Urease, selectivity

Directions are provided for constructing and characterizing an ammonium ion-selective electrode. The electrode is then modified to respond to urea by adding a few milligrams of urease and covering with a section of dialysis membrane. Directions for determining urea in serum also are provided. 

Following this procedure urea can be determined with a linear calibration graph from 0.143 p.g-ml To 1.43 p.g-ml and a detection limit of 0.04 p.g-ml based on 3o criterion. Results show precision, as well as a satisfactory analytical recovery. The selectivity of the kinetic method itself is improved due to the great specificity that urease has for urea. There were no significant interferences in urea determination among the various substances tested. Method was applied for the determination of urea in semm. 

Such electrodes make use of an enzyme to convert the substance to be determined into an ionic product which can itself be detected by a known ion-selective electrode. A typical example is the urea electrode, in which the enzyme urease is employed to hydrolyse urea ... 

FIGURE 6-11 Urea electrode, based on the immobilization of urease onto an ammonium ion-selective electrode. 

An enzyme deposited on the LAPS surface allows one to observe the spatial distribution of a specific substrate. In a urea-selective sensor urease was immobilized on a pH-selective LAPS [75],... 

Such a generalization is useful in that it provides clues to the. nature of the participation of metal ions in a reaction, when the order of catalytic effect of the various metals is known. As a first example of such an approach, let us consider the case in which the order of inhibition correlates with the order of complex stability such an order is frequently observed—e.g., urease (39). It may be concluded, in such an instance, that the metals do not activate the reaction, but inhibit it only. As a second example, if metal ions activate a reaction, but they do so in inverse order from that of complex stability, it follows that inhibition competes effectively with activation. Such an order is observed with enolase (Mg+2 >Zn+2>Mn+2>Fe+2>Co+2>Ni+2) (35, 53) presumably the inhibitory effect of the more strongly binding metals is responsible for the selection of the less active Mg+2 in the natural enzyme. Probably such effects are not as important in aconitase, making it possible for a stronger chelating metal to activate that enzyme. 

In principle, the specificity of an electrode is obtained when a membrane can selectively recognise the species to be measured in a sample solution. Biosensors have be developed in which the measuring electrode can detect a particular compound formed through a biochemical enzymatic reaction. For example, if urease is fixed to a membrane sensitive to the ammonium ion, this membrane will be able to detect urea since urease is an enzyme that will decompose urea to form ammonium ions. However, such electrodes do not presently exist in a commercial form. 

Phase transition in gels in response to biochemical reactions [27,28]. Polymer gels were synthesized in which an enzyme (urease) or a biologically active protein (lectin) was immobilized. The volume phase transitions were observed in such gels when biochemical reactions took place. Such mechano-biochemical gels will be used in devices such as, sensors, selective absorbers, and biochemically controlled drug release. 

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 ... 

The electrode is an ammonium ion-selective electrode surrounded by a gel impregnated with the enzyme urease [Fig. 6.13 (34)]. The generated ammo-... 

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 ... 

Electrodes with enzymes (e.g., urease) incorporated into a gel membrane. They are selective for biochemical compounds such as urea, glucose, L-amino acids, and penicillin (see - biosensors). 

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. 

Wire that senses pH changes such as antimony metal can be coated with urease (65) and used to determine urea in pure blood (66). Covering the antimony metal with a gas perm-selective membrane (67) improves the selectivity. The microsensors respond from 0.1 to 10 mM urea in 30-45 s. This ammonia sensor has a faster baseline recovery than commercial gas membrane electrodes. 

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... 

A new development in the field of potentiometric enzyme sensors came in the 1980s from the work of Caras and Janata (72). They describe a penicillin-responsive device which consists of a pH-sensitive, ion-selective field effect transistor (ISFET) and an enzyme-immobilized ISFET (ENFET). Determining urea with ISFETs covered with immobilized urease is also possible (73). Current research is focused on the construction and characterization of ENFETs (27,73). Although ISFETs have several interesting features, the need to compensate for variations in the pH and buffering capacity of the sample is a serious hurdle for the rapid development of ENFETs. For detailed information on the principles and applications of ENFETs, the reader is referred to several recent reviews (27, 74) and Chapter 8. 

Ideally, the sensor used to sense the biocatalyzed reaction should not react with other substances in the sample. This requirement is not always met using either potentiometric or amperometric methods. For example, immobilized urease electrodes operating with a cation glass sensor measuring the NHj are inadequate for blood and urine assays because they also respond to Na+ and K+ (59, 60). However, a glass electrode sensor (165) or, better, a solid antibiotic nonactin electrode (61) gives more selective response. The latter has a selectivity of NHt/K+ of 6.5 and NHt/Na+ of 0.075. 

Highly selective L-arginine biosensors are described where the ammonia liberated in the reaction sequence catalyzed by the enzymes arginase-urease is monitored potentiometrically. The probes exhibit linear responses in the range of arginine concentrations of 0.1 mM to 0.01 M (140) and 30 pM to 3 mM (287). 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

Enzyme sensors can measure analytes that are the substrates of enzymatic reactions. Thermometric sensors can measure the heat produced by the enzyme reaction [31], while optical or electrochemical transducers measure a product produced or cofactor consumed in the reaction. For example, several urea sensors are based on the hydrolysis of urea by urease producing ammonia, which can be detected by an ammonium ion-selective ISE or ISFET [48] or a conductometric device [49]. Amperometric enzyme sensors are based on the measurement of an electroactive product or cofactor [50] an example is the glucose oxidase-based sensor for glucose, the most commercially successful biosensor. Enzymes are incorporated in amperometric sensors in functionalised monolayers [51], entrapped in polymers [52], carbon pastes [53] or zeolites [54]. Other catalytic biological systems such as micro-organisms, abzymes, organelles and tissue slices have also been combined with electrochemical transducers. 

The use of enzyme labels in place of radioisotopes for the measurement of antigens, antibodies, and haptens has stimulated the new and expanding field of enzyme immunoassay (EIA). This technique has been the focus of several recent reviews, - and its merits compared to radioimmunoassay (RIA) have been discussed. In many cases, EIA can match RIA in terms of sensitivity and selectivity, yet has advantages of speed, convenience, and reduced cost. EIA sensitivity and simplicity is, however, dependent on the choice of enzyme label. It is the purpose of this work to introduce urease as a new enzyme label and to demonstrate the... 

Figure 6 shows a typical calibration curve obtained for the inhibition of binding of a urease-cAMP conjugate to anti-cAMP antibody as determined by the ammonia electrode. One hundred percent of activity refers to blank tubes, which had rates of 11-12 mV/min in the absence of cAMP. Selectivity of the assay over structurally similar cGMP is also shown in Fig. 6. It takes approximately 1000 times more cGMP than...

An electrode for measuring urea has been described (Gll), consisting of a thin film of urease, immobilized in acrylamide gel, on the surface of a glass electrode responsive to NH. Conditions are carefully selected to ensure stability of the enzyme, and the potential developed is proportional to the logarithm of the urea concentration. Blood glucose and lactate have been determined with a membrane electrode in which the enzyme (glucose oxidase or lactate dehydrogenase) is trapped in a porous or jellied layer at the membrane surface (W20). 

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