Enzyme Sequence Electrodes

The degradation of cholesterol leads to the production of bile acids which are structurally closely related to various steroid hormones. (3-Hydroxysteroid dehydrogenase (EC 1.1.1.51) catalyzes the NAD+-de-pendent oxidation of 3(3-, 17(3- and some l6(3-hydroxysteroids to the respective ketosteroids. The enzyme has been adsorbed on a carbon electrode modified by NMP+TCNQ and the NADH liberated in the reaction oxidized anodically (Albery et al., 1987a). Campanella et al. (1984) employed an enzyme sequence electrode composed of NAD+-de-pendent steroid dehydrogenase and horseradish peroxidase for assay of 7a-hydroxysteroids. 

In potentiometric enzyme electrodes lyases producing carbon dioxide or ammonia are used as terminal enzymes of sequences. In fact, the term enzyme sequence electrode was introduced on the occasion of the design of a potentiometric D-gluconate sensor containing gluconate kinase (EC 2.7.1.12) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) (Jensen and Rechnitz, 1979). The authors found that for such a sensor to function the optimal pH values of the enzymes and the transducer should be close to each other. Furthermore, cofactors, if necessary, must not react with one another nor with constituents of the sample. It was concluded that the rate of substance conversion in multiple steps cannot exceed that of the terminal enzyme reaction. A linear concentration dependence is obtained when an excess of all enzymes of the sequence is provided, i.e. complete conversion occurs of all substrates within the enzyme membrane. Different permeabilities of the different substrates results in different sensitivities. This is particularly important with combinations of disaccharidases and oxidases, where the substrate is cleaved to two monosaccharides of approximately the same molecular size. The above... 

Wollenberger et al. (1983) combined COD and CEH immobilized on Spheron particles in a sensor for total cholesterol. The sensor showed no response when separately fixed enzymes were used, but was active with coimmobilized enzymes. In terms of bound activity CEH was 6 times less active than COD. The dependences of the current signal of the enzyme sequence electrode for free and esterified cholesterol were equal for aqueous standard solutions and serum samples (Fig. 90). This indicates a sufficient CEH activity in the immobilized preparation. 

Fig. 95. Enzyme sequence electrode for maltose determination containing a hexokinase anti-interference layer for glucose.

An enzyme sequence electrode for phosphate assay based on AP and GOD has been devised by Guilbault and Nanjo (1975b). Glucose-6-phosphate (G6P) was used as the substrate for AP ... 

Tab. 5 Enzyme sequence electrodes with oxidase indicator enzyme... 

This line has been initiated in the late 70s by Rechnitz group (1), who introduced enzyme sequences into enzyme electrodes. The field has been considerably widened by introducing the competitive (parallel), recycling and accumulation sensors. The following main merits of such sensors shall be pointed out ... 

The development of analytically useful enzyme electrodes is limited by the availability of purified and stable enzyme preparations. In an effort to extend the range of measurable species using ISE devices further, Rechnitz and co-workers (Rl) recently introduced bacterial- and tissue-based bio-selective electrode systems. These sensors are prepared in much the same manner as the enzyme probes except that whole intact cells are utilized as the immobilized reagents. There are several potential advantages to this novel approach, including (1) no need to extract and purify the enzymes involved, i. e., low cost (2) enzymes which are unstable when extracted from the cell may be used in situ to maximize and preserve their activity (3) if desired enzyme reactions require cofactors, these co ctors need not be added to the assay mixture because they are already present in the intact cell and (4) analytical reactions involving multistep enzyme sequences already present in the cells may be used to detect given analytes. 

The combination of the creatinine-converting enzymes with sensors indicating primary reaction products, such as ion sensitive electrodes, NH3 gas sensors, or thermistors, is an effective alternative to enzyme sequence sensors (see Section 3.2.1). Enzyme reactors as well as true biosensors for creatinine have been described. 

In addition to such an extension of the spectrum of measurable compounds, enzyme sequences are also useful to enhance the selectivity of indicator reactions, e.g., in GOD-HRP electrodes, and to enhance the sensitivity, e.g., in thermistors using a GOD-catalase sequence. These... 

Matsumoto et al. (1985) developed a sensor with the same enzyme sequence by immobilizing the enzymes covalently to silanized glassy carbon by glutaraldehyde. The sensor had a half-life of 7 weeks. Electrochemical interferences were compensated for by use of an additional, enzyme-free electrode. 

With a constant of K = 2.7640-5 mol/1 (pH 7.0, 25°C) the equilibrium of the LDH-catalyzed reaction lies far to the lactate side. This means that whereas for lactate sensors based on LDH the forward reaction has to be forced by alkaline buffer and pyruvate- or NADH-trapping agents, the reduction of pyruvate proceeds spontaneously under normal conditions. This direction of the reaction has been used in a sequence electrode for pyruvate assay (Weigelt et al., 1987b). In the presence of lactate monooxygenase (LMO) lactate formed from pyruvate by LDH is oxidized by molecular oxygen, the consumption of which was indicated at a Clark-type electrode. The enzymes were immobilized in a gelatin membrane. Of course such a sensor measures the concentration of lactate in the sample, too. Therefore it is suited to the determination of the lactate/pyruvate ratio, which is a clinically important parameter. Pro-... 

The successive determination of both transaminases has also been carried out with a sequence electrode containing oxaloacetate decarboxylase and pyruvate oxidase (Kihara et al., 1984b). The enzymes were coadsorbed on a PVC membrane of 40 pm thickness in the presence of thiamine pyrophosphate, FAD, and MgC. ... 

The above authors coimmobilized choline oxidase and AChE on a nylon net which was fixed to a hydrogen peroxide probe so that the esterase was adjacent to the solution. The apparent activities were 200-400 mU/cm2 for choline oxidase and 50-100 mU/cm2 for AChE. The sensitivity of the sequence electrode for ACh was about 90% of that for choline, resulting in a detection limit of 1 pmol/l ACh. The response time was 1-2 min. The parameters of this amperometric sensor surpass those of potentiometric enzyme electrodes for ACh (see Section 3.1.25). Application to brain extract analysis has been announced. 

Pyruvate kinase (PK) activity in hemolyzed erythrocytes has been determined by using an LDH-lactate monooxygenase sequence electrode (Weigelt et al., 1988). The enzymes were immobilized in gelatin and attached to an oxygen probe. Since the sample material contains only... 

The measurement of the lactate/pyruvate ratio in plasma is possible by using a lactate dehydrogenase-lactate monooxygenase sequence electrode [373]. The sensor is equally sensitive to lactate and pyruvate (Figure 14-34), because of the high enzyme loading and the... 

When the three-enzyme sequence based on creatinine amidohydrolase is used, any creatine present can interfere with the determination of creatinine, so two sensors are used one to determine the total creatine plus creatinine and one to determine just creatine (by only using creatine amidinohydrolase and sarcosine oxidase). Creatinine is determined by difference. Amperometric sensors are generally based on this sequence and do not suffer from interferences. They are usually designed to respond to peroxide, though some have used oxygen electrodes. Typically, Pt electrodes are used. A sensor for just creatine only requires the creatine amidinohydrolase and sarcosine oxidase sequence. 

See also DNA Sequencing. Enzymes Enzyme-Based Electrodes. Forensic Sciences Blood Analysis. Immunoassays, Techniques Enzyme Immunoassays. Microelectrodes. Polarography Techniques Organic Applications. Purines, Pyrimidines, and Nucleotides. Sensors Chemically Modified Electrodes. Voltammetry Organic Compounds. 

The basic glucose sensor may be extended to multiple enzyme electrodes for the measurement of the concentration of various analytes which may be converted to glucose (i.e., oligo-, polysaccharides, a- and jS-glycosides). A few examples of such sequence electrodes are given below [160, 161] ... 

Most of the amperometric biosensors described until now are constructed either by crosslinking a suitable redox enzyme within a polymeric gel on the electrode surfiu e or by assembling a preformed enzyme-containing membrane on top of the electrode. Although this approach has led to amperometric biosensors for substrates of most oxidases or enzyme sequences involving oxidases, besides the inherent thermal instability of proteins two major drawbacks have to be fi cused on. 

Shu, H-C. and N-P. Wu. 2001. A chemically modified carbon paste electrode with D-lactate dehydrogenase and alanine aminotranferase enzyme sequences for D-lactic acid analysis. Talanta 54 361-368. 

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