Immobilisation and bioelectrochemistry of proteins on nanoporous TiO2 and ZnO films

In this paper we compare the use of nanoporous TiO2 and ZnO films as substrates for protein immobilisation and as electrodes for electrochemical reduction of the absorbed protein. These two metal oxides are of particular interest as at physiological pH values their surface charges are counterpoised, their isoelectric points (IEPs) being 6 and 9.5 for TiO2 and ZnO, respectively. We find that protein immobilisation on both films may be readily achieved from pH 7 aqueous solutions at 4 °C with a high binding stability and no detectable protein denaturation. However, consistent with the difference in surface charge of the two metal oxides at this pH, we find that immobilisation of cytochrome c (Cyt-c, IEP 10.5) is an optimum on TiO2 films, whereas, green fluorescence protein (GFP, IEP 4.5) binds optimally to the ZnO film. We conclude that, ZnO films may be particularly attractive for the immobilisation of low IEP proteins. Cyclic voltammetry and spectroelectrochemistry are employed to investigate reduction of immobilised Cyt-c on both metal oxides. Control studies in the absence of adsorbed protein indicate the ZnO film to be more conductive at moderate applied potentials than the TiO2 film. Studies with adsorbed Cyt-c indicate that the direct reduction of Cyt-c is possible on both films without the addition of electron transfer promoters or mediators and that all the protein on both films is electroactive. However, consistent with the difference in conductivity of the two films, Cyt-c reduction is observed at less negative potentials for the ZnO film relative to TiO2. This observation suggests that the use of ZnO films may be advantageous for the development of nanoporous biosensors employing reductive electrochemistry.