Photoelectrochemical study of Zn cytochrome-c immobilised on a nanoporous metal oxide electrode Emmanuel Topoglidis, Colin J. Campbell,† Emilio Palomares and James R. Durrant* Department of Chemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, UK SW7 2AY. E-mail: j.durrant@ic.ac.uk Received (in Cambridge, UK) 9th April 2002, Accepted 30th May 2002 First published as an Advance Article on the web 13th June 2002 Transient optical spectroscopies and photocurrent action spectra are used to demonstrate photoinduced charge separation between zinc-substituted cytochrome c and a nanocrystalline TiO 2 electrode. There is wide interest in the functionalisation of porous, nanocrystalline metal oxide electrodes by the adsorption of molecular species to their surface. The high surface area of such films makes them attractive for applications ranging from dye sensitised solar cells to electrochromic mirrors. 1 We have recently shown that the functionalisation of such nanocrystal- line films may furthermore be extended to the adsorption of biological macromolecules such as proteins and have demon- strated that the high protein loading achieved by this approach is particularly attractive for a range of bioanalytical applica- tions. 2–4 Electron transfer reactions at protein/electrode interfaces are of fundamental importance both for bioelectrochemical studies of protein function and also for technological applications such as biosensors and bioelectrocatalytic systems. 5,6 Such inter- facial electron transfer reactions are typically studied by electrochemical techniques. We demonstrate here that the optical transparency and high protein loading achieved with nanoporous TiO 2 results in such films being particularly amenable to photoelectrochemical studies of interfacial electron transfer processes, and moreover opens up the possibility of the development of optically driven bioelectrochemical devices. Transient optical techniques have been widely employed to resolve ultrafast electron injection from photogenerated dye excited states into the conduction band of the metal oxide. 7 However, with the exception of a report by McLendon and coworkers, 8 such techniques have not previously been applied to studies of protein/electrode electron transfer events. The strategy employed in this study is illustrated in Fig. 1. The protein employed is zinc-substituted cytochrome-c (Zn Cyt-c). Zinc-substituted Cyt-c is employed due to its long singlet excited state lifetime ( ~ 3.5 ns) relative to native Fe Cyt- c. Interfacial electron transfer is initiated by optical excitation of the Zn Cyt-c, resulting in electron injection into the TiO 2 conduction band. Anatase, nanocrystalline TiO 2 films were fabricated as reported previously 2 (nanoparticle diameter ~ 15 nm, film thickness 8 mm, pore volume fraction ~ 40%). Structurally analogous ZrO 2 films were fabricated by a sol–gel route as detailed previously, 9 these films were employed for control experiments as the higher conduction band edge of this metal oxide precludes electron injection from the Zn Cyt-c excited state. Zn Cyt-c was prepared 10 and immobilised 2–4 on the TiO 2 films according to the published procedures. The adsorption of Zn Cyt-c on the nanoporous TiO 2 films was confirmed by UV– visible absorption spectroscopy. Employing an extinction coefficient of 243 000 M 21 cm 21 at 423 nm for Zn Cyt-c, 11 we estimated a protein loading of 3.7 nmol cm 22 . The high protein loading, corresponding to a loading 160 times greater than monolayer coverage of a flat electrode surface (assuming a cross-sectional area for Zn Cyt-c of 7 nm 2 ), results from protein adsorption within the nanoporous film. Fig. 2 shows a photocurrent action spectrum for a Zn Cyt-c/ TiO 2 film. Data are plotted as the quantum efficiency for the conversion of incident photons to electrons flowing through the external circuit (IPCE). The Zn Cyt-c/TiO 2 IPCE spectrum exhibits maxima at 410 and 550 nm, in agreement with the UV– visible absorption maxima of the adsorbed Zn Cyt-c and consistent with this photocurrent deriving from photon absorp- tion by the Zn Cyt-c followed by electron injection into the TiO 2 electrode. 11 Time resolved emission spectroscopy was employed to investigate the quenching of the Zn Cyt-c singlet excited state due to electron injection into the TiO 2 electrode. Typical data are shown in Fig. 3. The emission decay for the control Zn Cyt-c/ZrO 2 film fitted well to a monoexponential decay with lifetime of 3.5 ns, in good agreement with the 3.2 ns lifetime of the Zn Cyt-c singlet excited state reported in previous solution phase studies of this protein. 8 Absorption of the Zn Cyt-c to the TiO 2 electrode results in clear quenching of the emission, with a monoexponential fit to the data yielding a lifetime of 0.9 ns (a biexponential analysis yielded a significantly better fit with † Current address, NTera Ltd, GSK Facility, Grange Road, Rathfarnham, Dublin 16, Eire. Fig. 1 Illustration of functionalisation of a nanoporous TiO 2 film with Zn Cyt-c. Optical excitation of the Zn Cyt-c results in electron injection into the semiconducting electrode. Fig. 2 Photocurrent action spectrum for a Zn Cyt-c/TiO 2 film incorporated as the working electrode of a three-electrode photoelectrochemical cell held at 0 V vs. a Ag/AgCl reference electrode, with an aqueous 2.2 mM K 4 Fe(CN) 6 electrolyte, pH 7 and Pt mesh counter electrode. Negligible photocurrent was observed for TiO 2 electrodes in the absence of Zn Cyt- c. This journal is © The Royal Society of Chemistry 2002 1518 CHEM. COMMUN. , 2002, 1518–1519 DOI: 10.1039/b203448d