Enzyme electrodes surface, activation

The high specific activity of enzymes and tfie tfieoretical possibility of using them to conduct electrochemical reactions are topics of great scientific interest. However, it is difficult to envisage prospects for a practical nse of enzymes for an acceleration and intensification of industrial electrode processes. The difficulty resides in the fact that enzymes are rather large molecnles, and on the surface of an enzyme electrode, fewer active sites are available than on other electrodes. Per unit snrface area, therefore, the effect expected from the nse of enzymes is somewhat rednced. 

Potcntiomctric Biosensors Potentiometric electrodes for the analysis of molecules of biochemical importance can be constructed in a fashion similar to that used for gas-sensing electrodes. The most common class of potentiometric biosensors are the so-called enzyme electrodes, in which an enzyme is trapped or immobilized at the surface of an ion-selective electrode. Reaction of the analyte with the enzyme produces a product whose concentration is monitored by the ion-selective electrode. Potentiometric biosensors have also been designed around other biologically active species, including antibodies, bacterial particles, tissue, and hormone receptors. 

The final method of coupling enzyme reactions to electrochemistry is to immobilize an enzyme directly at the electrode surface. Enzyme electrodes provide the advantages already discussed for immobilization of enzymes. In addition, the transport of enzyme product from the enzyme active site to the electrode surface is greatly enhanced when the enzyme is very near to the electrode. The concept of combining an enzyme reaction with an amperometric probe should offer all of the advantages discussed earlier for ion-selective (potentiometric) electrodes with a much higher sensitivity. In addition, since the response of amperometric electrodes is linear, background can be selected. 

In some instances, the design of an amperometric immunosensor may be such that the enzyme is located some distance away from the electrode surface, or the presence of interfering substances in biological samples may require using an alternative electron transfer pathway. This usually involves a redox-active species with a small molecular... 

Mainly, three approaches have been used to immobilize the enzyme on transducer or electrode surface, single layer, bilayer, and sandwich configurations [69, 98], In some studies enzymes are covalently linked with sol-gel thin films [99], Sol-gel thin films are highly convenient for fast, large, and homogeneous electron transfer [17]. With an increase in gel thickness the signal decays and diffusion of analytes to biomolecule active site becomes difficult eventually these factors lead to poor response. By employing thin films various biosensors such as optical and electrochemical biosensors have been reported. 

Electrochemical cofactor reduction can be achieved by direct reduction of the cofactor at the electrode surface, or indirectly by using a mediator molecule to shuttle electrons between the electrode and the cofactor. For details on the direct approach the reader is referred elsewhere [31, 32], since here no transition-metal complexes are involved. One point to be considered in the direct approach is the issue of selectivity. Whereas direct cofactor oxidation can be successfully achieved, special care must be taken to produce enzyme active reduced cofactors by direct electrolysis. 

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. 

Almost all the FDH molecules on the electrode surface seemed to retain the enzyme activity because of the mild immobilization at less extreme potential. The enzyme activity of immobilized FDH was dependent on the thickness of polypyrrole membrane because a thicker membrane could prevent the enzyme substrate from diffusing into the membrane matrix. Therefore, it was very important to make the polypyrrole membrane as thin as possible to minimize the effect on substrate diffusion and to ensure the complete coverage of the enzyme layer. 

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. 

However, because of the mostly very slow electron transfer rate between the redox active protein and the anode, mediators have to be introduced to shuttle the electrons between the enzyme and the electrode effectively (indirect electrochemical procedure). As published in many papers, the direct electron transfer between the protein and an electrode can be accelerated by the application of promoters which are adsorbed at the electrode surface [27], However, this type of electrode modification, which is quite useful for analytical studies of the enzymes or for sensor applications is in most cases not stable and effective enough for long-term synthetic application. Therefore, soluble redox mediators such as ferrocene derivatives, quinoid compounds or other transition metal complexes are more appropriate for this purpose. 

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