Urease 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. 

Jackbean urease was immobilized on kaolinite and montmorillonite [98]. The amounts of urease required for maximum immobilization were 70 and 90 mg g 1 of kaolinite and montmorillonite, respectively. The Km values of immobilized urease (25.1-60.8 mM) were of the same order of magnitude as that of free urease (29.4 mM) but one order of magnitude higher than those of soil urease (1.77-2.90 mM). Immobilization of urease on clay surfaces leads to increases in the kinetic constants. 

Fig. 15.4 (A) Urease adsorption isotherms for (a) Zn2AI-CI, (b) Zn3AI-CI, (c) Zn4AI-CI (B) amount of immobilized urease in Zn3-AI by the coprecipitation method.

Figure 3.21 — (A) Integrated FET with two hydrogen ion-sensitive FET elements. (B) Structure of enzyme-modified FET sensor S plastic card FET enzyme-modified FET chip lUM, immobilized urease membrane. (C) Flowthrough cell Bl fixed sensor cell block B2 movable sensor cell block SC flowthrough cell EC electrical connector RP silicone rubber sheet AMP amplifier. (Reproduced from [151] with permission of Elsevier Science Publishers).

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. 

Huang, T.C. and Chen, D.H., Kinetic studies on urea hydrolysis by immobilized urease in a batch squeezer and flow reactor, Biotechnol. Bioeng., (1992) 40,10,1203-09. Storey, K.B., Duncan, J.A., and Chakrabarti, A., Immobilization of amyloglucosidase using two forms of polyurethane polymer, Appl. Biochem. Biotechnol., (1990) 23, 3, 221-36. 

Urease (EC 3.5.1.5 Type IX, Sigma-Aldrich from Jack Beans) was used throughout the experiments. Before immobilizing urease onto the microreactor systems, the enzyme was evaluated for activity in the chosen buffer system (Tris[hydroxymethyl]aminomethane [THAM]). Free enzyme tests of the urease showed an approximate activity of 44,800 U/g of solid. 

Continuous-flow microreactors were successfully fabricated by etching channels in silicon and immobilizing urease onto channel surfaces by a layer-by-layer self-assembly technique. Preliminary results show urea conversion. The potential advantages of this surface-coating technique in microreactors warrant continued investigation. 

Figure 2 shows the results obtained when this polymer, containing dispersed hydrocortisone, is surrounded by a hydrogel containing immobilized urease and release of hydrocortisone is studied as a function of external urea concentration (2). Although this system has very little therapeutic relevance, it did establish the feasibility of this concept and served as the basis for the development of a more useful system. 

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. 

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. 

Another less-utilized transduction mechanism for biosensors involves the acoustoelectric effect. In principle, any biochemical process that produces a change in the electrical properties of the solution, can be monitored by observing changes in the frequency and/or attenuation of the device if its surface is not metallized. For example, a SH-SAW device has been reported for the detection of pH changes associated with the enzyme-catalyzed hydrolysis of urea [235]. Using an immobilized urease membrane on the sensor surface, it was anticipated that urea concentrations as small as 3 /u.M could be reliably detected. 

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,... 

The feedback-regulated drug delivery concept has been applied to the development of a bioerosion-regulated CrDDS by Heller and Trescony. " This CrDDS consists of a drug-dispersed bioerodible matrix fabricated from poly(vinyl methyl ether) half-ester, which was coated with a layer of immobilized urease (Fig. 26). In a solution with near neutral pH, the polymer only erodes very slowly. In the presence of urea, urease at... 

Fig. 26 Cross-sectional view of a bioerosion-regulated hydrocortisone delivery system, a feedback-regulated drug delivery system, showing the drug-dispersed monolithic bioerodible polymer matrix with surface-immobilized ureases. The mechanism of release and time course for the urea-activated release of hydrocortisone are also shown. (From Ref > 1)...

The conditions under which chemical indicator reactions are used often necessitates the use of postcolumn addition, however. Figure 4.11 shows an experimental setup for urea assays using an immobilized urease reactor.30 The postcolumn addition of sodium hydroxide allows the NH produced by the reactor to be detected as NH3 at an ammonia gas-sensing electrode placed in a flow cell. 

Figure 4.11. Enzyme reactor system for urea based on immobilized urease and potentio-metric detection.30...

Figure 12 shows a possible set-up for hemodialysis monitoring. Patients blood is pumped through a dialysis cell, and low molecular weight substances including urea are removed by a semipermeable membrane (cut off 10 kD) and dialysis buffer. The urea enriched dialysate passes through an injection valve and enters a waste container. Due to switching the valve, a defined sample volume is pumped to the ET. Here, enzymatic conversion takes place via immobilized urease and provides information about the current urea concentration. Thus, the hemodialysis effect is automatically monitored via urea analysis and makes an individual treatment possible. 

Recently, the ET was presented as an on-line monitor for biotransformation processes (Lammers and Scheper, 1996). Here, three different enzyme-catalyzed processes of industrial interest were investigated. In the first example, the enzymatic production of L-ornithine was monitored via urea analysis. In this process, arginase hydrolyzes L-arginine with the release of urea and L-ornithine. Immobilized urease was set into the ET in order to monitor urea release, and on-line data informed us of the progress of production. Moreover, a computer-controlled set-up (Fig. 15) allowed us to remove the product at a nominal value and to add fresh substrate (Fig. 16). 

Bataillard et al. (1993) described the integration of a series of thermocouples (thermopiles) on a silicon chip in order to increase the voltage output. The chip consists of an array of p-type silicon/aluminium thermocouples, connected in series and integrated in a n-type silicon epoxy layer grown on the silicon wafer. The overall size of the chip is only 5x5 mm. Several enzymes are immobilized on the array, and the chips are inserted into a FIA system. Glucose is monitored with coimmobilized glucose oxidase and catalase ranging from 2 to 100 mmol/1, urea (1-1000 mmol/1) with immobilized urease and penicillin (1-1000 mmol/1) with immobilized //-lactamase. 

Mattiasson B, Rieke E, Munnecke D, Mosbach K (1979) Enzyme analysis of organophosphate insecticides using an enzyme thermistor. J Solid-Phase Biochem 4 263-270 Mattiasson B, Danielsson B, Hermansson C, Mosbach K (1977b) Enzyme thermistor analysis of heavy metal ions with use of immobilized urease. FEBS Lett 85 203-206 Mattiasson B, Danielsson B (1982) Calorimetric analysis of sugars and sugar derivatives with aid of an enzyme thermistor. Carbohydr Res 102 273-283 Mattiasson B, Danielsson B,Winquist F, Nilsson H, Mosbach K (1981) Enzyme thermistor analysis of penicillin in standard solutions and fermentation broth. Appl Environm Microbiol 41(4) pp 903-908... 

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. 

II. lanniello, R. M., and Yacynych, A. M., Urea sensor based on iridium dioxide electrodes with immobilized urease. Anal. Chhn. Acta 146, 249-253 (1983). 

Potentiometric gas sensors for the reaction products, NH3 and CO2, have also been employed. Since these measurements are based on gas diffusion through a hydrophobic membrane, no direct disturbances by sample constituents occur. As early as 1969, Guilbault et al. coupled immobilized urease with a carbon dioxide sensor. Anfalt et al. (1973) applied an ammonia gas sensitive electrode to urea assay. A major drawback of these sensors is their long response time which is due to the slow diffusion of the gases. Since it takes several additional minutes to reach a new baseline after each measurement, only a few samples can be processed per hour. Guilbault et al. (1985) therefore tried an NH3 electrode, the interned buffer of which was exchanged after each measurement (double injection electrode). This approach led to a substantial decrease of the washing time. 

GC mode SECM experiments have been used to detect localized activity of a variety of enzymes (see Table 2). Since microelectrodes are used in the life sciences to detect neurotransmitters (36), metal cations (37), and free radicals (38), a wide variety of probes may be combined with SECM positioning technology to obtain spatially resolved information. The GC mode experiments may be carried out with potentiometric or amperometric tips depending on the species to be sensed. The tip must be chosen to determine a product or reactant consumed by the enzyme reaction, e.g., H+ for immobilized urease, H202 for immobilized glucose oxidase, or 4-aminophen-olate for alkaline phosphatase. A list of enzymes and the tips employed is given in Table 2. 

Kokufuta E, Matsukawa S. Construction of a biochemo-mechanical system using inhomogeneous polyelectrolyte gels with immobilized urease. Ber Bun-senges Phys Chem 1996 100 1073-1078. 

First potentiometric biosensor acrylamide-immobilized urease on an ammonia electrode to detect urea [9]... 

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