Enzyme electrodes applications

The choice of immobilization strategy obviously depends on the enzyme, electrode surface, and fuel properties, and on whether a mediator is required, and a wide range of strategies have been employed. Some general examples are represented in Fig. 17.4. Key goals are to stabilize the enzyme under fuel cell operating conditions and to optimize both electron transfer and the efficiency of fuel/oxidant mass transport. Here, we highlight a few approaches that have been particularly useful in electrocatalysis directed towards fuel cell applications. 

Enzyme electrodes belong to the family of biosensors. These also include systems with tissue sections or immobilized microorganism suspensions playing an analogous role as immobilized enzyme layers in enzyme electrodes. While the stability of enzyme electrode systems is the most difficult problem connected with their practical application, this is still more true with the bacteria and tissue electrodes. 

S.A. Jaffari and A.P.F. Turner, Novel hexacyanoferrate(III) modified graphite disc electrodes and their application in enzyme electrodes.1. Biosens. Bioelectr. 12,1—9 (1997). 

J.G. Zhao, J.R O Daly, R.W. Henkens, J. Stonehuemer, and A.L. Crumbliss, Axanthine oxidase/colloi-dal gold enzyme electrode for amperometric biosensor applications. Biosens. Bioelectron. 11, 493—502 (1996). 

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. 

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. 

In an early application, an enzyme electrode system was reported for the determination of creatinine and creatine, using a combination of creatinine amidohy-drolase, creatine amidinohydrolase and sarcosine oxidase, co-immobilized on an asymmetric cellulose acetate membrane. Thus, the hydrogen peroxide produced was detected to give a quantitative measure of creatine and creatinine in biological fluids [70]. 

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... 

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... 

Besides the broad applications of electrically contacted enzyme electrodes as amperometric biosensors, substantial recent research efforts are directed to the integration of these functional electrodes as biofuel cell devices. The biofuel cell consists of an electrically contacted enzyme electrode acting as anode, where the oxidation of the fuel occurs, and an electrically wired cathode, where the biocatalyzed reduction of the oxidizer proceeds (Fig. 12.4a). The biocatalytic transformations occurring at the anode and the cathode lead to the oxidation of the fuel substrate and the reduction of the oxidizer, with the concomitant generation of a current through the external circuit. Such biofuel cells can, in principle, transform chemical energy stored in biomass into electrical energy. Also, the use... 

Analytes are also used to specify the application. Glucose enzyme sensor is an enzyme biosensor measuring the glucose. Characteristics and commercial varieties of enzyme electrodes, especially using glucose oxidase, have been extensively reviewed by Kuan and Guilbault (17). 

Table 2 summarizes different possible applications of photoswitchable biomaterials, while detailing the nature of the biomaterial, the area of application, and, when possible, specific examples. Reversible light-induced activation and deactivation of redox proteins (enzymes) corresponds to write - read - erase functions. The photonic activation of the biomaterial corresponds to the write function, whereas the amperometric transduction of the recorded optical information represents the read function of the systems. Switching off of the redox functions of the proteins erases the stored photonic information and regenerates the photosensory biomaterial. These integrated, photoswitchable redox enzyme electrode assemblies mimic logic functions of computers, and may be considered as first step into the era of biocomputers. 

Highest sensitivity is reached when high enzyme activity within a thin layer is used and effective external mass transfer is provided. Under these conditions, substrate measurement can be managed down to the range of 1 micromolar with imprecision below 2 %. Therefore, owing to their limited sensitivity, "normal" enzyme electrodes are applicable only to metabolites present in the micro and millimolar concentration range. 

In recent years the electrochemistry of the enzyme membrane has been a subject of great interest due to its significance in both theories and practical applications to biosensors (i-5). Since the enzyme electrode was first proposed and prepared by Clark et al. (6) and Updike et al. (7), enzyme-based biosensors have become a widely interested research field. Research efforts have been directed toward improved designs of the electrode and the necessary membrane materials required for the proper operation of sensors. Different methods have been developed for immobilizing the enzyme on the electrode surface, such as covalent and adsorptive couplings (8-12) of the enzymes to the electrode surface, entrapment of the enzymes in the carbon paste mixture (13 etc. The entrapment of the enzyme into a conducting polymer has become an attractive method (14-22) because of the conducting nature of the polymer matrix and of the easy preparation procedure of the enzyme electrode. The entrapment of enzymes in the polypyrrole film provides a simple way of enzyme immobilization for the construction of a biosensor. It is known that the PPy-... 

In this paper we report the electrochemical polymerization of the PPy-GOD film on the glassy carbon (GC) electrode in enzyme solution without other supporting electrolytes and the electrochemical behavior of the synthesized PPy-GOD film electrode. Because the GOD enzyme molecules were doped into the polymer, the film electrode showed a different cyclic voltammetric behavior from that of a polypyrrole film doped with small anions. The film electrode has a good catalytic behavior to glucose, which is dependent on the film thickness and pH. The interesting result observed is that the thin PPy-GOD film electrode shows selectivity to some hydrophilic pharmaceutical drugs which may result in a new analytical application of the enzyme electrode. 

The most relevant fields of practical application of enzyme electrodes are medical diagnostics, followed by process control, food analysis, and environmental monitoring. 

Refs. [i] Clark LC, Lyons C (1962) Ann NY Acad Sci 102 29 [ii] Turner APT, Karuhe I, Wilson GS (1987) Biosensors fundamentals and applications. Oxford University Press, Oxford [in] Scheller FS, Schubert F (1992) Biosensors. Elsevier, Amsterdam [iv] Heller A (1990) Acc Chem Res 23 128 [v] Scheller F, Wollenberger U (2002) Enzyme electrodes. Ire Bard AJ, Stratmann M, Wilson GS (eds) Bioelectrochemistry. Encyclopedia of electrochemistry, vol. 9. Wiley-VCH, Weinheim [vi] Scheller F, LisdatF, Wollenberger U (2005) Application of electrically contacted enzymes for biosensors. In Willnerl, KatzE (eds) Bioelectronics from theory to applications. Wiley-VCH, Weinheim [vii] Cass AEG (ed) (1990) Biosensors a practical approach. Oxford University Press, Oxford... 

Collagen membranes also bind a variety of enzymes (141). The binding procedure is particularly mild because the enzyme never comes in contact with the chemical resents, avoiding all risks of denaturation. Such membranes, however are too thick and too fragile, especially at 37 °C, to be recommended for in vivo applications of enzyme electrodes (142). Several commercial preactivated membranes are available that provide simple and fast procedures for immobilizing membranes (90-92, 143). The stability of the enzymatic membranes were excellent More than 400 cissays were performed within 50 days. 

---