Electrodes, enzyme

Potcntiomctric Biosensors Potentiometric electrodes for the analysis of molecules of biochemical importance can be constructed in a fashion similar to that used for gas-sensing electrodes. The most common class of potentiometric biosensors are the so-called enzyme electrodes, in which an enzyme is trapped or immobilized at the surface of an ion-selective electrode. Reaction of the analyte with the enzyme produces a product whose concentration is monitored by the ion-selective electrode. Potentiometric biosensors have also been designed around other biologically active species, including antibodies, bacterial particles, tissue, and hormone receptors. 

One example of an enzyme electrode is the urea electrode, which is based on the catalytic hydrolysis of urea by urease... 

Potentiometric electrodes also can be designed to respond to molecules by incorporating a reaction producing an ion whose concentration can be determined using a traditional ion-selective electrode. Gas-sensing electrodes, for example, include a gas-permeable membrane that isolates the ion-selective electrode from the solution containing the analyte. Diffusion of a dissolved gas across the membrane alters the composition of the inner solution in a manner that can be followed with an ion-selective electrode. Enzyme electrodes operate in the same way. 

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

Wang, J. Macca, C. Use of Blood-Glucose Test Strips for Introducing Enzyme Electrodes and Modern Biosensors, ... 

Enzyme catalysis Enzyme electrode Enzyme immobilization Enzyme immunoassay Enzyme inhibitors... 

Fig. 5. Detection methods for glucose enzyme electrode based on (a) oxygen, (b) hydrogen peroxide, and (c) a mediator. See text.

A compound which is a good choice for an artificial electron relay is one which can reach the reduced FADH2 active site, undergo fast electron transfer, and then transport the electrons to the electrodes as rapidly as possible. Electron-transport rate studies have been done for an enzyme electrode for glucose (G) using interdigitated array electrodes (41). The following mechanism for redox reactions in osmium polymer—GOD biosensor films has... 

The next generation of amperomethc enzyme electrodes may weU be based on immobilization techniques that are compatible with microelectronic mass-production processes and are easy to miniaturize (42). Integration of enzymes and mediators simultaneously should improve the electron-transfer pathway from the active site of the enzyme to the electrode. 

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. 

Guilbaut, G. G. Enzyme electrodes in analytical chemistry, in Comprehensive Analytical Chemistry (ed.) Svehla, G. S., Amsterdam, Pergamon Press 1977... 

Ammann, D. et al. in Ion and Enzyme Electrodes in Biology and Medicine (eds.) Kessler, M. et al., Miinchen—Berlin—Wien, Urban Schwarzenberg 1976... 

The enzyme is used in an enzyme electrode in which a tube is sealed at its lower end with a cellulose acetate membrane. An outer membrane of collagen is also attached to the end of the electrode tube and glucose oxidase enzyme is contained in the space between the two diaphragms. 

J Koryta (Ed), Use of Enzyme Electrodes in Biomedical Investigations, Wiley, Chichester, 1980... 

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 are based on the coupling of a layer of an enzyme with an appropriate electrode. Such electrodes combine the specificity of the enzyme for its substrate with the analytical power of electrochemical devices. As a result of this coupling, enzyme electrodes have been shown to be extremely useful for monitoring a wide variety of substrates of analytical importance in clinical, environmental, and food samples. 

Impractical and Theoretical Considerations The operation of an enzyme electrode is illustrated in Figure 6-1. The immobilized enzyme layer is chosen to catalyze a reaction, which generates or consumes a detectable species ... 

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... 

FIGURE 6-1 Enzyme electrode based on a biocatalytic layer immobilized on an electrode transducer. 

FIGURE 6-2 Steps in the preparation of an ainperometric enzyme electrode with simple enzyme immohilization hy trapping between an inner cellulose acetate and outer collagen membrane, cast on the electrode body. (Reproduced with permission from reference 1.)... 

The response characteristics of enzyme electrodes depend on many variables, and an understanding of the theoretical basis of their function would help to improve their performance. Enzymatic reactions involving a single substrate can be formulated in a general way as... 

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.)... 

Enzyme electrodes for other substrates of analytical significance have been developed. Representative examples are listed in Table 6-1. Further advances in enzyme technology, and particularly the isolation of new and more stable enzymes, should enhance the development of new biocatalytic sensors. New opportunities (particularly assays of new environments or monitoring of hydrophobic analytes) derive from the finding that enzymes can maintain then biocatalytic activity in organic solvents (31,32). 

How would you extend the linear range of calibration plots based on the use of enzyme electrodes ... 

Suggest an enzyme electrode-based procedure for detecting organophosphate pesticides. 

Give example of an enzyme electrode based on an ion-selective electrode transducer. What is the relationship between the substrate concentration and the potential response ... 

Explain clearly why and how the response of glucose oxidase-based enzyme electrodes is influenced by fluctuations in the oxygen tension. 

Use equations to explain why and how an increase in the sensitivity of an enzyme electrode is often coupled to a narrower linear range. 

Electroosmotic flow, 195 End column detection, 89 Energy barrier, 16 Enzyme electrodes, 172, 174 Enzyme immunoassays, 185 Enzyme inhibition, 181 Enzyme reconstitution, 178 Enzyme wiring, 178 Equilibrium potential, 15 Ethanol electrodes, 87, 178 Exchange current, 14... 

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