Urease electrode

Tor [7] developed a new method for the preparation of thin, uniform, self-mounted enzyme membrane, directly coating the surface of glass pH electrodes. The enzyme was dissolved in a solution containing synthetic prepolymers. The electrode was dipped in the solution, dried, and drained carefully. The backbone polymer was then cross-linked under controlled conditions to generate a thin enzyme membrane. The method was demonstrated and characterized by the determination of acetylcholine by an acetylcholine esterase electrode, urea by a urease electrode, and penicillin G by a penicillinase electrode. Linear response in a wide range of substrate concentrations and high storage and operational stability were recorded for all the enzymes tested. 

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. 

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. 

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,... 

Fig. 67. Influence of sample pH on the measuring signal of a urease electrode 30 s after addition of 13.3 mmol/1 urea to the measuring solution.

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]. 

Figure 10.10 Potentiometric response of PVCZ/urease electrodes with different urea...

Rgure 1 Calibration of a potentiometric urease electrode for urea. Curves are related to different loadings. 

These fall into two classes, namely those where other substrates are present in the sample and those involving base enzyme activation or inhibition. With some enzymes, such as urease, the only substrate that reacts at a reasonable rate is urea hence, a urease electrode is almost specific for urea. Likewise, uricase is almost specific for uric acid. 

Fig. 41 Cyclic voltammograms of PANI/Mn02/urease electrode...

Interference at the transducer — Whatever the nature of the transducer, it can never be completely specific to one product. The transducer is much more likely to be selective, which means that it is also sensitive to other substances. The urease electrode uses an electrode that is sensitive to monovalent cations to detect NH4+[99]. This electrode is also sensitive to Li, Na+, and H, hence the idea of using a pCX>2 gas diffusion electrode [30] to eliminate interference from ionic compounds. 

Figure 4.18 Etayme electrodes that are sensitive to reversible inhibitors, (a) the AChE electrode for nicotine determination, and (b) the urease electrode for the determination of fluoride ions.

Figure 4.19 Influence of the substrate (urea) concentration on the calibration curve of an enzyme (urease) electrode designed for the determination of uncompetitive inhibitors (NaF).

Figure 4.21 Effect of incubation time, T, on the response of a urease electrode that is sensitive to fluoride ions. The dotted line represents the response of the enzyme sensor with respect to its substrate without incubation, before and after addition of fluoride ions.

Figure 4.23 Determination of several metals present in the same solution ([Co J = 10 M, [Ag ] = /0- M, [H ] = 5x lOr M) using a urease electrode which is iitiiibited and reactivated in several steps by the selective complexing agents KI, (NH4)2S and EDTA.

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