Enzyme electrodes glucose 6-phosphate

Figure 19. (A) The reconstitution of apo-glucose oxidase on a PQQ-FAD monolayer assembled on an Au electrode. (B) Cyclic voltammograms of the PQQ-FAD-reconstituted glucose oxidase on an Au electrode (a) in the absence of glucose (b) with glucose, 80 mM. Recorded in 0.1 M phosphate buffer, pH 7.0, under Ar, at 35°C potential scan rate, 5 mV s. Inset calibration curve corresponding to the amperometric responses (measured by chronoamperometry, = 0.2 V vs. SCE) of the PQQ-FAD-reconstituted glucose oxidase enzyme electrode at different concentrations of glucose.

Binyamin, Chen and Heller reported that wired enzyme electrodes constituted of glassy carbon electrodes coated with poly(4-vinylpyridine) complexed with [Os(bpy)2Cl] and quarternized with 2-bromoethylamine or poly[(iV-vinylimidazole) complexed with [Os(4,4 -dimethyl-2,2 -bypyridine)2Cl] or poly(vinylpyridine) complexed with [Os(4,4 -dimethoxy-2,2 -bypyridine)2Cl] quaternized with methyl groups lost their electrocatalytic activity more rapidly in serum or saline phosphate buffer (pH 7.2) in the presence of urate and transitional metal ions such as Zn and Fe " " than in plain saline phosphate buffer (pH 7.2). It was reported that as much as two-thirds of the current is lost in 2 h in some anodes. However, when a composite membrane of cellulose acetate, Nafion, and the polyaziridine-cross-linked co-polymer of poly(4-vinyl pyridine) quaternized with bromoacetic acid was applied, the glucose sensor stability in serum was improved and maintained for at least 3 days [27,50]. 

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 ... 

Renneberg et al. (1983a) described an enzyme electrode-based assay of factor VIII, which is important for blood coagulation diagnostics. AP was used as the marker enzyme and the hydrolysis of glucose-6-phosphate was measured with a glucose electrode. This combination allowed the determination of 1.6-16 ng of factor VIII in human plasma. 

Enzyme electrode Orthophosphate 775 25 Biosensor based on glucose-6 -phosphate inhibition of hydrolysis by potato acid phosphatase, high selectivity for F [121]... 

Enzyme electrode Orthophosphate 0.31 0.01 Amperometric detection of H2O2 produced by interaction of phosphate with maltose phosphorylase, acid phosphatase, glucose oxidase, and mutarotase immobilized on cellulose membrane. [182]... 

When the electrode is placed in an aqueous solution of glucose which has been suitably diluted with a phosphate buffer solution (pH 7.3), solution passes through the outer membrane into the enzyme where hydroxen peroxide is produced. Hydrogen peroxide can diffuse through the inner membrane which, however, is impermeable to other components of the solution. The electrode vessel contains phosphate buffer, a platinum wire and a silver wire which act as electrodes. A potential of 0.7 volts is applied to the electrodes (the apparatus shown in Fig. 16.17 is suitable) with the platinum wire as anode. At this electrode the reaction H202->02 + 2H+ +2e takes place, and the oxygen produced is reduced at the silver cathode ... 

Homogeneous electrochemical enzyme immunoassays for both phenytoin and digoxin have been developed. In both cases the label was glucose-6-phosphate dehydrogenase, which catalyzes the reduction of NAD to NADH. The NADH produced was detected by LCEC at a carbon paste electrode. 

Many dehydrogenase enzymes catalyze oxidation/reduction reactions with the aid of nicotinamide cofactors. The electrochemical oxidation of nicotinamide adeniiw dinucleotide, NADH, has been studied in depthThe direct oxidation of NADH has been used to determine concentration of ethanol i s-isv, i62) lactate 157,160,162,163) pyTuvate 1 ), glucose-6-phosphate lactate dehydrogenase 159,161) alanine The direct oxidation often entails such complications as electrode surface pretreatment, interferences due to electrode operation at very positive potentials, and electrode fouling due to adsorption. Subsequent reaction of the NADH with peroxidase allows quantitation via the well established Clark electrode. 

Figure 17.17 Schematic representation of a single-compartment glucose/02 enzyme fuel cell built from carbon fiber electrodes modified with Os -containing polymers that incorporate glucose oxidase at the anode and bilirubin oxidase at the cathode. The inset shows power density versus cell potential curves for this fuel cell operating in a quiescent solution in air at pH 7.2, 0.14 M NaCl, 20 mM phosphate, and 15 mM glucose. Parts of this figure are reprinted with permission from Mano et al. [2003]. Copyright (2003) American Chemical Society.

None of the systems lost any enzyme activity during 24 h of continuous pumping of glucose solution (1 mM) at 2.3 ml/min. Moreover the various membranes when stored at 4P C in sodium dihydrogen phosphate (pH 7) still responded to substrate (70% of the signal for a new electrode) with intermittent use over a period of about 4 months after the fabrication (4). 

Figure 17. (A) The preparation of an electrically wired enzyme by the reconstitution technique, involving the removal of the native FAD cofactor from the enzyme (e.g., GOx) and the incorporation of the artificial FAD-ferrocene dyad into the apo-enzyme. (B) Cyclic voltammograms of a system consisting of ferrocene-FAD-reconstituted GOx (1.75 mg mL ) at various concentrations of glucose (a) 0, (b) 1, (c) 3 and (d) 20.5 mM. Experiments were performed in 0.1 M phosphate buffer, pH 7.3, at 35 C, using a cystamine-modified Au electrode, potential scan rate 2 mV s , under argon. Inset calibration curve of the biocatalytic current (0.5 V vs. SCE) at different glucose concentrations.

The remanent activity of glucose oxidase (GOD) membranes has been determined by measuring the initial rate of H2O2 formation with a Pt electrode polarized to +600 mV, using a known amount of resolubilized membrane in 5 mmol/1 glucose solution at 37°C (Scheller et al., 1988). Thus, 70-90% of the initial activity has been found with gelatin-entrapped enzyme. The GOD membrane was solubilized in 0.05 mol/1 phosphate buffer, pH 5.5, at 40°C. 

ADP AFP ab as ALAT AP ASAT ATP BQ BSA CEH CK CME COD con A CV d D E E EC ECME EDTA EIA /e FAD FET FIA G GOD G6P-DH HBg HCG adenosine diphosphate a-fetoprotein antibody antigen alanine aminotranferase alkaline phosphatase aspartate aminotransferase adenosine triphosphate benzoquinone bovine serum albumin cholesterol ester hydrolase creatine kinase chemically modified electrode cholesterol oxidase concanavalin A coefficient of variation (relative standard deviation) layer thickness diffusion coefficient enzyme potential Enzyme Classification enzyme-chemically modified electrode ethylene diamine tetraacetic acid enzyme immunoassay enzyme loading factor flavin adenine dinucleotide field effect transistor flow injection analysis amplification factor glucose oxidase glucose-6-phosphate dehydrogenase hepatitis B surface antigen human chorionic gonadotropin... 

Figure 17.3 The pH dependence of a glucose oxidase membrane electrode at different enzyme loadings and different glucose concentrations electrode surface, 0.22 mm electrode potential, +600 mV against Ag/AgCl phosphate buffer, 0.66 molT (reproduced with the permission of Elsevier Science Publishers BV). 

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