Urease, enzyme electrode

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

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. 

Hydrolase enzymes catalyze the hydrolysis of a substrate and are most commonly coupled with potentiometericela trodes The pioneering work in this field focussed on de loping an enzyme electrode for the determination of urea. Urease catalyzes the hydrolysis of urea to ammonium and bicarbonate ions according to the reaction detailed below. 

Enzyme electrodes. Guilbault52 was the first to introduce enzyme electrodes. The bulb of a glass electrode was covered with a homogeneous enzyme-containing gel-like layer (e.g., urease in polyacrylamide) and the layer was protected with nylon gauze or Cellophane foil when placed in a substrate solution (e.g., urea) an enzymatic conversion took place via diffusion of substrate into the enzymatic layer. 

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. 

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 ... 

Enzyme electrodes that use urease attached with glutaraldehyde and a cation-selective glass electrode sensor have a range of 10 pM to 0.1 M (68). They have... 

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. 

Such interference falls into two classes competitive substrates and substances that either aaivate or inhibit the enzyme. With some enzymes, such as urease, the only substrate that reacts at reasonable rate is urease hence, the urease-coated electrode is specific for use (59, 165). Likewise, uricase acts almost specifically on uric acid (167), and aspartase on aspartic acid (8, 168). Others, such as penicillinase and amino oxidase, are less specific (63,169,170). Alcohol oxidase responds to methanol, ethanol, and allyl alcohol (171, 172). Hence, in using electrodes of these enzymes, the analyte must be separated if two or more are present (172). Assaying L-amino acids by using either the decarboxylative or the deaminating enzymes, each of which acts specifically on a different amino... 

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. 

Garry A. Rechnitz together with S. Katz introduced one of the first papers in the field of biosensors with the direct potenfiometric determination of urea after urease hydrolysis. At that fime the term biosensor had not yet been coined. Thus, these types of devices were called enzyme electrodes or biocatalyfic membrane electrodes [100[. 

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. 

Owing to differences in the Kyi values and the layer thickness, the transient from kinetic to diffusion control of different enzyme electrodes takes place at rather different enzyme activities. Gelatin-entrapped enzymes exhibit transient values of 0.17 U/cm2 (uricase,iifM= 17 pmol/1), 16 U/cm2 (urease, Kyi = 2 mmol/1) and 1.0 U/cm2 (lactate monooxygenase, Km = 7.2 mmol/1). 

There are over 300 enzymes available commercially, and many of these can be used in one way or another for analytical purposes. One of methods of use involves the determination of an analyte or substrate by means of the enzyme which reacts specifically with that substrate. Examples are 1. Glucose and glucose oxidase, O2 released, measurement by O2-ISE 2. Urea and urease, NHs and CO2 released, measurement by an NH4 - or CO2-ISE 3. Pectin and pectin-esterase, titration of H+ released. In chnical work, the opposing approach is often used, and the amount of an enzyme determined by adding the proper substrate to a solution of the enzyme and measuring with an ion-selective electrode a product of the reaction. Early work in this area, and in that of immobihsed enzyme electrodes, was carried out by Katz and Rechnitz [15,16], Guilbault and his co-workers [17] and Guilhault and Montalvo [18]. 

The first potentiometric enzyme electrode, aimed at monitoring urea, was developed by Guilbault and Montalvo [11]. In this case, urease was entrapped... 

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 ... 

Possible applications of enzyme electrodes include the determination of urea in blood (Equation (20)) and for which the optimum pH is about 7 at which 99.7 per cent of the ammonia product exists as NH4 cation, the only form among the species generated that elicits a potential response from the Beckman cation-selective glass electrode. Any sodium or potassium ion interference introduced with the enzyme (or substrate) can easily be detected because of the large positive potential reading that it causes. In such instances Dowex 50 cation exchanger may be used to remove the interferences beforehand [372]. Average differences between the urea values in blood and urine as determined by the urea-urease electrode and a spectrophotometric technique were 2.8 and 2.3 per cent m/m, respectively [373]. 

The potentiometric sensing of urea was also demonstrated by entrapping the enzyme urease within a polypyrrole membrane electrosynthesized on a platinum electrode [75]. The bioactive sensing membrane was synthesized by potentiostatic electropolymerization of pyrrole monomer in a solution containing the urease enzyme and a nucleophilic electrolyte such as NaOH, NaCOs, or NaHCOs. This biosensor exhibited a good Nemstian response to urea, with a slope of 31.8 mV/decade over an mea concentration range of 1.0x10 mo]/dm -0.3 mol/dm. ... 

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. 

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 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. 

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