or Pt mesh. 5 As diffusion by large proteins is relatively slow, these experiments typically employ faster diffusing chemical mediators to relay electrons to protein active sites, thus giving up the ability to precisely control protein potential. Alternatively, they suffer from sluggish Received: December 21, 2010 Revised: February 22, 2012 Published: February 27, 2012 Article pubs.acs.org/Langmuir © 2012 American Chemical Society 5861 dx.doi.org/10.1021/la300404r | Langmuir 2012, 28, 5861-5871