Christian Bourdillon

Immobilization of glucose oxidase on a carbon surface derivatized by electrochemical reduction of diazonium salts

Glassy carbon disk electrodes grafted with superficial 4-phenylacetic groups through electrochemical reduction of the corresponding diazonium salt were used for glucose oxidase covalent immobilization. Protein attachment was confirmed by means of X-ray photoelectron spectroscopy and the activity of the enzyme modified electrode was ascertained by performing enzymatic electrocatalysis. The present method does not cause any roughening of the electrode surface and concomitant increase of a background current thus allowing precise measurements using transient electrochemical techniques such as cyclic voltammetry. As diazonium salts (which upon reaction provide the anchoring site) can be synthesized with various substituents, it is conceivable that customized attachment of enzymes and biochemical reagents could be designed.

Electrochemical study of d-glucose oxidase autoinactivation

The immobilization of a d-glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) monolayer onto a glassy carbon rotating disc electrode allows the measurement of concentrations in the enzymes microenvironment and, hence, gives a method of easily following its activity. As it functions, d-glucose oxidase undergoes an autoinactivation which is clearly distinct from inactivation by H2O2, and which is more severe as the concentration of the two substrates is increased. It appears that the number of catalytic cycles is fixed at 107, irrespective of the concentrations of the two substrates. Kinetically, it is the enzyme-substrate complex which seems to be inactivated. Scavengers of toxic species of O2 have no effect on the kinetics of autoinactivation. Identical results were found in solution.

An electrochemical approach of the redox behavior of water insoluble ubiquinones or plastoquinones incorporated in supported phospholipid layers.

Physiological mole fractions of long isoprenic chain ubiquinone (UQ[10]) and plastoquinone (PQ9) were incorporated inside a supported bilayer by vesicle fusion. The template of the bilayer was an especially designed microporous electrode that allows the direct electrochemistry of water insoluble molecules in a water environment. The artificial structure, made by self-assembly procedures, consisted of a bilayer laterally in contact with a built-in gold electrode at which direct electron transfers between the redox heads of the quinones molecules and the electrode can proceed. The mass balances of quinone and lipid in the structure were determined by radiolabeling and spectrophotometry. A dimyristoyl phosphatdylcholine stable surface concentration of 250 +/- 50 pmol x cm(-2), unaffected by the presence of the quinone, was measured in the fluid monolayer. The mole fraction of quinone was between 1 and 3 mol%, remaining unchanged when going from the vesicles to the supported layers. The lipid molecules and the quinone pool were both laterally mobile, and cyclic voltammetry was used to investigate the redox properties of UQ10 and PQ9 over a wide pH range. Below pH 12, the two electrons-two protons electrochemical process at the gold electrode appeared under kinetic control. Thus all thermodynamic deductions must be anchored in the observed reversibility of the quinone/hydroquinol anion transformation at pH > 13. Within the experimental uncertainty, the standard potentials and the pK(a)s of the pertinent redox forms of UQ10 and PQ9 were found to be essentially identical. This differs slightly from the literature in which the constants were deduced from the studies of model quinones in mixed solvents or of isoprenic quinones without a lipidic environment.

Automatic apparatus for heterogeneous enzyme immunoassays based on electrocatalytic detection of the enzyme and electrochemical regeneration of the solid phase

Abstract A glassy carbon electrode, anodized for a short time at about 2.2 V vs. SCE, is a very convenient regenerable solid phase for the successive stages of an automated enzyme-linked immunosorbent assay. The anodized surface adsorbs the primary antibody and the blocking proteins in a similar manner to classical polystyrene solid phases; the detection of the immobilized glucose oxidase used as the label can be obtained from an electrocatalytic process occurring directly on the electrode; and the protein stacking, accumulated during the assay, can be eliminated by a simple re-anodization at the end of the cycle, allowing the adsorption of a new primary antibody. An automatic apparatus has been developed round an electrochemical flow cell fed with samples and reagents according to the principles of flow-injection analysis. Using rabbit IgG as analyte and glucose oxidase as label, series of 150 successive assays were conducted on the same electrode before it was polished again. The overall time of one assay, including the time for primary antibody adsorption and electrode regeneration, was 55 min.

Immobilization of glucose oxidase on carbon electrodes

Abstract The procedures tor glucose oxidase immobilization based on simple adsorption on pyrolytic graphite or on covalent attachment on glassy carbon using the carbodiimide activation method are compared. The catalytic current produced in the enzymatic oxidation of glucose is non-persistent when using the pyrolytic graphite electrode modified by the adsorption of glucose oxidase. In the case of glassy carbon electrodes, the successive steps (polishing, oxidation, activation, enzyme immobilization and detection) of the experimental procedure have been examined using electrochemical (cyclic and differential pulse voltammetry, controlled-potential amperometry) techniques. The influence of several parameters (incubation time, concentration, pH, temperature) on the electrode response has been investigated.

Construction of multicomponent catalytic films based on avidin-biotin technology for the electroenzymatic oxidation of molecular hydrogen

Two methods based on the avidin-biotin technology were developed for the multimonolayer immobilization of Desulfovibrio gigas hydrogenase on glassy carbon or gold electrodes. In both methods the molecular structure of the modified interface was the result of a step-by-step process. The first method alternates monolayers of avidin and biotinylated hydrogenase, the mediator (methyl viologen) being free to diffuse in the structure. In the second method, the avidin monolayers were used to immobilize both the biotinylated enzyme and a long-chain biotinylated viologen derivative. The viologen head of this hydrophilic arm shuttles the electrons between the electrode and the enzyme. The modified electrodes were evaluated for the electroenzymatic oxidation of molecular hydrogen, which has interest for the development of enzymatic fuel cells. The parameters that affect the current density of mediated oxidation of H(2) at the modified electrodes was studied. The second structure, which has given typical catalytic currents of 25 microA per cm(2) for 10 monolayers, was found clearly less efficient than the first structure (500 microA per cm(2) for 10 monolayers). In both methods the catalytic currents increased linearly with the number of monolayers of hydrogenase immobilized, which indicates that the multilayer structures are spatially ordered.

Direct electrochemical determination of glucose oxidase in biological samples

An electrochemical method for the quantitation of glucose oxidase in murine plasma and tissues has been developed. Instead of oxygen, this method uses benzoquinone as an artificial cosubstrate of glucose oxidase. The quantitative detection of the enzymatically produced hydroquinone by controlled-potential amperometry allows measurement of glucose oxidase concentrations in biological samples. The use of an internal standard corrects for all possible interfering effects. We demonstrated a 10-fold increase in sensitivity, as well as the ability to work in turbid media, in comparison to spectrophotometric methods.

Electrochemical regeneration of NAD+. A new evaluation of its actual yield

Owing to its actual yield being better than 99.99%, i.e. a coenzyme turnover number > 10 000, the electrochemical oxidation of NADH must be considered as one of the best methods of regeneration of enzymatically active NAD+ in terms of efficiency. Owing to its intrinsic simplicity of execution it could be very useful in the development of selective oxidations involving dehydrogenases. The sole cause of deactivation of the coenzyme is the acid-catalysed hydrolysis of NADH, a reaction not related to the method of regeneration.

A heterogeneous immunoassay performed on a rotating carbon disk electrode with electrocatalytic detection mass transfer control of the capture of an enterotoxin

An ELISA procedure for the determination of enterotoxin A from Staphylococcus aureus conducted on the surface of a glassy carbon electrode is described. The electrocatalytic detection of the immobilized labelled second antibody is based upon the electrochemical reaction and the enzymatic catalysis occurring on the same surface. The indirect quantification of the bound antigen is, therefore, very sensitive (10(-15) mol cm-2). This heterogeneous technique was used to study the kinetics of antigen binding to the immunological solid phase, the mass transfer of the antigen being controlled under well-defined hydrodynamic conditions. The experiments were performed with a rotating solid phase disk in such a way that thickness of the diffusion layer was known. We found that the capture of the antigen by the immobilized monoclonal antibody was solely limited by diffusion. A simple theoretical model permitted the amount of bound antigen and the sensitivity of the method to be predicted as a function of the incubation time, the rotational speed of the solid phase and the volume of the sample. Both the theory and the experimental results indicate that the assay may be performed with the sample volume undefined.

Kinetics of redox conversion at a gold electrode of water-insoluble ubiquinone (UQ(10)) and plastoquinone (PQ(9)) incorporated in supported phospholipid layers

The cyclic voltammetry of UQ(10) and PQ(9) incorporated in a supported lipid bilayer and reacting at a gold electrode is quantitatively analyzed assuming that the electrochemical process consists of a nine-member square scheme and using the approach developed earlier by Laviron. The striking change affecting the shape of the cathodic peak in the cyclic voltammograms when pH is increased from less than 7.5 to higher values reflects a shift from kinetic control of the cathodic process by the equivalent first electron exchange to kinetic control by the equivalent second electron exchange. Reasonable values are found for the thermodynamic and kinetic characteristics of the electrochemical process.