EP1504116A1 - Method and reagent system having an inactivated enzyme - Google Patents

Method and reagent system having an inactivated enzyme

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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Withdrawn
Application number
EP03752757A
Other languages
German (de)
French (fr)
Inventor
Carina Horn
Joachim Hoenes
Wolfgang-Reinhold Knappe
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.)
F Hoffmann La Roche AG
Roche Diagnostics GmbH
Original Assignee
F Hoffmann La Roche AG
Roche Diagnostics GmbH
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
Priority claimed from DE2002121845 external-priority patent/DE10221845A1/en
Priority claimed from DE2002121840 external-priority patent/DE10221840A1/en
Priority claimed from DE2002121846 external-priority patent/DE10221846A1/en
Application filed by F Hoffmann La Roche AG, Roche Diagnostics GmbH filed Critical F Hoffmann La Roche AG
Publication of EP1504116A1 publication Critical patent/EP1504116A1/en
Withdrawn legal-status Critical Current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
    • C12Q1/26—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.

Abstract

The invention relates to a method and reagent system for detecting an analyte in a sample by means of an enzymatic reaction, involving the use of a detection reagent containing a coenzyme and a catalytically inactive coenzyme-binding protein.

Description

Verfahren und Reagenzsystem mit inaktiviertem Enzym Process and reagent system with inactivated enzyme
Beschreibungdescription
Die Erfindung betrifft ein Verfahren und ein Reagenzsystem zum Nachweis eines Analyten in einer Probe durch eine enzymatische Reaktion, umfassend die Verwendung eines Nachweisreagenz, das ein Coenzym und ein katalytisch inaktives Coenzym-bindendes Protein enthält.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.
Der Nachweis von Analyten, beispielsweise Glucose in Blut, durch enzymatische Methoden ist bekannt. Dabei wird der zu bestimmende Analyt mit einem Nachweisreagenz, das ein durch eine enzymatische Reaktion nachweisbares, z.B. reduzierbares oder oxidierbares, Coenzym enthält, in Kontakt gebracht. Die bei Reduktion bzw. Oxidation des Coenzyms entstehenden Redoxäquivalente können auf Mediatoren übertragen werden, die dann in einem weiteren Schritt elektrochemisch oder photometrisch erfasst werden. Eine Kalibrierung liefert einen direkten Zusammenhang des Messwerts mit der Konzentration des zu bestimmenden Analyten.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. 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.
Ein beim Nachweis der enzymatischen Reaktion oft auftretendes Problem besteht darin, dass Mediatoren einerseits den Einsatz komplexer Reaktionsgemische erfordern, die zu einer geringen Stabilität und einer hohen Störanfälligkeit der Nachweisreaktion führen, andererseits werden Mediatoren oftmals benötigt, um überhaupt einen Nachweis durchführen zu können oder eine ausreichende Nachweissensitivität zu erreichen.A problem that often arises in the detection of the enzymatic reaction is that 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.
Die der vorliegenden Erfindung zugrunde liegende Aufgabe bestand darin, die geschilderten Nachteile des Standes der Technik mindestens teilweise zu vermeiden. Insbesondere sollte ein unempfindliches und sensitives Verfahren zum Nachweis von Analyten bereitgestellt werden, welches auch in Abwesenheit von Mediatoren zu zuverlässigen Messergebnissen führt.The object underlying the present invention was to at least partially avoid the disadvantages of the prior art described. In particular, an insensitive and sensitive Methods for the detection of analytes are provided, which leads to reliable measurement results even in the absence of mediators.
Diese Aufgabe wird dadurch gelöst, dass den übrigen Bestandteilen eines enzymatischen Nachweisreagenz ein katalytisch inaktives Coenzym- bindendes Protein zugesetzt wird. Insbesondere ist das katalytisch inaktive Protein in der Lage, ein durch Reaktion des Analyten verändertes Coenzym zu binden und somit dessen Nachweisbarkeit, insbesondere durch optische Methoden zu verbessern.This object is achieved in that a catalytically inactive coenzyme-binding protein is added to the other constituents of an enzymatic detection reagent. In particular, 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.
Ein Gegenstand der Erfindung ist somit ein Verfahren zum Nachweis eines Analyten in einer Probe durch eine enzymatische Reaktion, umfassend die Schritte:The invention thus relates to a method for the detection of an analyte in a sample by an enzymatic reaction, comprising the steps:
(a) Inkontaktbringen der Probe mit einem Nachweisreagenz, umfassend ein Coenzym und ein katalytisch inaktives Coenzym-bindendes(a) contacting the sample with a detection reagent comprising a coenzyme and a catalytically inactive coenzyme-binding
Protein, wobei das Coenzym durch Reaktion mit dem Analyten verändert wird und das veränderte Coenzym an das katalytisch inaktive Protein bindet, undProtein, wherein the coenzyme is changed by reaction with the analyte and binds the changed coenzyme to the catalytically inactive protein, and
(b) Nachweisen einer Reaktion des Analyten durch Veränderung des Coenzyms.(b) Detecting a reaction of the analyte by changing the coenzyme.
Ein weiterer Gegenstand der Erfindung ist ein Reagenzsystem zum Nachweis eines Analyten in einer Probe, umfassend: (a) ein Coenzym und (b) ein katalytisch inaktives Coenzym-bindendes Protein.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.
Die vorliegende Erfindung ermöglicht eine einfache qualitative oder quantitative Bestimmung von Analyten durch eine enzymatische Reaktion. Das Verfahren eignet sich zum Nachweis beliebiger Analyten, die durch eine enzymatische Reaktion unter Beteiligung eines Coenzyms nachgewiesen werden können. Das erfindungsgemäße Verfahren zeichnet sich gegenüber bekannten Verfahren dadurch aus, dass dem Nachweisreagenz ein katalytisch inaktives Coenzym-bindendes Protein zugesetzt wird, wobei eine verbesserte Nachweissensitivität bewirkt wird. Das Nachweisreagenz enthält das katalytisch inaktive Protein in einer ausreichenden Menge, um entsprechend dem gewünschten Testformat eine verbesserte Sensitivität bei einer qualitativen oder/und quantitativen Bestimmung des Analyten zu ermöglichen. Da bei dem erfindungsgemäßen Verfahren bevorzugt ein direkter Nachweis des durch die enzymatische Reaktion veränderten Coenzyms erfolgt, ist eine Anwesenheit von Mediatoren oder anderen Substanzen, die eine Regeneration des Coenzyms bewirken können, in vielen Fällen nicht erforderlich.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.
Das Verfahren und das Nachweissystem erlauben die Verwendung von kleinsten Probenmengen, beispielsweise Probenvolumina < 1 μ\, vorzugsweise <0, 1 μ\. Gegebenenfalls kann die Probe vor dem Inkontaktbringen mit dem Nachweisreagenz noch verdünnt werden.The method and the detection system allow the use of very small sample quantities, for example sample volumes <1 μ \, preferably <0, 1 μ \. If necessary, the sample can be diluted before contact with the detection reagent.
Das erfindungsgemäße Verfahren und Nachweissystem eignet sich zur Bestimmung beliebiger Analyten, beispielsweise Parametern in biologischen Proben, wie etwa Körperflüssigkeiten, wie etwa Blut, Serum, Plasma oder Urin, aber auch in Abwasserproben oder Lebensmitteln. Das Verfahren kann sowohl als Nasstest, z.B. in einer Küvette, oder als Trockentest auf einem entsprechenden Reagenzträger durchgeführt werden.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.
Als Analyten können beliebige biologische oder chemische Substanzen ausgewählt werden, die durch eine enzymatische Reaktion bestimmt werden können, wie etwa Enzyme oder Enzymsubstrate, wobei die Reaktion, insbesondere eine Redoxreaktion umf asst. Beispiele für geeignete Analyten sind etwa Glucose, Milchsäure, Äpfelsäure, Glycerin, Alkohol, Cholesterin, Triglyceride, Ascorbinsäure, Cystein, Glutathion, Peptide etc.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. Examples of suitable analytes are, for example, glucose, lactic acid, malic acid, glycerol, alcohol, cholesterol, triglycerides, ascorbic acid, cysteine, glutathione, peptides etc.
Die enzymatische Reaktion ist vorzugsweise eine Redoxreaktion, bei der eine Reduktion oder Oxidation des nachzuweisenden Coenzyms erfolgt. Für eine derartige Reaktion zum Nachweis von Enzymsubstraten wird als Enzym im Nachweisreagenz vorzugsweise eine Oxidoreduktase verwendet. Besonders bevorzugt verwendet man als Enzym eine Dehydrogenase, beispielsweise ausgewählt aus einer Glucose-Dehydrogenase (E.C.1 .1 .1 .47), Lactat-Dehydrogenase (E.C.1 .1 .1 .27, 1 .1 .1 .28), Malat- Dehydrogenase {E.C.1 .1 .1 .37), Glycerin-Dehydrogenase (E.C.1 .1 .1 .6), Alkohol-Dehydrogenase (E.C.1 .1 .1 .1 ) oder Aminosäure-Dehydrogenase, z.B. L-Aminosäure-Dehydrogenase (E.C.1 .4.1 .5). Weitere geeignete Enzyme sind Oxidasen, wie etwa Glucoseoxidase (E.C.1 .1 .3.4) oder Cholesterinoxidase (E.C.1 .1 .3.6).The enzymatic reaction is preferably a redox reaction in which the coenzyme to be detected is reduced or oxidized. For such a reaction for the detection of enzyme substrates, 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).
Wenn als Analyt ein Enzym nachgewiesen werden soll, ist die Anwesenheit eines Enzyms im Nachweisreagenz oftmals niGht erforderlich. In diesem Fall wird eine durch das in der Probe als Analyt vorhandene Enzym bewirkte Veränderung des Coenzyms nachgewiesen. Auch in diesem Falle ist die Nachweisreaktion bevorzugt eine Reduktion oder Oxidation und als Enzym wird eine Oxidoreduktase nachgewiesen.If an enzyme is to be detected as an analyte, the presence of an enzyme in the detection reagent is often not necessary. In this case, a change in the coenzyme caused by the enzyme present as analyte in the sample is detected. In this case too, the detection reaction is preferably a reduction or oxidation and an oxidoreductase is detected as an enzyme.
Coenzyme im Sinne der vorliegenden Erfindung sind vorzugsweise organische Moleküle, die kovalent oder nichtkovalent an ein Enzym gebunden sind und durch die Umsetzung des Analyten verändert, beispielsweise oxidiert oder reduziert werden. Bevorzugte Beispiele für Coenzyme sind Flavin-, Nicotin- und Chinonderivate, beispielsweise Flavinnukleosidderivate, wie etwa FAD, FADH2, FMN, FMNH2, etc., Nicotinnukleosidderivate wie etwa NAD + , NADH/FT, NADP+, NADPH/FT etc. oder Ubichinone, wie etwa Coenzym Q, PQQ etc. Besonders bevorzugt ist NADH/H+ als Coenzym.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.
Die Veränderung des Coenzyms durch Reaktion mit dem Analyten kann grundsätzlich auf beliebige Art und Weise nachgewiesen werden. Hier können grundsätzlich alle aus dem Stand der Technik bekannten Methoden zum Nachweis enzymatischer Reaktionen eingesetzt werden. Vorzugsweise wird jedoch die Veränderung des Coenzyms durch optische Methoden nachgewiesen. Optische Nachweismethoden umfassen beispielsweise die Messung von Absorption, Fluoreszenz, Circulardichroismus (CD), optische Rotationsdispersion (ORD) oder Refraktometrie. Besonders bevorzugt wird die Veränderung des Coenzyms durch Messung der Fluoreszenz nachgewiesen. Die Fluoreszenzmessung ist hochsensitiv und ermöglicht den Nachweis selbst geringer Konzentrationen des Analyten in miniaturisierten Systemen.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.
Durch Bindung des Coenzyms an das katalytisch inaktive Protein wird eine Verbesserung der Nachweisbarkeit, insbesondere durch optische Methoden bewirkt. So führt die Bindung des Coenzyms an ein katalytisch inaktives Protein insbesondere zu einer verbesserten Fluoreszenzausbeute des Coenzyms.By binding the coenzyme to the catalytically inactive protein, 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.
Das erfindungsgemäße Verfahren oder Nachweissystem kann einen Flüssigtest umfassen, wobei das Reagenz z.B. in Form einer Lösung oder Suspension in einer wässrigen oder nichtwässrigen Flüssigkeit oder als Pulver oder Lyophilisat vorliegt. Vorzugsweise beinhaltet das erfiήdungsgemäße Verfahren und Nachweissystem jedoch einen Trockentest, wobei das Reagenz auf einem Träger aufgebracht ist. Der Träger kann beispielsweise einen Teststreifen, umfassend ein saugfähiges oder/und quellbares Material, umfassen, das von der zu untersuchenden Probeflüssigkeit benetzt wird.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. However, 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.
Das katalytisch inaktive Coenzym-bindende Protein ist in der Lage, das als Produkt der enzymatischen Nachweisreaktion entstehende Coenzym zu binden, wobei durch die Bindung des Coenzyms eine verbesserte Nachweisbarkeit des Coenzym-Reaktionsprodukts bewirkt wird. Das katalytisch inaktive Protein ist vorzugsweise ein inaktiviertes Enzym oder ein Fragment eines inaktivierten Enzyms, welche nicht katalytisch aktiv ist, aber noch eine Coenzym-Bindungsstelle enthält. Vorzugsweise verwendet man eine inaktivierte Oxidoreduktase, z. B. eine inaktivierte Dehydrogenase. Besonders bevorzugt ist die Verwendung einer inaktivierten NADH-bindenden Dehydrogenase, wie etwa Glucose- Dehydrogenase.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. Preferably used an inactivated oxidoreductase, e.g. B. an inactivated dehydrogenase. The use of an inactivated NADH-binding dehydrogenase, such as glucose dehydrogenase, is particularly preferred.
Die Inaktivierung von Enzymen kann durch Mutationen in der Aminosäuresequenz, beispielsweise Deletionen, Insertionen oder/und Substitutionen einzelner Aminosäuren oder Abschnitten von mehreren Aminosäuren, bewirkt werden. In einer besonders bevorzugten Ausführungsform erfolgt eine Mutation im katalytischen Zentrum des Enzyms. So kann beispielsweise Glucose-Dehydrogenase an dem für die katalytische Aktivität essenziellen Histidinrest an Possition 1 47 mutagenisiert werden, beispielsweise zu Serin oder Tryptophan. DasInactivation 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. In a particularly preferred embodiment, a mutation takes place in the catalytic center of the enzyme. For example, 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
Ergebnis ist ein NADH-bindendes Protein ohne substanzielle katalytische Aktivität. Andererseits kann die Inaktivierung des Enzyms auch durch chemische Modifikationen, beispielsweise chemische Modifikationen im katalytischen Zentrum, erfolgen, durch die die katalytische Aktivität zumindest weitgehend beseitigt wird, während die Fähigkeit zur Bindung des Coenzyms erhalten bleibt.The result is a NADH-binding protein without substantial catalytic activity. On the other hand, 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.
In einer besonders bevorzugten Ausführungsform wird das Nachweisreagenz in einer Matrix eingelagert, beispielsweise in einem saugfähigen Material oder in einer Gelmatrix, verwendet. Die Gelmatrix weist vorzugsweise eine Schichtdicke < 50 μm, insbesondere < 5 μm, auf und ist auf einem Träger, beispielsweise einem zumindest teilweise optisch transparenten Träger aufgebracht. Die Gelmatrix ist vorzugsweise ein Polymer, das auf Basis von photopolymerisierbaren Monomeren, wie etwa acrylischen Monomeren, z.B. Acrylamid oder/und Acrylsäureestern, wie Polyethylenglykoldiacrylat, oder vinylaromatischen Monomeren, z.B. 4- Vinylbenzolsulfonsäure, oder Kombinationen davon, aufgebaut ist. Zur Herstellung einer derartigen Gelmatrix kann eine Flüssigkeit, die das Reagenz, umfassend ein Coenzym, ein katalytisch inaktives Protein, ein oder mehrere photopolymerisierbare Monomere sowie gegebenenfalls Enzym, Photoinitiator oder/und nichtreaktive Bestandteile, enthält, auf einen zumindest teilweise optisch transparenten Träger, beispielsweise auf eine Plastikfolie, aufgetragen und z.B. mit UV-Licht von der Rückseite her bestrahlt werden, so dass eine Polymerisation des Monomers oder der Monomere auf dem Träger bis zu einer vorbestimmten Schichtdicke erfolgt. Die Schichtdicke kann durch Zugabe von absorbierenden Substanzen zum Reagenz oder/und durch die Bestrahlungsdauer bzw. -intensität gesteuert werden. Überschüssiges flüssiges Reagenz kann nach der Polymerisation entfernt und erneut eingesetzt werden.In a particularly preferred embodiment, 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 μm, in particular <5 μm, 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.
Andererseits kann die Matrix auch durch konventionelle Beschichtungsprozeduren hergestellt werden, wobei das flüssige Reagenz auf einen Träger aufgetragen, dort mit geeigneten Methoden, z.B. mit einem Rakel, auf die gewünschte Dicke gebracht und dann getrocknet oder vollständig polymerisiert wird.On the other hand, 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.
Nach Einpolymerisierung in die Gelmatrix befinden sich das katalytisch inaktive Protein und gegebenenfalls das Enzym in einer geschützten Mikroumgebung. Bei ausreichender Vernetzung der polymeren Gelmatrix liegen die Proteinmoleküle in einer immobilisierten Form vor. Niedermolekulare Substanzen bzw. Glucose oder andere Analyten oder auch Coenzyme können frei durch das Polymernetzwerk diffundieren.After polymerization into the gel matrix, 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.
Das katalytisch inaktive Protein und gegebenenfalls das Enzym können zusammen mit dem Coenzym in die Matrix einpolymerisiert werden oder die Matrix kann nach der Polymerisation mit einer Lösung des Coenzym in Kontakt gebracht werden, so dass dieses in die Matrix eindiffundieren kann. Das durch die Reaktion veränderte, z.B. reduzierte oder oxidierte, Coenzym ist durch Bindung an das inaktive Protein und gegebenenfalls zusätzlich durch Einlagerung in die Gelmatrix optimal vor Störeinflüssen geschützt. Weiterhin soll die vorliegende Erfindung durch das nachfolgende Beispiel näher erläutert werden.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.
Beispiel: Lactatnachweis über NADH-Fluoreszenz mit inaktivierter Glucose-Dehydrogenase/NAD+/Lactat-Dehydrogenase in einerExample: Lactate detection via NADH fluorescence with inactivated glucose dehydrogenase / NAD + / lactate dehydrogenase in one
Küvettecuvette
Inaktivierung von Glucose-Dehvdroqenase (GlucDH)Inactivation of Glucose Dehvdroqenase (GlucDH)
Das essentielle Histidin der GlucDH an Position 147 wurde nach bekannten Verfahren gegen Serin ausgestauscht.The essential histidine of GlucDH at position 147 was exchanged for serine by known methods.
LactatnachweisLactatnachweis
100 mg/ml der inaktivierten GlucDH wurden in Puffer von pH 7 gelöst und mit der entsprechenden Menge NAD+ und einer katalytischen Menge Lactat-Dehydrogenase versetzt. Bei Zugabe von ansteigenden Mengen Lactat ließ sich visuell ein Anstieg der Fluoreszenz unter einer UV-Lampe (Anregungswellenlänge 366 nm) erkennen. Ohne inaktiviertes Enzym war keine vergleichbare Fluoreszenz beobachtbar. 100 mg / ml of the inactivated GlucDH were dissolved in pH 7 buffer and the appropriate amount of NAD + and a catalytic amount of lactate dehydrogenase were added. When increasing amounts of lactate were added, an increase in fluorescence under a UV lamp (excitation wavelength 366 nm) could be seen visually. No comparable fluorescence was observed without the inactivated enzyme.

Claims

Ansprüche Expectations
1 . Verfahren zum Nachweis eines Analyten in einer Probe durch eine enzymatische Reaktion, umfassend die Schritte:1 . A method for the detection of an analyte in a sample by an enzymatic reaction, comprising the steps:
(a) Inkontaktbringen der Probe mit einem Nachweisreagenz, umfassend ein Coenzym und ein katalytisch inaktives Coenzym-bindendes Protein (Bindungsprotein), wobei das Coenzym durch Reaktion mit dem Analyten verändert wird und das veränderte Coenzym an das katalytisch inaktive(a) contacting the sample with a detection reagent comprising a coenzyme and a catalytically inactive coenzyme-binding protein (binding protein), the coenzyme being changed by reaction with the analyte and the changed coenzyme to the catalytically inactive
Protein bindet undProtein binds and
(b) Nachweisen einer Reaktion des Analyten durch Veränderung des Coenzym-Bindungsprotein-Komplexes.(b) Detecting a reaction of the analyte by changing the coenzyme-binding protein complex.
2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass die enzymatische Reaktion eine Redoxreaktion umfasst.2. The method according to claim 1, characterized in that the enzymatic reaction comprises a redox reaction.
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass das Nachweisreagenz weiterhin ein Enzym enthält.3. The method according to claim 1 or 2, characterized in that the detection reagent further contains an enzyme.
4. Verfahren nach Anspruch 3, dadurch gekennzeichnet, dass man als Enzym eine Oxidoreduktase verwendet und eine4. The method according to claim 3, characterized in that an oxidoreductase is used as the enzyme and a
Veränderung des Coenzyms durch Oxidation oder Reduktion nachweist.Changes in the coenzyme detected by oxidation or reduction.
5. Verfahren nach Anspruch 4, dadurch gekennzeichnet, dass man als Enzym eine Dehydrogenase verwendet, ausgewählt aus einer Glucose-Dehydrogenase (E.C.1 .1 .1 .47), Lactat- Dehydrogenase (E.C.1 .1 .1 .27, 1 .1 .1 .28), Malat-Dehydrogenase (E.C.1 .1 .1 .37), Glycerin-Dehydrogenase (E.C.1 .1 .1 .6), Alkohol- Dehydrogenase (E.C.1 .1 .1 .1 ) oder Aminosäure-Dehydrogenase, z.B. L-Aminosäure-Dehydrogenase (E.C.1 .4.1 .5).5. The method according to claim 4, characterized in that a dehydrogenase is used as the enzyme, 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).
6. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass man ein Coenzym ausgewählt aus Flavin-, Nicotin- und Chinonderivaten verwendet.6. The method according to any one of the preceding claims, characterized in that one uses a coenzyme selected from flavin, nicotine and quinone derivatives.
7. Verfahren nach Anspruch 6, dadurch gekennzeichnet, dass man das Coenzym auswählt aus FAD, FADH2, FMN, FMNH2, Coenzym Q, PQQ, NAD + , NADH/H+, NADP+ und NADPH/H + .7. The method according to claim 6, characterized in that one selects the coenzyme from FAD, FADH 2 , FMN, FMNH 2 , coenzyme Q, PQQ, NAD + , NADH / H + , NADP + and NADPH / H +.
8. Verfahren nach einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, dass man als katalytisch inaktives Coenzym-bindendes Protein ein inaktiviertes Enzym verwendet.8. The method according to any one of claims 1 to 7, characterized in that an inactivated enzyme is used as the catalytically inactive coenzyme-binding protein.
9. Verfahren nach Anspruch 8, dadurch gekennzeichnet, dass man eine inaktivierte Oxidoreduktase verwendet.9. The method according to claim 8, characterized in that one uses an inactivated oxidoreductase.
1 0. Verfahren nach einem der Ansprüche 8 oder 9, dadurch gekennzeichnet, dass man eine inaktivierte Dehydrogenase verwendet.1 0. The method according to any one of claims 8 or 9, characterized in that an inactivated dehydrogenase is used.
1 1 . Verfahren nach einem der Ansprüche 8 bis 10, dadurch gekennzeichnet, dass das inaktivierte Enzym eine Mutation in der Aminosäuresequenz aufweist. 1 1. Method according to one of claims 8 to 10, characterized in that the inactivated enzyme has a mutation in the amino acid sequence.
1 2. Verfahren nach Anspruch 1 1 , dadurch gekennzeichnet, dass eine Mutation im katalytischen Zentrum vorliegt.1 2. The method according to claim 1 1, characterized in that a mutation is present in the catalytic center.
13. Verfahren nach einem der Ansprüche 8 bis 10, dadurch gekennzeichnet, dass das inaktivierte Enzym eine chemische Modifikation enthält.13. The method according to any one of claims 8 to 10, characterized in that the inactivated enzyme contains a chemical modification.
14. Verfahren nach Anspruch 13, dadurch gekennzeichnet, dass eine chemische Modifikation im katalytischen Zentrum vorliegt.14. The method according to claim 13, characterized in that a chemical modification is present in the catalytic center.
1 5. Verfahren nach Anspruch 1 bis 14, dadurch gekennzeichnet, dass man die Veränderung des Coenzyms durch optische Methoden nachweist.1 5. The method according to claim 1 to 14, characterized in that the change in the coenzyme is detected by optical methods.
1 6. Verfahren nach Anspruch 1 5, dadurch gekennzeichnet, dass man die Veränderung des Coenzyms durch Messung der1 6. The method according to claim 1 5, characterized in that the change in the coenzyme by measuring the
Fluoreszenz nachweist.Detects fluorescence.
1 7. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass man einen Analyten in einer Körperflüssigkeit bestimmt.1 7. The method according to any one of the preceding claims, characterized in that one determines an analyte in a body fluid.
1 8. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass man einen Analyten, ausgewählt aus Enzymen und Enzymsubstraten, bestimmt. 1 8. The method according to any one of the preceding claims, characterized in that an analyte selected from enzymes and enzyme substrates is determined.
1 9. Verfahren nach Anspruch 17 oder 18, dadurch gekenzeichnet, dass man eine Bestimmung von Glucose im Blut durchführt.1 9. The method according to claim 17 or 18, characterized in that one carries out a determination of glucose in the blood.
20. Verfahren nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass man das Nachweisreagenz in einer Gelmatrix eingelagert verwendet.20. The method according to any one of the preceding claims, characterized in that the detection reagent is used embedded in a gel matrix.
21 . Verfahren nach Anspruch 20, dadurch gekennzeichnet, dass die Gelmatrix auf einem zumindest teilweise optisch transparenten Träger aufgebracht wird.21. A method according to claim 20, characterized in that the gel matrix is applied to an at least partially optically transparent carrier.
22. Verfahren nach Anspruch 1 7 oder 18, dadurch gekennzeichnet, dass die Gelmatrix eine Schichtdicke von < 50 μm, insbesondere von < 5 μm aufweist.22. The method according to claim 1 7 or 18, characterized in that the gel matrix has a layer thickness of <50 microns, in particular of <5 microns.
23. Reagenzsystem zum Nachweis eines Analyten in einer Probe, umfassend:23. A reagent system for the detection of an analyte in a sample, comprising:
(a) ein Coenzym und(a) a coenzyme and
(b) ein katalytisch inaktives Coenzym-bindendes Protein.(b) a catalytically inactive coenzyme binding protein.
24. Reagenzsystem nach Anspruch 23 weiterhin umfassend ein Enzym.24. The reagent system of claim 23 further comprising an enzyme.
25. Reagenzsystem nach Anspruch 23 oder 24 weiterhin umfassend einen Träger zur Aufnahme des Nachweisreagenz.25. Reagent system according to claim 23 or 24 further comprising a carrier for receiving the detection reagent.
26. Reagenzsystem nach einem der Ansprüche 23 bis 25, dadurch gekennzeichnet, dass das Nachweisreagenz in einer Gelmatrix eingelagert ist. 26. Reagent system according to one of claims 23 to 25, characterized in that the detection reagent is embedded in a gel matrix.
27. Reagenzsystem nach einem der Ansprüche 23 bis 26, dadurch gekennzeichnet, dass der Träger zumindest teilweise optisch transparent ist.27. Reagent system according to one of claims 23 to 26, characterized in that the carrier is at least partially optically transparent.
28. Verwendung eines Reagenzsystems nach einem der Ansprüche 23 bis 27 in einem Verfahren nach einem der Ansprüche 1 bis 22. 28. Use of a reagent system according to one of claims 23 to 27 in a method according to one of claims 1 to 22.
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DE2002121840 DE10221840A1 (en) 2002-05-16 2002-05-16 Production of polymer layers on a transparent support, for use in sensors, e.g. for blood analysis, comprises coating the support with a photopolymerizable liquid composition and irradiating the liquid through the support
DE2002121846 DE10221846A1 (en) 2002-05-16 2002-05-16 Detecting an analyte by enzymatic reaction, useful specifically for measuring glucose in blood, based on reaction with coenzyme and inactive coenzyme-binding protein
DE10221845 2002-05-16
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