Oxygen-Based Enzyme Electrodes

The earliest design of the glucose electrode (which is generally applicable to any oxidase and uses oxygen as the ultimate electron acceptor) is based on the differential measurement of oxygen deficiency at the oxygen electrode, caused by the 

There are two major problems encountered with this approach. First is the selfinhibition caused by the hydrogen peroxide this has been mentioned before. It is less acute in this case than in the hydrogen peroxide sensor because the catalase substantially eliminates any excess of H2O2. The second problem is encountered when the electrode is used in vivo. The tissue or blood concentration of oxygen, which is the cosubstrate, is low and at high glucose concentrations the current becomes limited by the availability of oxygen. 

An elegant solution to this problem has been proposed (Gough et al., 1985). By design, the mass transport of oxygen has been increased, relative to that of glucose, by cylindrical diffusion into the enzyme layer and the transport of glucose restricted to the linear diffusion through the distal end of the sensor (Fig. 7.14). In other words, 

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Explain clearly why and how the response of glucose oxidase-based enzyme electrodes is influenced by fluctuations in the oxygen tension. 

Fig. 5. Detection methods for glucose enzyme electrode based on (a) oxygen, (b) hydrogen peroxide, and (c) a mediator. See text.

S. Akgol and E. Dinckaya, A novel biosensor for specific determination of hydrogen peroxide catalase enzyme electrode based on dissolved oxygen probe. Talanta 48, 363-367 (1999). 

Biosensors Biosensors are used in the final measurement stage of immunoassays such as an electrochemistry-based enzyme-linked immunoassay (ELISA) or the measurement of catalyse-labelled antigens at an antibody-coated oxygen electrode. 

Gorton et al. reported carbon paste electrodes based on Toluidine Blue O (TBO)-methacrylate co-polymers or ethylenediamine polymer derivative and NAD" " with yeast alcohol dehydrogenase for the analysis of ethanol [152,153] and with D-lactate dehydrogenase for the analysis of D-lactic acid [154]. Use of electrodes prepared with dye-modified polymeric electron transfer systems and NAD+/NADH to detect vitamin K and pyruvic acid has also been reported by Okamoto et al. [153]. Although these sensors showed acceptable performances, insensitivity to ambient oxygen concentration, sensor stability and lifetime still need to be improved to obtain optimal dehydrogenase based enzyme biosensors. 

Several other sensors are available that are based on the amperometric measurement of hydrogen peroxide produced by enzymatic reactions. The analytes measured include sucrose, lactose, ethanol, and L-lactate. A different enzyme is, of course, required for each species. In some cases, enzyme electrodes can be based on measuring oxygen or on measuring pH. 

The biosensors were constructed with optical sensor for oxygen based on optical fibre Avaspec -Oxy (Avantes, Holland). The membrane with immobilised enzyme was attached to the electrodes with a dialysis membrane and a pipette tip. 

Phenol-2-hydroxylase also oxidizes NADPH with the formation of H2O2. Various monosubstituted phenols are also oxidized, but at a lower rate than phenol itself. Therefore the sensitivity of phenol hydroxylase enzyme electrodes based on O2 indication as described by Kjellen and Neujahr (1980) is different for substituted and unsubstituted phenols. Consequently, in mixtures of phenols the oxygen consumption does not reflect the real phenol concentration. 

An enzyme electrode based on coimmobilized cytochrome b2 and laccase (Scheller et al., 1987b) allows an explanation of the principle of substrate recycling in enzyme electrodes in greater detail (Fig. 100). The advantage of this system is that the cosubstrate, oxygen, as well as the analytes, hydroquinone and benzoquinone, are electrochemically active. This permits one to study different parts of the recycling process. Recycling of the analyte in the presence of the substrate of cytochrome b2, lactate, results in an increase in the sensitivity by a factor of 500 as compared with lactate-free operation. Under conditions that are optimal for laccase the analyte is almost completely in the oxidized state, i.e. it... 

Other examples of amperometric enzyme electrodes based on the measurement of oxygen or hydrogen peroxide include electrodes for the measurement of galactose in blood (galactose oxidase,enzyme), oxalate in urine (oxalate oxidase), and cholesterol in blood serum (cholesterol oxidase). Ethanol is determined by reacting with a cofactor, nicotinamide adenine dinucleotide (NAD" ) in the presence of the enzyme alcohol dehydrogenase to produce the reduced form of NAD", NADH, which is electrochemically oxidized. Lactate in blood is similarly determined (lactate dehydrogenase enzyme). 

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