Electrodes Carrying Enzymes

More recently, nanotechnology has faciUtated progress in miniaturizing redox enzyme electrodes and extending their application. In order to achieve contact between the active site of the redox enzyme where electron transfer takes place, usually buried within the protein structure, and the electrode electrical contact, cofactor-functionaUzed nanomaterials have been developed [75]. Diffusible cofactors such as FAD can be used as the relay system for carrying electrons to electrical... 

Enzyme Electrode with Magnetic Beads Carrying the Enzyme Activity... 

Replacing the enzyme component of tm enzyme electrode in a convenient and reproducible way is often desirable. The use of magnetic beads carrying the immobilized enzymes in combination with an electrode placed in a homo-... 

Enzyme-linked electrochemical techniques can be carried out in two basic ways. The first approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. In the second approach, the enzyme is immobilized at the electrode. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of the substrate to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system that is amenable to in vivo analysis. Alternatively, the transport of enzyme product from the enzyme active site to the electrode surface is greatly enhanced when the enzyme is very near the electrode. Enzyme electrodes are an extremely important area of bioelectrochemical analysis, and many reviews are available in the literature. ... 

The sensor mentioned under (iii) uses an ammonium ion selective electrode carrying a PVC nitrobenzene-dibenzo-18-crown-6 gel. The enzyme was immobilized by crosslinking with glutaraldehyde on a Teflon membrane. After addition of urea to the measuring solution the ammonia produced in the enzymatic reaction reaches the internal solution layer (pH 9.7) of the sensor, where it is partially converted to ammonium ion which is transferred into the nitrobenzene phase. As a result, at 370 mV a current flow was observed which was related to urea concentration. The peak current was proportional to the concentration of urea between 1 pmol/1 and 2 mmol/1. The response time of the sensor was about 1 min. 

There are over 300 enzymes available commercially, and many of these can be used in one way or another for analytical purposes. One of methods of use involves the determination of an analyte or substrate by means of the enzyme which reacts specifically with that substrate. Examples are 1. Glucose and glucose oxidase, O2 released, measurement by O2-ISE 2. Urea and urease, NHs and CO2 released, measurement by an NH4 - or CO2-ISE 3. Pectin and pectin-esterase, titration of H+ released. In chnical work, the opposing approach is often used, and the amount of an enzyme determined by adding the proper substrate to a solution of the enzyme and measuring with an ion-selective electrode a product of the reaction. Early work in this area, and in that of immobihsed enzyme electrodes, was carried out by Katz and Rechnitz [15,16], Guilbault and his co-workers [17] and Guilhault and Montalvo [18]. 

A further development of the wall-jet system is to insert a packed-bed electrode upstream of the jet to make a packed-bed wall-jet electrode (PBWJE) [5-7]. The packed bed can be used to generate reactant, to generate a fresh electrode surface or, if the bed has immobilised enzyme, to carry out an enzymatic reaction. The packed-bed wall-jet electrode is illustrated in Fig. 3. 

Viologen-acrylamide copolymers have been used in a similar manner to entrap enzymes and carry out direct reduction of the active site by bound viologen moieties. In these studies the polymer was reduced chemically rather than electrochemically, but there appears no reason in principle why this same chemistry could not be carried out on an electrode. These viologen-acrylamide copolymers have been shown to reduce nitrate reductase (EC 1.9.6.l/ and glutathione reductase (EC 1.6.4.2). 2 >... 

In order to transfer electrons directly between the electrodes and enzymes, an electron relay that transfers redox equivalents (electrons) from the active site of GOD s cofactor to the electrode surface is needed. The choice for an artificial electron relay depends on a molecule s ability to reach the reduced flavin adenine dinucleotide, FADH2 (in close proximity to the GOD active site), undergo fast electron transfer, and then transport electrons to the electrodes as rapidly as possible. Surridge and co-workers have carried out electron-transport rate studies on an enzyme electrode for glucose using interdigited array electrodes [16]. 

Other electrodes electrodes modified by enzymes electrodes modified by current-carrying polymers electrodes modified by phthalocyanines, ferrocene, etc. 

Nature uses an array of enzymes to carry out a large number of organic reactions. In a sense, a redox enzyme is a surface which provides or accepts electrons, much like an electrode surface (Fig. 1), The most striking similarity between the two is that electrons are transferred one at a time in both systems. This has been pointed out by a number of workers, most recently by Guengerich and MacDonald (8) in a study of cytochrome P-450 systems. The extent to which an electrode actually functions as surface will be discussed in the latter part of this paper. The biggest difference between the two systems is that an enzyme surface has a more sophisticated structure which holds substrate molecules in certain ways or conformations so that unique reactions may take place. A second major difference is that an enzyme is part of a system to transfer electrons between a substrate and a secondary redox reagent such as air or peroxide (in oxidations). In an electrochemical system, the electrons go into the circuit. 

Immobilization of polyphenol oxidase (PPO) was achieved by electropolymerization of pyrrole on a previously poly (TATE) coated platinum electrode. The solution consists 0.3 mg ml polyphenol oxidase, 0.6 mg ml supporting electrolyte (sodium dodecyl sulfate) and O.OIM pyrrole and 10 ml citrate buffer (pH=6.5). Immobilization was performed in a typical three electrode cell, consisting of Pt working and counter electrodes and a Ag/Ag reference electrode. Immobilization was carried out at a constant potential of -r 1.0 V for 20 min at room temperature. Enzyme electrodes were kept at 4°C in citrate buffer solution when not in use. 

The research in Chapter 6 was carried out to evaluate the phenolic capacity of two red wines produced in Turkey. Analysis was performed by using enzyme electrodes constructed by the immobilization of tyrosinase in condueting eopolymers. 

The amperometric measurements were carried out with a computer aided Electrochemical Measuring System ECM 700 from the Academy of Sciences of the GDR. A potential of +400 mV vs SCE for H2Q detection and of +100 mV for BQ reduction was applied at the enzyme electrode. Hexacyanoferrate(III) reduction was monitored at +150 mV. The electrodes were inserted into a measuring cell containing 2 ml of stirred buffer at room temperature. 

Biocatalyst electrodes, that is, modified electrodes carrying an immobilized enzyme in which the electrode behaves as a substitute for a chemical electron acceptor or donor in the enzyme reaction can be used in such novel applications as biosensors, bioreactors and biofuel cells 2 ... 

Figure 11.39 summarizes the reactions taking place in this amperometric sensor. FAD is the oxidized form of flavin adenine nucleotide (the active site of the enzyme glucose oxidase), and FAD1T2 is the active site s reduced form. Note that O2 serves as a mediator, carrying electrons to the electrode. Other mediators, such as Fe(CN)6 , can be used in place of O2. 

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