WO2001077656A1 - Biosensor and method for making same - Google Patents

Biosensor and method for making same Download PDF

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Publication number
WO2001077656A1
WO2001077656A1 PCT/FR2001/001133 FR0101133W WO0177656A1 WO 2001077656 A1 WO2001077656 A1 WO 2001077656A1 FR 0101133 W FR0101133 W FR 0101133W WO 0177656 A1 WO0177656 A1 WO 0177656A1
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Prior art keywords
bio
molecule
sensor
solution
electrode
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PCT/FR2001/001133
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French (fr)
Inventor
Guillaume Herlem
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Guillaume Herlem
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Priority to AU52327/01A priority Critical patent/AU5232701A/en
Publication of WO2001077656A1 publication Critical patent/WO2001077656A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood

Definitions

  • the invention relates to a method of manufacturing a bio-sensor of a specific molecule present in an analyte, said bio-sensor, of potentiometric type, comprising at least one reference electrode and at least one measurement electrode. of said molecule, said reference electrode and said measurement electrode each being covered by a polymeric coating, said measurement electrode further comprising an enzyme reacting specifically with said molecule to be detected.
  • the invention also relates to a bio-sensor resulting from such a process.
  • the invention relates to the field of intended bio-sensors capable of detecting a specific molecule within an analyte.
  • the invention proposes to respond to the concern of these people through a potentiometric bio-sensor capable of detecting, on the surface of the skin, in saliva or in urine for example, the presence of a substance determined, its proportion and its evolution over time.
  • a bio-sensor comprising a reference electrode and a measurement electrode, the latter being covered with a mixture of an oxidation-reduction enzyme. and an electron acceptor, this bio-sensor being used for the detection of albumin.
  • the measured values depend on the variation of the pH in a given sample.
  • a biosensor intended for measuring fructose in food and the measuring electrode is prepared from the enzyme fructose-dehydrogenas (FDH) in a pH 5 buffer containing a surfactant.
  • FDH fructose-dehydrogenas
  • a drying operation takes place under reduced pressure, between 3 and 8 ° C.
  • An aqueous solution of polyethylene imine (PEI) at pH 5 is then added, before another drying operation takes place between 3 and 8 ° C.
  • Mixing is then carried out with graphite at a temperature of approximately 650 ° C. for one minute and paraffin oil is added to form a paste from which the electrode is wetted.
  • DE-A-29 620 371 and DE-A-106 39 224 also disclose an optical device for measuring the blood sugar level, which is integrated in a watch.
  • the display device can be connected either to the measuring device or to the watch mechanism by pressing a button.
  • the device is more particularly intended for diabetic people who have to control their sugar level several times a day. It will be observed that in the context of this state of the art, a certain number of bio-sensors of the amperometric type are also known, requiring the application of a constant potential, between the electrode for detecting an analyte. and an auxiliary electrode or counter electrode, for measuring the current response of the electrochemical transformation, in particular of glucose into gluconic acid plus hydrogen peroxide.
  • the document US Pat. No. 5,683,563 relates to an amperometric bio-sensor, trapping in its polymer matrix on the surface of an electrode, the suitable enzyme: lactase oxidase.
  • the polymer can be polyethyleneimine synthesized organically and deposited by simple spreading on an electrode surface.
  • the present invention is intended to provide a solution to the problem of manufacturing, at a reduced cost price while being very reliable, of a potentiometric biosensor capable of detecting, with precision and very great linearity, the presence, within an analyte, of a specific molecule through a pH measurement.
  • the invention relates to a method of manufacturing a bio-sensor of at least one molecule present in an analyte, said potentiometric bio-sensor comprising at least a reference electrode and at least one measurement electrode made of an electrically conductive or semiconductor material and each covered with a polymeric coating, said measurement electrode also carrying an enzyme capable of reacting specifically with said molecule to be detected, characterized in that:
  • said polymeric coating is obtained on said reference and measurement electrodes by electrochemical means, by soaking at least partially said reference and measurement electrodes in a chemical solution based on at least one saturated aliphatic diamine and by bringing to a potential sufficient to reach that corresponding to the electrochemical oxidation of said chemical solution;
  • the chemical solution is a pure saturated aliphatic diamine or else a mixture of pure saturated aliphatic diamines, knowing that it can also be in the form of one or of a mixture of these diamines in solution in a solvent more difficult to oxidize electrochemically than the diamine or diamines contained in this solution.
  • the invention also relates to a bio-sensor conforming to this manufacturing process and comprising at least one reference electrode and at least one measurement electrode made of an electrically conductive or semiconductor material and each covered with a coating.
  • polymer said measuring electrode carrying, again, an enzyme capable of reacting specifically with said molecule to be detected, characterized in that it comprises a processing unit provided capable of measuring a potential difference across the reference and measuring electrodes and to transmit, by means of visual and / or audible signaling means, clear interpretation information concerning the specific molecule or molecules detected through said measurement of potential difference.
  • this bio-sensor can be integrated into the case of a wristwatch.
  • FIG. 1 is a schematic representation of the operating principle of the bio-sensor, therefore of the electrochemical capture at the electrode / sample interface for the detection of a specific molecule A.
  • FIG. 2 illustrates, diagrammatically, a bio-sensor comprising a reference electrode and several measurement electrodes.
  • Figure 3 is a schematic representation of a bio-sensor according to the invention.
  • the present invention relates, in particular, to the field of potentiometric bio-sensors and to their manufacturing process.
  • the invention relates to a potentiometric biosensor of at least one molecule present within an analyte.
  • such a bio-sensor 1 comprises at least one reference electrode 2 and at least one measurement electrode 3.
  • a bio- sensor comprising several measurement electrodes 3A, 3B, 3C, 3D capable, possibly, of detecting, each, a different molecule.
  • These reference 2 and measurement 3 electrodes are made of an electrically conductive or semiconductor material and are each covered with a polymeric coating 4, said measurement electrode 3 also carrying an enzyme reacting specifically with said molecule to be detected.
  • the polymeric coating 4 at the electrodes 2, 3 is obtained by electrochemical means.
  • said electrodes 2, 3 are at least partially soaked in a chemical solution based on at least one saturated aliphatic diamine and these electrodes are brought to a sufficient potential to reach that corresponding to the electrochemical oxidation of said solution. chemical.
  • Figure 1 the principle of electrochemical capture at the electrode-sample interface is illustrated for the detection of a specific molecule, marked A.
  • the solution in which the electrodes 2, 3 are soaked to obtain the polymeric coating by electrochemical means it may be composed of one or more pure saturated aliphatic diamines or in solutions in a solvent more difficult to oxidize electrochemically than this or these diamines.
  • this or these diamines are characterized in that they comprise at least two primary amine functions having, in each of them, an alkyl group.
  • this chemical solution is based on ethylenediamine or else on diethylenetriamine or even a solution of one and / or the other of these components, for obtaining a layer of linear polyethyleneimine as a polymeric coating. .
  • this chemical solution is based on another saturated, non-aromatic aliphatic diamine, 1, 3-diaminopropane for obtaining, as polymeric coating after anodic oxidation, polypropylenimine.
  • the modification of the polymeric coating 4 finally obtained by an enzyme reacting specifically with the molecule to be detected may result from a simple operation of quenching said measurement electrode 3 in a solution of said enzyme until impregnation of this polymeric coating 4, just as it can come from the deposition of said enzyme, in gel form, on this polymeric coating 4.
  • the polymeric coating 4 of several measurement electrodes 3, 3A, 3B, 3C, 3D is modified by different enzymes reacting specifically, respectively, to one of the molecules to be detected.
  • the bio-sensor 1 also comprises a processing unit 5 to which these reference 2 and measurement 3 electrodes are connected and which is provided capable, on the one hand, of measuring a potential difference by through the latter and, on the other hand, to interpret the measurement (s) carried out with a view to transmitting information clearly understandable to a user by means of visual and / or sound signaling means 6, again connected to this processing unit 5.
  • a processing unit 5 to which these reference 2 and measurement 3 electrodes are connected and which is provided capable, on the one hand, of measuring a potential difference by through the latter and, on the other hand, to interpret the measurement (s) carried out with a view to transmitting information clearly understandable to a user by means of visual and / or sound signaling means 6, again connected to this processing unit 5.
  • Such visual signaling means 6 can be in the form of a display screen 7 capable of displaying, in the form of a biochemical value of interpretation accessible to the user, the potential differential measurement (s) performed by the processing unit 5 via the electrodes 2, 3.
  • this display screen 7 can be in the form of a graphic screen or the like.
  • such visual and / or audible signaling means 6 are provided capable of emitting visually identifiable signaling or by audible signal in the event of detection, via the processing unit 5, of exceeding one or more predetermined measurement thresholds, known in the memory of this processing unit 5.
  • these visual or audible signaling means 6 can be in the form of a simple indicator light or an activated buseur in the event that the potential differential measured by the processing unit 5 is expressed by a value biochemical above or below a normal value.
  • this biochemical value can correspond to the level of urea or to the level of sugar in the blood which, precisely, can be determined through the measurement of the pH of the skin and therefore of the sweat of the wearer of the bio-sensor 1.
  • this biosensor is in no way limited to the detection of the biochemical values which have just been mentioned.
  • the advantages which arise from such a potentiometric bio-sensor 1, designed according to the invention consist in that the polymeric coating 4 of the electrodes is very adherent. It has been observed that the adhesion of the polymeric coating 4 was further improved on conductive or semiconductor materials, composing these reference 2 and measurement 3 electrodes, such as platinum, gold, vitreous carbon, carbon fibers, silicon.
  • the present invention is however not limited to this enumeration of conductive or semiconductor materials capable of constituting said electrodes.
  • said polymeric coating is not toxic to humans, while being non-corrosive, whereas on the contrary and it should be remembered, the amines which are at the origin of this coating are.
  • Electrodes have, moreover, a potential which responds linearly to the pH of aqueous solutions, in a pH range from 2 to 13 approximately.
  • this response of the electrodes is not Nernstian, it is linear, with a variation of the order of 40 to 45 V per unit of pH at ordinary temperature.
  • a bio-sensor 1 in accordance with the invention, easily lends itself to miniaturization making it possible to envisage its integration, for the detection of substance in sweat, for example in the case of a watch, taking into account , moreover, that the signal it delivers is a measurable voltage, almost without consumption of electrical energy.
  • the display dial of such a watch 8 can then advantageously constitute the visual signaling means 6 previously mentioned.
  • This watch 8 includes, under these conditions, one or more control buttons, for example similar to a watch winder, making it possible to select its operating mode as a watch or as a bio-sensor.
  • the invention is in no way limited to such an embodiment, since the bio-sensor 1 can borrow many other embodiments, in particular that of a pen, more suitable for the detection of a molecule in saliva, even urine.
  • a pen can, of course, receive any one of the sound and / or visual signaling means 6 to which reference has been made above.
  • the present invention comes to respond advantageously to the various problems currently encountered in this field of biosensors.

Abstract

The invention concerns a method for making a biosensor, said biosensor comprising at least a reference electrode and at least a measuring electrode, each coated with a polymeric coating. Said method is characterised in that: said polymeric coating is obtained on said reference and measuring electrodes by electrochemical process, by soaking at least partly said reference and measuring electrodes in a chemical solution based at least on a saturated aliphatic diamine and by bringing them to sufficient potential to reach that corresponding to electrochemical oxidation of said chemical solution; modifying said polymeric coating of the measuring electrode by depositing an enzyme specific to said molecule to be detected in the analyte.

Description

BIO-CAPTEUR ET PROCEDE DE FABRICATION BIO-SENSOR AND MANUFACTURING METHOD
L'invention concerne un procédé de fabrication d'un bio-capteur d'une molécule spécifique présente au sein d'un analyte, ledit bio-capteur, de type potentiométrique, comprenant au moins une électrode de référence et au moins une électrode de mesure de ladite molécule, ladite électrode de référence et ladite électrode de mesure étant recouverte, chacune, par un revêtement polymérique, ladite électrode de mesure comportant, en outre, une enzyme réagissant spécifiquement avec ladite molécule à détecter. L'invention concerne encore un bio-capteur issu d'un tel procédé.The invention relates to a method of manufacturing a bio-sensor of a specific molecule present in an analyte, said bio-sensor, of potentiometric type, comprising at least one reference electrode and at least one measurement electrode. of said molecule, said reference electrode and said measurement electrode each being covered by a polymeric coating, said measurement electrode further comprising an enzyme reacting specifically with said molecule to be detected. The invention also relates to a bio-sensor resulting from such a process.
L' invention concerne le domaine des bio-capteurs prévus aptes à détecter une molécule spécifique au sein d'un analyte.The invention relates to the field of intended bio-sensors capable of detecting a specific molecule within an analyte.
Il existe, pour l'heure, très peu de capteurs de pH fiables, faciles à réaliser et à mettre en oeuvre dans des applications en tant que bio-capteur spécifique d'une substance à détecter grâce à une enzyme adaptée. De tels capteurs sont, qui plus est, particulièrement onéreux.For the time being, there are very few reliable pH sensors, which are easy to produce and implement in applications as a specific bio-sensor for a substance to be detected using an adapted enzyme. What is more, such sensors are particularly expensive.
Il convient d'observer que de nombreuses substances, telles que 1' urée , le glucose et autres , ont une influence sur l' acidité en surface de notre peau. Parallèlement, il est très fréquent que des personnes, atteintes de certaines pathologies, soient amenées, pour cette raison, de maintenir, continuellement, sous surveillance la concentration de l'une ou l'autre de ces substances, afin d'en garantir le maintien dans des proportions déterminées pour ne pas aggraver leur situation. En fonction de ces concentrations, ces personnes sont, par ailleurs, amenées, la plupart du temps , à prendre certains médicaments , souvent à caractère régulateur, précisément dans le but de compenser une déficience de l'organisme assurant, habituellement, lui-même cette régulation. A ce propos, l'invention se propose de répondre au souci de ces personnes au travers d'un bio-capteur potentiométrique capable de détecter, en surface de peau, dans la salive ou dans les urines par exemple, la présence d'une substance déterminée, sa proportion et son évolution dans le temps.It should be observed that many substances, such as urea, glucose and the like, have an influence on the surface acidity of our skin. At the same time, it is very frequent that people, affected by certain pathologies, are brought, for this reason, to maintain, continuously, under surveillance the concentration of one or the other of these substances, in order to guarantee the maintenance of it in determined proportions so as not to worsen their situation. Depending on these concentrations, these people are, moreover, brought, most of the time, to take certain drugs, often of a regulatory nature, precisely with the aim of compensating for a deficiency in the organism which usually ensures itself this regulation. In this regard, the invention proposes to respond to the concern of these people through a potentiometric bio-sensor capable of detecting, on the surface of the skin, in saliva or in urine for example, the presence of a substance determined, its proportion and its evolution over time.
Il est déjà connu, notamment par le document JP-A-9 222 412, un bio-capteur comportant une électrode de référence et une électrode de mesure, celle-ci étant recouverte d'un mélange d'une enzyme d' oxydation-réduction et d'un accepteur d'électrons, ce bio-capteur étant utilisé pour la détection de 1' albumine . Les valeurs mesurées dépendent de la variation du pH dans un échantillon déterminé.It is already known, in particular from document JP-A-9 222 412, a bio-sensor comprising a reference electrode and a measurement electrode, the latter being covered with a mixture of an oxidation-reduction enzyme. and an electron acceptor, this bio-sensor being used for the detection of albumin. The measured values depend on the variation of the pH in a given sample.
De même, il est connu par le document ES-A-2 091 714, un biocapteur destiné à la mesure du fructose dans les aliments et l'électrode de mesure est préparée à partir de l'enzyme fructose-deshydrogénas (FDH) dans un tampon de pH 5 contenant un tensioactif . Dans une seconde étape intervient une opération de séchage sous pression réduite, entre 3 et 8°C. Il est ensuite ajouté une solution aqueuse de polyéthylène imine (PEI) à pH 5, avant qu' intervienne une autre opération de séchage entre 3 et 8°C. On procède, alors, à un mélangeage avec du graphite à une température d'environ 650°C pendant une minute et on ajoute de l'huile de paraffine pour former une pâte à partir de laquelle on mouille l'électrode.Similarly, it is known from document ES-A-2 091 714, a biosensor intended for measuring fructose in food and the measuring electrode is prepared from the enzyme fructose-dehydrogenas (FDH) in a pH 5 buffer containing a surfactant. In a second step, a drying operation takes place under reduced pressure, between 3 and 8 ° C. An aqueous solution of polyethylene imine (PEI) at pH 5 is then added, before another drying operation takes place between 3 and 8 ° C. Mixing is then carried out with graphite at a temperature of approximately 650 ° C. for one minute and paraffin oil is added to form a paste from which the electrode is wetted.
Il est encore connu par les documents DE-A-29 620 371 et DE-A-106 39 224 un dispositif optique de mesure du taux de sucre dans le sang, lequel est intégré dans une montre. Le dispositif d'affichage peut être relié, soit au dispositif de mesure, soit au mécanisme de la montre par action sur une touche . Le dispositif est plus particulièrement destiné aux personnes diabétiques qui sont amenées à contrôler leur taux de sucre plusieurs fois par jour. L'on observera que dans le cadre de cet état de la technique, l'on connaît également un certain nombre de bio-capteurs de type ampérométriques nécessitant l'application d'un potentiel constant, entre l'électrode de détection d'un analyte et une électrode auxiliaire ou contre-électrode, pour mesurer la réponse en courant de la transformation électrochimique, en particulier du glucose en acide gluconique plus eau oxygénée.DE-A-29 620 371 and DE-A-106 39 224 also disclose an optical device for measuring the blood sugar level, which is integrated in a watch. The display device can be connected either to the measuring device or to the watch mechanism by pressing a button. The device is more particularly intended for diabetic people who have to control their sugar level several times a day. It will be observed that in the context of this state of the art, a certain number of bio-sensors of the amperometric type are also known, requiring the application of a constant potential, between the electrode for detecting an analyte. and an auxiliary electrode or counter electrode, for measuring the current response of the electrochemical transformation, in particular of glucose into gluconic acid plus hydrogen peroxide.
A ce propos, l'on se référera au document US-A-5.540.828 où il est question de bio-capteurs comportant une électrode de mesure sur laquelle est greffée l'enzyme au carbone réticulé avant que le polymère soit électrodéposé . En fait, il s'agit, ici, d'un monomère, le 1,2-diaminobenzène, qui est dissout en faible quantité dans une solution tampon aqueuse phosphatée de pH 6,5. L' oxydation anodique de cette électrode entraîne la formation du polymère qui est le poly 1 ,2-diaminobenzène .In this regard, reference will be made to document US-A-5,540,828 where it is a question of bio-sensors comprising a measurement electrode on which the enzyme crosslinked carbon is grafted before the polymer is electrodeposited. In fact, it is a monomer here, 1,2-diaminobenzene, which is dissolved in small amounts in a phosphate-buffered aqueous buffer solution of pH 6.5. The anodic oxidation of this electrode leads to the formation of the polymer which is poly 1, 2-diaminobenzene.
Le document US-A-5.683.563 concerne, lui, un bio-capteur ampérométrique, piégeant dans sa matrice polymère à la surface d'une électrode, l'enzyme idoine : la lactase-oxydase. Le polymère peut être de la polyéthylèneimine synthétisée par voie organique et déposée par simple étalement sur une surface d' électrode.The document US Pat. No. 5,683,563 relates to an amperometric bio-sensor, trapping in its polymer matrix on the surface of an electrode, the suitable enzyme: lactase oxidase. The polymer can be polyethyleneimine synthesized organically and deposited by simple spreading on an electrode surface.
Au vu de cet état de la technique, la présente invention se veut à même d'apporter une solution au problème de la fabrication, à un coût de revient réduit tout en étant d'une très grande fiabilité, d'un bio-capteur potentiométrique capable de détecter, avec précision et une très grande linéarité, la présence, au sein d'un analyte, d'une molécule spécifique au travers d'une mesure de pH.In view of this state of the art, the present invention is intended to provide a solution to the problem of manufacturing, at a reduced cost price while being very reliable, of a potentiometric biosensor capable of detecting, with precision and very great linearity, the presence, within an analyte, of a specific molecule through a pH measurement.
A cet effet, l'invention concerne un procédé de fabrication d'un bio-capteur d'au moins une molécule présente au sein d'un analyte, ledit bio-capteur potentiométrique comprenant au moins une électrode de référence et au moins une électrode de mesure en un matériau conducteur d'électricité ou semi-conducteur et recouverte, chacune, d'un revêtement polymérique, ladite électrode de mesure portant, encore, une enzyme susceptible de réagir spécifiquement avec ladite molécule à détecter, caractérisé en ce que :To this end, the invention relates to a method of manufacturing a bio-sensor of at least one molecule present in an analyte, said potentiometric bio-sensor comprising at least a reference electrode and at least one measurement electrode made of an electrically conductive or semiconductor material and each covered with a polymeric coating, said measurement electrode also carrying an enzyme capable of reacting specifically with said molecule to be detected, characterized in that:
- l'on obtient ledit revêtement polymérique sur lesdites électrodes de référence et de mesure par voie électrochimique, en venant tremper au moins partiellement lesdites électrodes de référence et de mesure dans une solution chimique à base d' au moins une diamine aliphatique saturée et en les portant à un potentiel suffisant pour atteindre celui correspondant à l'oxydation électrochimique de ladite solution chimique ;said polymeric coating is obtained on said reference and measurement electrodes by electrochemical means, by soaking at least partially said reference and measurement electrodes in a chemical solution based on at least one saturated aliphatic diamine and by bringing to a potential sufficient to reach that corresponding to the electrochemical oxidation of said chemical solution;
- l'on modifie ledit revêtement polymérique de l'électrode de mesure par un dépôt d'une enzyme spécifique de ladite molécule à détecter dans l' analyte.- Modifying said polymeric coating of the measuring electrode by depositing an enzyme specific for said molecule to be detected in the analyte.
Avantageusement, la solution chimique est une diamine aliphatique saturée pure ou encore un mélange de diamines aliphatiques saturées pures, sachant qu'elle peut encore se présenter sous forme d'une ou d'un mélange de ces diamines en solution dans un solvant plus difficile à oxyder de manière électrochimique que la ou les diamines que contient cette solution.Advantageously, the chemical solution is a pure saturated aliphatic diamine or else a mixture of pure saturated aliphatic diamines, knowing that it can also be in the form of one or of a mixture of these diamines in solution in a solvent more difficult to oxidize electrochemically than the diamine or diamines contained in this solution.
L' invention concerne encore un bio-capteur conforme à ce procédé de fabrication et comportant au moins une électrode de référence et au moins une électrode de mesure en un matériau conducteur d'électricité ou semi-conducteur et recouverte, chacune, d'un revêtement polymérique, ladite électrode de mesure portant, encore, une enzyme susceptible de réagir spécifiquement avec ladite molécule à détecter, caractérisé en ce qu' il comporte une unité de traitement prévue apte à mesurer une différence de potentiel au travers des électrodes de référence et de mesure et à transmettre, par l'intermédiaire de moyens de signalisation visuelle et/ou sonore, une information d'interprétation claire concernant la ou les molécules spécifiques détectées au travers de la dite mesure de différence de potentiel.The invention also relates to a bio-sensor conforming to this manufacturing process and comprising at least one reference electrode and at least one measurement electrode made of an electrically conductive or semiconductor material and each covered with a coating. polymer, said measuring electrode carrying, again, an enzyme capable of reacting specifically with said molecule to be detected, characterized in that it comprises a processing unit provided capable of measuring a potential difference across the reference and measuring electrodes and to transmit, by means of visual and / or audible signaling means, clear interpretation information concerning the specific molecule or molecules detected through said measurement of potential difference.
Selon un exemple de réalisation, ce bio-capteur peut être intégré dans le boîtier d'une montre bracelet.According to an exemplary embodiment, this bio-sensor can be integrated into the case of a wristwatch.
L'invention sera mieux comprise à la lecture de la description qui va suivre et du dessin joint en annexe se rapportant à un mode de réalisation.The invention will be better understood on reading the description which follows and the appended drawing relating to an embodiment.
La figure 1 est une représentation schématisée du principe de fonctionnement du bio-capteur donc de la capture électrochimique à l'interface électrode/échantillon pour la détection d'une molécule spécifique A.FIG. 1 is a schematic representation of the operating principle of the bio-sensor, therefore of the electrochemical capture at the electrode / sample interface for the detection of a specific molecule A.
La figure 2 illustre, de manière schématisée, un bio-capteur comportant une électrode de référence et plusieurs électrodes de mesure.FIG. 2 illustrates, diagrammatically, a bio-sensor comprising a reference electrode and several measurement electrodes.
La figure 3 est une représentation schématisée d'un bio-capteur conforme à l' invention.Figure 3 is a schematic representation of a bio-sensor according to the invention.
La présente invention a trait, tout particulièrement, au domaine des bio-capteurs potentiométriques et à leur procédé de abrication.The present invention relates, in particular, to the field of potentiometric bio-sensors and to their manufacturing process.
Plus particulièrement, l'invention concerne un bio-capteur potentiométrique d' au moins une molécule présente au sein d' un analyte.More particularly, the invention relates to a potentiometric biosensor of at least one molecule present within an analyte.
Ainsi, tel que visible dans les différentes figures 1 à 3 du dessin ci-joint, un tel bio-capteur 1 comporte au moins une électrode de référence 2 et au moins une électrode de mesure 3.Thus, as visible in the various figures 1 to 3 of the attached drawing, such a bio-sensor 1 comprises at least one reference electrode 2 and at least one measurement electrode 3.
A ce propos, il a été représenté, dans la figure 2, un bio- capteur comportant plusieurs électrodes de mesure 3A, 3B, 3C, 3D susceptibles, éventuellement, de détecter, chacune, une molécule dif éren e .In this connection, a bio- sensor comprising several measurement electrodes 3A, 3B, 3C, 3D capable, possibly, of detecting, each, a different molecule.
Ces électrodes de référence 2 et de mesure 3 sont conçues en un matériau conducteur d' électricité ou semi-conducteur et sont recouvertes, chacune, d'un revêtement polymérique 4, ladite électrode de mesure 3 portant, encore, une enzyme réagissant spécifiquement avec ladite molécule à détecter.These reference 2 and measurement 3 electrodes are made of an electrically conductive or semiconductor material and are each covered with a polymeric coating 4, said measurement electrode 3 also carrying an enzyme reacting specifically with said molecule to be detected.
En fait, selon le procédé de fabrication d'un tel bio-capteur, le revêtement polymérique 4 au niveau des électrodes 2,3 est obtenu par voie électrochimique. Pour cela on trempe au moins partiellement lesdites électrodes 2, 3 dans une solution chimique à base d'au moins une diamine aliphatique saturée et l'on vient porter ces électrodes à un potentiel suffisant pour atteindre celui correspondant à l' oxydation électrochimique de ladite solution chimique.In fact, according to the method of manufacturing such a bio-sensor, the polymeric coating 4 at the electrodes 2, 3 is obtained by electrochemical means. For this, said electrodes 2, 3 are at least partially soaked in a chemical solution based on at least one saturated aliphatic diamine and these electrodes are brought to a sufficient potential to reach that corresponding to the electrochemical oxidation of said solution. chemical.
Puis , dans une dernière étape, on vient modifier ce revêtement polymérique 4 au travers de ladite enzyme réagissant spécifiquement avec la molécule à détecter.Then, in a last step, we just modify this polymeric coating 4 through said enzyme reacting specifically with the molecule to be detected.
Dans la figure 1 il est illustré le principe de la capture électrochimique à l'interface électrode-échantillon pour la détection d'une molécule spécifique, repérée A.In Figure 1 the principle of electrochemical capture at the electrode-sample interface is illustrated for the detection of a specific molecule, marked A.
Ainsi, comme visible sur cette figure, lors d'une réaction chimique entre deux réactifs, il y a un échange d'électrons entre eux. Si une molécule A vient réagir sur une autre molécule B piégée ou greffée dans un polymère P à la surface d'une électrode de mesure EM, il y a modification de la densité de la charge à la surface de cette électrode EM par transfert électronique des molécules A sur les molécules B. L' électrochimie inter aciale permet d' étudier ce type de réaction à la jonction d'une électrode en contact avec un liquide ou un gaz contenant l'échantillon de molécules à doser. Cela est rendu possible en enregistrant un différentiel de potentiel par comparaison du signal électrique de l'électrode spécifique de mesure EM à celui d'une électrode de référence ΞR, celle-ci, bien que de même nature que l'électrode de mesure EM ne contenant pas de molécules B dans le polymère P qui recouvre sa surface.Thus, as can be seen in this figure, during a chemical reaction between two reactants, there is an exchange of electrons between them. If a molecule A reacts on another molecule B trapped or grafted in a polymer P on the surface of an EM measurement electrode, there is a change in the density of the charge on the surface of this EM electrode by electronic transfer of the molecules A on molecules B. Interacial electrochemistry makes it possible to study this type of reaction at the junction of an electrode in contact with a liquid or a gas containing the sample of molecules to be assayed. This is made possible by recording a potential differential by comparing the electrical signal of the specific measurement electrode EM with that of a reference electrode ΞR, the latter, although of the same nature as the measurement electrode EM does containing no B molecules in the polymer P which covers its surface.
A noter qu'en ce qui concerne la solution dans laquelle l'on vient tremper les électrodes 2, 3 pour l'obtention du revêtement polymérique par voie électrochimique, elle peut être composée d'une ou plusieurs diamines aliphatiques saturées pures ou en solutions dans un solvant plus difficile à oxyder de manière électrochimique que cette ou ces diamines.It should be noted that as regards the solution in which the electrodes 2, 3 are soaked to obtain the polymeric coating by electrochemical means, it may be composed of one or more pure saturated aliphatic diamines or in solutions in a solvent more difficult to oxidize electrochemically than this or these diamines.
Par ailleurs, cette ou ces diamines se caractérisent en ce qu'elles comportent au moins deux fonctions aminés primaires ayant en α de chacune d' entre elles un groupement alkyl .Furthermore, this or these diamines are characterized in that they comprise at least two primary amine functions having, in each of them, an alkyl group.
Préférentiellement, cette solution chimique est à base d' éthylènediaminé ou encore de diéthylènetriamine voire une solution de l'un et/ou l'autre de ces composants, pour 1' obtention, en tant que revêtement polymérique, d'une couche de polyéthylènimine linéaire.Preferably, this chemical solution is based on ethylenediamine or else on diethylenetriamine or even a solution of one and / or the other of these components, for obtaining a layer of linear polyethyleneimine as a polymeric coating. .
Selon un autre mode de réalisation, cette solution chimique est à base d'une autre diamine aliphatique saturée, non aromatique, la 1 ,3-diaminopropane pour l'obtention, en tant que revêtement polymérique après oxydation anodique, de polypropylènimine .According to another embodiment, this chemical solution is based on another saturated, non-aromatic aliphatic diamine, 1, 3-diaminopropane for obtaining, as polymeric coating after anodic oxidation, polypropylenimine.
En ce qui concerne la modification du revêtement polymérique 4 finalement obtenu par une enzyme réagissant spécifiquement avec la molécule à détecter, elle peut résulter d'une simple opération de trempe de ladite électrode de mesure 3 dans une solution de ladite enzyme jusqu'à imprégnation de ce revêtement polymérique 4 , tout comme elle peut provenir du dépôt de ladite enzyme , sous orme de gel, sur ce revêtement polymérique 4.As regards the modification of the polymeric coating 4 finally obtained by an enzyme reacting specifically with the molecule to be detected, it may result from a simple operation of quenching said measurement electrode 3 in a solution of said enzyme until impregnation of this polymeric coating 4, just as it can come from the deposition of said enzyme, in gel form, on this polymeric coating 4.
Bien entendu, pour la détection de plusieurs molécules différentes , le revêtement polymérique 4 de plusieurs électrodes de mesure 3, 3A, 3B, 3C, 3D est modifié par des enzymes différentes réagissant spécifiquement, respectivement, à l'une des molécules à détecter.Of course, for the detection of several different molecules, the polymeric coating 4 of several measurement electrodes 3, 3A, 3B, 3C, 3D is modified by different enzymes reacting specifically, respectively, to one of the molecules to be detected.
Selon l'invention, le bio-capteur 1 comporte, encore, une unité de traitement 5 à laquelle sont raccordées ces électrodes de référence 2 et de mesure 3 et qui est prévue apte, d'une part, à mesurer une différence de potentiel par l' intermédiaire de ces dernières et, d'autre part, à interpréter la ou les mesures effectuées en vue d' émettre une information clairement compréhensible pour un usager au travers de moyens de signalisation visuelle et/ou sonore 6, là encore raccordés à cette unité de traitement 5.According to the invention, the bio-sensor 1 also comprises a processing unit 5 to which these reference 2 and measurement 3 electrodes are connected and which is provided capable, on the one hand, of measuring a potential difference by through the latter and, on the other hand, to interpret the measurement (s) carried out with a view to transmitting information clearly understandable to a user by means of visual and / or sound signaling means 6, again connected to this processing unit 5.
De tels moyens de signalisation visuelle 6 peuvent se présenter sous forme d'un écran d' a fichage 7 apte à af icher, sous forme d'une valeur biochimique d'interprétation accessible à l'usager, la ou les mesures de différentiel de potentiel effectuées par l'unité de traitement 5 par l'intermédiaire des électrodes 2, 3. Ainsi , cet écran d' af ichage 7 peut se présenter sous forme d'un écran graphique ou autre.Such visual signaling means 6 can be in the form of a display screen 7 capable of displaying, in the form of a biochemical value of interpretation accessible to the user, the potential differential measurement (s) performed by the processing unit 5 via the electrodes 2, 3. Thus, this display screen 7 can be in the form of a graphic screen or the like.
Selon un autre mode de réalisation, de tels moyens de signalisation visuelle et/ou sonore 6 sont prévus aptes à émettre une signalisation repérable visuellement ou par voie sonore en cas de détection, par l'intermédiaire de l'unité de traitement 5, d'un dépassement d'un ou plusieurs seuils de mesure prédéterminés, connus en mémoire de cette unité de traitement 5. A titre d'exemple, ces moyens de signalisation visuelle ou sonore 6 peuvent se présenter sous forme d'un simple voyant ou d'un buseur activé au cas où le différentiel de potentiel mesuré par l'unité de traitement 5 se traduit par une valeur biochimique supérieure ou inférieure à une valeur normale.According to another embodiment, such visual and / or audible signaling means 6 are provided capable of emitting visually identifiable signaling or by audible signal in the event of detection, via the processing unit 5, of exceeding one or more predetermined measurement thresholds, known in the memory of this processing unit 5. By way of example, these visual or audible signaling means 6 can be in the form of a simple indicator light or an activated buseur in the event that the potential differential measured by the processing unit 5 is expressed by a value biochemical above or below a normal value.
Ainsi, cette valeur biochimique peut correspondre au taux d'urée ou au taux de sucre dans le sang qui, précisément, peut être déterminé au travers de la mesure du pH de la peau donc de la sueur du porteur du bio-capteur 1.Thus, this biochemical value can correspond to the level of urea or to the level of sugar in the blood which, precisely, can be determined through the measurement of the pH of the skin and therefore of the sweat of the wearer of the bio-sensor 1.
Comme cela ressort déjà de la description qui précède, ce biocapteur n' est nullement limité à la détection des valeurs biochimiques qui viennent d' être citées .As is already apparent from the above description, this biosensor is in no way limited to the detection of the biochemical values which have just been mentioned.
Par ailleurs, on observera que les avantages qui découlent d'un tel bio-capteur 1 potentiométrique, conçu selon l'invention, consistent en ce que le revêtement polymérique 4 des électrodes est très adhérent. L'on a pu observer que l'adhérence du revêtement polymérique 4 était encore améliorée sur des matériaux conducteurs ou semi-conducteurs, composant ces électrodes de référence 2 et de mesure 3, tels que le platine, l'or, le carbone vitreux, les fibres de carbone, le silicium. La présente invention n' est toutefois pas limitée à cette énumération de matériaux conducteurs ou semi-conducteurs susceptibles de constituer les dites électrodes .Furthermore, it will be observed that the advantages which arise from such a potentiometric bio-sensor 1, designed according to the invention, consist in that the polymeric coating 4 of the electrodes is very adherent. It has been observed that the adhesion of the polymeric coating 4 was further improved on conductive or semiconductor materials, composing these reference 2 and measurement 3 electrodes, such as platinum, gold, vitreous carbon, carbon fibers, silicon. The present invention is however not limited to this enumeration of conductive or semiconductor materials capable of constituting said electrodes.
De plus, ledit revêtement polymérique n'est pas toxique pour les humains, tout en étant non corrosif, alors qu'au contraire et il convient de le rappeler, les aminés qui sont à l'origine de ce revêtement le sont.In addition, said polymeric coating is not toxic to humans, while being non-corrosive, whereas on the contrary and it should be remembered, the amines which are at the origin of this coating are.
Ces électrodes possèdent, en outre, un potentiel qui répond linéairement au pH de solutions aqueuses, dans une gamme de pH de 2 à 13 environ. Ainsi, bien que cette réponse des électrodes ne soit pas Nernstienne, elle est linéaire, avec une variation de l'ordre de 40 à 45 V par unité de pH à température ordinaire.These electrodes have, moreover, a potential which responds linearly to the pH of aqueous solutions, in a pH range from 2 to 13 approximately. Thus, although this response of the electrodes is not Nernstian, it is linear, with a variation of the order of 40 to 45 V per unit of pH at ordinary temperature.
Finalement, un bio-capteur 1, conforme à l'invention, se prête, aisément, à une miniaturisation permettant d'envisager son intégration, pour la détection de substance dans la sueur, par exemple dans le boîtier d'une montre, tenant compte, par ailleurs, que le signal qu'il délivre est une tension mesurable, quasiment sans consommation d'énergie électrique. A noter que le cadran d'a ichage d'une telle montre 8 peut, alors, constituer, avantageusement, les moyens de signalisation visuelle 6 préalablement cités . Cette montre 8 comporte, dans ces conditions, un ou plusieurs boutons de commande, par exemple similaire à un remontoir, permettant de sélectionner son mode de fonctionnement en tant que montre ou de bio-capteur.Finally, a bio-sensor 1, in accordance with the invention, easily lends itself to miniaturization making it possible to envisage its integration, for the detection of substance in sweat, for example in the case of a watch, taking into account , moreover, that the signal it delivers is a measurable voltage, almost without consumption of electrical energy. Note that the display dial of such a watch 8 can then advantageously constitute the visual signaling means 6 previously mentioned. This watch 8 includes, under these conditions, one or more control buttons, for example similar to a watch winder, making it possible to select its operating mode as a watch or as a bio-sensor.
Bien sûr, l'invention n'est nullement limitée à un tel mode de réalisation, puisque le bio-capteur 1 peut emprunter bien d'autres formes de réalisation, en particulier celle d'un stylo, plus adapté pour la détection d'une molécule dans la salive, voire les urines. Un tel stylo peut, bien évidemment, recevoir l'un quelconque des moyens de signalisation sonore et/ou visuelle 6 auxquels il a été fait référence plus haut.Of course, the invention is in no way limited to such an embodiment, since the bio-sensor 1 can borrow many other embodiments, in particular that of a pen, more suitable for the detection of a molecule in saliva, even urine. Such a pen can, of course, receive any one of the sound and / or visual signaling means 6 to which reference has been made above.
Tel que cela ressort de la description qui précède, la présente invention vient répondre de manière avantageuse aux différents problèmes actuellement rencontrés dans ce domaine des biocapteurs . As appears from the above description, the present invention comes to respond advantageously to the various problems currently encountered in this field of biosensors.

Claims

REVENDICATIONS
1) Procédé de fabrication d'un bio-capteur (1) d'au moins une molécule présente au sein d'un analyte, ledit bio-capteur (1) de type potentiométrique comprenant au moins une électrode de référence (2) et au moins une électrode de mesure (3, 3A, 3B, 3C, 3D) en un matériau conducteur d'électricité ou semiconducteur et recouverte, chacune, d'un revêtement polymérique (4) , ladite électrode de mesure portant, encore, une enzyme susceptible de réagir spécifiquement avec ladite molécule à détecter, caractérisé en ce que :1) Method for manufacturing a bio-sensor (1) of at least one molecule present within an analyte, said bio-sensor (1) of potentiometric type comprising at least one reference electrode (2) and at least one least one measuring electrode (3, 3A, 3B, 3C, 3D) made of an electrically conductive or semiconductor material and each covered with a polymeric coating (4), said measuring electrode also carrying an enzyme capable of to react specifically with said molecule to be detected, characterized in that:
- l'on obtient ledit revêtement polymérique (4) sur lesdites électrodes de référence (2) et de mesure (3, 3A, 3B, 3C, 3D) par voie électrochimique, en venant tremper au moins partiellement lesdites électrodes de référence et de mesure dans une solution chimique à base d'au moins une diamine aliphatique saturée et en les portant à un potentiel suffisant pour atteindre celui correspondant à l'oxydation électrochimique de ladite solution chimique ;- said polymeric coating (4) is obtained on said reference (2) and measurement electrodes (3, 3A, 3B, 3C, 3D) electrochemically, by at least partially soaking said reference and measurement electrodes in a chemical solution based on at least one saturated aliphatic diamine and bringing them to a potential sufficient to reach that corresponding to the electrochemical oxidation of said chemical solution;
- l'on modifie ledit revêtement polymérique (4) de l'électrode de mesure par un dépôt d'une enzyme spécifique de ladite molécule à détecter dans l' analyte .- said polymeric coating (4) of the measuring electrode is modified by depositing an enzyme specific to said molecule to be detected in the analyte.
2) Procédé selon la revendication 1, caractérisé en ce que la solution chimique est une diamine aliphatique saturée pure.2) Method according to claim 1, characterized in that the chemical solution is a pure saturated aliphatic diamine.
3) Procédé selon la revendication 1, caractérisé en ce que la solution chimique est un mélange de diamines aliphatiques saturées pures .3) Method according to claim 1, characterized in that the chemical solution is a mixture of pure saturated aliphatic diamines.
4) Procédé selon la revendication 1, caractérisé en ce que la solution chimique est une diamine aliphatique saturée en solution dans un solvant plus difficile à oxyder de manière électrochimique que la diamine que contient ladite solution. 5) Procédé selon la revendication 1, caractérisé en ce que la solution est un mélange de diamines aliphatiques saturées en solution dans un solvant plus difficile à oxyder de manière électrochimique que les diamines que contient ladite solution.4) Method according to claim 1, characterized in that the chemical solution is a saturated aliphatic diamine in solution in a solvent more difficult to oxidize electrochemically than the diamine contained in said solution. 5) Method according to claim 1, characterized in that the solution is a mixture of saturated aliphatic diamines in solution in a solvent more difficult to oxidize electrochemically than the diamines contained in said solution.
6) Procédé selon l'une quelconque des revendications précédentes , caractérisé par le fait que la solution chimique est à base d' éthylènediamine et/ou de 1,3-diaminopropane pour l'obtention après oxydation anodique de polyéthylènimine et/ou de polypropylènimine, respectivement.6) Process according to any one of the preceding claims, characterized in that the chemical solution is based on ethylenediamine and/or 1,3-diaminopropane to obtain, after anodic oxidation, polyethylenimine and/or polypropylenimine, respectively.
7) Bio-capteur potentiométrique obtenu par le procédé de fabrication selon l'une quelconque des revendications précédentes, et comportant au moins une électrode de référence7) Potentiometric bio-sensor obtained by the manufacturing process according to any one of the preceding claims, and comprising at least one reference electrode
(2) et au moins une électrode de mesure (3, 3A, 3B, 3C, 3D) en un matériau conducteur d'électricité ou semi-conducteur et recouverte, chacune, d'un revêtement polymérique (4) , ladite électrode de mesure (3, 3A, 3B, 3C, 3D) portant, encore, une enzyme susceptible de réagir spécifiquement avec ladite molécule à détecter, caractérisé en ce qu'il comporte une unité de traitement (5) prévue apte à mesurer une différence de potentiel au travers des électrodes de référence (2) et de mesure (3, 3A, 3B, 3C, 3D) et à transmettre, par l'intermédiaire de moyens de signalisation visuelle et/ou sonore (6) , une information d' interprétation claire concernant la ou les molécules spécifiques détectées au travers de la dite mesure de différence de potentiel.(2) and at least one measuring electrode (3, 3A, 3B, 3C, 3D) made of an electrically conductive or semiconducting material and each covered with a polymeric coating (4), said measuring electrode (3, 3A, 3B, 3C, 3D) carrying, again, an enzyme capable of reacting specifically with said molecule to be detected, characterized in that it comprises a processing unit (5) provided capable of measuring a potential difference at through reference (2) and measurement electrodes (3, 3A, 3B, 3C, 3D) and to transmit, via visual and/or audible signaling means (6), clear interpretation information concerning the specific molecule(s) detected through said potential difference measurement.
8) Bio-capteur potentiométrique selon la revendication 7, caractérisé en ce qu'il est intégré dans le boîtier d'une montre bracelet (8) .8) Potentiometric bio-sensor according to claim 7, characterized in that it is integrated into the case of a wristwatch (8).
9) Bio-capteur potentiométrique selon la revendication 7 , caractérisé par le fait qu'il emprunte la forme d'un stylo intégrant lesdits moyens de signalisation visuelle et/ou sonore. 9) Potentiometric bio-sensor according to claim 7, characterized in that it takes the shape of a pen integrating said visual and/or audible signaling means.
PCT/FR2001/001133 2000-04-12 2001-04-12 Biosensor and method for making same WO2001077656A1 (en)

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