Enzyme electrodes stability

The success of the enzyme electrode depends, in part, on the immobilization of the enzyme layer. The objective is to provide intimate contact between the enzyme and the sensing surface while maintaining (and even improving) the enzyme stability. Several physical and chemical schemes can thus be used to immobilize the enzyme onto the electrode. The simplest approach is to entrap a solution of the... 

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. 

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. 

Biosensors fabricated on the Nafion and polyion-modified palladium strips are reported by C.-J. Yuan [193], They found that Nafion membrane is capable of eliminating the electrochemical interferences of oxidative species (ascorbic acid and uric acid) on the enzyme electrode. Furthermore, it can restricting the oxidized anionic interferent to adhere on its surface, thereby the fouling of the electrode was avoided. Notably, the stability of the proposed PVA-SbQ/GOD planar electrode is superior to the most commercially available membrane-covered electrodes which have a use life of about ten days only. Compared to the conventional three-dimensional electrodes the proposed planar electrode exhibits a similar... 

The stability of enzyme electrodes is difficult to define because an enzyme can lose some of its activity. Deterioration of immobilized enzyme in the potentiometric electrodes can be seen by three changes in the response characteristics (a) with age the upper limit will decrease (e.g., from 10-2 to 10 3 moll-1), (b) the slope of the analytical (calibration) curve of potential vs. log [analyte] decrease from 59.2 mV per decade (Nernstian response) to lower value, and (c) the response time of the biosensor will become longer as the enzyme ages [59]. The overall lifetime of the biosensor depends on the frequency with which the biosensor is used and the stability depends on the type of entrapment used, the concentration of enzyme in the tissue or crude extract, the optimum conditions of enzyme, the leaching out of loosely bound cofactor from the active site, a cofactor that is needed for the enzymatic activity and the stability of the base sensor. 

D. Moscone, Prussian Blue and enzyme bulk-modified screen-printed electrodes for hydrogen peroxide and glucose determination with improved storage and operational stability, Anal. Chim. Acta, 485 (2003) 111-120. A. Lupu, D. Compagnone and G. Palleschi, Screen-printed enzyme electrodes for the detection of marker analytes during winemaking, Anal. Chim. Acta, 513 (2004) 67-72. 

Tissue and Bacteria Electrodes The limited stability of isolated enzymes, and the fact that some enzymes are expensive or even not available in the pure state, has prompted the use of cellular materials (plant tissues, bacterial cells, etc.) as a source of enzymatic activity (48). For example, the banana tissue (which is rich with polyphenol oxidase) can be incorporated by mixing within the carbon paste matrix to yield a fast-responding and sensitive dopamine sensor (Fig. 6.14). These biocatalytic electrodes function in a manner similar to that for conventional enzyme electrodes (i.e., enzymes present in the tissue or cell produce or consume a detectable species). 

The loading is crucial for the response characteristics and the stability of the enzyme electrode. The choice of the enzyme determines the chemical selectivity of the measurement due to the specificity of the signal-producing interaction of the enzyme with the analyte. The selection of the indicator electrode is largely determined by the species involved in the enzyme reaction. 

Piovesta s alcohol oxidase, from the yeast Pichia pastor is. Described as "equally active on methanol and ethanol" (D. Ranasiak arid T. Hopkins, Provest a, personal communication) it is "one-third as active no ethanol than on methanol.." on an enzyme electrode (Hopkins and Muller (in )). The commercial product is virtually devoid of catalase and has a pH stability profile (5. -8. 3) that see/n.s to rule out its use in beer or wines. Alcohol oxidase is still of interest... 

The choice of an appropriate electrochemical sensor is governed by several requirements (1) the nature of the substrate to be determined (ions or redox species) (2) the shape of the final sensor (microelectrodes) (3) the selectivity, sensitivity, and speed of the measurements and (4) the reliability and stability of the probe. The most frequently used sensors operate under potentiometric or amperometric modes. Amperometric enzyme electrodes, which consume a specific product of the enzymatic reaction, display an expanded linear response... 

Amperometric sensors monitor current flow, at a selected, fixed potential, between the working electrode and the reference electrode. In amperometric biosensors, the two-electrode configuration is often employed. However, when operating in media of poor conductivity (hydroalcoholic solutions, organic solvents), a three-electrode system is best (29). The amperometric sensor exhibits a linear response versus the concentration of the substrate. In these enzyme electrodes, either the reactant or the product of the enzymatic reaction must be electroactive (oxidizable or reducible) at the electrode surface. Optimization of amperometric sensors, with regard to stability, low background currents, and fast electron-transfer kinetics, constitutes a complete task. 

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. 

Fig. 10.6. Change of the rate hmiting step in t5rrosinase carbon paste electrodes, (a) ind (b) show the flow rate dependence of steady-state currents for unmediated (a) emd mediated (b) enzyme electrodes. Applied potential — 0.05 V vs. Ag/AgCl for ( ) 1 p.M phenol ind ( ) 0.5 mM ferrocyanide as control of diffusion limited response. Error bars show the standard deviation of six electrodes. The cheinge from kinetic to dififtisional control in mediated electrodes results in higher sensitivity and improved operational stability as demonstrated in (c) where (1) represents the FIA response of mediated electrodes and (2) unmediated with consecutive 20 /rl injections of 10 p,M phenol in a thin-layer cell. Applied potential - 0.05 V vs. Ag/AgCl, mobile phase 0.25 M phosphate buffer pH 6.0 ind flow rate 0.7 ml min . ...

Enzyme biosensors containing pol3mieric electron transfer systems have been studied for more than a decade. One of the earlier systems was first reported by Degani and Heller [1,2] using electron transfer relays to improve electrochemical assay of substrates. Soon after Okamoto, Skotheim, Hale and co-workers reported various flexible polymeric electron transfer systems appUed to amperometric enz5une biosensors [3-16], Heller and co-workers further developed a concept of wired amperometric enzyme electrodes [17—27] to increase sensor accuracy and stability. 

Binyamin, Chen and Heller reported that wired enzyme electrodes constituted of glassy carbon electrodes coated with poly(4-vinylpyridine) complexed with [Os(bpy)2Cl] and quarternized with 2-bromoethylamine or poly[(iV-vinylimidazole) complexed with [Os(4,4 -dimethyl-2,2 -bypyridine)2Cl] or poly(vinylpyridine) complexed with [Os(4,4 -dimethoxy-2,2 -bypyridine)2Cl] quaternized with methyl groups lost their electrocatalytic activity more rapidly in serum or saline phosphate buffer (pH 7.2) in the presence of urate and transitional metal ions such as Zn and Fe " " than in plain saline phosphate buffer (pH 7.2). It was reported that as much as two-thirds of the current is lost in 2 h in some anodes. However, when a composite membrane of cellulose acetate, Nafion, and the polyaziridine-cross-linked co-polymer of poly(4-vinyl pyridine) quaternized with bromoacetic acid was applied, the glucose sensor stability in serum was improved and maintained for at least 3 days [27,50]. 

The measurements performed with the two types of biosensors show that the linear range of this type of enzyme electrodes using natural oxygen mediator manifest a wide range of measurement. The immobilised enzyme showed high operative stability, which makes the measurements easily reproducible. Both electrodes have very good correlation coefficients and a small standard deviation. 

The next stage was achieved in 1967 by Updike and Hicks, who entrapped GOD in a gel of polyacrylamide, thus increasing the operational stability of the enzyme and simplifying the sensor preparation. Further investigations by Reitnauer (1972) enabled the successful application of an enzyme electrode in a prototype blood glucose analyzer. In 1975 Yellow Springs Instrument Co. (USA) commercialized a glucose analyzer (model 23 A) which was based on a patent by Clark (1970). The Lactate Analyzer LA 640 by La Roche (Switzerland) followed one year later. In this instrument the enzyme is dissolved in a buffer in a reaction chamber placed in front of the electrode. 

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