Immobilization immobilized acid-urease

Satoh I, Akahane M and Matsumoto K 1991 Analytical applications of immobilized acid urease for urea in flow streams Sensors Actuators B 5 241 Danielsson B 1995 Handbook of Analytical Sciences (London Academic) at press Xie B, Hedberg (Harbom) U, Mecklenburg M and Danielsson B 1993 Fast determination of whole blood glucose with a calorimetric micro-biosensor Sensors Actuators B 15-16 141-4... 

In addition to nucleic acids, enzymes have also been incorporated into conducting a polymer matrix for biosensor applications. As a transducer, a CP can convert the chemical response into an electric current. To enhance the sensitivity and the response time, fabrication of CPs/enzyme nanocomposites with large surface area is a meaningful objective. Syu and Chang demonstrated the immobilization of urease onto PPy nanotubes over carbon paper substrate by a physical entrapment approach [124]. The composite electrodes exhibited a detection sensitivity for the determination of urea of 53.74 mVdecade and a detection limit on the urea concentration of 1.0 pM. Furthermore, the composite electrode shows rapid response, storage stability and reusability. Lipase can also be covalently immobilized... 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

Figure 5.19 — Flow-through biochemical sensor based on the twofold immobilization of the catalyst (urease) and reagent (an acid-base azo dye) in the sensing microzone for the determination of urea in kidney dialysate. (A) Sensing microzone held in a microcircuit. (B) Valveless flow injection manifold. P pumps T timer S sample W waste. For details, see text. (Reproduced from [57] 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. 

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). 

Recently, a renewable potentiometric urease inhibition biosensor based on self-assembled gold nanoparticles has been developed by Yunhui et al. for the determination of mercury ions [43]. The advantages of self-assembled immobilization are low detection limit (0.05 iM), fast response, and relatively easy regeneration of the biosensors. The assembled gold nanoparticles and inactivated enzyme layers denatured by Hg2+ can be rinsed out via a saline solution with acid and alkali successively. 

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,... 

Electrodes based on the immobilization of enzymes at the electrode surface have been reported for a few pharmaceutical applications. Seegopaul and Rechnitz developed a CO2 electrode that responds to methotrexate due to the inhibition of dihydrofolate reductase by the analyte. Additional applications have included the detection of penicillin consumption by penicillinase as well as urea by urease with a pH electrode and analysis of L-ascorbic acid by ascorbate oxidase.f ... 

Diagnosis of renal problems, xanthinuria, and toxemia of pregnancy via determination of the ratio of hypoxanthine to xanthine in plasma is facilitated by the use of biosensors. Xanthine oxidase immobilized on aminopropyl-CPG (controlled pore glass) activated with glutaraldehyde oxidizes hypoxanthine first to xanthine and then to uric acid. Use of an IMER with biosensors for hypoxanthine, xanthine, and uric acid provides the necessary data. Pre- or postcolumn enzymatic reactions catalyzed by creatinine deiminase, urease, alkaline phosphatase, ATPase, inorganic pyrophosphatase, or arylsufatase facilitate analysis of uremic toxins (simultaneous detection of electrolytes, serum urea, uric acid, creatinine, and methylguanidine). 

Yeast invertase,30 32 33 acid phosphatase,29 32 35 37 39 urease,29 /3-glucosi-dase,29 dCMP-amino hydrolase31 and malic enzyme34 36 have been immobilized in gel form on both flat and capillary membranes. Cellulosic and polyamide polymers have been used as supporting membrane matrices. In all instances, immobilized enzymes behave in a manner almost identical to their behavior in homogenous solution, independent of the nature of the polymer. Neither allosteric nor pseudo-allosteric enzymes, proteins whose kinetic behavior is affected by the presence of particular compounds in the reaction environment (ligands), show different kinetic behavior, as they do when subjected to less gentle immobilization procedures.31 34 36... 

Many other similar applications have been reported such as the electrochemical determination of electroinactive cationic medicines,313 determination of urea,314 uric acid,315 and application to glucose biosensors to decrease interference of ascorbate, urate, and acetaminophen.316 Enzyme immobilized membranes are also sensing membranes, e.g. urea responsive membranes, poly(carboxylic acid) membranes in which urease is immobilized,317 fructose responsive membranes, and polyion complex membranes in which fructose dehydrogenase is immobilized.318 Such applications will expand further in the future and contribute to human life. 

K. Ishihana, N. Muramoto, H. Fujii and I. Shinohara, Preparation and permeability of urea-responsive polymer membrane consisting of immobilized urease and poly-(aromatic carboxylic acid), J. Polym. Sci., Polym. Lett. Ed., 1985, 23, 531-535. 

The next group of multimembrane systems comprises membranes sensitized biologically using immobilized enzymes or microorganisms. Species that are directly sensed by an ISE are produced in the enzymatic reaction of the analyte. Examples of such sensors are those used for determination of urea in milk, based on immobilized urease and measurement of a pH change. An example of the application of bacteria strains is the use of immobilized recombinant Escherichia coli coupled with a pH electrode. Such electrodes have been used for determination of cephalosporins. When this bacterial strain is coupled with a CO2 gas sensor, glutamic acid determination can be carried out. 

Fig. 2. Immobilized enzymes. Schematic representation of an artificial cell, containing urease and albumin-coated active charcoal as an absorbent for uric acid, ammonia and creatinine. A 10 ml suspension of these 20 pm-diameter urease capsules corresponds to a surface area of 20,000 cm, which Is larger than that of the conventional artificial kidney.

Na+, NHJ (urea), K+ Dilution, treatment with immobilized urease reactor Ion exchange 5 itiM nitric acid Indirect conductivity 26... 

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