EP0666981A1 - Detecteur pour detecter des substances decomposables biologiquement - Google Patents

Detecteur pour detecter des substances decomposables biologiquement

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Publication number
EP0666981A1
EP0666981A1 EP93923968A EP93923968A EP0666981A1 EP 0666981 A1 EP0666981 A1 EP 0666981A1 EP 93923968 A EP93923968 A EP 93923968A EP 93923968 A EP93923968 A EP 93923968A EP 0666981 A1 EP0666981 A1 EP 0666981A1
Authority
EP
European Patent Office
Prior art keywords
membrane
electrode
sensor according
enzyme
membranes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP93923968A
Other languages
German (de)
English (en)
Inventor
Gerald Dr. Urban
Gerhard Jobst
Artur Jachimowicz
Peter Svasek
Manfred Rakohl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP0666981A1 publication Critical patent/EP0666981A1/fr
Withdrawn legal-status Critical Current

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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
    • 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
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes

Definitions

  • the invention relates to a sensor for detecting biologically, in particular enzymatically, convertible substances of a biological substrate with an electrode and at least one membrane covering the electrode.
  • glucose sensors A particularly suitable way of determining glucose is the enzymatic reaction of glucose with glucose oxidase, in which gluconolactone and hydrogen peroxide are formed while consuming oxygen.
  • the water formed in this way • ⁇ • peroxide can be at an anode under dissipation of electrons back to form oxygen, the anode current can be measured and can be regarded as a measure for the oxidase by the Glucose ⁇ converted glucose.
  • other biological or enzymatic reactions are conceivable, in which the enzymatic reaction product can be used to obtain a corresponding signal on an electrode.
  • the invention now aims to further develop a sensor of the type mentioned in the introduction so that with high measurement signals and good linearity over a large concentration ""
  • the senor according to the invention is essentially characterized in that at least three membranes are provided, of which at least two each have at least one enzyme for the conversion of the biological substance to be determined and / or one reactant for a reaction product of the enzymatic Contain reaction and that at least one further membrane is arranged for setting a distance between the aforementioned membranes and / or as a permselective membrane.
  • oxidase or peroxidase in particular glucose oxidase or lactate oxidase
  • a catalase-containing membrane is arranged at a greater distance from the electrode than an oxidase-containing membrane the adjustability of this distance between the two membranes makes it possible to optimize the available signal and to ensure that the greater part of the hydrogen peroxide formed is actually converted back to oxygen at the anode and not directly about the return path via the membrane containing catalase decomposes the hydrogen peroxide to water and oxygen.
  • the reaction at the anode leads to the decomposition of one molecule of hydrogen peroxide to one molecule of oxygen, whereas the catalase reaction with two molecules of hydrogen peroxide only allows one molecule of oxygen to recede into the surrounding substrate.
  • glucose oxidase as well as, for example, a lactate oxidase each form a molecule of hydrogen peroxide while consuming a molecule of oxygen, the fact that a larger part of the converted hydrogen peroxide is converted back directly under the action of the catalase would lead to a depletion of oxygen in the Lead environment and at the same time, of course, to a substantial reduction in the useful signal at the anode, or a reduction in the linear measuring range.
  • a particularly stable and high sensitivity can be achieved according to the invention in that the further membrane determining the distance between the membranes containing enzymes determines about 5 to 250%, preferably 50 to 150%, the strength of the membrane carrying an enzyme, which membrane is closer to the electrode be ⁇ contributes.
  • the design is advantageously made such that the electrode is covered by a semi-permeable membrane with an exclusion limit between 50 and 150 Daltons.
  • the design is advantageously made such that the membrane determining the distance between the enzyme-bearing membranes consists of hydrogels, furthermore preferably the enzyme-bearing membranes consist of radically crosslinked polymers, in particular of photocrosslinked polymers.
  • the use of free-radically crosslinked polymers as a matrix for the membranes carrying the enzymes has the advantage that the enzymes are incorporated into the membranes be particularly protected and maintain their high activity unhindered.
  • An improvement in the stability of the signal and a greater independence of the signal from the oxygen concentration in the measuring medium can be achieved by executing the membrane which creates the distance between the two enzyme membranes from a membrane for gases such as e.g. Oxygen, permeable, for the substance to be reacted, in particular glucose but impermeable material, preferably polysiloxanes, reach, such a membrane for the passage of glucose must have openings which are preferably larger than the thickness of the underlying membrane. In such openings one can be used for both oxygen and e.g. Glucose permeable membrane can be used.
  • gases such as e.g. Oxygen, permeable
  • glucose but impermeable material preferably polysiloxanes
  • An improvement in the stability of the enzymes in the manufacture of the membranes can be achieved in that the enzymes are mixed with polymeric substances, e.g. Polyethylene glycol, dissolved or dispersed in a membrane precursor and used in this form in the manufacture of the membrane.
  • polymeric substances e.g. Polyethylene glycol
  • the design for these purposes is advantageously such that at least one membrane containing a reactant for an enzymatic reaction product is arranged between a membrane containing an enzyme and the electrode, the reaction between said reactants and the enzymatic reaction product being electrical must be reversible.
  • the design is advantageously made such that the electrode for anodic Settlement of H2O2 is formed in 02 with derivation of electrons, noble metal, in particular platinum, being advantageous as suitable materials for such an electrode.
  • the working electrodes can be polarized relative to a reference electrode, a silver-silver chloride electrode being used as the reference electrode, for example.
  • the design is preferably such that the outer membrane, which contains enzymes, in particular catalase, for the higher molecular weight reaction products of an inner, enzyme-carrying membrane, in particular gluco - nolactone or pyruvate, is permeable.
  • FIG. 1 shows a section through a first one
  • FIG. 2 shows a schematic representation of the individual layers of the sensor shown in FIG. 1, the most important implementation reactions being indicated;
  • Fig. 3 in a to Fig. Similar representation shows a section through a modified embodiment of a sensor according to the invention;
  • Fig. 4 shows a section through a design of a sensor according to the invention, which is particularly suitable as an immunosensor, and
  • FIG. 5 shows a section through a modified design of a sensor.
  • a work piece made of platinum for example, is located on a substrate 1.
  • electrode 2 arranged.
  • a semipermeable membrane 3 is directly adjacent to the working electrode 2, to which a membrane 4 containing an enzyme, for example glucose oxidase or lactate oxidase, is connected.
  • This membrane 4 is followed by a further membrane 5, which is intended to increase the distance between the first membrane 4 containing an enzyme and a second membrane 6 containing another enzyme, for example catalase.
  • insulation for example made of a polyimide 7, is also indicated.
  • the glucose oxidase-containing membrane 4 converts the glucose with oxygen to gluconolactone + hydrogen peroxide, while when lactate is used, lactate oxidase is used in the membrane 4 to convert it to pyruvate and hydrogen peroxide .
  • the hydrogen peroxide passes through the semipermeable membrane 3 to the anode formed, for example, by platinum, the hydrogen peroxide being converted on this electrode 2 and the current caused by the released electrons being processed into a measurement signal.
  • the external for example catalytic, provided membrane 6, in which a conversion of hydrogen peroxide into water and oxygen takes place.
  • an additional membrane 5 is provided, which delays the diffusion of the hydrogen peroxide formed in the membrane 4 into the membrane 6 containing catalase, so that relatively more hydrogen peroxide molecules are seen towards the working electrode Long as such molecules in turn are broken down in the membrane 6.
  • the membrane 5 has a thickness, which is between approximately 5 and 250% of the thickness of the membrane 4 closer to the electrode, so that it immediately results that the relative dimensions shown in the drawing are not to scale.
  • the semipermeable membrane 3 immediately adjacent to the electrode 2 essentially serves to prevent molecules other than hydrogen peroxide from reaching the surface of the electrode and thus poisoning it.
  • This mode of operation of the membrane 3 is indicated in FIG. 2 by the arrow 8, the arrow 8 representing, for example, acetaminophen, uric acid or the like.
  • the membrane 5 the strength of which is selected as a function of the substances to be determined and the further boundary conditions with a view to optimizing the signal, can also have hydrophobic properties, for example, so that the diffusion of oxygen is less impeded than the diffusion ⁇ sion of glucose, so that overall a balanced balance between glucose and oxygen is always ensured in the area of the membrane 4 in order to ensure complete conversion of the glucose.
  • peroxidase or a porous conductive membrane can also be provided in the outermost membrane 6, it being necessary in each case in this membrane 6 to ensure that peroxide is broken down. Before they are in a porous conductive membrane, the hydrogen peroxide can thus be broken down electrically by catalytic cleavage.
  • the membrane 5 causes a flattening of the gradient for the diffusion of hydrogen peroxide through or to the catalase membrane, so that on the one hand more hydrogen peroxide reaches the electrode 2. Furthermore, as can be seen directly from the most important reaction equations above, a conversion of 100% of the oxygen takes place at the electrode, while only 50% of the originally present oxygen can be used again by the conversion in the catalase membrane.
  • the membrane 4 can contain a reactant for an enzymatic reaction product, so that the membrane 4 can be used as a storage medium for a quantity to be measured, the reaction product being formed in the outer membrane 6 .
  • a membrane which is permeable for the reaction product of the enzymatic reaction on the substrate side but impermeable for the reaction product of the second reactant to be accumulated is advantageously inserted between the two layers mentioned above.
  • a significant improvement in the stability of the enzymes in the production of the membranes can be achieved in that the solution which is the membrane precursor and which contains the enzyme contains, apart from water and the enzyme, predominantly polymeric components * .
  • the membrane formers are preferably dissolved in a mixture of water and polyethylene glycol and the enzyme is likewise dissolved or dispersed therein.
  • the membrane 5 which represents a specific embodiment, has a non-uniform membrane 5.
  • the membrane 5 consists of a hydrophobic sieve-like membrane 9, which is only permeable to gases.
  • stoppers 10 are used in the openings of the sieve-like membrane 9, which in particular consist of polyhydroxyethyl methaacrylate and which stoppers are permeable both for gases and for glucose.
  • the other reference numerals in FIG. 5 were chosen as in FIG.
  • Precursor 2 96% precursor 1 and 4% glucose oxidase
  • PS851 poly (dimethyl-2 -3% methyl methacryloxypropyl siloxane); Petrarch), 1% w.w '-dimethoxy w-phenylacetophenone and 50% toluene
  • Example 1 Integrated glucose / lactate sensor for in vivo
  • a flexible electrode structure is used as the electrochemical transducer, which is described in Urban G., Jobst G., Keplinger F., Aschauer E., Jachimowicz A., Kohl F. (1992); Biosensors & Bioelectronic 7: 733-739 and two platinum working electrodes with a surface area of 0.4 mm 2 which are provided with an electropolymerized permselective layer, a platinum counter electrode and a silver / silver chloride quasi reference electrode, which contains a flexible Polymer film are present and are insulated with a photostructurable polyimide insulation lacquer.
  • the wafer which contains a large number of electrode structures described above, is coated with precursor 2 by means of a centrifugal process.
  • Those working electrodes which are to be designed as glucose electrodes are irradiated with ultraviolet light for one minute through a photomask using a mask adjusting device.
  • This process forms an insoluble polymer network, which physically includes the enzymes it contains.
  • the unexposed and therefore not networked parts of the Precursors are dissolved in a mixture of polyethylene glycol / water 1: 1 with ultrasound support.
  • the catalase membrane is produced by the same process using Precursor 4 and covers the entire
  • the layer thicknesses of the membranes can be controlled in a simple manner, which in the example described is 4 ⁇ m for the glucose oxidase membrane, 6 ⁇ m for the lactate oxidase membrane, 6 ⁇ m for the intermediate membrane and 6 ⁇ m for the catalase membrane.
  • a platinum working electrode with a surface of 0.96 mm 2 which is provided with a permselective layer, is used as the electrochemical transducer, which is produced on silicon wafers together with a platinum counter electrode and a silver / silver chloride quasi reference electrode by means of thin-film technology processes.
  • the working electrodes of the wafer which contains a large number of electrode structures described above, are placed on the computer using a computer-controlled nanoliter metering device and a computer-controlled XY table which controls the metering device. Positioned over the individual working electrodes, 80 nl precursor 5 applied and then the entire wafer exposed to ultraviolet light. After the uncrosslinked precursor material has been removed with a mixture of polyethylene glycol / water 1: 1, an enzyme membrane with a layer thickness of 8 ⁇ m remains. This process is repeated with 40 ml precursor 1 and 40 ml precursor 6, which leads to the formation of the desired intermediate layer with a thickness of 4 ⁇ m and for the formation of the desired membrane destroying glutamate and water peroxide with a thickness of 4 ⁇ m.
  • the sensors according to this exemplary embodiment show the desired selectivity against glutamine even in the presence of glutamate, as a result of which no simultaneous determination of glutamate is necessary in order to obtain the correct glutamine concentration.
  • An electrode arrangement produced by means of conventional screen printing technology is used as the electrochemical transducer, which has working electrodes made of a mixture of graphite paste with horseradish peroxidase and binder and silver chloride counterelectrodes.
  • the thixotropic precursor 9 is applied to the working electrodes by means of a screen printing process and dried at room temperature.
  • the intermediate membrane is made of precursor 8 and the catalase membrane is made of precursor 10.
  • the electrical signal is not obtained by oxidation of hydrogen peroxide but by reduction of the enzyme mercury peroxidase adhering to the graphite electrode, which in turn is pxidized by hydrogen peroxide.
  • Example 4 Affinity sensor with intermediate product accumulation
  • Example 2 The electrode structure described in Example 2 is used as the electrochemical transducer.
  • a 3 ⁇ m thick horseradish peroxidase membrane is formed above the working electrode, which in this case has no permselective membrane, in accordance with the method described in Example 1 using precursor 11.
  • This membrane is impregnated with a 1% aqueous solution of 2,5-dihydroxy benzoic acid and in turn covered with a 2 ⁇ m thick gas-permeable membrane which is produced from precursor 7.
  • precursor 12 a third approximately 0.5 ⁇ m thick membrane is formed over the gas-permeable membrane using the same method, to the primary amino groups of which the desired antibody or antigen is bound by means of glutardialdehyde coupling.
  • the enzymatic activity of this membrane only develops in the course of the affinity test, which is carried out as a classic "sandwich" test with glucose oxidase as the marker enzyme.
  • the electrical signal is obtained by reducing the p-benzoquinonecarboxylic acid, which in turn is oxidized by hydrogen peroxide under the catalytic effect of horseradish peroxidase.
  • An advantage of the arrangement described is the possibility of accumulating the resulting oxidized intermediate product by keeping the working electrode used currentless. After a certain period of time has elapsed, by means of which the sensitivity of the sensor can be controlled in the simplest way, a corresponding cathodic polarization voltage is applied to the working electrode and thus the quinone reduced, the measurement signal being obtained at the same time and the sensor returning to its initial state.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Dans un détecteur visant à détecter des substances décomposables biologiquement, notamment par voie enzymatique, d'un substrat biologique à l'aide d'une électrode (2) et d'au moins une membrane qui recouvre l'électrode (2), il est prévu au moins trois membranes (3, 4, 5, 6) dont au moins deux contiennent chacune au moins une enzyme pour la décomposition de la substance biologique à identifier et/ou un réactif pour un produit de réaction de la réaction enzymatique. Au moins une autre membrane (3, 5) sert à instaurer un écart entre les deux membranes précitées (4, 6) et/ou à servir de membrane permsélective.
EP93923968A 1992-10-29 1993-10-29 Detecteur pour detecter des substances decomposables biologiquement Withdrawn EP0666981A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AT214292A AT399511B (de) 1992-10-29 1992-10-29 Sensor zur erfassung von biologisch umsetzbaren substanzen
AT2142/92 1992-10-29
PCT/AT1993/000168 WO1994010560A1 (fr) 1992-10-29 1993-10-29 Detecteur pour detecter des substances decomposables biologiquement

Publications (1)

Publication Number Publication Date
EP0666981A1 true EP0666981A1 (fr) 1995-08-16

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Application Number Title Priority Date Filing Date
EP93923968A Withdrawn EP0666981A1 (fr) 1992-10-29 1993-10-29 Detecteur pour detecter des substances decomposables biologiquement

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EP (1) EP0666981A1 (fr)
AT (1) AT399511B (fr)
WO (1) WO1994010560A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19621241C2 (de) * 1996-05-25 2000-03-16 Manfred Kessler Membranelektrode zur Messung der Glucosekonzentration in Flüssigkeiten
US6764581B1 (en) 1997-09-05 2004-07-20 Abbott Laboratories Electrode with thin working layer
WO2004007756A1 (fr) * 2002-07-12 2004-01-22 Novo Nordisk A/S Procede servant a ameliorer l'efficacite de detecteurs de glucose in vivo
US8165651B2 (en) 2004-02-09 2012-04-24 Abbott Diabetes Care Inc. Analyte sensor, and associated system and method employing a catalytic agent
US7699964B2 (en) 2004-02-09 2010-04-20 Abbott Diabetes Care Inc. Membrane suitable for use in an analyte sensor, analyte sensor, and associated method
US7885698B2 (en) 2006-02-28 2011-02-08 Abbott Diabetes Care Inc. Method and system for providing continuous calibration of implantable analyte sensors
CN105899132B (zh) 2013-12-31 2020-02-18 雅培糖尿病护理公司 自供电分析物传感器以及使用其的装置

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0617889B2 (ja) * 1984-11-27 1994-03-09 株式会社日立製作所 生物化学センサ
CA1249333A (fr) * 1985-06-28 1989-01-24 James E. Jones Capteur elrectrochimique, et sa membrane
GB8522834D0 (en) * 1985-09-16 1985-10-23 Ici Plc Sensor
US4891104A (en) * 1987-04-24 1990-01-02 Smithkline Diagnostics, Inc. Enzymatic electrode and electrode module and method of use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9410560A1 *

Also Published As

Publication number Publication date
ATA214292A (de) 1994-10-15
WO1994010560A1 (fr) 1994-05-11
AT399511B (de) 1995-05-26

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