Immobilized enzyme electrodes

Mifflin, T. E. Andriano, K. M. Robbins, W. B. Determination of Penicillin Using an Immobilized Enzyme Electrode, /. Chem. Educ. 1984, 61, 638-639. 

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). 

Fig. 6. Diagram of an immobilized enzyme electrode, where S is the substrate and P is the enzyme-bound substrate product.

The response of the immobilized enzyme electrode can be made independent of the enzyme concentration by using a large excess of enzyme at the electrode surface. The electrode response is limited by the mass transport of the substrate. Using an excess of enzyme often results in longer electrode lifetimes, increased linear range, reduced susceptibiUty to pH, temperature, and interfering species (58,59). At low enzyme concentrations the electrode response is governed by the kinetics of the enzyme reaction. 

Several techniques have been developed for the determination of purine and pyrimidine derivatives in food sample and in particular for hypoxanthine quantification biosensors (220-223) and electrochemical methods making use of immobilized enzyme electrode (224 -227), electrochemical enzymatic-based HA methods (228,229), enzyme reaction with fluorimetric detection (230), radioimmunoassay (231), colorimetric methods (232), capillary electrophoresis (233), and TLC (234). Many HPLC methods have also been developed and are reported in Table 4 (235-247) the most recent ones are described next. 

In the field of biosensor technology, immobilized enzyme electrode development occupies a place of prominence due to the attractive performance of this hybrid device. Coupling an immobilized enzyme layer with an electrochemical sensor combines the advantages of using an insolubilized enzyme system (see below) with the sensitivity of readily available potentiometric and amperometric electrodes. The resulting biosensor enables direct, reliable, and reproducible... 

A potentiometric L-lhreonine selective sensor for determining L-threonine in biological fluids and foods utilizes threonine deaminase in conjunction with an NH3 gas-sensing electrode. The biosensor exhibits a linear response to l-threonine concentration over the 0.1-200 mM range (292). Comparing l-tryptophan bacteria and immobilized enzyme electrodes shows that the enzyme probe is stable for less than 5 days but that the bacterial probe functions for approximately 3 weeks (293). 

A distribution coefficient is used widely in various areas involving two-phase systems [43,44] to describe behaviour of immobilized enzymes, electrode systems, different kinds of chromatographic separation and, in particular, makes it possible to correlate analytically parameters describing equilibria on a surface with parameters of column and thin-layer chromatography, whose success is determined mostly by extensive use of pristine and modified silicas as adsorbents and supports. 

After a short historical survey the fundamentals of signal transducers and the present state of thermometric, optoelectronic, and piezoelectric biosensors are presented. The most relevant electrochemical techniques are outlined in detail because electrochemical transducers are predominant. The aim of the second section is to provide information on the function of the biocomponents used in biosensors, primarily enzymes, but also antibodies and chemoreceptors. Special attention is paid to the methods of immobilization of the biomaterial and to the discussion and mathematical modeling of the interplay of biochemical reactions with mass transfer processes in immobilized enzyme electrodes. 

Immobilized enzyme electrode for the determination of D-glucose Active immobilized enzyme for clinical analysis of D-glucose used in microcolumn form as part of an Autoanalyzer continuous flow system Immobilized enzyme reactor for determination of D-glucose in a continuous flow analyzer... 

D-Glucose oxidase has been immobilized for use in a number of analytical systems. Immobilization of the enzyme onto nonporous glassy carbon electrodes by carbodi-imide-mediated coupling to superficial oxides generated by anodic oxidation has afforded an immobilized enzyme electrode with which hydrogen peroxide released enzymically from D-glucose may be measured amperometri-cally. Properties of the enzyme immobilized on amino-organo-sylochrome have been studied. 

P.N. Bartlett, K.F.E. Pratt. Theoretical treatment of diffusion and kinetics in amperometiic immobilized enzyme electrodes. 1. Redox mediator entrapped within the film. J Electroanal Chem. 397 61 (1995). 

Electrocatalytic enzyme mediation has been demonstrated using quinones, viol-ogens, 2,2-azinobis(3-ethylbenzothiazohne-6-sulfonate) (ABTS), and complexes of iron, ruthenium, cobalt, osmium, and many other compounds [22-24]. Much early work concerned the GOx anode, intended for a glucose sensor. In 1974, Schlapfer et al. tested 11 different mediators for a GOx electrode with a semipermeable membrane [25]. Ten years later, Cass et al. reported membrane-bound electrodes that operated in whole blood [26]. In 1986, Bourdillon et al. presented an analysis arguing that immobilized enzyme electrodes have higher efficiency than those with free enzyme in solution [27]. These examples demonstrate several possible enzyme/ mediator configurations. Both enzyme and mediator can exist as firee species in the liquid electrolyte, or one or both can be immobilized on the electrode surface. As an alternative to immobilization, enzyme and mediator can also be confined near the electrode by a semipermeable membrane. 

This equation represents a useful model for interpreting data from flow experiments on immobilized enzyme electrodes (in this case, aleohol dehydrogenase) in which an electron mediator (i.e., NAD" ) is limiting and the substrate (i.e., ethanol) is present in excess. In this scenario, bulk NAD is pumped to the electrode and its reduced counterpart (NADH) is measured spectrophotometrically in the outflow. Specifically, half of the equation is measured experimentally and the remainder is plotted in the form of a Lineweaver-Burk plot, wherein a value for the combined mass transfer parameter Mnad+is assumed. As the equation has three unknowns. 

TABLE 12.1 Model Kinetic Parameters as a Function of Flow Rate for Immobilized Enzyme Electrodes [24]... 

Effect of pH — Enzymes are generally only active in limited pH ranges, and each enzyme has its own optimal pH value. The presence of an inhibitor changes the different ionization states of the system governing the enzymatic activity, which results in a variation in the behavior of the inhibited enzyme with respect to pH from one inhibitor to another. This phenomenon can be studied using an immobilized enzyme electrode, the response of which gives the enzymatic activity in... 

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