Immobilized enzyme membrane electrodes

Enzymatic sensor A membrane electrode that has been coated with an immobilized enzyme the electrode responds to the amount of analyte in the sample. 

The physical combination of immobilized enzymes and electrodes can be realized in several different ways. The most common are those in which the enzyme is immobilized in a membrane that is held in close proximity to the electrode surface, immobilized directly on the electrode surface with retained activity, or immobilized in an electrically conducting paste comprising a carbon-oil mixture, known as a carbon paste electrode. The enzyme substrate (and cosubstrate, if required) will diffuse from the... 

The designation membrane is used here in a very general sense, as many materials are used. Commercially available electrodes include liquid membrane units, solid-state electrodes, glass membrane electrodes, and plastic membrane electrodes. General classes of ion specific electrodes in addition to those cited are immobilized-liquid membrane electrodes, mixed-crystal membrane electrodes, enzyme electrodes, and antibiotic electrodes (Rechnitz, 1973). Certain of these membrane electrodes are now discussed in some detail. A generalized membrane electrode is illustrated in Figure 6.1a. 

Mifflin and associates described a membrane electrode for the quantitative analysis of penicillin in which the enzyme penicillinase is immobilized in a polyacrylamide gel that is coated on a glass pH electrode. The following data were collected for a series of penicillin standards. 

The enzyme can be immobilized on the electrode by several techniques (53). The simplest method, first used in 1962, is to trap an enzyme solution between the electrode surface and a semipermeable membrane. Another technique is to immobilize the enzyme in a polymer gel such as polyacrylamide which is coated on the electrode surface. Very thin-membrane films can be obtained by electropolymerization techniques (49,54,55) using polypyrrole, polyindole, or polyphenylenediamine films, among others. These thin films (qv) offer the advantage of improved diffusion of substrate and product that... 

Biocatalytic membrane electrodes have an ISE or a gas sensing electrode in contact with a thin layer of biocatalytic material, which can be an immobilized enzyme, bacterial particles or a tissue slice, as shown in Fig. 3 The biocatalyst converts substrate (the analyte) into product, which is measured by the electrode. Electrodes of this type are often referred to as biosensors . 

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-... 

The first enzyme biosensor was a glucose sensor reported by Clark in 1962 [194], This biosensor measured the product of glucose oxidation by GOD using an electrode which was a remarkable achievement even though the enzyme was not immobilized on the electrode. Updark and Hicks have developed an improved enzyme sensor using enzyme immobilization [194], The sensor combined the membrane-immobilized GOD with an oxygen electrode, and oxygen measurements were carried out before and after the enzyme reaction. Their report showed the importance of biomaterial immobilization to enhance the stability of a biosensor. 

The techniques developed in enzyme immobilization have facilitated the development of enzyme electrodes and of novel enzyme -based, automated, analytical methods (l6,17,l8). Enzyme electrodes have resulted from the combination of an enzyme membrane and an ion-selective electrode they were used successfully to assay directly appropriate substrates. Enzyme columns or enzyme tubes, prepared in a conventional manner, were used as a specific auxiliary component in the indirect assay of substrates in many of the novel automated analytical procedures. 

Immobilized enzymes used in conjunction with ion-selective electrodes provide very convenient methods of analysis. The immobilized enzyme may be held in a gel or membrane around the electrode and the substance to be measured diffuses into the enzyme gel. Its conversion to the product alters the ionic equilibrium across the ion-selective membrane (Figure 8.23). It is important that the enzyme layer is thin, to minimize any problems caused by slow diffusion rates through the layer. 

The classic potentiometric enzyme electrode is a combination of an ion-selective electrode-based sensor and an immobilized (insolubilized) enzyme. Few of the many enzyme electrodes based on potentiometric ion- and gas-selective membrane electrode transducers have been included in commercially available instruments for routine measurements of biomolecules in complex samples such as blood, urine or bioreactor media. The main practical limitation of potentiometric enzyme electrodes for this purpose is their poor selectivity, which does not arise from the biocatalytic reaction, but from the response of the base ion or gas transducer to endogenous ionic and gaseous species in the sample. 

Many different types of techniques for protein immobilization have been developed using, in most cases, enzyme sensors. Early studies of enzyme biosensors often employed thick polymer membranes (thickness 0.01-1 mm) in which enzymes are physically entrapped or chemically anchored. The electrode surface was covered with the enzyme-immobilized polymer membranes to prepare electrochemical enzyme sensors. Although these biosensors functioned appropriately to... 

Immobilized enzyme (wide range) membranes with oxygen, ammonia, or carbon dioxide electrodes Universal Sensors, USA... 

In the previous papers(12,13), we reported on the vessel access type, i.e. tubular type, glucose sensor. It consisted of a glucose electrode system with a GOX enzyme immobilized Nylon membrane and a glucose semipermeable membrane, and a reference oxygen electrode system. The sensor could directly measure up to 700 mg/dl of BGL in an arterial blood stream when it was placed into an external A-V shunt. This sensor, however, has some problems such as thrombus during in vivo testing without heparin and clinical complexity associated with implanting the sensor in a blood stream. 

A sensitized electrode is a composite device that combines a membrane reactor and a primary electrode. The membrane reactor converts a specific analyte into products that are measurable by the primary electrode. Membrane reactors containing immobilized enzymes, cells, or neutral carriers are capable of very selective conversion of sugars, amino acids, organic acids, and alcohols, and... 

During the past 40 years there have been numerous exciting extensions of electrochemistry to the field of analytical chemistry. A series of selective-ion potentiometric electrodes have been developed, such that most of the common ionic species can be quantitatively monitored in aqueous solution. A highly effective electrolytic moisture analyzer provides continuous online assays for water in gases. Another practical development has been the voltammetric membrane electrode for dioxygen (02), which responds linearly to the partial pressure of 02, either in the gas phase or in solution. The use of an immobilized enzyme (glucose oxidase) on an electrode sensor to assay glucose in blood is another extension of electrochemistry to practical analysis. 

In recent years the electrochemistry of the enzyme membrane has been a subject of great interest due to its significance in both theories and practical applications to biosensors (i-5). Since the enzyme electrode was first proposed and prepared by Clark et al. (6) and Updike et al. (7), enzyme-based biosensors have become a widely interested research field. Research efforts have been directed toward improved designs of the electrode and the necessary membrane materials required for the proper operation of sensors. Different methods have been developed for immobilizing the enzyme on the electrode surface, such as covalent and adsorptive couplings (8-12) of the enzymes to the electrode surface, entrapment of the enzymes in the carbon paste mixture (13 etc. The entrapment of the enzyme into a conducting polymer has become an attractive method (14-22) because of the conducting nature of the polymer matrix and of the easy preparation procedure of the enzyme electrode. The entrapment of enzymes in the polypyrrole film provides a simple way of enzyme immobilization for the construction of a biosensor. It is known that the PPy-... 

Cytochrome c oxidase can be immobilized in an electrode-supported lipid bilayer membrane. The area of the electrode used is 0.2 cm2, and the diameter of the oxidase is 80 A. This enzyme is electroactive and yields the cyclic voltammetric response shown in Fig. P14.2. 

Enzyme-selective electrodes (Fig. 17.11) have been made as a membrane containing immobilized enzymes placed over a pH electrode or over a gas electrode such as an ammonia electrode for potentiometric detection, or over an oxygen electrode for amperometric detection. The products of the reaction of enzyme with substrate are detected by the electrode. 

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