Operational stability, enzyme electrodes

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. 

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

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. 

F. Ricci, A. Amine, C.S. Tuta, A.A. Ciucu, F. Lucarelli, G. Palleschi and 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(1) (2003) 111-120. 

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. 

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 . ...

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. 

In enzyme electrodes, which are deliberately operated under conditions of diffusion control, the diffusion limits the sensitivity. Here, the coupling of cyclic enzyme reactions gives rise to a sensitivity enhancement by overcoming the limit set by diffusion. The excess of enzyme present in the membrane is included in the substrate conversion. On the other hand, the upper limit of linearity and the operational stability are decreased. 

Usually the imprecision found with enzyme electrodes ranges from 0.5% to 5%, which is, of covuse, dependent on the analytical setup used (e.g., manual sample injection versus flow injection). The lifetime of enzyme electrodes depends on several factors including the specific enzyme activity, the eventual formation of inactivating reaction products, and the operation conditions of the sensor. Generally, a high enzyme excess in the biocatalytic membrane is desirable in order to obtain a high stability. Lifetimes are in the range of several days or months, or between some hundreds and several thousand measurements. 

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