Stable mediated amperometric biosensors using a graphite electrode embedded with tetrathiafulvalene in silicone oil

A new method has been developed to incorporate the mediator, tetrathiafulvalene (TTF), to the electrode/solution interface of an amperometric biosensor. TTF was dissolved in methylphenyl polysiloxane (silicone oil) and embedded in a graphite disc electrode. The mediator was able to diffuse to the electrode surface at an electrocatalytically significant speed. The storage of TTF in the inert polysiloxane provided a long-lasting and stable mediator supply.

TTF-silicone oil electrodes with immobilized glucose oxidase, xanthine oxidase, or amino acid oxidase exhibited sensitive, fast and reproducible responses. The glucose oxidase electrode was very stable for at least 2 months when stored at 4°C. Together with flow injection analysis (FIA), the enzyme electrodes were reused for at least 500 repeated analyses during a 25 h operation without losing their initial activity.     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

Complexes of glucose oxidase with chitosan and dextran possessing enhanced stability

Abstract

In this study, the different mole ratios of glucose oxidase/chitosan/dextran–aldehyde and glucose oxidase/chitosan/dextran–sulfate complexes were synthesized. The modification of glucose oxidase by non-covalent complexation with dextran and chitosan in different molar ratios was studied in order to increase the enzyme activity. The enzyme/polymer complexes obtained were investigated by UV spectrophotometer and dynamic light scattering. Activity determination of synthesized complexes and free enzyme were performed at a temperature range. The best results were obtained by C_chitosan/C_{dextran–aldehyde} = 10/1 ratio and C_chitosan/C_{dextran–sulfate} = 1/5 ratio that were used in thermal stability, shelf life, salt stress, and ethanol effect experiments. The results demonstrated that both complexes were thermally stable at 60?°C and had superior storage stability compared to the free glucose oxidase. Complexes showed higher enzymatic activity than free enzyme in the organic solvent environment using 10% ethanol. The complexes were resistant to salt stress containing 0.1?M NaCl or CaCl₂. The particle size distribution results of the triple complex evaluated the complexation of the chitosan, dextran derivative, and glucose oxidase. The average size of the triple complex in diameter was found to be 325.8?±?9.3?nm. Overall findings suggest that the complexes of glucose oxidase, chitosan, and dextran showed significant enhancement in the enzyme activity.     

Pt based enzyme electrode probes assembled with Prussian Blue and conducting polymer nanostructures

Conductive polymer nanotubules of 1,2-diaminobenzene (1,2-DAB) were prepared using a porous polycarbonate membrane template, placed on a Pt foil and used to support the polymer, then, the electropolymerisation was performed by chronocoulometry. The obtained conductive polymer nanostructures were then placed on Pt electrode and used to support highly dispersed prussian blue (PB), which acts as the active component for H2O2 detection. The observed good stability of PB as catalyst of H2O2 was related to the presence of organic non-conventional conducting polymers in a composite nanostructured film. These nanostructured polymer/PB composite films were also characterised by scanning electron microscopy (SEM) and Raman spectroscopy. The non-conventional conducting polymer nanotubules/PB modified Pt electrodes were tested by cyclic voltammeter for stability at different pH values, then, by amperometry, for hydrogen peroxide, ascorbic acid, acetaminophen, uric acid and acetylcholine. Glucose oxidase (GOD), lactate oxidase (LOD), L-amino acid oxidase (L-AAOD), alcohol oxidase (AOD), glycerol-3-phosphate oxidase (GPO), lysine oxidase (LyOx), and choline oxidase (ChOx) were immobilised on PB layer supported on 1,2-diaminobenzene (1,2-DAB) nanotubules onto the Pt electrodes. Different strategies for enzyme immobilisation were performed and used. Analytical parameters such as reproducibility, interference rejection, response time, storage and operational stability of the sensors have been studied and optimised. Results provide a guide to design high sensitive, stable and interference-free biosensors. The glucose biosensors assembled with nanostructured poly(1,2-DAB) showed a detection limit of 5 x 10(-5) mol l(-1), a wide linearity range (5 x 10(-5) to 5 x 10(-3) mol l(-1)), a high selectivity, a stability of 3 months at 4 degrees C, and at least 4 weeks at room temperature. Similar analytical parameters and stability were also studied for L-(+)-lactic acid, L-leucine, ethanol, glycerol-3-phosphate, lysine, and choline biosensors.     

Glucose oxidase-polypyrrole electrodes synthesized in <Emphasis Type="Italic">p</Emphasis>-toluenesulfonic acid and sodium <Emphasis Type="Italic">p</Emphasis>-toluenesulfonate

Amperometric glucose biosensors have been developed based on entrapment on platinum (Pt) electrode using cyclic voltammetry technique in glucose oxidase (GOD) and pyrrole containing p-toluenesulfonic acid (pTSA) or sodium p-toluenesulfonate (NapTS) as supporting electrolyte solutions. Both of electrolyte solutions were suitable media for the formation and deposition of polypyrrole-GOD (PPy-GOD) layers on Pt substrate. Pt/PPy-GOD electrodes brought about in different morphological properties as well as different electrochemical and biochemical response. The highest responses obtained in pTSA and NapTS electrolytes were observed at pH of 4.5 and 7.0 for Pt/PPy-GOD electrodes, respectively. While linearity was observed between 0.0–1.0 mM glucose substrate for both electrodes, I _max value of Pt/PPy-GOD_NapTS electrode was approximately twice as high as that of Pt/PPy-GOD_pTSA electrode as 25.4 and 14.2 μA, respectively. Five commercial drinks were tested with enzyme electrodes and compared with results obtained spectropho-tometrically using glucose kit. Results revealed that Pt/PPy-GOD_NapTS electrode exhibited better biosensor response.     

Disposable, enzymatically modified printed film carbon electrodes for use in the high-performance liquid chromatographic–electrochemical detection of glucose or hydrogen peroxide from immobilized enzyme reactors

Disposable screen-printed, film carbon electrodes (PFCE) were modified with cast-coated Osmium–polyvinylpyrridine-wired horse radish peroxidase gel polymer (Os-gel-HRP) to enable the detection of the reduction at 0 mV of hydrogen peroxide (H₂O₂) derived from a post-column immobilized enzyme reactor (IMER) containing acetylcholinesterase and choline oxidase. In another series of experiments PFCE were initially modified with cast-coated Os-gel-HRP and then treated with glucose oxidase in bovine serum albumin (BSA) and cross-linked with glutaraldehyde to form a bi-layer glucose–Os-gel-HRP PFCE. This bi-layer glucose–Os-gel-HRP PFCE generated a reduction current at 0 mV to H₂O₂ derived from the reaction of glucose oxidase and glucose in solution. These enzyme-modified PFCE were housed in a radial flow cell and coupled with cation-exchange liquid chromatographic methods to temporally separate substrates in solution for the determination of acetylcholine (ACh) and choline (Ch) in the first experimental series, or glucose in the second experimental series. These two disposable enzyme-modified PFCE exhibited linear current vs. substrate relations, were durable, being usable for approximately 40 determinations, and were sufficiently sensitive to be employed in biological sampling. Both assays utilized the same HPLC equipment. The limit of detection for ACh was 16 fmol/10 μl and that for glucose was 12 μmol/7.5 μl. ACh and Ch were measured from a microdialysate from the frontal cortex of a rat. Glucose in human urine was determined using the bi-layer glucose oxidase–Os-gel-HRP PFCE.     

Optimization of culture conditions for the production of rifamycin oxidase by Curvularia lunata

Maximum activity (8.9 IU/ml) of rifamycin oxidase in Curvularia lunata, grown in shake-flask culture at 28°C and pH 6.5, was after 96 h. Nearly all the glucose was used in 72 h. An initial culture pH of 6.5 and 28°C were optimum for the growth and enzyme production. Among various carbon and organic nitrogen sources, carboxymethylcellulose and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when yeast extract was also added to the medium. The optimum medium for the production of rifamycin oxidase contained 10 g each of yeast extract, peptone and carboxymethylcellulose/l and 0.04% (NH₄)₂SO₄.The author is with the Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Post Box 1304, Sector 39-A, Chandigarh 160 014, India     

Enzyme electrode formed by evaporative concentration and its performance characterization

A highly concentrated immobilized enzyme layer was formed on a small working electrode, and the behavior of the electrode as an amperometric sensor was examined. To this end, a super-hydrophobic layer was formed in an area other than the sensitive area by using polytetrafluoroethylene (PTFE) beads. A small droplet of an enzyme solution containing glucose oxidase (GOD) and bovine serum albumin (BSA) was placed on the sensitive area, concentrated by evaporation, and crosslinked with glutaraldehyde. With the same enzyme activity per unit area, the current density increased with smaller working electrodes. Also, the current density increased with higher enzyme loadings up to a limiting value. In addition, the linear range of the calibration plot was expanded to higher glucose concentrations. The enzyme electrode fabricated by the novel method was incorporated in a micro-flow channel. Compared with large enzyme electrodes with the same enzyme activity per unit area, smaller electrodes showed a significant increase in the current density and a decrease in the flow dependence. The conversion efficiency could be improved by narrowing the flow channel and increasing the number of electrodes, which was comparable with a large electrode placed in a shallow flow channel.     

Optimisation of the composition of a screen-printed acrylate polymer enzyme layer with respect to an improved selectivity and stability of enzyme electrodes

Glucose oxidase (GOD) was immobilized on screen-printed platinum electrodes by entrapment in a screen printable paste polymerized by irradiation with UV-light. The influences of different additives, in particular polymers and graphite, on the sensitivity and stability of the sensor and the permeability of the enzyme layer for a possible electrochemical interferent were investigated. The chosen additives were Gafquat 755N, poly-L-lysine, bovine serum albumin (BSA), sodium dodecylsulfate (SDS), polyethylene glycol (PEG), Nafion and graphite. All additives led to increases of glucose signals, i.e. improved the sensitivity of glucose detection with Gafquat 755N, poly-L-lysine, SDS and graphite showing the strongest influences with increases by a factor 4, 6.5, 5 and 10, respectively. Ascorbic acid was used as a model interferent showing the influence of the enzyme layer composition on the selectivity of the sensor. The addition of Gafquat 755N or poly-L-lysine led to higher signals not only for glucose, but also for ascorbic acid. SDS addition already reduced the influence of ascorbic acid, which was almost completely eliminated when Nafion (5%) and PEG (10%) were added. A comparable beneficial effect on the selectivity of the sensors was also observed for the addition of 0.5% graphite. Thus, the enzyme electrodes with PEG, Nafion or graphite as additives in the enzyme layer were applied to glucose determinations in food samples and samples obtained from E. coli cultivations.     

Bienzyme biosensors for glucose,ethanol and putrescine built on oxidase and sweet potato peroxidase

Amperometric biosensors for glucose, ethanol, and biogenic amines (putrescine) were constructed using oxidase/peroxidase bienzyme systems. The H(2)O(2) produced by the oxidase in reaction with its substrate is converted into a measurable signal via a novel peroxidase purified from sweet potato peels. All developed biosensors are based on redox hydrogels formed of oxidases (glucose oxidase, alcohol oxidase, or amine oxidase) and the newly purified sweet potato peroxidase (SPP) cross-linked to a redox polymer. The developed electrodes were characterized (sensitivity, stability, and performances in organic medium) and compared with similarly built ones using the 'classical' horseradish peroxidase (HRP). The SPP-based electrodes displayed higher sensitivity and better detection limit for putrescine than those using HRP and were also shown to retain their activity in organic phase much better than the HPR based ones. The importance of attractive or repulsive electrostatic interactions between the peroxidases and oxidases (determined by their isoelectric points) were found to play an important role in the sensitivity of the obtained sensors.     

Colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of enzyme electrodes by electrodeposition

Glucose oxidase, horseradish peroxidase, xanthine oxidase, and carbonic anhydrase have been adsorbed to colloidal gold sols with good retention of enzymatic activity. Adsorption of xanthine oxidase on colloidal gold did not result in a change in enzymatic activity as determined by active site titration with the stoichiometric inhibitor pterin aldehyde and by measurement of the apparent Michaelis constant (K'(M)). Gold sols with adsorbed glucose oxidase, horseradish peroxidase, and xanthine oxidase have also been electrodeposited onto conducting matrices (platinum gauze and/or glassy carbon) to make enzyme electrodes. These electrodes retained enzymatic activity and, more importantly, gave an electrochemical response to the enzyme substrate in the presence of an appropriate electron transfer mediator. Our results demonstrate the utility of colloidal gold as a biocompatible enzyme imobilization matrix suitable for the fabrication of enzyme electrodes. (c) 1992 John Wiley & Sons, Inc.     

Electrically Contacted Bienzyme‐Functionalized Mesoporous Carbon Nanoparticle Electrodes: Applications for the Development of Dual Amperometric Biosensors and Multifuel‐Driven Biofuel Cells

The capping of electron relay units in mesoporous carbon nanoparticles (MPC NPs) by crosslinking of different enzymes on MPC NPs matrices leads to integrated electrically contacted bienzyme electrodes acting as dual biosensors or as functional bienzyme anodes and cathodes for biofuel cells. The capping of ferrocene methanol and methylene blue in MPC NPs by the crosslinking of glucose oxidase (GOx) and horseradish peroxidase (HRP) yields a functional sensing electrode for both glucose and H₂O₂, which also acts as a bienzyme cascaded system for the indirect detection of glucose. A MPC NP matrix, loaded with ferrocene methanol and capped by GOx/lactate oxidase (LOx), is implemented for the oxidation and detection of both glucose and lactate. Similarly, MPC NPs, loaded with 2,2′‐azino‐bis(3‐ethylbenzo­thiazoline‐6‐sulphonic acid), are capped with bilirubin oxidase (BOD) and catalase (Cat), to yield a bienzyme O₂ reduction cathode. A biofuel cell that uses the bienzyme GOx/LOx anode and the BOD/Cat cathode, glucose and/or lactate as fuels, and O₂ and/or H₂O₂ as oxidizers is assembled, revealing a power efficiency of ≈90 μW cm^?2 in the presence of the two fuels. The study demonstrates that multienzyme MPC NP electrodes may improve the performance of biofuel cells by oxidizing mixtures of fuels in biomass.     

Highly stable enzyme precipitate coatings and their electrochemical applications

This paper describes highly stable enzyme precipitate coatings (EPCs) on electrospun polymer nanofibers and carbon nanotubes (CNTs), and their potential applications in the development of highly sensitive biosensors and high-powered biofuel cells. EPCs of glucose oxidase (GOx) were prepared by precipitating GOx molecules in the presence of ammonium sulfate, then cross-linking the precipitated GOx aggregates on covalently attached enzyme molecules on the surface of nanomaterials. EPCs-GOx not only improved enzyme loading, but also retained high enzyme stability. For example, EPC-GOx on CNTs showed a 50 times higher activity per unit weight of CNTs than the conventional approach of covalent attachment, and its initial activity was maintained with negligible loss for 200 days. EPC-GOx on CNTs was entrapped by Nafion to prepare enzyme electrodes for glucose sensors and biofuel cells. The EPC-GOx electrode showed a higher sensitivity and a lower detection limit than an electrode prepared with covalently attached GOx (CA-GOx). The CA-GOx electrode showed an 80% drop in sensitivity after thermal treatment at 50°C for 4 h, while the EPC-GOx electrode maintained its high sensitivity with negligible decrease under the same conditions. The use of EPC-GOx as the anode of a biofuel cell improved the power density, which was also stable even after thermal treatment of the enzyme anode at 50°C. The excellent stability of the EPC-GOx electrode together with its high current output create new potential for the practical applications of enzyme-based glucose sensors and biofuel cells.     

Immobilized glucose dehydrogenase for cofactor regeneration in heme-catalyzed demethylation and hydroxylation reactions

Glucose dehydrogenase (E.C. 1.1.1.47) from B. megaterium M 1286 was immobilized together with mutarotase (E.C. 5.1.3.3) on several organic carriers and by different methods. The storage stability of the enzyme at pH-values > 6 is slightly improved by immobilization and the pH-optimum is shifted from 8.3 to 8.0. Kinetic constants of the immobilized enzyme are: K′_M(NAD⁺) = 5.36 × 10^?4 mol/l K′_M(glucose) = 3.76 · 10^?2 mol/l and V′_max = 5.54 · 10^?5 mol/(l min g carrier) for the most active preparation (2.16 mg enzyme/g carrier). In reactor experiments the immobilized glucose dehydrogenase was used with glucose to regenerate NADPH in NADPH-dependent iron-III-protoporphyrin-IX-imidazole catalyzed hydroxylation and demethylation of model substrates of cytochrome P-450. The advantages of the coupling of both reactions with cofactor recycling are shown and discussed.     

Glucose oxidase immobilized polyethylene-g-acrylic acid membrane for glucose oxidase sensor

A polyethylene-g-acrylic acid (PE-g-AA) graft copolymer was prepared via gamma-ray-irradiation-induced postirradiation procedures, and was used as support material for the immobilization of glucose oxidase. Soluble carbodiimides were used as the coupling agent. Reasonable yields were obtained with CMC but not with EDAC, EEDQ, or WRK. A number of factors were studied. (1) The use of water-soluble carbodiimides as condensing agent was attempted and the optimum condition for coupling glucose oxidase to PE-g-AA was established; (2) the effect of pH and temperature on the reactivity of native and immobilized glucose oxidase was studied. When exposed to temperatures in excess of 60 degrees C, the immobilized glucose oxidase was less sensitive to thermal inactivation than the native enzyme. The optimum pH value for the performance of the enzyme-immobilized membrane was 5. 6. For 200 tests, the response error of glucose sensor was less than 4% and its linear detected range was 0-1000 ppm. The obtained glucose oxidase-immobilized PE-g-AA membranes were kept in pH 5. 6 acetate buffer solution at 4 degrees C. The glucose oxidase activity of the membrane was determined at sevenday intervals. The membranes still have 92% glucose oxidase activity even after eight weeks of storage.     

Behavior of glucose oxidase immobilized in various electropolymerized thin films

Glucose oxidase was immobilized by electropolymerization into films of polyaniline, polyindole, polypyrrole, poly(o-phenylediamine), and polyaniline crosslinked with p-phenylenediamine. The kinetics and the behavior of the entrapped enzyme toward elevated temperature, organic solvent denaturation, and pH were investigated, along with the response of the films to electroactive species such as acetaminophen, ascorbate, cysteine, and uric acid. For most of the films, linearity to glucose extended from 7 to 10 mM. The poly(o-phenylenediamine)/glucose oxidase film gave the best signal/noise ratio and polypyrrole/glucose oxidase film gave the most reproducible current responses. No significant shift of the optimum reaction pH (5.5), except for polypyrrole (5.0), was observed after immobilization of glucose oxidase in the various films. Enzymatic activity decreased rapidly when pH was raised above 7.5. Thermodeactivation studies at 55 degrees , 60 degrees , and 65 degrees C have shown polypyrrole/and poly(o-phenylediamine)/glucose oxidase films to be the most resistant enzymatic films. Poly(o-phenylenediamine) films offered the best protection against glucose oxidase deactivation in hexane, chloroform, ether, THF, and acetonitrile when compared with the other electropolymerized films. All the enzymatic films exhibited permselection toward electroactive species. (c) 1996 John Wiley & Sons, Inc.     

Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense

Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH₄)₂SO₄, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the V _max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V _max and a 2-fold decrease in K _m were observed. Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997     

Glucose oxidase bienzyme electrodes for ATP, NAD+, starch and disaccharides

Based on a glucose oxidase sensor for determination of glucose several glucoseoxidase bioenzyme electrodes have been developed. Enzymes producing glucose by hydrolysis of saccharides (glucamylase, invertase, cellulase) as well as glucose consuming systems (hexo-kinase, glucose dehydrogenase) have been coupled to glucose oxidase. The function of the bienzyme systems was demonstrated by concentration measurements (blood glucose, maltose, ATP, NAD+, starch) and enzyme activity measurements (alpha-amylase, ATPase, lactate dehydrogenase).     

Effect of salts and Triton X-100 on ultrafiltration purification and properties of extracellular glucose oxidase from <Emphasis Type="Italic">Penicillium adametzii</Emphasis> LF F-2044.1

We compared the effectiveness of glucose oxidase isolation from the culture fluid of Penicillium adametzii LF F-2044.1 in the presence of ammonium sulfate, ammonium chloride, and Triton X-100. Ammonium chloride inhibited glucose oxidase in the culture fluid. This compound increased K _M (by 1.2–1.3 times), but decreased V _max for D-glucose oxidation (by 1.7–1.8 times). Ammonium sulfate had little effect on kinetic parameters. Combined treatment with salts and Triton X-100 was followed by a significant increase in the effectiveness of ultrafiltration purification of the culture fluid. The samples of glucose oxidase were electrophoretically characterized. The dependence of kinetic parameters on glucose oxidase concentration during oxidation of D-glucose was evaluated. The catalytic constant and k _cat/K _M ratio for glucose oxidase samples from the culture fluid isolated in the presence of additives significantly surpassed those for enzyme samples, which were obtained by ultrafiltration of the culture fluid with no additives and chromatography on aluminum oxide. The activity of glucose oxidase isolated from the culture fluid in the presence of ammonium chloride was lower compared to that of the enzyme obtained in the presence of ammonium sulfate. This agent is preferable for ultrafiltration of the culture fluid.     

Covalent immobilization of FAD and glucose oxidase on carbon electrodes

The effectiveness of attaching flavin adenine dinucleotide (FAD) via a C bridge to Teflon-bonded carbon black (CB), and the subsequent immobilization of glucose oxidase on the FAD-modified electrodes has been studied by cyclic voltammetry. When FAD alone is bound to the electrode, it undergoes reduction and oxidation at -0.62 and -0.5 V, respectively-values similar to those obtained with free FAD. Compared to the free enzyme, the reduction of FAD as part of the immobilized enzyme is 200 mV more cathodic, while the oxidation potential remains the same in both cases.