EP1504116A1 - Procede et systeme de reactifs contenant une enzyme inactivee - Google Patents
Procede et systeme de reactifs contenant une enzyme inactiveeInfo
- Publication number
- EP1504116A1 EP1504116A1 EP03752757A EP03752757A EP1504116A1 EP 1504116 A1 EP1504116 A1 EP 1504116A1 EP 03752757 A EP03752757 A EP 03752757A EP 03752757 A EP03752757 A EP 03752757A EP 1504116 A1 EP1504116 A1 EP 1504116A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- coenzyme
- enzyme
- dehydrogenase
- analyte
- detection
- 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- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 46
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 34
- 102000004190 Enzymes Human genes 0.000 title claims description 31
- 108090000790 Enzymes Proteins 0.000 title claims description 31
- 239000005515 coenzyme Substances 0.000 claims abstract description 54
- 238000001514 detection method Methods 0.000 claims abstract description 40
- 239000012491 analyte Substances 0.000 claims abstract description 23
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 14
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 11
- 108091008324 binding proteins Proteins 0.000 claims abstract description 11
- 239000011159 matrix material Substances 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 102000004169 proteins and genes Human genes 0.000 claims description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- 230000003197 catalytic effect Effects 0.000 claims description 8
- 101710088194 Dehydrogenase Proteins 0.000 claims description 7
- 108090000854 Oxidoreductases Proteins 0.000 claims description 6
- 102000004316 Oxidoreductases Human genes 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 6
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 6
- 230000003287 optical effect Effects 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 5
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims description 4
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims description 4
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 claims description 4
- 238000007385 chemical modification Methods 0.000 claims description 4
- 230000035772 mutation Effects 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 235000017471 coenzyme Q10 Nutrition 0.000 claims description 3
- 229960002715 nicotine Drugs 0.000 claims description 3
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 claims description 3
- 230000003647 oxidation Effects 0.000 claims description 3
- 238000007254 oxidation reaction Methods 0.000 claims description 3
- 238000006479 redox reaction Methods 0.000 claims description 3
- 239000000758 substrate Substances 0.000 claims description 3
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 claims description 2
- 102000007698 Alcohol dehydrogenase Human genes 0.000 claims description 2
- 108010021809 Alcohol dehydrogenase Proteins 0.000 claims description 2
- YPZRHBJKEMOYQH-UYBVJOGSSA-N FADH2 Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1COP(O)(=O)OP(O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C(NC(=O)NC2=O)=C2NC2=C1C=C(C)C(C)=C2 YPZRHBJKEMOYQH-UYBVJOGSSA-N 0.000 claims description 2
- YTNIXZGTHTVJBW-SCRDCRAPSA-N FMNH2 Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2NC2=C1NC(=O)NC2=O YTNIXZGTHTVJBW-SCRDCRAPSA-N 0.000 claims description 2
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 claims description 2
- 108030000198 L-amino-acid dehydrogenases Proteins 0.000 claims description 2
- 102000013460 Malate Dehydrogenase Human genes 0.000 claims description 2
- 108010026217 Malate Dehydrogenase Proteins 0.000 claims description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 claims description 2
- 210000001124 body fluid Anatomy 0.000 claims description 2
- 239000010839 body fluid Substances 0.000 claims description 2
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 claims description 2
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 claims description 2
- 150000004059 quinone derivatives Chemical class 0.000 claims description 2
- 229940088598 enzyme Drugs 0.000 description 21
- 235000018102 proteins Nutrition 0.000 description 12
- 239000000523 sample Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 6
- 239000000178 monomer Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000002250 absorbent Substances 0.000 description 3
- 230000002745 absorbent Effects 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- -1 flavin nucleoside derivatives Chemical class 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000006116 polymerization reaction Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000002983 circular dichroism Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- MAGFQRLKWCCTQJ-UHFFFAOYSA-N 4-ethenylbenzenesulfonic acid Chemical compound OS(=O)(=O)C1=CC=C(C=C)C=C1 MAGFQRLKWCCTQJ-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 108010089254 Cholesterol oxidase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 102000024135 NADH binding proteins Human genes 0.000 description 1
- 108091012742 NADH binding proteins Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 229960002433 cysteine Drugs 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 125000004386 diacrylate group Chemical group 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 150000002303 glucose derivatives Chemical class 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 235000003969 glutathione Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- 210000002966 serum Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 150000003669 ubiquinones Chemical class 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
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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Abstract
Applications Claiming Priority (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10221840 | 2002-05-16 | ||
DE2002121846 DE10221846A1 (de) | 2002-05-16 | 2002-05-16 | Verfahren und Reagenzsystem mit inaktiviertem Enzym |
DE2002121840 DE10221840A1 (de) | 2002-05-16 | 2002-05-16 | Verfahren zur Herstellung von Polymerschichten |
DE10221845 | 2002-05-16 | ||
DE2002121845 DE10221845A1 (de) | 2002-05-16 | 2002-05-16 | Verfahren und Reagenzsystem mit nicht-regenerierbarem Enzym-Coenzym-Komplex |
DE10221846 | 2002-05-16 | ||
PCT/EP2003/005177 WO2003097863A1 (fr) | 2002-05-16 | 2003-05-16 | Procédé et système de réactifs contenant une enzyme inactivée |
Publications (1)
Publication Number | Publication Date |
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EP1504116A1 true EP1504116A1 (fr) | 2005-02-09 |
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ID=29553736
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP03752757A Withdrawn EP1504116A1 (fr) | 2002-05-16 | 2003-05-16 | Procede et systeme de reactifs contenant une enzyme inactivee |
EP03730061A Expired - Lifetime EP1504113B1 (fr) | 2002-05-16 | 2003-05-16 | Procede de fabrication de couches polymeres |
EP03732396A Withdrawn EP1504115A1 (fr) | 2002-05-16 | 2003-05-16 | Procede et systeme de reactifs contenant un complexe enzyme-coenzyme non regenerable |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
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EP03730061A Expired - Lifetime EP1504113B1 (fr) | 2002-05-16 | 2003-05-16 | Procede de fabrication de couches polymeres |
EP03732396A Withdrawn EP1504115A1 (fr) | 2002-05-16 | 2003-05-16 | Procede et systeme de reactifs contenant un complexe enzyme-coenzyme non regenerable |
Country Status (15)
Country | Link |
---|---|
US (3) | US7341830B2 (fr) |
EP (3) | EP1504116A1 (fr) |
JP (4) | JP5118288B2 (fr) |
KR (2) | KR101164048B1 (fr) |
CN (2) | CN1653189B (fr) |
AT (1) | ATE345396T1 (fr) |
AU (3) | AU2003232790A1 (fr) |
BR (2) | BR0311175A (fr) |
CA (2) | CA2493918C (fr) |
DE (1) | DE50305687D1 (fr) |
DK (1) | DK1504113T3 (fr) |
ES (1) | ES2275095T3 (fr) |
HK (2) | HK1081599A1 (fr) |
MX (2) | MXPA04011220A (fr) |
WO (3) | WO2003097859A2 (fr) |
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- 2003-05-16 WO PCT/EP2003/005178 patent/WO2003097864A1/fr active Application Filing
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