FR2656423A1 - Electrochemical biosensor - Google Patents

Electrochemical biosensor Download PDF

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FR2656423A1
FR2656423A1 FR8917069A FR8917069A FR2656423A1 FR 2656423 A1 FR2656423 A1 FR 2656423A1 FR 8917069 A FR8917069 A FR 8917069A FR 8917069 A FR8917069 A FR 8917069A FR 2656423 A1 FR2656423 A1 FR 2656423A1
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biosensor
electrode
polymer particles
biosensor according
anode
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Bardeletti Gilbert
D Urso Edith
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Rhodia Chimie SAS
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Rhone Poulenc Chimie SA
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001Enzyme electrodes
    • C12Q1/002Electrode membranes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087Acrylic polymers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001Enzyme electrodes

Abstract

Electrochemical biosensor consisting of a single-piece electrode comprising an anode and a cathode; a cap provided with a central perforation and a neutral membrane permitting the diffusion of substrate is attached to the end of the sensor; the biosensor contains, in the space available between the cap and the body of the electrode and adjacent to the working electrode, enzymes immobilised on particles of a polymer which is non-soluble in the reaction medium. Use for the assay of organic substrates liberating hydrogen peroxide by enzymatic reaction.

Description

BIOCAPTEUR ELECTROCHIMIQUE
La présente invention a pour objet un biocapteur constitué d'une électrode enzymatique et son application pour le dosage de substrats organiques.
ELECTROCHEMICAL BIOSENSOR
The present invention relates to a biosensor consisting of an enzymatic electrode and its application for the determination of organic substrates.

I1 est connu de doser le glucose par exemple à l'aide d'un capteur ampérométrique à H202 plongé dans une cuve dans laquelle est réalisée l'oxydation enzymatique du glucose en acide gluconique avec libération de
H202 ; la quantité de H202 formée est directement proportionnelle à la quantité de glucose mise en oeuvre. L'H202 est détectée ampérométri quementv : une différence de potentiel (ddp) fixe, d'environ 600 mV, entre une électrode de travail (platine) et une électrode de référence (Ag/AgCl) permet l'oxydation électrochimique de H202 sur l'anode de platine ; une variation de courant anodique proportionnelle à la concentration d'H202 formée par la réaction enzymatique peut être ainsi mise en évidence sur un enregistreur.Une telle méthode faisant intervenir un enzyme relativement peu fragile comme la glucose oxydase est particulièrement performante.
I1 is known to measure glucose for example using an amperometric sensor to H2O2 immersed in a tank in which is carried out the enzymatic oxidation of glucose to gluconic acid with release of
H2O2; the amount of H2O2 formed is directly proportional to the amount of glucose used. H202 is detected amperometrically v: a fixed potential difference (ddp), of approximately 600 mV, between a working electrode (platinum) and a reference electrode (Ag / AgCl) allows the electrochemical oxidation of H202 on l 'platinum anode; a variation in anode current proportional to the concentration of H 2 O 2 formed by the enzymatic reaction can thus be demonstrated on a recorder. Such a method involving a relatively not very fragile enzyme such as glucose oxidase is particularly effective.

Par contre celle-ci est mal adaptée aux dosages faisant intervenir des enzymes fragiles entraînant des phénomènes d'inactivation ou d'interaction entre substrat et produit de la réaction enzymatique. I1 en est ainsi du dosage du pyruvate à l'aide de la pyruvate oxydase. En effet il a été constaté que le substrat (pyruvate) et l'eau oxygénée formée interagissent avec formation d'un peracide.

Figure img00010001
By cons it is poorly suited to dosages involving fragile enzymes causing inactivation phenomena or interaction between substrate and product of the enzymatic reaction. This is the case with the determination of pyruvate using pyruvate oxidase. Indeed it has been found that the substrate (pyruvate) and the hydrogen peroxide formed interact with the formation of a peracid.
Figure img00010001

Si la pyruvate oxydase est introduite dans la cuve de mesure, l'eau oxygénée formée réagit rapidement avec le pyruvate ; il en résulte une diminution de la concentration des deux espèces dans la cuve ; le dosage est alors inexact.If pyruvate oxidase is introduced into the measuring tank, the hydrogen peroxide formed reacts quickly with the pyruvate; this results in a decrease in the concentration of the two species in the tank; the dosage is then inaccurate.

La demanderesse a résolu ce problème en introduisant les enzymes à l'intérieur du capteur électrochimique. The Applicant has solved this problem by introducing the enzymes inside the electrochemical sensor.

Selon l'invention il s'agit d'un biocapteur électrochimique constitué d'une électrode monocorps comprenant une anode et une cathode, et se fixant à l'extrémité du capteur, d'un embout muni d'une perforation centrale et d'une membrane neutre permettant la diffusion du substrat, biocapteur caractérisé en ce qu'il renferme dans l'espace disponible entre l'embout et le corps de l'électrode et avoisinant l "'électrode de travail" des enzymes immobilisés sur des particules de polymère non soluble dans le milieu réactionnel.According to the invention, it is an electrochemical biosensor consisting of a single-body electrode comprising an anode and a cathode, and attaching to the end of the sensor, a nozzle provided with a central perforation and a neutral membrane allowing the diffusion of the substrate, biosensor characterized in that it contains in the space available between the tip and the body of the electrode and bordering the "working electrode" of the enzymes immobilized on particles of polymer not soluble in the reaction medium.

Le biocapteur de l'invention basé sur une détection ampérométrique est tout particulièrement intéressant pour le dosage de substrats organiques libérant par réaction enzymatique de l'eau oxygénée. L'"électrode de travail", l'anode, est constituée d'un disque de platine où se réalise la réaction électrochimique.The biosensor of the invention based on amperometric detection is very particularly advantageous for the determination of organic substrates releasing by enzymatic reaction of oxygenated water. The "working electrode", the anode, consists of a platinum disc where the electrochemical reaction takes place.

L'électrode de référence, la cathode, est constituée d'un fil spiralé d'argent recouvert de chlorure d'argent.The reference electrode, the cathode, consists of a silver spiral wire covered with silver chloride.

Un électrolyte présent dans l'espace entre l'embout et le corps de l'électrode assure la conductivité entre l'anode et la cathode ; la présence de chlorure de potassium dans l'électolyte assure la chloruration permanente de l'électrode de référence.An electrolyte present in the space between the tip and the body of the electrode ensures the conductivity between the anode and the cathode; the presence of potassium chloride in the electolyte ensures permanent chlorination of the reference electrode.

Le corps et l'embout de l'électrode sont de préférence en résine synthétique du type polychlorure de vinyle.The body and the tip of the electrode are preferably made of synthetic resin of the polyvinyl chloride type.

La membrane est neutre ; elle peut être constituée en fibres synthétiques (polyester, polyamide...) ; son épaisseur est généralement de l'ordre de 50 à 150 micromètres avec un diamètre de pores inférieur à celui des particules de polymère et généralement de l'ordre de 0,1 à 0,8 micromètres.The membrane is neutral; it can be made of synthetic fibers (polyester, polyamide ...); its thickness is generally of the order of 50 to 150 micrometers with a pore diameter smaller than that of the polymer particles and generally of the order of 0.1 to 0.8 micrometers.

Les polymères pouvant constituer les particules support dérivent de monomères non miscibles à l'eau (c'est-à-dire de solubilité dans l'eau inférieure à 5 % en poids).The polymers which can constitute the support particles are derived from water-immiscible monomers (that is to say solubility in water of less than 5% by weight).

Parmi ces monomères on peut citer
- les monomères vinylaromatiques (styrène, vinyltoluène...)
- les alkylesters d'acides a-ss insaturés (acrylates et méthacrylates de méthyle, éthyle...)
- les esters d'acides carboxyliques insaturés (acetate de vinyle...)
- le chlorure de vinyle ; le chlorure de vinylidène
- les diènes (butadiène...)
- ceux présentant des fonctions nitriles (acrylonitrile...)
- les siloxanes.
Among these monomers, mention may be made
- vinyl aromatic monomers (styrene, vinyltoluene ...)
- alkyl esters of unsaturated a-ss acids (methyl, ethyl acrylates and methacrylates ...)
- esters of unsaturated carboxylic acids (vinyl acetate, etc.)
- vinyl chloride; vinylidene chloride
- dienes (butadiene ...)
- those with nitrile functions (acrylonitrile ...)
- siloxanes.

La composition monomère dont dérive ledit polymère contient en outre jusqu'à 10 % de son poids (de préférence jusqu'à 4 % de son poids) d'au moins un monomère portant des groupes ionogènes ou réactifs tels que - S03H, - OSO3H, - NR3, - COOH, - OH, - NH2, - NR2,
CONH2, - SH,

Figure img00030001

- COOR, - PO(OR)2,
Figure img00030002

- # CH2C1,
R représentant un radical alkyle en Cl - C4, de préférence en Cl - C2
A titre d'exemple on peut citer
- le vinylbenzène sulfonate, les sulfoalkylesters d'acides insaturés (2-sulfoéthylméthacrylates...)
- les acides carboxyliques insaturés (acide acrylique, méthacrylique, maléfique, itaconique...)
- les hydroxyalkylacrylates ou méthacrylates (acrylate d'hydroxyéthyle, hydroxypropyle...)
- les aminoalkylesters d'acides insaturés (2-aminoéthylméthacrylate...)
- l'acrylamide
- le chlorure de vi nyl benzène
- le méthacrylate de glycidyle.The monomer composition from which said polymer is derived also contains up to 10% of its weight (preferably up to 4% of its weight) of at least one monomer carrying ionogenic or reactive groups such as - SO 3 H, - OSO 3 H, - NR3, - COOH, - OH, - NH2, - NR2,
CONH2, - SH,
Figure img00030001

- COOR, - PO (OR) 2,
Figure img00030002

- # CH2C1,
R representing a C1 - C4, preferably C1 - C2 alkyl radical
As an example we can cite
- vinylbenzene sulfonate, sulfoalkyl esters of unsaturated acids (2-sulfoethylmethacrylates ...)
- unsaturated carboxylic acids (acrylic, methacrylic, malefic, itaconic ...)
- hydroxyalkylacrylates or methacrylates (hydroxyethyl acrylate, hydroxypropyl ...)
- aminoalkylesters of unsaturated acids (2-aminoethylmethacrylate ...)
- acrylamide
- vi nyl benzene chloride
- glycidyl methacrylate.

Lesdites particules de polymère peuvent présenter une granulométrie de l'ordre de 0,2 à 20 microns et de préférence de l'ordre de 0,2 à 3 microns.Said polymer particles can have a particle size of the order of 0.2 to 20 microns and preferably of the order of 0.2 to 3 microns.

Une variante consiste à utiliser comme particules support des particules polymère non hydrosoluble magnétisable.A variant consists in using, as support particles, non-water-soluble magnetizable polymer particles.

Les particules de polymère magnétisable contiennent de 0,5 à 70 % en poids (de préférence de 5 à 50 %) d'une charge magnétique dont la taille est inférieure à l ian et de préférence comprise entre 0,002-0,02 ian ; la charge magnétique est bien évidemment suffisamment fine pour pouvoir être incluse dans les particules de polymère. Cette charge magnétique peut être constituée par exemple par
- des métaux ou leur alliages tels que : fer, fer-silicium, nickel, cobalt, samarium ou leurs alliages avec du molybdène, du chrome, du cuivre, du vanadium, du manganèse, de l'aluminium, du titane, neodyme...
The particles of magnetizable polymer contain from 0.5 to 70% by weight (preferably from 5 to 50%) of a magnetic charge whose size is less than 1 ian and preferably between 0.002-0.02 ian; the magnetic charge is obviously fine enough to be able to be included in the polymer particles. This magnetic charge can be constituted for example by
- metals or their alloys such as: iron, iron-silicon, nickel, cobalt, samarium or their alloys with molybdenum, chromium, copper, vanadium, manganese, aluminum, titanium, neodymium, etc. .

- des oxydes de fer : Fe304 ou y-Fe03 pur ou en combinaison ou en mélange avec d'autres oxydes comme les oxydes de cobalt, manganèse, zinc, baryum, terres rares
- du dioxyde de chrome.
- iron oxides: Fe304 or y-Fe03 pure or in combination or in mixture with other oxides such as cobalt, manganese, zinc, barium, rare earth oxides
- chromium dioxide.

Les particules de polymère magnétisable peuvent être obtenues sous forme de latex selon des procédés connus par exemple selon les procédés décrits dans le brevet européen ne 38.730 ou le brevet américain n 4.157.323.The particles of magnetizable polymer can be obtained in the form of latex according to known methods for example according to the methods described in European patent no 38,730 or American patent n 4,157,323.

L'immobilisation des enzymes sur les particules de polymère magnétisable ou non magnétisable peut être réalisée par adsorption ou bien par couplage, la réaction de couplage faisant intervenir les groupes superficiels réactifs ou fonctionnels du polymère et les groupements fonctionnels de l'enzyme à fixer.The immobilization of the enzymes on the particles of magnetizable or non-magnetizable polymer can be carried out by adsorption or else by coupling, the coupling reaction involving the reactive or functional surface groups of the polymer and the functional groups of the enzyme to be fixed.

La réaction de couplage peut être réalisée selon des méthodes bien connues par exemple en faisant appel le plus souvent à l'activation des fonctions du polymère en utilisantdes agents de couplage (diazotation, bromure de cyanogène, chlorure de tosyle, glutaraldehyde, carbodiimide hydrosoluble,
N-hydroxybenzotriazole...) avec ou sans l'utilisation de spacers du type 1-6 diaminohexane, polysaccharide..., puis réaction avec l'enzyme à fixer.
The coupling reaction can be carried out according to well-known methods, for example by most often calling on the activation of the polymer functions using coupling agents (diazotization, cyanogen bromide, tosyl chloride, glutaraldehyde, water-soluble carbodiimide,
N-hydroxybenzotriazole ...) with or without the use of spacers of type 1-6 diaminohexane, polysaccharide ..., then reaction with the enzyme to be fixed.

Des exemples de réactions de couplage sont donnés dans les brevets américains ne 3.857.931, 4.045.384, 4.140.662, 4.046.723, 4.421.896, anglais n0 2.004.892, français n" 2.331.567, 2.345.459, 2.378.094, européen ne 15.841...Examples of coupling reactions are given in the US patents no 3,857,931, 4,045,384, 4,140,662, 4,046,723, 4,421,896, English no. 2,004,892, French no. 2,333,567, 2,345,459, 2,378,094, European no 15,841 ...

Le protocole d'immobilisation doit être adapté en fonction des caractéristiques propres de l'enzyme utilisé (pH optimum, activité spécifique...)
La quantité d'enzyme immobilisée sur les particules de polymère est généralement de l'ordre de 10 à 500 unités d'enzyme par gramme de polymère (de préférence de 100 à 200).
The immobilization protocol must be adapted according to the specific characteristics of the enzyme used (optimum pH, specific activity, etc.)
The amount of enzyme immobilized on the polymer particles is generally of the order of 10 to 500 units of enzyme per gram of polymer (preferably from 100 to 200).

Le biocapteur faisant l'objet de l'invention étant particulièrement intéressant pour les dosages de substrats organiques libérant par réaction enzymatique de l'eau oxygénée, les enzymes mis en oeuvre sont en particulier les oxydases comme par exemple
- la lactate oxydase pour doser le L-lactate
- la glucose oxydase pour doser le glucose
- la pyruvate oxydase pour doser le pyruvate
- l'alcool oxydase pour doser les alcools courts (methanol, ethanol, propanol...)
Lors de son utilisation, le biocapteur relié à un polarostat imposant une différence de potentiel entre l'anode et la cathode, est plongé dans une cuve thermostatée (généralement vers 30') contenant le substrat à doser.
The biosensor forming the subject of the invention being particularly advantageous for the assays of organic substrates releasing by enzymatic reaction of oxygenated water, the enzymes used are in particular the oxidases such as for example
- lactate oxidase to measure L-lactate
- glucose oxidase to measure glucose
- pyruvate oxidase to dose pyruvate
- alcohol oxidase to measure short alcohols (methanol, ethanol, propanol ...)
During its use, the biosensor connected to a polarostat imposing a potential difference between the anode and the cathode, is immersed in a thermostated tank (generally around 30 ') containing the substrate to be assayed.

Les enzymes immobilisés à l'intérieur du biocapteur ne pouvant diffuser à l'extérieur de l'électrode, l'eau oxygénée se forme au voisinage de l'anode.Since the enzymes immobilized inside the biosensor cannot diffuse outside the electrode, hydrogen peroxide is formed in the vicinity of the anode.

Dans le cas du pyruvate le schéma réactionnel est le suivant pyruvate+O2+phosphate

Figure img00050001
In the case of pyruvate the reaction scheme is as follows pyruvate + O2 + phosphate
Figure img00050001

<tb> pyruvate <SEP> oxydase,
<tb> acetylphosphate+C02+H202
La quantité de H202 formée est directement proportionnelle à celle du pyruvate présent.
<tb> pyruvate <SEP> oxidase,
<tb> acetylphosphate + C02 + H202
The amount of H2O2 formed is directly proportional to that of the pyruvate present.

L'eau oxygénée, au fur et à mesure de sa formation, est oxydée à l'anode selon la réaction
ddp (+ 500 mV) H2O2

Figure img00050002

02 + 2H+ + 2e
Ag/Ag Cl/C1
Un enregistreur relié au polarostat permet de lire la variation du courant en fonction du temps.Oxygenated water, as it is formed, is oxidized at the anode according to the reaction
ddp (+ 500 mV) H2O2
Figure img00050002

02 + 2H + + 2nd
Ag / Ag Cl / C1
A recorder connected to the polarostat makes it possible to read the variation in current as a function of time.

La même opération réalisée avec un biocapteur classique (ne renfermant pas l'enzyme immobilisé) plongé dans une cuve contenant du pyruvate et de la pyruvate oxydase immobilisée sur des particules de polymère ne peut conduire à un dosage satisfaisant du substrat car ce dernier intéragit sur l'eau oxygénée formée.The same operation carried out with a conventional biosensor (not containing the immobilized enzyme) immersed in a tank containing pyruvate and pyruvate oxidase immobilized on polymer particles cannot lead to a satisfactory assay of the substrate because the latter interacts on the hydrogen peroxide formed.

La mise en oeuvre de ce biocapteur est aisée et rapide ; celui-ci peut être réutilisé. The implementation of this biosensor is easy and rapid; it can be reused.

Claims (8)

REVENDICATIONS 1) Biocapteur électrochimique constitué d'une électrode monocorps comprenant une anode, une cathode, et se fixant à l'extrémité du capteur, d'un embout muni d'une perforation centrale et d'une membrane neutre permettant la diffusion du substrat, biocapteur caractérisé en ce qu'il renferme dans l'espace disponible entre l'embout et le corps de l'électrode et avoisinant l'électrode de travail des enzymes immobilisés sur des particules de polymère non soluble dans le milieu réactionnel.1) Electrochemical biosensor consisting of a single-body electrode comprising an anode, a cathode, and attaching to the end of the sensor, a nozzle provided with a central perforation and a neutral membrane allowing the diffusion of the substrate, biosensor characterized in that it contains in the space available between the tip and the body of the electrode and surrounding the working electrode of the enzymes immobilized on polymer particles not soluble in the reaction medium. 2) Biocapteur selon la revendication 1 caractérisé en ce que l'électrode de travail est l'anode et en ce qu'elle est constituée d'un disque de platine.2) Biosensor according to claim 1 characterized in that the working electrode is the anode and in that it consists of a platinum disc. 3) Biocapteur selon la revendication 1 ou 2 caractérisé en ce que l'électrode de référence est la cathode et en ce qu'elle est constituée d'un fil d'argent recouvert de chlorure d'argent.3) Biosensor according to claim 1 or 2 characterized in that the reference electrode is the cathode and in that it consists of a silver wire covered with silver chloride. 4) Biocapteur selon l'une quelconque des revendications précédentes caractérisé en ce que les particules de polymère sur lesquelles sont immobilisés les enzymes dérivent d'une composition monomère non miscible à l'eau contenant jusqu'à 10 % de son poids d'au moins un monomère portant des groupes ionogènes ou réactifs.4) Biosensor according to any one of the preceding claims, characterized in that the polymer particles on which the enzymes are immobilized derive from a water-immiscible monomer composition containing up to 10% by weight of at least a monomer carrying ionogenic or reactive groups. 5) Biocapteur selon la revendication 4 caractérisé en ce que les particules de polymère ont une granulométrie de l'ordre de 0,2 à 20 microns.5) Biosensor according to claim 4 characterized in that the polymer particles have a particle size of the order of 0.2 to 20 microns. 6) Biocapteur selon la revendication 5 caractérisé en ce que les particules de polymère contiennent de 0,5 à 70 % de leur poids d'une charge magnétisable.6) Biosensor according to claim 5 characterized in that the polymer particles contain from 0.5 to 70% of their weight of a magnetizable charge. 7) Biocapteur selon l'une quelconque des revendications précédentes caractérisé en ce que la quantité d'enzyme immobilisée sur les particules de polymère estde l'ordre de 10 à 500 unités d'enzyme par gramme de polymère.7) Biosensor according to any one of the preceding claims, characterized in that the quantity of enzyme immobilized on the polymer particles is of the order of 10 to 500 units of enzyme per gram of polymer. 8) Utilisation du biocapteur faisant l'objet de l'une quelconque des revendications précédentes pour le dosage de substrats organiques libérant par réaction enzymatique de l'eau oxygénée. 8) Use of the biosensor which is the subject of any one of the preceding claims for the determination of organic substrates releasing by enzymatic reaction of hydrogen peroxide.
FR8917069A 1989-12-22 1989-12-22 Electrochemical biosensor Withdrawn FR2656423A1 (en)

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WO2001006245A1 (en) * 1999-07-15 2001-01-25 Iit Limited Polymeric compositions and sensor devices
EP1011425A4 (en) * 1997-03-04 2004-06-16 Dexcom Inc Device and method for determining analyte levels
US6862465B2 (en) 1997-03-04 2005-03-01 Dexcom, Inc. Device and method for determining analyte levels
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