Glucose oxidase, electron transfer

Further improvements can be achieved by replacing the oxygen with a non-physiological (synthetic) electron acceptor, which is able to shuttle electrons from the flavin redox center of the enzyme to the surface of the working electrode. Glucose oxidase (and other oxidoreductase enzymes) do not directly transfer electrons to conventional electrodes because their redox center is surroimded by a thick protein layer. This insulating shell introduces a spatial separation of the electron donor-acceptor pair, and hence an intrinsic barrier to direct electron transfer, in accordance with the distance dependence of the electron transfer rate (11) ... 

With regard to biosensor applications, a wide variety of electrochemically active species (ferrocene, ruthenium complexes, or carbon and metal (Pt, Pd, Au...) [185,186] were also introduced into the sol-gel matrices or adsorbed to improve the electron transfer from the biomolecules to the conductive support [187,188]. For instance, glucose oxidase has been trapped in organically modified sol-gel chitosan composite with adsorbed ferrocene to construct a low-cost biosensor exhibiting high sensitivity and good stability [189]. 

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Electron transfer of the glucose oxidase/polypyrrole on the electrode surface was confirmed by differential pulse voltammetiy and cyclic voltammetry. The glucose oxidase clearly exhibited both reductive and oxidative current peaks in the absence of dissolved oxygen in these voltammograms. These results indicate that electron transfer takes place from the electrode to the oxidized form of glucose oxidase and the reduced form is oxidized by electron transfer to the electrode through polypyrrole. It may be concluded that polypyrrole works as a molecular wire between the adsorbed glucose oxidase and the platinum electrode. 

In contrast to the molecular wire of molecular interface, electron mediators are covalently bound to a redox enzyme in such a manner as an electron tunneling pathway is formed within the enzyme molecule. Therefore, enzyme-bound mediators work as molecular interface between an enzyme and an electrode. Degani et al. proposed the intramolecular electron pathway of ferrocene molecules which were covalently bound to glucose oxidase [ 4 ]. However, few fabrication methods have been developed to form a monolayer of mediator-modified enzymes on the electrode surface. We have succeeded in development of a novel preparation of the electron transfer system of mediator-modified enzyme by self-assembly in a porous gold-black electrode as schematically shown in Fig.12 [14]. 

It is noted that the anodic peak current prominently increases with an increase in the molar ratio of ferrocene to glucose oxidase whilst the amount of enzyme self-assembled on the electrode surface is fixed as presented in Figs. 14-16. This indicates that each modified ferrocene may contribute to electron transfer between the enzyme and the electrode in the case of gold-black electrode, the ferrocene-modified enzyme could form multi electron transfer paths on the porous gold-black electrode. 

The porous matrix of gold-black electrode has enabled ferrocene-modified glucose oxidase to perform the smooth electron transfer by means of easy access between self-assembled molecules and electrode surface. 

Electron mediators successfully used with oxidases include 2,6-dichlorophenolindophol, hexacyanoferrate-(III), tetrathiafulvalene, tetracyano-p-quinodimethane, various quinones and ferrocene derivatices. From Marcus theory it is evident that for long-range electron transfer the reorganization energies of the redox compound have to be low. Additionally, the redox potential of the mediator should be about 0 to 100 mV vs. standard calomel electrode (SCE) for a flavoprotein (formal potential of glucose oxidase is about -450 mV vs SCE) in order to attain rapid vectrial electron transfer from the active site of the enzyme to the oxidized form of the redox species. 

Mizutani et al. [16] have demonstrated that ferrocene derivatives, attached by means of covalent bonds to the surface of bovine serum albumin, have been able to mediate the electron transfer between the glucose oxidase and the electrode through the osemium complex. 

In contrast to the mediator-modified electrodes, Degani et al. modified glucose oxidase itself by means of covalently bound ferrocene [4]. After modifying enzymes with ferrocene carboxylic acid, they observed direct electron transfer from the active site of the enzyme to a gold or platinum... 

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