Biocatalysis on Nanostructured Surfaces: Investigation and Application of Redox Proteins using Spectro-Electrochemical Methods

In this thesis, different aspects within the research field of protein spectro- and electro-chemistry on nanostructured materials are addressed. On the one hand, this work is related to the investigation of nanostructured transparent and conductive metal oxides as platform for the immobilization of electroactive enzymes. On the other hand the second part of this work is related to the immobilization of sulfite oxidase on gold nanoparticles modified electrode. Finally direct and mediated spectroelectrochemistry protein with high structure complexity such as the xanthine dehydrogenase from Rhodobacter capsulatus and its high homologues the mouse aldehyde oxidase homolog 1.

Stable immobilization and reversible electrochemistry of cytochrome c in a transparent and conductive tin-doped and tin-rich indium oxide film with a well-defined mesoporosity is reported. The transparency and good conductivity, in combination with the large surface area of these materials, allow the incorporation of a high amount of electroactive biomolecules (between 250 and 2500 pmol cm-2) and their electrochemical and spectroscopic investigation. Both, the electrochemical behavior and the immobilization of proteins are influenced by the geometric parameters of the porous material, such as the structure and pore shape, the surface chemistry, as well as the protein size and charge. UV-Vis and Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of cytochrome c immobilized in the mesoporous indium tin oxide and reveal no perturbation of the structural integrity of the redox protein. A long term protein immobilization is reached using these unmodified mesoporous indium oxide based materials, i.e. more than two weeks even at high ionic strength.
The potential of this modified material as an amperometric biosensor for the detection of superoxide anions is demonstrated.
In addition an electrochemical switchable protein-based optical device is designed with the core part composed of cytochrome c immobilized on a mesoporous indium tin oxide film. A color developing redox sensitive dye is used as switchable component of the system. The cytochrome c-catalyzed oxidation of the dye by hydrogen peroxide is spectroscopically investigated. When the dye is co-immobilized with the protein, its redox state is easily controlled by application of an electrical potential at the supporting material. This enables to electrochemical reset the system to the initial state and repetitive signal generation.
The modification of an indium tin oxide film with a positive charged polymer and the employment of a antimony doped tin oxide film were investigated in this work in order to overcome the repulsion induced by similar charges of the protein and electrode. Human sulfite oxidase and its separated heme-containing domain are able to direct exchange electrons with the supporting material.

A study of a new approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase immobilized on a gold nanoparticles modified electrode is reported. The spherical gold nanoparticles were prepared via a novel method by reduction of HAuCl4 with branched poly(ethyleneimine) in an ionic liquid resulting in particles of about 10 nm in hydrodynamic diameter.
An enhanced interfacial electron transfer and electrocatalysis is therefore achieved. UV-Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, were employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response. The investigated system provides remarkable advantages, since it works at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples.

Finally protein with high structure complexity such as the xanthine dehydrogenase from Rhodobacter capsulatus and the mouse aldehyde oxidase homolog 1 were spectroelectrochemically studied. It could be demonstrated that different cofactors present in the protein structure, like the FAD and the molybdenum cofactor, are able to directly exchange electrons with an electrode and are displayed as a single peak in a square wave voltammogram. Protein mutants bearing a serine substituted to the cysteines, bounding to the most exposed iron sulfur cluster additionally showed direct electron transfer which can be attributable to this cluster. On the other hand a mediated spectroelectrochemical titration of the protein bound FAD cofactor was performed in presence of transparent iron and cobalt complex mediators. The results showed the formation of the stable semiquinone and the fully reduced flavin. Two formal potentials for each single electron exchange step were then determined.