Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING 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
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
  • C12Q1/002—Electrode membranes
• • 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
  • G01N27/403—Cells and electrode assemblies
  • G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
  • G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors

Definitions

• the present invention belongs to the general technical field of integrated electrochemical sensors, based on the principle of dosing a substrate using an enzyme and a field effect transistor, sensitive to at least one given ionic species ( Ion Sensitive Field Effect Transistor, ISFET).
• ISFET Ion Sensitive Field Effect Transistor
• ENFET ENzymatic Field Effect Transistor
• the invention relates to an electrochemical sensor for enzymatic assay of the ENFET type, in which the selective grid, sensitive to at least one given ionic species, is at least partially covered:
• At least one internal membrane comprising at least one enzyme capable of catalyzing the decomposition of the substrate (or substrates) to be assayed, by forming and / or consuming the aforementioned given ionic species,
• This type of sensor exploits the fact that the enzyme which it contains causes the hydrolysis of the substrate to be assayed and, consequently, a modification of the pH of the assay solution in the vicinity of the surface of the sensor, for example.
• the iSFET used is an ISFET for measuring pH. The pH variations thus determined are directly related to the concentration of the substrate to be assayed.
• ENFET sensors have the advantage, among other things, of having a relatively small size, of being of a low cost price, of offering a very good measurement sensitivity, of giving access to multiple possibilities of assays. different substrates and to be biocompatible.
• glucose and urea which involve, respectively, glucose oxidase (GOD) and urease.
• ENFETs Another obstacle to the development of ENFETs is that their dynamic measurement interval, i. e. the substrate concentration range in which the response of the sensor is linear, is relatively limited. Often, this dynamic measurement interval is well below normal biological concentrations of substrates, such as glucose or urea.
• ENFET sensors for glucose measurement and comprising an enzyme, glucose oxidase, immobilized in a polyacrylamide gel or else in bovine serum albumin crosslinked with glutaraldehyde.
• ENFETs do not make it possible to eliminate the penalizing dilution operation, any more than the harmful influence of the buffer.
• the effect of ionic strength which is weak in the case of biological samples, must be neutralized by adding NaCl.
• Such an arrangement makes it possible to restrict the diffusion of glucose from the assay medium to the enzymatic membrane and thus to significantly increase the dynamic measurement interval of the sensor.
• ENFET REFET differential circuit is also used in the experiments described in this document, in order to limit the influence of pH variations in the dosing medium. In any event, this variant in the mode of support of the enzyme does not resolve anything with regard to the major problem of the harmful influence of the buffer.
• One of the objects of the present invention is to provide an electrochemical enzyme dosing sensor, of the ENFET type, in which the negative effect of the buffer, possibly present in the dosing medium, is not felt, in particular with regard to the threshold of detection of the substrate considered.
• Another object of the invention is to provide an ENFET sensor having a wide dynamic measurement interval, as well as a certain insensitivity to variations in pH of the assay medium; but on the other hand a sensitivity for measuring the enzymatic activity, therefore the concentration of substrate to be assayed, high.
• Another object of the invention is to provide an ENFET sensor in which the enzymatic reaction of hydrolysis of the substrate takes place without disturbance, resulting, for example, from the ionic strength of the assay medium.
• Another object of the invention is to provide an ENFET sensor of limited cost price.
• the Applicant has had the merit of highlighting that the nature of the material constituting the additional membrane is preponderant with regard to the insensitivity of the sensor vis-à-vis the buffer. It was after much research and experience that she was able to isolate the class of ionomeric materials as being particularly suitable for the additional membrane.
• the present invention achieves the above objects, among others, by proposing an electrochemical sensor for enzymatic assay, of the field effect transistor type (ENFET), in which the selective grid, sensitive to at least one ionic species given, is at least partially covered by at least one internal enzymatic layer comprising at least one enzyme capable of catalyzing the decomposition of the substrate to be assayed, by forming and / or consuming the aforementioned given ionic species and by at least one additional external membrane, superimposed on the enzymatic layer, the originality of this sensor coming, essentially, from the fact that this additional membrane is based on at least one ionomer.
• ENFET field effect transistor type
• ionomer designates ionic polymers having inorganic salt groups linked to the polymer chain. This definition corresponds to that given in "Encyclopedia of Polymer
• the ionomer constituting the additional membrane, is chosen from the following families of ionomers:
• polyacylates such as polyacetate or butyrate and polyvinylimidazole
• Perfluorinated, perfluorinated ionomers being preferred and, among them, perfluorosulfonates being particularly preferred.
• One of the most suitable ionomers is a copolymer of tetrafluoroethylene and perfluorinated vinyl ether, having a fluorosulfonate group.
• polyvinyl butyral which is a linear copolymer of butyral vinyl / vinyl alcohol / vinyl acetate.
• the respective proportions of these comonomers vary from 60 to 90/9 to 33/1 to 7, preferably from 70 to 80/28 to 19/1 to 2, the 75/22/3 ratio being particularly preferred.
• polyvinylimidazole which is a positively charged ionic polymer.
• the polymers marketed under the name EASTMAN AQ polymers by the company EASTMAN belong to this family of amorphous polyesters.
• the ENFET thus produced, has an extremely limited sensitivity to the surrounding buffer capacity. It should also be noted that this ENFET has a relatively wide dynamic measurement interval and, moreover, it allows the enzymatic reaction to be carried out under optimal conditions. According to the invention, the additional ionomer membrane has a thickness which is the result of a compromise between the insensitivity vis-à-vis the target buffer and the measurement speed.
• the thickness of the additional membrane can be between 0, 1 and 10 micrometers, preferably between 0.5 and 5 micrometers and, more preferably still, between 0.5 and 2.5 micrometers.
• the internal enzymatic layer and in accordance with a first embodiment, it is constituted by a membrane forming a matrix in which the enzyme is included and immobilized.
• the constituent material of this membrane is, advantageously, at least one macromolecular compound, preferably chosen from proteins, polysaccharides, synthetic polymers and copolymers.
• non-enzymatic proteins such as collagens, albumin, preferably crosslinked.
• polysaccharides the illustration can be perfected by mentioning alginates, chitin, cellulose and its derivatives, such as nitro cellulose or cellulose esters and ethers, or even starches and derivatives.
• polyacrylamide gels vinyl polymers, in particular polyvinyl acetates, polyvinyl alcohols, preferably polyvinyl butyral alcohol.
• polyurethanes or polysiloxanes, of the heteropolysiloxane type carrying functional groups, such as amines, are also suitable.
• the crosslinking takes place advantageously using glutaraldehyde and, in this regard, it is interesting to note that the level of crosslinking has an effect on the extension of the dynamic interval. of measurement.
• This level of crosslinking is linked to the time of exposure to glutaraldehyde, which is preferably chosen from 20 to 60 minutes and, more preferably still, to 30 minutes at room temperature.
• the ratio R expressed in% by mass, varies from 5 to 95, preferably from 8 to 50 and, more preferably still, from 9 to 11.
• the internal membrane contains at at least one polyol, preferably glycerol, sucrose or sorbitol, glycerol being particularly preferred.
• This polyol has the effect of improving the surface condition, the mechanical and adhesion properties of the enzymatic membrane.
• the ratio R 2 of macromolecular compounds / polyols, expressed in% by volume, is between 2.5 and 20, preferably between 5 and 15 and, more preferably still, is of the order of 10;
• the enzyme (or enzymes) is directly covalently linked to the selective grid.
• the ENFET sensor according to the invention has, for example, a general structure of the type of that of known ISFET or ENFET sensors.
• an enzymatic electrochemical dosing device using at least one ENFET measurement sensor, as described above, as well as at least one reference sensor (REFET) similar to the above-mentioned measurement sensor , except that it does not contain an enzyme.
• ENFET ENFET measurement sensor
• REFET reference sensor
• Fig. 1 Graphs of the voltage U measured in millivolts by ENFET glucose oxidase sensors allowing the measurement of glucose, as a function of the glucose concentration in millimoles.
• the curves Ia, Ib correspond to a sensor 1 according to the prior art membrane and free of additional areas 2c, 2d to a sensor 2 with an additional membrane made of Nafion ® in accordance with the invention.
• the measurements were made in a medium titrating 1 millimole / 1 of phosphate buffer and a pH equal to 7.4, while for measurements b and d, the buffer concentration is 10 millimoles / 1 for a 7.4 pH.
• Fig. 2 Graph of dU / dT (mV.m 1 ) as a function of the glucose concentration (millimoles) for sensors 1 and 2 under conditions a, b and c, d above.
• Fig. 3 Graph of the measurement sensitivity of a sensor 3 with additional polyvinyl butyral membrane according to the invention, as a function of the glucose concentration in millimoles / 1.
• enzymatic ENFET sensors comprising glucose oxidase and intended for the determination of glucose.
• a reference ENFET 1 is produced in accordance with the prior art and having no additional membrane in addition to the enzymatic membrane and, on the other hand, an ENFET 2 sensor having an additional membrane based on perfluorosulfonate ionomer sold under the registered trademark Nafion ® .
• the ENFET sensors used are of a perfectly known type and are obtained from ISFET pH sensors, such as those described in Sensors and
• the glucose oxidase (GOD) used comes from vital Penicillium, having an activity of 168 IU / mg and coming from the factory COSARSKY (CHERKASY, Ukraine).
• bovine serum albumin is that sold by the SIGMA Company.
• Glutaraldehyde is presented in a solution containing 25% and sold by the company MERCK.
• the Nafion ® used is the commercial product consisting of a 5% solution in a hydroalcoholic mixture containing 10% water and sold by the company ALDRICH under the reference 27,470-4, page 937 catalog.
• the conventional procedure consists in preparing a phosphate buffer solution (KH 2 PO 4 - NaOH) pH 7.4, titrating 20 millimoles and containing
• the membrane is obtained, first of all, by depositing a drop of the previous solution on the sensitive part of the ENFET sensor, the REFET being treated with the same enzyme-free solution, then by exposing the sensor to vapors of glutaraldehyde for 30 min.
• the sensors thus prepared are then dried at room temperature for 10 to 15 min. They are stored before use, in 20 mM phosphate buffer at a pH of 7.4 or in the dry state. In the latter case, the addition of a preservative is not necessary.
• the measurements are made at room temperature.
• Fig. 1 shows the surprising and significant efficiency of sensor 2 according to H
• the polyvinyl butyral solution used for the deposition is ethanolic and has a titer of 5%. It is clear from fig. 3 that the ENFET sensor 3 according to the invention makes it possible to obtain a curve of measurement sensitivity (microvolts / s) / glucose concentration (millimoles / 1), approximately linear from 1 millivolt to 50 millivolts. It can therefore be deduced therefrom that the additional membranes based on ionomers of the sensors according to the invention have the additional advantage, but not negligible, of allowing the extension of the dynamic measurement interval of the sensor. This facilitates the measurements since it is no longer necessary to dilute the dosing medium.

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• 1993
  • 1993-05-12 FR FR9305941A patent/FR2705148B1/fr not_active Expired - Fee Related
• 1994
  • 1994-05-10 WO PCT/FR1994/000552 patent/WO1994026871A1/fr not_active Application Discontinuation
  • 1994-05-10 EP EP94915608A patent/EP0651782A1/de not_active Withdrawn

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