Enzyme electrodes activation

Monolayer- or Multilayer-enzyme Electrodes Activated by DifTusional Mediators... 

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. 

A compound which is a good choice for an artificial electron relay is one which can reach the reduced FADH2 active site, undergo fast electron transfer, and then transport the electrons to the electrodes as rapidly as possible. Electron-transport rate studies have been done for an enzyme electrode for glucose (G) using interdigitated array electrodes (41). The following mechanism for redox reactions in osmium polymer—GOD biosensor films has... 

The next generation of amperomethc enzyme electrodes may weU be based on immobilization techniques that are compatible with microelectronic mass-production processes and are easy to miniaturize (42). Integration of enzymes and mediators simultaneously should improve the electron-transfer pathway from the active site of the enzyme to the electrode. 

Enzyme electrodes for other substrates of analytical significance have been developed. Representative examples are listed in Table 6-1. Further advances in enzyme technology, and particularly the isolation of new and more stable enzymes, should enhance the development of new biocatalytic sensors. New opportunities (particularly assays of new environments or monitoring of hydrophobic analytes) derive from the finding that enzymes can maintain then biocatalytic activity in organic solvents (31,32). 

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. 

Another area exists, however, where the nse of enzyme electrodes olfers great promise. Owing to their high selectivity and their capability of reacting only in the presence of specific substrates, they can be nsed as sensors for the presence of par-ticnlar, biologically active snbstances in a solntion, and in certain cases for determining the concentration of these snbstances. 

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. 

Kessler, M., Hajek, K., Simon, W. Four-Barreled Microelectrode for the Measurement of Potassium, Sodium, and Calcium-Ion Activity, in Ion and Enzyme Electrodes in Biology and Medicine (Kessler, M., Clark, Jr, L. C., Lubbers, D, W., Silver, I. A., Simon, W., eds.) Munich Urban and Schwarzenberg, 1976, p. 136... 

Important inherent characteristics of an enzyme that should be considered are the substrate affinity, characterized by the Michaelis constant the rate of turnover fecat> providing the catalytic efficiency fecat/ M. and the catalytic potential. Several attempts to compare enzyme catalysis with that of platinum have been published. Direct comparisons are difficult, because enzyme electrodes must be operated in aqueous electrolyte containing dissolved substrate, whereas precious metal electrodes aie often supplied with a humidified gaseous stream of fuel or oxidant, and produce water as steam. It is not straightforward to compare tme optimal turnover rates per active site, as it is often unclear how many active sites are being engaged in a film of enzyme on an electrode. 

A critical factor for biotechnology application is the stability of the enzyme electrode. Hydrogenase immobilized into carbon filament material has high level of both operational and storage stability. Even after the half year of storage with periodical testing, the enzyme electrode preserved more than 50 % of its initial activity [9,10], Thus, it is possible to achieve appropriate stability of the enzyme electrode, suitable for hydrogen fuel cells development. 

Hydrogenase based enzyme electrode was not inhibited, when CO content in the mixture was less than 0.1 %. In the presence of 1 % CO the rate of hydrogen oxidation was decreased by 10 % and zero-current potential was shifted positively for 30 mV. The steady-state Currents were achieved in a few minutes [10], An important advantage of the hydrogen enzyme electrode is completely reversible nature of inhibition by CO. Like the soluble hydrogenase the enzyme electrode recovered 100 % of its initial activity as soon as the atmosphere of pure carbon monoxide was changed back to hydrogen. 

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. 

Examples of surface-immobilized mediators are electropolymerized azines for electro-oxidation of The extreme form of this approach is formation of biocatalytic monolayer, comprising a surface-bound mediator species that is itself bound to a single enzyme molecule. Katz et al. report a complete cell based on novel architecture at both electrodes (Figure 7). On the anode side, the FAD center of glucose oxidase is removed from the enzyme shell and covalently attached to a pyrroloquinoline quinone (PQQ) mediator species previously immobilized on a gold surface. The GOx apoenzyme (enzyme with active center removed) is reintroduced in solution and selectively binds to FAD, resulting in a PQQ-... 

More recently, nanotechnology has faciUtated progress in miniaturizing redox enzyme electrodes and extending their application. In order to achieve contact between the active site of the redox enzyme where electron transfer takes place, usually buried within the protein structure, and the electrode electrical contact, cofactor-functionaUzed nanomaterials have been developed [75]. Diffusible cofactors such as FAD can be used as the relay system for carrying electrons to electrical... 

Microbial sensors offer a number of assets, namely (a) they are less sensitive to inhibition by solutes and more tolerant to suboptimal pH and temperature values than are enzyme electrodes b) they have longer lifetimes than enzymes and (c) they are less expensive than enzyme electrodes as they require no active enzyme to be isolated. On the other hand, they lag behind enzyme electrodes in a few other respects thus, (a) some have longer response times than their enzyme counterparts b) baseline restoration after measurement typically takes longer and (c) cells contain many enzymes and due care must be exercised to ensure adequate selectivity e.g. by optimizing the storage conditions or using specific enzyme reactions) —some mutant microorganisms lack certain enzymes. 

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... 

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