Guy Fortier

Electrochemistry of the polypyrrole glucose oxidase electrode

Abstract A polypyrrole-glucose oxidase electrode prepared by anodic polymerization of pyrrole and glucose oxidase with KCl aqueous electrolyte on a platinum electrode showed redox behaviour in aqueous pure supporting electrolyte. The electrochemistry of the polypyrrole-glucose oxidase electrode resembled closely that of a polypyrrole electrode prepared from an aqueous solution containing only pyrrole and KC1. The amperometric response to glucose was generated by electrochemical oxidation of enzymatically produced hydrogen peroxide and was observed only after the loss of polypyrrole electroactivity. The results presented in this paper indicate that polypyrrole served only as an immobilization matrix to glucose oxidase in the polypyrrole-glucose oxidase electrode.

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of +0.65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentiostating the enzyme electrode at a potential of +0.7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determine the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0.3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0.01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0.17 microns (75 mC/cm2) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1.0 mM to 7.5 mM glucose and kinetic parameters determined for the optimized biosensors were 33.4 mM for the Km and 7.2 microA for the Imax. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.

Development of Biosensors Based on Immobilization of Enzymes in Eastman AQ Polymer Coated with a Layer of Nafion

Abstract A fast and simple procedure to prepare enzyme electrode is presented herein. A blend of amorphous polyester cationic exchangers (AQ 29D:AQ 55D; ratio 1:1) dispersed in water has been used for the immobilization of l-amino acid, choline, galactose or glucose oxidase enzymes at the surface of a platinum electrode. The resulting polymer-enzyme film was covered by a thin layer of Nafion to avoid its subsequent dissolution in water. The assays are based on the electrochemical detection of enzymatically generated hydrogen peroxide. Good amperometric responses were obtained with each of these enzyme electrodes. The major advantage in using this water dispersed polymer lies in its ability to dissolve the enzyme without any significative loss of enzymatic activity.

Immobilization of native and poly(ethylene glycol)‐treated (‘PEGylated’) bovine serum amine oxidase into a biocompatible hydrogel

Native bovine serum amine oxidase (BSAO) and poly(ethylene glycol) (PEG)‐treated (‘PEGylated’) BSAO were immobilized into a hydrogel during its synthesis. The hydrogel was obtained by cross‐linking of BSA with PEG di‐nitrophenyl carbonates with a molecular mass of 10 kDa. Approx. 60% of the amino groups at the surface of BSAO were modified by monomethoxy‐PEG with a molecular mass of 5 kDa when the reaction was carried out for 5 h in borate buffer, pH 9. The number of anchorage points of BSAO in the matrix, which was determined as minimal when PEGylated BSAO was used or maximal when native BSAO was used, did not influence the apparent Km and Vmax values of the different preparations. The apparent Km values of both forms of the enzyme were decreased due to preconcentration of benzylamine substrate by the negatively charged hydrogel. Vmax values were generally lower upon immobilization. We can therefore conclude that the hydrogel swelling has no significant effect on the enzymes structure. The operational stability, evaluated in the presence of substrate, was generally increased upon enzyme immobilization into the hydrogel. Enzymic hydrogels were very stable during storage in solution at 4 °C, maintaining a high activity even after several weeks. The immobilization of both forms of BSAO did not improve their thermostability at 65 °C. The BSA‐PEG hydrogel is a good matrix for immobilization of enzymes with therapeutic potential such as BSAO.

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°, 60°, and 65°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.

Poly(Ethylene Glycol)-Serum Albumin Hydrogel as Matrix for Enzyme Immobilization: Biomedical Applications

Poly(ethylene glycol)-albumin hydrogels were implanted in mice in subcutaneous position to study their biocompatibility. After one month of implantation, the fibrous capsule formed around the implant was thin and the inflammatory tissue was limited. Acid phosphatase (AP) was selected to evaluate the hydrogel as matrix for enzyme immobilization. AP-hydrogels were prepared using activated PEG (PEGa) of different molecular weights (M.W. 4,600 to 20,000) to evaluate the effect of the matrix composition on the activity of AP. The apparent Km of the immobilized AP was 16 to 20 times higher than the Km of the soluble enzyme. The apparent Km value decreases with the increase of the chain length of the PEGa used. This can be correlated to an increase in the hydrogel porosity. The operational stability of the AP was markedly improved after immobilization by 110 to 160 times according to the PEGa molecular weight involved. Also, asparaginase (ASNase) was immobilized in PEGa (M.W. 10,000)-albumin-hydrogel as a model for in vivo bioreactor. ASNase hydrogels were implanted in the peritoneal cavity of rats; 7 days later, 75% of the initial enzyme activity were retrieved.

Electrochemical and Enzymatic Studies of Electron Transfer Mediation by Ferrocene Derivatives with Nafion-Glucose Oxidase Electrodes

Nafion-glucose oxidase-ferrocene electrodes have been used to study the electron transfer between an electrode and the active site of the enzyme. These electrodes were prepared by casting a mixture of Nafion and glucose oxidase, GOx, (both dissolved in methanol) at the surface of a platinum disk electrode. The ferrocene mediators were incorporated by either soaking Pt/Nafion and Pt/Nafion-GOx electrodes in an aqueous solutions of N′-dimethylaminomethylferrocene, DMAFc, or by depositing an aliquot of a ferrocene solution at the surface of the enzyme film electrode. The Nafion-GOx electrodes are characterized by a slower incorporation rate of the ferrocene than the plain Nafion electrode indicating that the affinity of Nafion for the ferrocene derivative is increased by the addition of the enzyme. Accordingly, the ion-exchange distribution coefficient is larger for plain Nafion (3.2×103) than for Nafion-GOx (7.2×102). The cyclic voltammetry of the Pt/Nafion-GOx electrode in the presence of glucose and in deaerated solution show a well developed catalytic wave indicating that the ferrocene acts as a mediator for the electron transfer between the enzyme and the platinum electrode. For a high glucose concentration (75 mM), the amperometric response of these electrodes increased and the apparent diffusion coefficient DMAFc decreased with an increase of DMAFc concentration in the enzymatic layer. These observations suggest that the glucose response is limited by the probability of an encounter between the reduced enzyme and the oxidized ferrocene rather than by the charge transfer between the reduced ferrocene and the electrode. The effective Michaelis constant, Km , for glucose and DMAFc were evaluated and values of 25 mM and 4 nmol/cm were obtained, respectively.

Fast and easy preparation of an amperometric glucose biosensor

Electrochemical polymerization of pyrrole in aqueous KCl solution containing glucose oxidase produces adherent films at platinum electrode surface. Such coated electrodes are prepared in 20 min and can determine glucose in the range 0 to 100mm. The useful lifetime of the electrode is 85 assays. Its stability is at least 75 days under storage in PBS at 4°C.

Enhancement of response by incorporation of platinum microparticles into a polypyrrole-glucose oxidase electrode

Abstract Platinum was incorporated into a polypyrrole/glucose oxidase electrode by immersion in a hexachloroplatinate(IV) solution after electrochemical oxidation of the polypyrrole film; dispersed platinum was then formed by electrochemical reduction. The platinized electrode reproducibly yielded a response to glucose (20 mM) which was typically about 40% higher than that obtained in the absence of platinum microparticles in the polypyrrole/glucose oxidase film.

New Bioartificial Polymeric Material: Poly(ethylene glycol) Cross-Linked with Albumin. I. Synthesis and Swelling Properties

A new family of translucide hydrogels obtained by cross-linking of bifunctionalized poly(ethylene glycol) (PEG) of various molecular masses (M, 2,000 to 35,000) with an albumin protein, namely bovine serum albumin (BSA), is described. The composition and the structure of these hydrogels have been investigated on the basis of the molar ratio of reagents, and through measurements of PEG and BSA released during washing and swelling steps. It appears that an excess of activated PEG is required in the reaction mixture to allow the gel formation with PEG of molecular mass ranging from 3,350 to 10,000. Above these Mr of PEG, smaller amounts of PEG are needed to carry out the gel formation. Experimental results suggest that the cross-link density decreases when the molecular mass of PEG increases. These hydrogels are characterized by a very high swelling ability with equilibrium water contents (EWC) ranging from 96.6 to 97.5% in distilled water containing NaN3 at 0.02% (w/v). The swelling factors (SF) ranged from 14 to 40 according to the incuba tion medium and to the PEG molecular mass involved. The swelling rates increased with increasing PEG molecular mass used to synthesize the hy drogel. This family of hydrogels possesses a good porosity since diffusion of bovine serum albumin out of the hydrogel network was observed in a long-term period.