Enzymes and electrochemical reactions

SCHEME 55.2. Enzyme and electrochemical reactions in glucose biosensor. 

Bioelectrocatalysis involves the coupling of redox enzymes with electrochemical reactions [44]. Thus, oxidizing enzymes can be incorporated into redox systems applied in bioreactors, biosensors and biofuel cells. While biosensors and enzyme electrodes are not synthetic systems, they are, essentially, biocatalytic in nature (Scheme 3.5) and are therefore worthy of mention here. Oxidases are frequently used as the biological agent in biosensors, in combinations designed to detect specific target molecules. Enzyme electrodes are possibly one of the more common applications of oxidase biocatalysts. Enzymes such as glucose oxidase or cholesterol oxidase can be combined with a peroxidase such as horseradish peroxidase. 

Construction principles and the mechanism for biosensors derived from enzymes. Combined enzymatic and electrochemical reactions proceeding on electrodes from various materials in electrolyte solutions promote development of many biosensor types for detection of glucose, amino acids, lactose, urea, pyruvate and other metabolites. Biosensors are successfully applied to environmental contamination control, medical diagnostics and the food industry. 

Association between enzymatic and electrochemical reactions has provided efficient tools not only for analytical but also for synthetic purposes. In the latter field, the possibilities of enzymatic electrocatalysis, e.g., the coupling of glucose oxidation (catalyzed either by glucose oxidase or glucose dehydrogenase) to the electrochemical regeneration of a co-substrate (benzoquinone or NAD+) have been demonstrated [171, 172]. An electroenzymatic reactor has also been developed ]172] to demonstrate how the enzyme-electrode association can be used to produce biochemicals on a laboratory scale. 

Enzyme electrodes comprising coimmobilized COD and CEH can be used for the determination of total cholesterol. On the other hand, coupling of COD with HRP enables cholesterol measurement at low electrode overvoltage, which avoids electrochemical interferences. Fig. 89 shows the diversity of the potential sequences of enzymatic and electrochemical reactions in cholesterol electrodes. 

It is also of interest to note that tyrosine hydroxylase catalyzes the hydroxylation of 7-hydroxychlorpromazine to 7,8-dihydroxychlorpromazine and then to 7,8-dioxochlorpromazine. Under similar conditions the electrochemical oxidation of 7-hydroxychlorpromazine progresses through a similar reaction seequence (see Figure 11 A) to the same product observed enzymically. A comparison of the information obtained from the enzymic and electrochemical oxidations strongly supports the conclusion that electrochemical techniques yield considerably more mechanistic information and allow the ready detection of transient intermediate species. The fact that the same products are formed electrochemically and enzymatically lends support to the view that the electrochemically generated intermediates may be real possibilities for involvement in the in vivo metabolic processes. 

The structure and physicochemical properties of the enzymes which have been used to date to promote electrochemical reactions are briefly outlined. Methods of their immobilization are described. The status of research on redox transformations of proteins and enzymes at the electrode-electrolyte interface is discussed. Current concepts on the ways of conjugation of enzymatic and electrochemical reactions are summarized. Examples of bioelectrocatalysis in some electrochemical reactions are described. Electrocatalysis by enzymes under conditions of direct mediatorless transport of electrons between the electrode and the enzyme active center is considered in detail. Lastly, an analysis of the status of work pertaining to the field of sensors with enzymatic electrodes and to biofuel cells is provided. 

In the course of work on bioelectrocatalysis all three ways of using enzymes to accelerate electrochemical reactions have been taken into account. These research areas cover the following electrochemical reactions hydrogen, oxygen, and oxidation reactions of certain organic compounds. In each case, different methods were employed to achieve the conjugation of enzymatic and electrochemical reactions. 

FIGURE 9.13. Model of an immobilzed enzyme layer at the electrode surface showing the various partition, diffusion, and reaction steps involved in the overall enzyme-catalyzed electrochemical reaction. 

To understand the interplay of enzyme catalysis and mass transfer within polymer film, it is essential to develop models that take account of these effects, then compare the models predictions with experiment. Fig. 9.13 illustrates the physicochemical processes involved in the enzymic turnover of substrate to product within a polymer film. Such processes include mass transport of substrate and product either to or from the film, partition of these species across the polymer-solution interface, transport of reactants and products within the film (by diffusion), and electrochemical reaction with enzymic products at the electrode surface. Effects of migration of charged species within the film are usually ignored. 

The inherent nature of the redox reaction makes it natural to couple these dehydrogenase-catalyzed reactions to electrochemical methods. The transfer of electron(s) from a substrate to an electrode (or the reverse) may then take place via electrochemical redox reactions of the coenzymes, as depicted in Fig. 2. The utility of combining enzymes and electrochemical methods for electroanalytical applications was predicted by Clark and Lyons by an enzyme electrode [28] and by Shaw in energy production by a biofuel cell anode [29] in the early 1960s. 

Immobilized Enzymes. The immobilized enzyme electrode is the most common immobilized biopolymer sensor, consisting of a thin layer of enzyme immobilized on the surface of an electrochemical sensor as shown in Figure 6. The enzyme catalyzes a reaction that converts the target substrate into a product that is detected electrochemicaHy. The advantages of immobilized enzyme electrodes include minimal pretreatment of the sample matrix, small sample volume, and the recovery of the enzyme for repeated use (49). Several reviews and books have been pubHshed on immobilized enzyme electrodes (50—52). 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

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. 

Because of this lack of resolving power, much electroanalytical research is aimed at providing increased selectivity. This can be accomplished in two ways. First, electrochemistry can be combined with another technique which provides the selectivity. Examples of this approach are liquid chromatography with electrochemical detection (LCEC) and electrochemical enzyme immunoassay (EEIA). The other approach is to modify the electrochemical reaction at the electrode to enhance selectivity. This... 

Monitoring enzyme catalyzed reactions by voltammetry and amperometry is an extremely active area of bioelectrochemical interest. Whereas liquid chromatography provides selectivity, the use of enzymes to generate electroactive products provides specificity to electroanalytical techniques. In essence, enzymes are used as a derivatiz-ing agent to convert a nonelectroactive species into an electroactive species. Alternatively, electrochemistry has been used as a sensitive method to follow enzymatic reactions and to determine enzyme activity. Enzyme-linked immunoassays with electrochemical detection have been reported to provide even greater specificity and sensitivity than other enzyme linked electrochemical techniques. 

Enzyme linked electrochemical techniques can be carried out in two basic manners. In the first approach the enzyme is immobilized at the electrode. A second approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of product to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system which is amenable to in vivo analysis. 

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