Enzyme electrode systems

The use of a catalyst with oxidase enzyme is an example of the use of a combined enzyme system, which illustrates the wide potential offered by multi-enzyme electrode systems. Various enzymes can be arranged to work sequentially to transform quite complex substances and eventually produce a measurable concentration-dependent change, which is detected by the output signal and recorded for analysis. 

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. 

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]. 

T. Hu, X.E. Zhang, Z.P. Zhang and L.Q. Chen, A screen-printed disposable enzyme electrode system for simultaneous determination of sucrose and glucose, Electroanalysis, 12 (2000) 868-870. 

Pollmann KH, Gerber MT, Kost KM, Ochs ML, Walljng PD, Bateson JE, Kuhn LS, Han CA (1994) US Patent 5,288 636, Enzyme electrode system. Boehringer Mannheim... 

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. 

C15. Crochet, K. L., and Montalvo, J. G., Enzyme electrode system for assay of serum cholinesterase. Atuil. Chim. Acta 66, 259-269 (1973). 

G. J. Moody, G. S. Sanghera, and J. D. R. Thomas, Amperometric Enzyme Electrode System for the Flow Injection Analysis of Glucose. Analyst, 111 (1986) 605. 

Danzer T. and Schwedt G., Chemometric methods for the development of a biosensor system and the evaluation of inhibition studies with solutions and mixtures of pesticides and heavy metals. Part 1. Development of an enzyme electrodes system for pesticides and heavy metals screening using selected chemometrics methods. Anal. Chim. Acta, 318, 275-286, 1996. 

Recently, a novel two electrode differential potentiometric cell for enzyme electrode systems has been described that provides enhanced substrate sensitivities compared to conventional cells composed of a single enzyme electrode and reference (Cha, G.S. Meyerhoff, M.E. Electroanalysis, in press). The cell employs two working enzyme electrodes, one which responds to the analyte in the positive potential direction via detection of cations, and one which responds to the same analyte but in a negative direction owing to anion detection. A similar approach can be applied in the design of new two electrode gas-selective sensors with enhanced gas sensitivity. 

While potentiometric enzymatic electrodes for detection of phosphate have been developed [121,122], no application has been made of these to water analysis because of the relatively poor sensitivity. However, amperometric enzyme electrodes have been reported that they have high sensitivity, selectivity, and long operational life and it is expected that the use of these for water analysis will become more widespread (see Table 8.2). For example, a sensitive enzyme electrode based on the amperometric detection of hydrogen peroxide produced by membrane coimmobilized nucleoside phosphorylase and xanthine oxidase has been reported for the detection of phosphate by D Urso and Coulet [123]. Other similar enzyme electrode systems suitable for water analysis are listed in Table 8.2. 

A three-enzyme electrode system, such as needed for creatinine measurement, poses a more difficult enzyme-immobilisation problem, in that different enzymes have different immobilisation requirements and their microenvironmental interrelationships need to be optimised. For one creatine sensor, the requisite creatine amidinohydrolase and sarcosine oxidase were immobihsed in polyurethane pre-polymer and PEG-hnked creatinine amidohydrolase was attached via diisocyanate pre-polymer to create a polyurethane adduct [14]. The likelihood of enzyme inactivation with chemical immobih-sation is high, but provided an enzyme preparation survives this, long-term stability is feasible. In the case of these three particular enzymes, a loss of activity resulted from silver ions diffusing from the reference electrode the material solution was to protect the enzyme layer with a diffusion-resisting cellulose acetate membrane. 

Amperometric glucose-sensing electrodes with modified enzymes enzyme electrode systems, 42-43 experimental description, 41 2 flow injection measurement of glucose for lipid-modified enzyme electrode, 44,45/,46f... 

The detection of phenols is not only of importance in industry but also in the medical field. Tyrosinase has been used as part of an enzyme-electrode system to detect catechols and assess catecholamines in the urine of patients with neural crest tumors [8],... 

Moody G.J., Sanghera G.S. and Thomas J.D.R. (1986) Amperometric enzyme electrode system for the flow-injection analysis of glucose. Analyst, 111, 605-609. 

Improved sensitivity and scope can be achieved by coupling two (or more) enzymatic reactions hi a chain, cycling, or catalytic mechanism (9). For example, a considerable enhancement of the sensitivity of enzyme electrodes can be achieved by enzymatic recycling of the analyte in two-enzyme systems. Such an amplification... 

FIGURE 6-5 Second-generation enzyme electrode sequence of events that occur in a mediated system. (Reproduced with permission from reference 12.)... 

The high specificity required for the analysis of physiological fluids often necessitates the incorporation of permselective membranes between the sample and the sensor. A typical configuration is presented in Fig. 7, where the membrane system comprises three distinct layers. The outer membrane. A, which encounters the sample solution is indicated by the dashed lines. It most commonly serves to eliminate high molecular weight interferences, such as other enzymes and proteins. The substrate, S, and other small molecules are allowed to enter the enzyme layer, B, which typically consist of a gelatinous material or a porous solid support. The immobilized enzyme catalyzes the conversion of substrate, S, to product, P. The substrate, product or a cofactor may be the species detected electrochemically. In many cases the electrochemical sensor may be prone to interferences and a permselective membrane, C, is required. The response time and sensitivity of the enzyme electrode will depend on the rate of permeation through layers A, B and C the kinetics of enzymatic conversion as well as the charac-... 

Fig. 7. Schematic diagram of an enzyme electrode. A, B and C comprise the membrane system, while D is the detector (either amperometric or potentiometric)...

Although the primary focus of oxidase based enzyme electrodes has been the determination of glucose, the list of extensions to other analytes is considerable. Systems have been described for cholesterol 123,132-136) galactose ii -i35-i37) dd i38.i39) lactatepyruvatecreatinine serum lipaseethanoland amino acids... 

Substrate Enzyme Substrate or product measured Electrode system... 

Equations 2.26 and 2.27 carmot be solved analytically except for a series of limiting cases considered by Bartlett and Pratt [147,192]. Since fine control of film thickness and organization can be achieved with LbL self-assembled enzyme polyelectrolyte multilayers, these different cases of the kinetic case-diagram for amperometric enzyme electrodes could be tested [147]. For the enzyme multilayer with entrapped mediator in the mediator-limited kinetics (enzyme-mediator reaction rate-determining step), two kinetic cases deserve consideration in this system in both cases I and II, there is no substrate dependence since the kinetics are mediator limited and the current is potential dependent, since the mediator concentration is potential dependent. Since diffusion is fast as compared to enzyme kinetics, mediator and substrate are both approximately at their bulk concentrations throughout the film in case I. The current is first order in both mediator and enzyme concentration and k, the enzyme reoxidation rate. It increases linearly with film thickness since there is no... 

This chapter is concerned with processes that lead to formation of ISE membrane potentials. The membrane potentials of electrodes with liquid membranes containing a dissolved ion-exchanger ion or a dissolved ionophore (ion carrier), and of electrodes with solid or glassy membranes will be considered. More complicated systems, for example ISEs with a gas gap and enzyme electrodes, will be discussed in chapters 4 and 9. 

Composite potentiometric sensors involve systems based on ion-selective electrodes separated from the test solution by another membrane that either selectively separates a certain component of the analyte or modifies this component by a suitable reaction. This group includes gas probes, enzyme electrodes and other biosensors. Gas probes are discussed in this section and chapter 8 is devoted to potentiometric biosensors. 

The ISE life-time is closely connected with the drift and is at least one year for good electrodes. With some systems, e.g. enzyme electrodes (see chapter 8), the life-time is only a few weeks. It follows from the results of Oesch and Simon [119] that the life-time of electrodes based on ionophores in solventpolymeric membranes depends on the kinetics of dissolution of the ionophore and the plasticiser in the analyte. If both the ionophore and the membrane solvent have distribution coefficients between water and the membrane greater than 10 , then the ISE life-time is at least one year. 

This system is also the most important enzyme electrode in practice. 

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. 

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... 

The sensing microzone of the flow-through sensor depicted in Fig. 5.9.B1 integrates gas-diffusion and detection with two analytical reactions [28], viz. (a) the urease-catalysed formation of ammonium ion by hydrolysis of urea (the analyte), which takes places on a hydrophilic enzyme membrane in contact with the sample-donor stream, which contains a gel where the enzyme is covalently bound and (b) an acid-b reaction that takes place at the microzone on the other side of the diffusion membrane and involves Bromothymol Blue as indicator. This is a sandwich-type sensor including a hydrophilic and a hydrophobic membrane across which the sample stream is circulated —whence it is formally similar to some enzyme electrodes. Since the enzymatic conversion of the analyte must be as efficient as possible, deteetion (based on fibre optics) is performed after the donor and acceptor streams have passed through the sensor. Unlike the previous sensor (Fig. 5.9.A), this does not rely on the wall-jet approach in addition, each stream has its own outlet and the system includes two sensing microzones... 

Urea in kidney dialysate can be determined by immobilizing urease (via silylation or with glutaraldehyde as binder) on commercially available acid-base cellulose pads the process has to be modified slightly in order not to alter the dye contained in the pads [57]. The stopped-flow technique assures the required sensitivity for the enzymatic reaction, which takes 30-60 s. Synchronization of the peristaltic pumps PI and P2 in the valveless impulse-response flow injection manifold depicted in Fig. 5.19.B by means of a timer enables kinetic measurements [62]. Following a comprehensive study of the effect of hydrodynamic and (bio)chemical variables, the sensor was optimized for monitoring urea in real biological samples. A similar system was used for the determination of penicillin by penicillinase-catalysed hydrolysis. The enzyme was immobilized on acid-base cellulose strips via bovine serum albumin similarly as in enzyme electrodes [63], even though the above-described procedure would have been equally effective. 

---