Preparation of Enzyme Electrodes

urease is dissolved in 1 ml phosphate buffer (pH 6.8), 1 ml of a 17.5% bovine albumin solution and 0.07 ml of a 25% glutaraldehyde solution are added, and the mixture is stirred for 2 minutes. The cation-selective electrode is washed with water, care- 

Substrate (substance to be determined) biologically active compound (activity to be determined) Product (indicated material) optimal Buffer and pH-value Indicator- electrode Working range Literature 

Acetylcholine (indirect organic phosphorous pesticides in the ng/ml-range) Acetylcholinesterase Choline, acetic acid physiol, salt soln pH 7.2 Acetylcholine liquid membrane IO- - lO- M [192, 193, 194, 195] 

5 -Adenosinemono-phosphate (5-AMP) AMP-Deaminase 5-Inosinmono-phosphate, NH3 0.05 M Tris pH 7.5 NH3 Gas-Sensor 10- - lO- M [215] 

Albumin (Human) Anti-Human-Serum-Albumin (Immuno-reaction) unreacted Antibodies 0.1 M NaOH Ag S 0.5-30 Mg/ml (1961 

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A second route for the preparation of enzyme electrodes starting with poly[3-(2-hydroxyethyl)thiophene] is the bromocyane method - also well known from protein chemistry. PT with pendant hydroxy groups is treated with bromocyane forming the reactive cyanate (see also Scheme 3, route b) which can be taken for the immobilization of enzymes, e.g. alcohol dehydrogenase [228, 230]. 

Kobos R.K., Eveleigh J.W., Stepler M.L., Haley B.J. and Papa S.L. (1988) Fluorocarbon based immobilization method for preparation of enzyme electrodes. Anal. Chem., 60, 1996-1998. 

The techniques developed in enzyme immobilization have facilitated the development of enzyme electrodes and of novel enzyme -based, automated, analytical methods (l6,17,l8). Enzyme electrodes have resulted from the combination of an enzyme membrane and an ion-selective electrode they were used successfully to assay directly appropriate substrates. Enzyme columns or enzyme tubes, prepared in a conventional manner, were used as a specific auxiliary component in the indirect assay of substrates in many of the novel automated analytical procedures. 

Figure 4-1 i Illustration of enzyme electrode prepared using oxidase enzyme immobilized at the surface of amperometric P02 sensor. Increase in substrate concentration S reduces the amount of oxygen present at the surface of the sensor. 

Also, third-generation biosensors for superoxide anion (O ) have been developed based on superoxide dismutase (SOD) immobilised by thin silica-PVA sol-gel film on a gold electrode surface [633]. The preparation of SOD electrode is easy and simple. The uniform porous structure of the silica-PVA sol-gel matrix results in a fast response rate of immobilised SOD and is very efficient for stabilising the enzyme activity. 

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. 

Such a simultaneous incorporation of CNT and glucose oxidase imparted bio-catalytic and electrocatalytic properties onto amperometric transducers and represented a facile and effective route for preparing an enzyme electrode. 

Many enzyme immobilization techniques developed in connection with the preparation of heterogeneous biocatalysts have been applied for the construction of enzyme electrodes as well as for other types of biosensors. The immobilization of enzymes or other biocomponents on different membranes is most frequently realized by crosslinking agents, first of all by glutaraldehyde, and with the addition of bovine serum albumin [169] or other proteins, with l,8-diamino-4-aminomethyloc-tane, [170], etc. 

Enzymes have also been immobilized on collagen membrane after its stepwise modification to esters, hydrazides and azides [171]. Another method of enzyme electrode preparation consists of enzyme immobilization on polyacryl acid modified with p-nitroaniline and by a subsequent reduction of N02-groups with titanous chloride and following diazota-tion of resulting aromatic amines [150]. An enzyme electrode has also been prepared by the direct immobilization of an enzyme on the surface of a Pt-electrode which was formerly modified first with 3-aminopropyl triethoxysilane and secondly with glutaraldehyde and bovine serum albumin [172]. Enzymes can also be immobilized on p-benzoquinone-carbon paste[173] or on the graphite electrode after its activation with cyanuric chloride [174]. In a similar way an enzyme electrode has been prepared by using iridium diiodide electrode as a support [175]. 

In our method [176] of enzyme electrode preparation the enzymes are immobilized on a partially hydrolyzed nylon net via Ugi s four-component reaction [177]. This reaction has been used in two different ways. In the first method the nylon net was partially hydrolyzed with hydrochloric acid and the enzymes were covalently bound on this activated net by the reaction with glutaraldehyde and cyclohexyl isocyanide. The existence of four amide bonds is the result of this reaction. The immobilization of enzymes by means of these amide bonds is more effective than via Shiff s bases which are produced in the most common method for enzyme electrode preparation. When glutaraldehyde alone was used for the immobilization, i.e., without cyclohexyl isocyanide, the resulting enzyme electrode showed an approximately fivefold lower activity. 

The second method of enzyme electrode preparation is based on the immobilization of glycoenzymes via their glycosidic component [178]. Here the carbohydrate moiety of glycoenzymes is activated by a periodate oxidation cleavage of vicinal diols of the carbohydrate units. The... 

FIGURE 6-2 Steps in the preparation of an ainperometric enzyme electrode with simple enzyme immohilization hy trapping between an inner cellulose acetate and outer collagen membrane, cast on the electrode body. (Reproduced with permission from reference 1.)... 

Figure 17.4 Cartoon representation of strategies for studying and exploiting enzymes on electrodes that have been used in electrocatalysis for fuel cells, (a) Attachment or physisorption of an enzyme on an electrode such that redox centers in the protein are in direct electronic contact with the surface, (b) Specific attachment of an enzyme to an electrode modified with a substrate, cofactor, or analog that contacts the protein close to a redox center. Examples include attachment of the modifier via a conductive linker, (c) Entrapment of an enzyme within a polymer containing redox mediator molecules that transfer electrons to/from centers in the protein, (d) Attachment of an enzyme onto carbon nanotubes prepared on an electrode, giving a large surface area conducting network with direct electron transfer to each enzyme molecule.

Ikariyama [2] described a unique method for the preparation of a glucose oxidase (GOD) electrode in their work. The method is based on two electrochemical processes, i.e. electrochemical adsorption of GOD molecules and electrochemical growth of porous electrode. GOD immobilized in the growing matrix of platinum black particles is employed for the microfabrication of the enzyme electrode. It demonstrated high performance with high sensitivity and fast responsiveness. 

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. 

For farther improvement of hydrogen enzyme electrode the commercial carbon filament materials were used as an electrode matrix. Such type of materials are accessible and well characterized, that provides the reproducibility of the results. A procedure for hydrogen enzyme electrode preparation included the pretreatment of electrode support with sulfuric acid followed by enzyme immobilization. This procedure is a critical step, since initially carbon filament material is completely hydrophobic [9]. 

The polypyrrole molecular interface has been electrochemically synthesized between the self-assembled protein molecules and the electrode surface for facilitating the enzyme with electron transfer to the electrode. Figure 9 illustrates the schematic procedure of the electrochemical preparation of the polypyrrole molecular interface. The electrode-bound protein monolayer is transferred in an electrolyte solution containing pyrrole. The electrode potential is controlled at a potential with a potentiostat to initiate the oxidative polymerization of pyrrole. The electrochemical polymerization should be interrupted before the protein monolayer is fully covered by the polypyrrole layer. A postulated electron transfer through the polypyrrole molecular interface is schematically presented in Fig. 10. 

In contrast to the molecular wire of molecular interface, electron mediators are covalently bound to a redox enzyme in such a manner as an electron tunneling pathway is formed within the enzyme molecule. Therefore, enzyme-bound mediators work as molecular interface between an enzyme and an electrode. Degani et al. proposed the intramolecular electron pathway of ferrocene molecules which were covalently bound to glucose oxidase [ 4 ]. However, few fabrication methods have been developed to form a monolayer of mediator-modified enzymes on the electrode surface. We have succeeded in development of a novel preparation of the electron transfer system of mediator-modified enzyme by self-assembly in a porous gold-black electrode as schematically shown in Fig.12 [14]. 

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