Glucose oxidase mediators

Figure 18 Glucose oxidase-mediated asymmetric oxidation of sulfide in IL...

FIGURE 5.7. Effect of changing the cosubstrate and the pH on the kinetics of an homogeneous redox enzyme reaction as exemplified by the electrochemical oxidation of glucose by glucose oxidase mediated by one-electron redox cosubstrates, ferricinium methanol ( ), + ferricinium carboxylate ( ), and (dimethylammonio)ferricinium ( ). Variation of the rate constant, k3, with pH. Ionic strength, 0.1 M temperature 25°C. Adapted from Figure 3 in reference 11, with permission from the American Chemical Society. 

Bourdillon, C. Demaille, C. Moiroux, J. Saveant, J. M. New insights into the enzymatic catalysis of the oxidation of glucose by native and recombinant glucose-oxidase mediated by electrochemically generated one-electron redox cosubstrates. J. Am. Chem. Soc. 1993, 115, 2-10. 

Some oxidoreductases have been reported to induce polymerization of vinyl monomers. HRP, xanthin oxidase, alcohol oxidase, chloroperoxidase, and laccase could catalyze the pol5merization of acrylamide and hydroxyethyl methacrylate (331,332). Glucose oxidase mediated the initiation of vinyl polymerization in the presence of Fe + and dissolved oxygen (333). 

The catalysis of the oxidation of D-glucose by native and by recombinant glucose oxidase, mediated by one-electron redox co-substrates, has been monitored by cyclic voltammetry. A study on the effect of pH on the Pd-catalysed oxidation of D-glucose revealed that in acidic noedia the product, free D-gluconic acid, reversibly inhibits the oxidation process. ... 

Figure 11.39 summarizes the reactions taking place in this amperometric sensor. FAD is the oxidized form of flavin adenine nucleotide (the active site of the enzyme glucose oxidase), and FAD1T2 is the active site s reduced form. Note that O2 serves as a mediator, carrying electrons to the electrode. Other mediators, such as Fe(CN)6 , can be used in place of O2. 

A second principle used widely for glucose analysis, is that of the oxidation of glucose enzymatically, mediated by the action of glucose oxidase with the formation of gluconic acid and hydrogen peroxide (22). In this procedure it is the hydrogen peroxide which is usually assayed for determination of glucose. This method suffers from the action of inhibitors which occur, particularly with patients in a diabetic coma and these need to be removed. 

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. 

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. 

To overcome the poor stability of ferrocene-mediated enzyme sensors, mediator-modified electrodes have been used. In the case of glucose oxidase, the cofactor FAD is deeply buried within the protein matrix. The depth of the active center is estimated to be 0.87 nm. Therefore, one cannot expect that the mediator covalently attached to the electrode surface via a short spacer retain the possibility of closely approaching the cofactor of the enzyme. 

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... 

Schuman et al. have synthesized ferrocene-modified glucose oxidase with the ferrocene derivatives bound via long and flexible chains directly to the outer surface of the enzyme [17]. A peripherally attached redox mediator may accept electrons through either an intramolecular or through an intermo-lecular process. 

Aizawa et al. [14] have immobilized mediator-modified glucose oxidase within micropores of a gold black electrode by self-assembling via the thiol-gold interaction. 

Alonso et al. prepared ferrocenyl silicon dendrimers [67], which could be used as mediators in glucose biosensors, based on glucose oxidase [68, 69], The ferrocenyl units are located at the end of long, flexible, silicon containing branches and serve to electrically connect the enzyme to the electrode. The flexibility of the dendrimer is proposed to play an important role in the interaction with the redox center of glucose oxidase. 

---