Electron mediated, enzymes

Apart from electron promoters a large number of electron mediators have long been investigated to make redox enzymes electrochemically active on the electrode surface. In the line of this research electron mediators such as ferrocene and its derivatives have successfully been incorporated into an enzyme sensor for glucose [3]. The mediator was easily accessible to both glucose oxidase and an electron tunnelling pathway could be formed within the enzyme molecule [4]. The present authors [5,6] and Lowe and Foulds [7] used a conducting polymer as a molecular wire to connect a redox enzyme molecule to the electrode surface. 

Electron mediator Either electrode of enzyme is modified by an electron mediator in various manners. 

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]. 

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. 

An alternative containment scheme is immobilization of active species on a surface" " or within a tethered polymer brush or network. ° Surface immobilization can achieve high surface utilization by locating mediators and biocatalysts within nanometers of conducting surfaces. Immobilization on polymer networks allows for dense packing of enzymes within electrode volumes at the expense of long-distance electron mediation between the enzyme active center and a conductive surface. Such mediation often represents the rate-limiting step in the overall electrode reaction. 

More complex reductions of CO2 by enzyme cascades have also been achieved. A combination of an electron mediator and two enzymes, formate dehydrogenase and methanol dehydrogenase, was used to reduce CO2 to methanol. This system operates with current efficiencies as high as 90% and low overpotentials (approximately —0.8 V vs. SCE at pH 7) [125]. The high selectivity and energy efficiency of this system indicate the potential of enzyme cascades. There are also drawbacks to these systems. In general, enzymes are... 

Another technique used is a cyclic voltammetry (CV) in the presence of electron mediator. In the GOx-catalyzed oxidation reaction of glucose, cofactor FAD, which is contained at the active center of GOx, oxidizes glucose to gluconolactone and resultant FADHj is converted back to the active FAD form by Oj. In the conventional type of enzyme sensors, the H2O2 generated from O2 is oxidized at the electrode surface. 

Willner and coworkers have extended this approach to electron relay systems where core-based materials facilitate the electron transfer from redox enzymes in the bulk solution to the electrode.56 Enzymes usually lack direct electrical communication with electrodes due to the fact that the active centers of enzymes are surrounded by a thick insulating protein shell that blocks electron transfer. Metallic NPs act as electron mediators or wires that enhance electrical communication between enzyme and electrode due to their inherent conductive properties.47 Bridging redox enzymes with electrodes by electron relay systems provides enzyme electrode hybrid systems that have bioelectronic applications, such as biosensors and biofuel cell elements.57... 

ZnS colloids were also used by Kuwabata et al. to photoreduce C02 to formate [130]. In this system, the ZnS colloids also reduced pyrroloquinoline quinone which served as an electron mediator to the enzyme, methanol dehydrogenase, which could then reduce formate to methanol. In C02-saturated aqueous solution at pH 7 and under far-UV (280nm) illumination, quantum efficiencies of 7% and 6% were achieved for formate and methanol, respectively. 

A further approach to controlling electrical communication between redox proteins and their electrode support through a photo-command interface includes photo stimulated electrostatic control over the electrical contact between the redox enzyme and the electrode in the presence of a diffusional electron mediator (Scheme 12).[58] A mixed monolayer, consisting of the photoisomerizable thiolated nitrospiropyran units 30 and the semi-synthetic FAD cofactor 25, was assembled on an Au electrode. Apo-glucose oxidase was reconstituted onto the surface FAD sites to yield an aligned enzyme-layered electrode. The surface-reconstituted enzyme (2 x 10-12 mole cm-2) by itself lacked electrical communication with the electrode. In the presence of the positively charged, protonated diffusional electron mediator l-[l-(dimethylamino)ethyl]ferrocene 29, however, the bioelectrocatalytic functions of the enzyme-layered electrode could be activated and controlled by the photoisomerizable component co-immobilized in the monolayer assembly (Figure 12). In the... 

Electron-Mediated Biosensor. Electropolymerization of 1,3-diaminobenzene (1,3-DAB), followed by adsorption of l,r-dimethylferrocene (1,1 -DMF), and immobilization of glucose oxidase, results in an easily and quickly (<2 h) constructed glucose biosensor with excellent linearity and stability (>3 months). Figure 5 shows a proposed schematic of the sensing layer consisting of film/mediator/enzyme. The ferrocene is depicted as circles... 

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