Enzyme electrodes requirements

The concentrations of a number of analytically relevant compounds, such as creatinine, pyruvate, hormones, and drugs, are often so low that their assay with enzyme electrodes requires large sample volumes or is totally impossible. The detection limit of usual enzyme electrodes is... 

Similar to other analyzers the Glukometer GKM (Fig. 128) is particularly well suited to the analysis of single samples and small sample series. The measuring and sample solutions are injected by using pipettes. The preparation of the enzyme electrode requires about 3 min the... 

Because of a build-up of product in the enzyme membrane, enzyme electrodes require washing prior to contact with the next sample. The washing time varies from just 20 s for urease in conjvmction with an ammonia electrode to as long as 10 min for urease with a pH electrode. The washing time increases with enzyme membrane thickness, as also observed for additional membranes discussed above. It will also depend on the enzyme used and on the characteristic of the base sensor itself, and will be affected by diffusion and kinetic effects. The use of flow injection analysis simplifies the procedures, since the carrier stream serves to wash out between samples. 

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 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. 

Most suitable would be the use of a perfectly NH4+ ion-selective glass electrode however, a disadvantage of this type of enzyme electrode is the time required for the establishment of equilibrium (several minutes) moreover, the normal Nernst response of 59 mV per decade (at 25° C) is practically never reached. Nevertheless, in biochemical investigations these electrodes offer special possibilities, especially because they can also be used in the reverse way as an enzyme-sensing electrode, i.e., by testing an enzyme with a substrate layer around the bulb of the glass electrode. 

In most cases the electronic connection between an immobilized redox enzyme and the electrode requires a mediator to shuttle the electrons to the prosthetic group or some type of wiring that plays the same role. There are cases, however, especially those involving relatively small enzymes, where direct electron transfer takes place between the electrode and the prosthetic group or some electronic relay in the enzyme. Analysis of the catalysis responses then follows the principles described and illustrated in Section 4.3.2. Somewhat more complicated schemes are treated in references7, where illustrative experimental examples can also be found. 

Microbial sensors offer a number of assets, namely (a) they are less sensitive to inhibition by solutes and more tolerant to suboptimal pH and temperature values than are enzyme electrodes b) they have longer lifetimes than enzymes and (c) they are less expensive than enzyme electrodes as they require no active enzyme to be isolated. On the other hand, they lag behind enzyme electrodes in a few other respects thus, (a) some have longer response times than their enzyme counterparts b) baseline restoration after measurement typically takes longer and (c) cells contain many enzymes and due care must be exercised to ensure adequate selectivity e.g. by optimizing the storage conditions or using specific enzyme reactions) —some mutant microorganisms lack certain enzymes. 

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. 

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-... 

A great number of enzymes have already been combined with electrodes. In some cases, several possible enzymatic routes may be selected, depending on the substrate to be analyzed. One such example is that used to determine aspartame (Fig. 3) (18-20). Enzyme electrodes use, ideally, only one enzyme and monitor the main substrate-enzyme reaction, for example, glucose oxidase-glucose. If the main substrate-enzyme reaction is not electrochemically detectable or if signal amplification is required, bi- or trienzymatic sequences may be applied. 

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... 

There appear to be two classes of redox enzymes intrinsic and extrinsic (5). With the former, the catalytic reaction between an enzyme and its substrates takes place within a highly localized assembly of redox-active sites. There need be no electron transfer pathways from these sites to the surface of the enzyme, where, it is presumed, it would interact with an electrode. For such intrinsic redox enzymes, electrode reactions may require (1) that the sites of the catalytic reaction be close to the protein surface, (2) that the enzyme can deform without loss of activity, (3) that the electrode surface projects into the enzyme. 

Several other sensors are available that are based on the amperometric measurement of hydrogen peroxide produced by enzymatic reactions. The analytes measured include sucrose, lactose, ethanol, and L-lactate. A different enzyme is, of course, required for each species. In some cases, enzyme electrodes can be based on measuring oxygen or on measuring pH. 

The development of analytically useful enzyme electrodes is limited by the availability of purified and stable enzyme preparations. In an effort to extend the range of measurable species using ISE devices further, Rechnitz and co-workers (Rl) recently introduced bacterial- and tissue-based bio-selective electrode systems. These sensors are prepared in much the same manner as the enzyme probes except that whole intact cells are utilized as the immobilized reagents. There are several potential advantages to this novel approach, including (1) no need to extract and purify the enzymes involved, i. e., low cost (2) enzymes which are unstable when extracted from the cell may be used in situ to maximize and preserve their activity (3) if desired enzyme reactions require cofactors, these co ctors need not be added to the assay mixture because they are already present in the intact cell and (4) analytical reactions involving multistep enzyme sequences already present in the cells may be used to detect given analytes. 

Besides the differential equations the complete formulation of the model requires a set of initial and boundary conditions. These must reflect the situation at the interface between measuring solution and enzyme electrode membrane and between membrane and sensor. For the models considered, it is assumed that the measuring solution is perfectly mixed and contains a large amount of substrate as compared to the substrate converted in the enzyme membrane. It has been shown experimentally (Carr and Bowers, 1980) that in measuring solutions diffusion is much more rapid than in membranes. A boundary layer effect is not considered. On the sensor side all electrode-inactive substances fulfill zero flux conditions. If the model contains more than one layer the transfer between the layers may be modeled by using relations of mass conservation. The respective equations will be given in the following sections. 

The determination of oxalate in urine is required for diagnosis of renal calculus and hyperoxaluria. For enzymatic oxalate determination, oxalate decarboxylase (EC 4.1.1.2) has been employed in enzyme thermistors (Danielsson etal., 1981), enzyme reactors (Lindberg, 1983), and potentiometric enzyme electrodes (Kobos and Ramsey, 1980). 

Such interferences can be compensated by using difference measurements, which usually require a reference transducer. An elegant alternative is the use of enzymatic anti-interference systems containing enzymes in front of the sensor that catalyze the conversion of the disturbing compounds to inert products. Such systems have been developed in conjunction with analytical enzyme reactors as well as enzyme electrodes. 

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