Emmanuel Topoglidis

Protein Adsorption on Nanocrystalline TiO2 Films: An Immobilization Strategy for Bioanalytical Devices

We have investigated the use of optically transparent, nanoporous TiO(2) films as substrates for protein immobilization. Immobilization on such films may be readily achieved from aqueous solutions at 4 °C. The nanoporous structure of the film greatly enhances the active surface area available for protein binding (by a factor of 150 for a 4-μm-thick film). We demonstrate that the redox state of immobilized cytochrome c may be modulated by the application of an electrical bias potential to the TiO(2) film and that the fluorescence yield of immobilized fluorophore-labeled maltose-binding protein may be used to monitor specifically maltose concentration. We conclude that nanoporous TiO(2) films may be useful both for basic studies of protein/electrode interactions and for the development of array-based bioanalytical devices employing both optical and electrochemical signal transduction methodologies.

Protein adsorption on nanoporous TiO2 films: a novel approach to studying photoinduced protein/electrode transfer reactions

We have investigated the use of nanoporous TiO2 films as substrates for protein immobilisation. Such films are of interest due to their high surface area, optical transparency, electrochemical activity and ease of fabrication. These films moreover allow detailed spectroscopic study of protein/electrode electron transfer processes. We find that protein immobilisation on such films may be readily achieved from aqueous solutions at 4 degrees C with a high binding stability and no detectable protein denaturation. The nanoporous structure of the film greatly enhances the active surface area available for protein binding (by a factor of up to 850 for an 8 microns thick film). We demonstrate that the redox state of proteins such as immobilised cytochrome-c (Cyt-c) and haemoglobin (Hb) may be modulated by the application of an electrical bias potential to the TiO2 film, without the addition of electron transfer mediators. The binding of Cyt-c on the TiO2 films is investigated as a function of film thickness, protein concentration, protein surface charge and ionic strength. We demonstrate the potential use of immobilised Hb on such TiO2 films for the detection of dissolved CO in aqueous solutions. We further show that protein/electrode electron transfer may be initiated by UV bandgap excitation of the TiO2 electrode. Both photooxidation and photoreduction of the immobilised proteins can be achieved. By employing pulsed UV laser excitation, the interfacial electron transfer kinetics can be monitored by transient optical spectroscopy, providing a novel probe of protein/electrode electron transfer kinetics. We conclude that nanoporous TiO2 films may be useful both for basic studies of protein/electrode interactions and for the development of novel bioanalytical devices such as biosensors.

Functionalizing Nanocrystalline Metal Oxide Electrodes With Robust Synthetic Redox Proteins

De novo designed synthetic redox proteins (maquettes) are structurally simpler, working counterparts of natural redox proteins. The robustness and adaptability of the maquette protein scaffold are ideal for functionalizing electrodes. A positive amino acid patch has been designed into a maquette surface for strong electrostatic anchoring to the negatively charged surfaces of nanocrystalline, mesoporous TiO2 and SnO2 films. Such mesoporous metal oxide electrodes offer a major advantage over conventional planar gold electrodes by facilitating formation of high optical density, spectroelectrochemically active thin films with protein loading orders of magnitude greater (up to 8 nmol cm−2) than that achieved with gold electrodes. The films are stable for weeks, essentially all immobilized‐protein display rapid, reversible electrochemistry. Furthermore, carbon monoxide ligand binding to the reduced heme group of the protein is maintained, can be sensed optically and reversed electrochemically. Pulsed UV excitation of the metal oxide results in microsecond or faster photoreduction of an immobilized cytochrome and millisecond reoxidation. Upon substitution of the heme‐group Fe by Zn, the light‐activated maquette injects electrons from the singlet excited state of the Zn protoporphyrin IX into the metal oxide conduction band. The kinetics of cytochrome/metal oxide interfacial electron transfer obtained from the electrochemical and photochemical data obtained are discussed in terms of the free energies of the observed reactions and the electronic coupling between the protein heme group and the metal oxide surface.

Interfacial electron transfer on cytochrome-c sensitised conformally coated mesoporous TiO2 films

Hybrid protein films incorporating Cyt-c immobilized on TiO(2) films were prepared and characterised optically with UV-visible spectroscopy and electrochemically with cyclic voltammetry, and their conductivity properties were studied in detail. In addition the effects of a thin overlayer coating of a second metal oxide such as SiO(2), Al(2)O(3), ZrO(2) and MgO(2) were studied and the effects over the electrochemical properties of the hybrid working electrodes were discussed.

Direct spectroelectrochemistry of peroxidases immobilised on mesoporous metal oxide electrodes: Towards reagentless hydrogen peroxide sensing.

In this paper, we employ two peroxidases (horseradish peroxidase, HRP and cytochrome c peroxidase, CcP) to demonstrate their ability to retain their redox and biological functions after their immobilisation on mesoporous TiO(2) and SnO(2) electrodes. We will also demonstrate the use of HRP immobilised on the metal oxide electrodes for the development of reagentless optical and electrochemical biosensors for the detection of hydrogen peroxide (H(2)O(2)) with low detection limit of 0.04 and 1 microM, respectively.

Photoelectrochemical study of Zn cytochrome-c immobilised on a nanoporous metal oxide electrode

Transient optical spectroscopies and photocurrent action spectra are used to demonstrate photoinduced charge separation between zinc-substituted cytochrome c and a nanocrystalline TiO2 electrode.

Use of microperoxidase-11 to functionalize tin dioxide electrodes for the optical and electrochemical sensing of hydrogen peroxide

In this paper, we employ microperoxidase MP-11 immobilized on mesoporous SnO(2) electrodes in order to study its peroxidase activity and reaction mechanism. We demonstrate the catalytic redox chemistry of the immobilized MP-11 via direct interfacial electron transfer without the use of electron mediators. By taking advantage from the optical transparency of the SnO(2) electrodes, optical absorbance spectroscopy is used in order to compliment the data information obtained from electrochemical techniques. The catalytic activity of the immobilized MP-11 is found to proceed via the Fenton reaction, yielding OH radical intermediates. We also demonstrate the viability of using this electrode system as a potential H(2)O(2) biosensor with a sensitivity range of 0.05-30 μM.

Nanostructured ZnO in a Metglas/ZnO/Hemoglobin Modified Electrode to Detect the Oxidation of the Hemoglobin Simultaneously by Cyclic Voltammetry and Magnetoelastic Resonance

In the present work, a nanostructured ZnO layer was synthesized onto a Metglas magnetoelastic ribbon to immobilize hemoglobin (Hb) on it and study the Hb’s electrochemical behavior towards hydrogen peroxide. Hb oxidation by H2O2 was monitored simultaneously by two different techniques: Cyclic Voltammetry (CV) and Magnetoelastic Resonance (MR). The Metglas/ZnO/Hb system was simultaneously used as a working electrode for the CV scans and as a magnetoelastic sensor excited by external coils, which drive it to resonance and interrogate it. The ZnO nanoparticles for the ZnO layer were grown hydrothermally and fully characterized by X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and photoluminescence (PL). Additionally, the ZnO layer’s elastic modulus was measured using a new method, which makes use of the Metglas substrate. For the detection experiments, the electrochemical cell was performed with a glass vial, where the three electrodes (working, counter and reference) were immersed into PBS (Phosphate Buffer Solution) solution and small H2O2 drops were added, one at a time. CV scans were taken every 30 s and 5 min after the addition of each drop and meanwhile a magnetoelastic measurement was taken by the external coils. The CV plots reveal direct electrochemical behavior of Hb and display good electrocatalytic response to the reduction of H2O2. The measured catalysis currents increase linearly with the H2O2 concentration in a wide range of 25–350 μM with a correlation coefficient 0.99. The detection limit is 25–50 μM. Moreover, the Metglas/ZnO/Hb electrode displays rapid response (30 s) to H2O2, and exhibits good stability and reproducibility of the measurements. On the other hand, the magnetoelastic measurements show a small linear mass increase versus the H2O2 concentration with a slope of 152 ng/μM, which is probably due to H2O2 adsorption in ZnO during the electrochemical reaction. No such effects were detected during the control experiment when only PBS solution was present for a long time.

Electrochemical and spectroelectrochemical characterization of different mesoporous TiO 2 film electrodes for the immobilization of Cytochrome c

In this work three different mesoporous TiO2 film electrodes were prepared and used for the immobilization of Cytochrome c (Cyt-c). Films prepared via a standard sol-gel route (SG-films) were compared with commercially available benchmark nanotitania materials, namely P25 Degussa (P25-films) and Dyesol nanopaste (Dyesol films). Their properties, film deposition characteristics and their abilities to adsorb protein molecules in a stable and functional way were examined. We investigated whether it is possible, rather than preparing TiO2 films using multistep, lengthy and not always reproducible sol-gel procedures, to use commercially available nanotitania materials and produce reproducible films faster that exhibit all the properties that make TiO2 films ideal for protein immobilization. Although these materials are formulated primarily for dye-sensitized solar cell applications, in this study we found out that protein immobilization is facile and remarkably stable on all of them. We also investigated their electrochemical properties by using cyclic voltammetry and spectroelectrochemistry and found out that not only direct reduction of Fe(III)-heme to Fe(II)-heme of immobilized Cyt-c was possible on all films but that the adsorbed protein remained electroactive.

A chemical sensor for CBr4 based on quasi-2D and 3D hybrid organic–inorganic perovskites immobilized on TiO2 films

The effective immobilization of hybrid organic–inorganic semiconductors (HOIS) or their blends over standard mesoporous, transparent, semiconducting TiO2 films in order to study their electrochemical behavior as sensors through a cyclic voltammetry technique (CV) has been reported for the first time. The CV technique is used in order to acquire a comprehensive understanding of the electron transport dynamics within the TiO2 film, loaded with perovskites, while the structural and optical properties can be easily extracted from X-ray diffraction, optical absorption and photoluminescence measurements. The electrochemical behavior of the adsorbed perovskites was studied as a function of the potential applied to the semiconductive film, which induces transition from an insulating to a conductive state. The localized traps in the bandgap of the composite semiconductive film predominantly govern the electron transfer process. The HOIS employed are either 3D or mixtures of quasi-2D perovskites, for which there is strong evidence that the lead bromide and chloride based HOIS exhibit cathodic peaks at positive voltages. In addition, exploiting the capability of ions to migrate and interact with the HOIS lattice, a perovskite-based electrode was successfully used as an (electro-)chemical sensor for CBr4. All electrochemical processes concurring on the composite hybrid film vary with respect to the CBr4 concentration in the tested solution, leading to a CBr4 detectability range of the order of 20 ppb mol mol−1.