Enzyme electrodes lactate

Kalger GmbH, Neuberg, Germany Microzym-L Lactate Enzyme electrode Food, biotechnology, medicine... 

Matsumoto et al (41) prepared a multi-enzyme electrode using glucose oxidase, invertase, mutarotase, fructose-5-dehydrogenase, and catalase to simultaneously detect glucose, fructose, and sucrose in fruit juices and soft drinks. Detection of multi-components by enzyme sensors was also reported in analysis of sucrose and glucose in honey (42) and drinks (43), and L-malate and L-lactate in wines (44). 

Sulfite modified enzyme electrode. (2) L-Lactate/L-malate/ sulfite multibiosensor L-lactate dehydrogenase/L-malate dehydrogenase/ sulfite oxidase surface-modified enzyme electrodes/enzymes were deposited on the composite electrodes and covered with a dialysis membrane ... 

A. Avramescu, T. Noguer, V. Magearu and J.-L. Marty, Chronoampero-metric determination of D-lactate using screen-printed enzyme electrodes, Anal. Chim. Acta, 433(1) (2001) 81-88. 

Torriero et al. [30] managed to estimate the concentration of lactate in untreated milk without the use of a microdialysis unit. Samples were fed into a reactor consisting of a rotating disc bearing lactate oxidase which by its motion ensured adequate mass transport of hydrogen peroxide to an enzyme electrode. The electrode consisted of horse-radish peroxidase immobilised over osmium on the surface of a glassy carbon electrode. Such an electrode can be poised at 0.0 Y so avoiding electrochemical... 

M. Kyrolainen, H. Hakanson, B. Mattiasson and P. Vadgama, Minimal fouling enzyme electrode for continuous flow measurement of whole blood lactate, Biosens. Bioelectron., 11 (1997) 1073-1081. 

M. Kyrolainen, S.M. Reddy and P.M. Vadgama, Blood compatibility and extended linearity of lactate enzyme electrode using polyCvinyl chloride) outer membranes, Anal. Chim. Acta, 353 (1997) 281-289. 

Setric G.I., France Microzym-L enzyme electrodes with NHg, O2, and H2O2 electrodes L-Lactate and D-glucose... 

Chemically binding enzymes to nylon net is very simple and gives strong mechanically resistant membranes (135). The nylon net is first activated by methylation and then quickly treated with lysine. Finally, the enzyme is chemically bound with GA. The immobilized disks are fixed direcdy to the sensor surface or stored in a phosphate buffer. GOD, ascorbate oxidase, cholesterol oxidase, galactose oxidase, urease, alcohol oxidase (135), and lactate oxidase (142) have been immobilized by this procedure and the respective enzyme electrode performance has been established. 

The activity of the enzyme is also strongly affected by the presence of inhibitors. Fluoride ions inhibit urease (173) and oxalate ions inhibit lactate oxidase (174), but the major inhibitors are heavy-metal ions, such as Ag+, Hg +, Cu " ", organophosphates, and sulfhydryl reagents (/i-chloromercuribenzoate and phenylmercury(II) acetate), which block the free thiol groups of many enzyme active centers, especially oxidase (69). Inhibiting the enzyme electrodes makes it possible to quantify the inhibitors themselves (69), for example, H2S and HCN detection using a cytochrome oxidase immobilized electrode (176). 

The commercial availability of various lactate-specific enzymes and the high demand for lactate analysis for clinical and food processing led to the construction of numerous lactate enzyme electrodes (Tkble 8). Depending on the selected enzyme—lactate oxidase (LOD), lactate mono-oxidase (LMOD), or lactate dehydrogenase (LDH)—several mechanistic approaches may be applied ... 

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,... 

Cunningham, D.D. Stephens, D. Analysis of the enantiomeric purity of lactate with an enzyme electrode. Anal. Lett. 1992, 25, 1813-1821. 

Fry has used a similar system for the enzymatic reduction of pyruvate to L-lactate. In this case, the one-electron transfer redox catalyst, methyl viologen, and the lipoamide dehydrogenase are coimmobilized within a Nafion cation-exchange layer on the surface of a reticulated vitreous carbon electrode. As production enzyme, L-lactate dehydrogenase (LDH) was employed [48], which was later stabilized considerably in the form of cross-... 

Ohara TJ, Rajagopalan R, Heller A. Wired enzyme electrodes for amperometric determination of glucose or lactate in the presence of interfering substances. Anal Chem 1994 66 2451-7. 

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. 

This method was applied to assemble integrated electrically-contacted NAD(P)-dcpcndcnt enzyme electrodes. The direct electrochemical reduction of NAD(l ) cofactors or the electrochemical oxidation of NAD(P)H cofactors are kineticaUy unfavored. Different diffusional redox mediators such as quinones, phenazine, phenoxazine, ferrocene or Os-complexes were employed as electrocatalysts for the oxidation of NAD(P)H cofactors An effective electrocatalyst for the oxidation of the NAD(P)H is pyrroloquinoline quinone, PQQ, (7), and its immobilization on electrode surfaces led to efficient electrocatalytic interfaces (particularly in the presence of Ca ions) for the oxidation of the NAD(P)H cofactors. This observation led to the organization of integrated electrically contacted enzyme-electrodes as depicted in Fig. 3-20 for the organization of a lactate dehydrogenase electrode. 

The next stage was achieved in 1967 by Updike and Hicks, who entrapped GOD in a gel of polyacrylamide, thus increasing the operational stability of the enzyme and simplifying the sensor preparation. Further investigations by Reitnauer (1972) enabled the successful application of an enzyme electrode in a prototype blood glucose analyzer. In 1975 Yellow Springs Instrument Co. (USA) commercialized a glucose analyzer (model 23 A) which was based on a patent by Clark (1970). The Lactate Analyzer LA 640 by La Roche (Switzerland) followed one year later. In this instrument the enzyme is dissolved in a buffer in a reaction chamber placed in front of the electrode. 

The direct anodic oxidation of cytochrome c at a bipyridyl-modified electrode has already been incorporated in enzyme electrodes for lactate, carbon monoxide, and hydrogen peroxide. Here, cytochrome c is reduced by cytochrome b2, CO oxidoreductase, or horseradish peroxidase and anodically reoxidized. Cytochrome c has also been applied to couple mitochondria and chloroplasts to redox electrodes (Albery et al. 1987). Although no practically applicable sensor has been constructed as yet, this principle offers a new avenue to the determination of inhibitors of photosynthesis or respiration (Cardosi and Turner, 1987). 

Owing to differences in the Kyi values and the layer thickness, the transient from kinetic to diffusion control of different enzyme electrodes takes place at rather different enzyme activities. Gelatin-entrapped enzymes exhibit transient values of 0.17 U/cm2 (uricase,iifM= 17 pmol/1), 16 U/cm2 (urease, Kyi = 2 mmol/1) and 1.0 U/cm2 (lactate monooxygenase, Km = 7.2 mmol/1). 

The different cosubstrate specificities of the lactate-oxidizing enzymes offer the use of a great variety of electrochemical indicator reactions in membrane sensors. In enzyme electrodes based on LDH the biochemical reaction has been coupled to the electrode via NADH oxidation, either directly or by using mediators or additional enzymes (see Section 3.2.1). This leads to a shift of the unfavorable reaction equilibrium by partial trapping of the reduced cofactor. Such a shift has also been achieved by using pyruvate oxidase coimmobilized with LDH (Mizutani, 1982). 

In most amperometric cytochrome b2 electrodes the reaction is followed by anodic oxidation of ferrocyanide at a potential of +0.25 V or above. The first of such sensors was assembled by Williams et al. (1970), who immobilized the enzyme (from baker s yeast) physically at the tip of a platinum electrode within a nylon net of 0.15 mm thickness. The large layer thickness resulted in a response time of 3-10 min. Owing to the low specific enzyme activity used, the sensor was kinetically controlled. Therefore the linear measuring range extended only up to 0.1 Km-A similar sensor has been applied by Durliat et al. (1979) to continuous lactate analysis. The enzyme was contained in a reaction chamber of 1 pi volume in front of the electrode. This principle has also been employed in the first commercial lactate analyzer using an enzyme electrode (Roche LA 640, see Section 5.2.3.3X With a sensor stability of 30 days and a C V below 5%, 20-30 samples/h can be processed with this device. 

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... 

Between 15 and 20 analyzers based on enzyme electrodes are on the market worldwide. They are one-parameter instruments for the measurement of glucose, galactose, uric add, choline, ethanol, lysine, lactate, pesti-ddes, sucrose, lactose, and the activity of a-amylase (Table 23). They provide for a negligible enzyme consumption of less than 1 pg per sample. 

Fig. 128. Manual glucose analyzer Glukometer GKM (Zentrum ftir Wissenschaftlichen Gerfltebau, Academy of Sciences of the GDR) consisting of a thermostat furnished with measuring cell and enzyme electrode (left), sample dispenser (center), and electronics (adapted to glucose, uric acid, and lactate measurement, right).

The first enzyme electrode-based lactate analyzer was developed in 1976 by La Roche (Switzerland) (see Table 23). It uses cytochrome b2 in a tiny reaction chamber on top of a platinum electrode polarized at +0.25-0.40V. The solution for blood sample pretreatment recommended by the manufacturer has been improved by Soutter et al. (1978) by addition of cetyltrimethylammonium bromide. This compound hemo-lyzes the sample, stabilizes the lactate content, and leads to a good correlation with the spectrophotometric reference method using deproteinized blood ... 

Fig. 137. Scheme of an on-line bedside analyzer for parallel determination of glucose, lactate, and pyruvate using enzyme electrodes. (Redrawn from Mascini, 1987). 

T. Yao, T. Yano, On-line microdialysis assay of L-lactate and pyruvate in vitro and in vivo by a flow-injection system with a dual enzyme electrode, Talanta 63 (2004) 771. 

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