Urease, enzyme electrode immobilization

Enzyme sensors are based primarily on the immobilization of an enzyme onto an electrode, either a metallic electrode used in amperometry (e.g., detection of the enzyme-catalyzed oxidation of glucose) or an ISE employed in potentiometry (e.g., detection of the enzyme-catalyzed liberation of hydronium or ammonium ions). The first potentiometric enzyme electrode, which appeared in 1969 due to Guilbault and Montalvo [140], was a probe for urea with immobilized urease on a glass electrode. Hill and co-workers [141] described in 1986 the second-generation biosensor using ferrocene as a mediator. This device was later marketed as the glucose pen . The development of enzyme-based sensors for the detection of glucose in blood represents a major area of biosensor research. 

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. 

An unusual type of derivative is the complex that forms between urease and bentonite in acid medium (61). The adsorbed form was found catalytically active. Similarly, urease immobilized in a polyacrylamide gel matrix has been used to prepare a urea-specific enzyme electrode (62). Yet another active water-insoluble derivative has been prepared (63) by allowing p-chloromercuribenzoate-treated urease to react with a diazotized copolymer of p-amino-D,L-Phe and L-Leu. Urease has been found to retain about 20% of its original activity when encapsulated in 100 n microcapsules of benzalkonium-heparin in collodion (64). 

The gas-sensing electrodes also are used for the potentiometric measurement of biologically important species. An enzyme is immobilized at or near the gas probe. The gas sensor measures the amount of characteristic gas produced by the reaction of the analyzed substance with the enzyme. For example, an enzyme electrode for urea [NH2C(0)NH2] determination is constructed by the immobilization of urease onto the surface of an ammonia-selective electrode. When the electrode is inserted into a solution that contains urea, the enzyme catalyzes its conversion to ammonia ... 

Chemically binding enzymes to nylon net is very simple and gives strong mechanically resistant membranes (135). The nylon net is first activated by methylation and then quickly treated with lysine. Finally, the enzyme is chemically bound with GA. The immobilized disks are fixed direcdy to the sensor surface or stored in a phosphate buffer. GOD, ascorbate oxidase, cholesterol oxidase, galactose oxidase, urease, alcohol oxidase (135), and lactate oxidase (142) have been immobilized by this procedure and the respective enzyme electrode performance has been established. 

The activity of the enzyme is also strongly affected by the presence of inhibitors. Fluoride ions inhibit urease (173) and oxalate ions inhibit lactate oxidase (174), but the major inhibitors are heavy-metal ions, such as Ag+, Hg +, Cu " ", organophosphates, and sulfhydryl reagents (/i-chloromercuribenzoate and phenylmercury(II) acetate), which block the free thiol groups of many enzyme active centers, especially oxidase (69). Inhibiting the enzyme electrodes makes it possible to quantify the inhibitors themselves (69), for example, H2S and HCN detection using a cytochrome oxidase immobilized electrode (176). 

Enzyme electrodes for lactate determination using immobilized lactate dehydrogenase 16), for urea determination using immobilized urease 17), for L-amino acids using immobilized L-amino acid oxidase 18), and for various amines using the appropriate immobilized deaminase system (19) have also been prepared. A urease electrode is commercially available from Beckman,... 

Figure 4 14 Potentiometric enzyme electrode for determination of biood urea, based on urease enzyme immobilized on the surface of an ammonium ion-seiective polymeric membrane electrode.

Techniques such as potentiometry, polarography, and microcalorimetry have been chosen in exploiting the benefits of immobilized enzymes (see Chapter 4). Enzymes incorporated into membranes form part of enzyme electrodes. The surface of an ion-sensitive electrode is coated with a layer of porous gel in which an enzyme has been polymerized. When the electrode is immersed in a solution of the appropriate substrate, the action of the enzyme produces ions to which the electrode is sensitive. For example, an oxygen electrode coated with a layer containing glucose oxidase can be used to determine glucose by the amount of oxygen consumed m the reaction, and urea can be estimated by the combination of a selective ammonium ion-sensitive electrode and a urease membrane. 

Another method of following the rate of urea hydrolysis is based on a specific-ion electrode for ammonium ions (see Section 21D). Here, the production of NH4 is monitored potentiometrically and is used to obtain the reaction rate. In yet another approach, the urease can be immobilized on the surface of a pH electrode and the rate of change of pH monitored. Many enzymes have now been immobilized onto supports such as gels, membranes, tubing walls, glass beads, polymers, and thin films. Immobilized enzymes often show enhanced stability over their soluble counterparts. In addition, they can be reused often for hundreds or thousands of analyses. 

The sensitive part of an electrode is covered with a membrane on which the enzyme is immobilized in immunocomplexes. The enzyme-catalyzed reaction takes place near the sensor (Mattiasson and Nilsson, 1977). The method is as fast as the thermometric assay but less sensitive. Electrode-based EIA using urease conjugates have been reviewed by Meyerhoff and Rechnitz (1980). This method has reasonably low detection limits. These promising potentiometric EIA are discussed by Boiteux et al. (1981) and Gabauer and Rechnitz (1982). 

The best-known enzyme electrode is that used to analyze for urea in blood. The enzyme urease is immobilized in a polyacrylamide hydrophilic gel and fixed at the bottom of a glass electrode whose characteristics make it an NH4 ISE. Alternatively, the ISE can be a composite system designed to detect NH3. In the presence of the enzyme, urea is hydrolyzed according to the reaction... 

While the majority of enzyme electrodes fabricated have been rather large devices, there have been some recent reports concerning the development of miniaturized and even microsensors. For example, MeyerhoflF (M5) prepared an essentially disposable urea sensor (tip diameter 3 mm) by immobilizing urease at the surface of a new type of polymer-membrane electrode-based ammonia sensor (see Fig. 4). Alexander and Joseph (Al) have also prepared a new miniature urea sensor by immobilizing urease at the surface of pH-sensitive antimony wire. Similarly, lannello and Ycynych (II) immobilized urease on a pH-sensitive iridium dioxide electrode. In these latter investigations, ammonia liberated from the enzyme-catalyzed reaction alters the pH in the thin film of enzyme adjacent to the pH-sensitive wire. 

Hamann et al. (1988) developed a urea sensor based on a pH glass electrode (Forschungsinstitut Meinsberg, GDR) and urease from soy beans with a specific activity of 2 U/mg. The enzyme was immobilized by dipping the wetted sensor tip first into crystalline urease and then into a solution of methylene chloride containing 21-25 mg/ml of cellulose triacetate. After evaporation of the solvent an amount of enzyme corresponding to 3 U remained at the sensor. The layer thickness and the diffusion coefficient of urea in the layer were dermined to be 30-50 pm... 

One of the primary applications of entrapment immobilization has been to prepare enzyme-electrode-based biosensors [27], and one of the first functional enzyme electrodes utilized urease entrapped in an acrylamide film to detect urea using an ammonium ion selective electrode [28]. Highly hydrophobic bilayer lipid membranes and liposomes have also been used to entrap highly labile biomolecules (see chapter 9). Such films and layers are, however, inherently unstable themselves and are useful primarily as research tools. 

A second application of immobilized enzymes involves enzyme electrodes. An illustrative example of kinetic fundamentals is provided by the urease electrode,which uses a urease membrane to cleave urea into bicarbonate and ammonium ion, coupled to an ammonium ion specific pontentiometric electrode. As with the glucose oxldase/catalase example above, the homogeneous phase kinetics can be applied to describe the response of the immobilized urease ... 

Immobihzation of urease onto an IME device can produce an impedimetric enzyme biosensor for determination of urea [4]. The enzyme urease can be immobilized by a similar covalent tethering strategy to the interdigit surface via 3-aminopropyltrimethoxysilane monolayer and sulfo-NHS and EDC as cross-linkers. This creates an enzyme layer which is very thin relative to the electrode dimensions. The iimnobihzed urease catalyzes the hydrolysis of neutral urea, leading to the formation of ammonium, bicarbonate, and hydroxide ions ... 

Guilbault and Montalvo were the first, in 1969, to detail a potentiometric enzyme electrode. They described a urea biosensor based on urease immobilized at an ammonium-selective liquid membrane electrode. Since then, over hundreds of different applications have appeared in the literature, due to the significant development of ion-selective electrodes (ISEs) observed during the last 30 years. The electrodes used to assemble a potentiometric biosensor include glass electrodes for the measurement of pH or monovalent ions, ISEs sensitive to anions or cations, gas electrodes such as the CO2 or the NH3 probes, and metal electrodes able to detect redox species some of these electrodes useful in the construction of potentiometric enzyme electrodes are listed in Table 1. 

The indirect method was applied to FIA technique for the simultaneous determination of urea and NH4 in agricultural irrigation waters [204] and natural waters [205]. The final concentrations of NH4 were determined by spectrophotometric and fluorimetric methods, respectively. The continuous-flow technique was proposed for the determination of urea in river and lake waters by Kara et al. [191] in the concentration range of 0.4-8.0 jxmol N L. This automated procedure included the removal of the NH4 initially present in the samples, the decomposition of urea by means of an immobilized urease enzyme reactor, and the final determination of NH4 by a gas-sensing membrane electrode detector system. 

Hara H., Kitagawa T., and Okabe Y. 1993. Continuous-flow determination of low concentrations of urea in natural water using an immobilized urease enzyme reactor and an ammonia gas-sensing membrane electrode detector system. Analyst 118 1317-1320. 

Other, practically useful, enzyme electrodes include those based on urease and penicillinase. The penicillin electrode utilizes a pH electrode with immobilized penicillinase. It is widely used to monitor the penidllin content of fermentation reaction mixtures ... 

Enzymes are substances that react very selectively with a substrate in a very specific reaction. Their immobilization on a membrane which is then placed over an electrode in a solution together with the substrate to be determined leads to reaction products that can be detected at the electrode covered by the membrane. An example is the degradation of urea by urease with an internal sensor element (i.e. ion-selective electrode) sensitive to ammonium ion ... 

The first electrode for urea was prepared by immobilizing urease in a poly-acrylcimide gel on nylon or Dacron nets. The nets were placed onto a Beckman electrode (NH J selective) (59). In a later development, the electrode was improved by covering the enzyme gel layer with a cellophane membrane to prevent leaching of urease into the solution (60). The urease electrode could be used for 21 days with no loss of activity. 

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