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
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
• • 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
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

• the invention relates to a method and a reagent system for the detection of an analyte in a sample by an enzymatic reaction, comprising the use of a detection reagent which contains a coenzyme and a catalytically inactive coenzyme-binding protein.
• the detection of analytes, for example glucose in blood, by enzymatic methods is known.
• the analyte to be determined is mixed with a detection reagent that contains a detectable by an enzymatic reaction, e.g. reducible or oxidizable, containing coenzyme, brought into contact.
• a detection reagent that contains a detectable by an enzymatic reaction, e.g. reducible or oxidizable, containing coenzyme, brought into contact.
• the redox equivalents that result from the reduction or oxidation of the coenzyme can be transferred to mediators, which are then recorded electrochemically or photometrically in a further step.
• a calibration provides a direct relationship between the measured value and the concentration of the analyte to be determined.
• mediators on the one hand require the use of complex reaction mixtures, which lead to a low stability and a high susceptibility to interference of the detection reaction, on the other hand mediators are often required in order to be able to carry out a detection or a sufficient one To achieve detection sensitivity.
• the object underlying the present invention was to at least partially avoid the disadvantages of the prior art described.
• an insensitive and sensitive Methods for the detection of analytes are provided, which leads to reliable measurement results even in the absence of mediators.
• a catalytically inactive coenzyme-binding protein is added to the other constituents of an enzymatic detection reagent.
• the catalytically inactive protein is able to bind a coenzyme modified by reaction of the analyte and thus to improve its detectability, in particular by optical methods.
• the invention thus relates to a method for the detection of an analyte in a sample by an enzymatic reaction, comprising the steps:
• the invention further relates to a reagent system for the detection of an analyte in a sample, comprising: (a) a coenzyme and (b) a catalytically inactive coenzyme-binding protein.
• the present invention enables simple qualitative or quantitative determination of analytes by an enzymatic reaction.
• the method is suitable for the detection of any analytes that can be detected by an enzymatic reaction with the participation of a coenzyme.
• the method according to the invention is distinguished from known methods in that the Detection reagent a catalytically inactive coenzyme-binding protein is added, whereby an improved detection sensitivity is brought about.
• the detection reagent contains the catalytically inactive protein in a sufficient amount to enable improved sensitivity in a qualitative and / or quantitative determination of the analyte in accordance with the desired test format. Since the method according to the invention preferably detects the coenzyme modified by the enzymatic reaction, in many cases the presence of mediators or other substances which can bring about regeneration of the coenzyme is not necessary.
• the method and the detection system allow the use of very small sample quantities, for example sample volumes ⁇ 1 ⁇ ⁇ , preferably ⁇ 0, 1 ⁇ ⁇ . If necessary, the sample can be diluted before contact with the detection reagent.
• the method and detection system according to the invention is suitable for determining any analyte, for example parameters in biological samples, such as body fluids, such as blood, serum, plasma or urine, but also in waste water samples or food.
• the method can be used both as a wet test, e.g. in a cuvette, or as a dry test on an appropriate reagent carrier.
• any biological or chemical substances that can be determined by an enzymatic reaction such as enzymes or enzyme substrates, can be selected as the analyte, the reaction comprising, in particular, a redox reaction.
• suitable analytes are, for example, glucose, lactic acid, malic acid, glycerol, alcohol, cholesterol, triglycerides, ascorbic acid, cysteine, glutathione, peptides etc.
• the enzymatic reaction is preferably a redox reaction in which the coenzyme to be detected is reduced or oxidized.
• an oxidoreductase is preferably used as the enzyme in the detection reagent.
• a dehydrogenase is particularly preferably used as the enzyme, for example selected from a glucose dehydrogenase (EC1 .1 .1 .47), lactate dehydrogenase (EC1 .1 .1 .27, 1 .1 .1 .28), malate dehydrogenase ⁇ EC1 .1 .1 .37), glycerol dehydrogenase (EC1 .1 .1 .6), alcohol dehydrogenase (EC1 .1 .1 .1) or amino acid dehydrogenase, e.g. L-amino acid dehydrogenase (EC1 .4.1 .5).
• Other suitable enzymes are oxidases, such as glucose oxidase (EC1 .1 .3.4) or cholesterol oxidase (EC1 .1 .3.6).
• the detection reaction is preferably a reduction or oxidation and an oxidoreductase is detected as an enzyme.
• Coenzymes in the sense of the present invention are preferably organic molecules which are covalently or non-covalently bound to an enzyme and which are changed, for example oxidized or reduced, by the reaction of the analyte.
• Preferred examples of coenzymes are flavin, nicotine and quinone derivatives, for example flavin nucleoside derivatives such as FAD, FADH 2 , FMN, FMNH 2 , etc., nicotine nucleoside derivatives such as NAD + , NADH / FT, NADP + , NADPH / FT etc. or Ubiquinones, such as coenzyme Q, PQQ etc.
• NADH / H + is particularly preferred as the coenzyme.
• the change in the coenzyme by reaction with the analyte can in principle be detected in any manner. In principle, all methods known from the prior art for the detection of enzymatic reactions can be used here. Preferably however, the change in the coenzyme is detected by optical methods. Optical detection methods include, for example, the measurement of absorption, fluorescence, circular dichroism (CD), optical rotation dispersion (ORD) or refractometry. The change in the coenzyme is particularly preferably detected by measuring the fluorescence. The fluorescence measurement is highly sensitive and enables the detection of even low concentrations of the analyte in miniaturized systems.
• the detectability is improved, in particular by optical methods.
• the binding of the coenzyme to a catalytically inactive protein leads in particular to an improved fluorescence yield of the coenzyme.
• the method or detection system according to the invention can comprise a liquid test, the reagent e.g. is in the form of a solution or suspension in an aqueous or non-aqueous liquid or as a powder or lyophilisate.
• the method and detection system according to the invention preferably includes a dry test, the reagent being applied to a support.
• the carrier can comprise, for example, a test strip comprising an absorbent and / or swellable material which is wetted by the sample liquid to be examined.
• the catalytically inactive coenzyme-binding protein is able to bind the coenzyme formed as the product of the enzymatic detection reaction, whereby the binding of the coenzyme results in an improved detectability of the coenzyme reaction product.
• the catalytically inactive protein is preferably an inactivated enzyme or a fragment of an inactivated enzyme which is not catalytically active but still contains a coenzyme binding site.
• an inactivated oxidoreductase e.g. B. an inactivated dehydrogenase.
• an inactivated NADH-binding dehydrogenase such as glucose dehydrogenase, is particularly preferred.
• Inactivation of enzymes can be brought about by mutations in the amino acid sequence, for example deletions, insertions and / or substitutions of individual amino acids or sections of several amino acids.
• a mutation takes place in the catalytic center of the enzyme.
• glucose dehydrogenase can be mutagenized on the histidine residue at position 14, which is essential for the catalytic activity, for example to serine or tryptophan.
• the enzyme can also be inactivated by chemical modifications, for example chemical modifications in the catalytic center, by which the catalytic activity is at least largely eliminated, while the ability to bind the coenzyme is retained.
• the detection reagent is stored in a matrix, for example in an absorbent material or in a gel matrix.
• the gel matrix preferably has a layer thickness of ⁇ 50 ⁇ m, in particular ⁇ 5 ⁇ m, and is applied to a carrier, for example an at least partially optically transparent carrier.
• the gel matrix is preferably a polymer which is based on photopolymerizable monomers, such as acrylic monomers, for example acrylamide or / and acrylic esters, such as polyethylene glycol diacrylate, or vinyl aromatic monomers, for example 4-vinylbenzenesulfonic acid, or combinations thereof.
• a liquid containing the reagent comprising a coenzyme, a catalytically inactive protein can be used to produce such a gel matrix or contains several photopolymerizable monomers and optionally enzyme, photoinitiator and / or non-reactive constituents, applied to an at least partially optically transparent support, for example on a plastic film, and irradiated, for example, with UV light from the back, so that the monomer polymerizes or the monomers on the carrier take place up to a predetermined layer thickness.
• the layer thickness can be controlled by adding absorbent substances to the reagent and / or by the duration or intensity of the irradiation. Excess liquid reagent can be removed after the polymerization and used again.
• the matrix can also be produced by conventional coating procedures, the liquid reagent being applied to a support, there using suitable methods, e.g. with a doctor blade, brought to the desired thickness and then dried or completely polymerized.
• the catalytically inactive protein and possibly the enzyme are in a protected microenvironment. If the polymeric gel matrix is sufficiently cross-linked, the protein molecules are in an immobilized form. Low molecular weight substances or glucose or other analytes or also coenzymes can diffuse freely through the polymer network.
• the catalytically inactive protein and optionally the enzyme can be polymerized into the matrix together with the coenzyme, or the matrix can be brought into contact with a solution of the coenzyme after the polymerization, so that it can diffuse into the matrix.
• the coenzyme modified, for example reduced or oxidized, by the reaction is optimally protected from interferences by binding to the inactive protein and, if appropriate, additionally by incorporation into the gel matrix. Furthermore, the present invention is to be explained in more detail by the following example.
• the essential histidine of GlucDH at position 147 was exchanged for serine by known methods.

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• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
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• 2003
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