Method for the colorimetric determination of analyte using enzymatic oxydation.

The invention relates to a method for the colorimetric determination of an analyte by means of enzymatic oxidation of the analyte in the presence of an electron acceptor and determination of the reduced electron acceptor by color formation as a measure of the amount of the analyte.

The invention further relates to a means for the colorimetric determination of an analyte by enzymatic oxidation of the analyte, containing an oxidoreductase and a color-forming electron acceptor.

The invention extends specifically to the use of a substance from the group of compounds with nitrogen in an oxidation state between +1 and -1 as a direct electron acceptor of an analyte / oxidase system or an analyte / NAD (P) independent dehydrogenase system.

In particular, the invention relates to the use of a substance from the group of nitroso compounds, hydroxylamines and oximes as a color-forming electron acceptor in the oxidation of an analyte by means of an oxidoreductase.

In analysis, enzymatic oxidations enable the detection and determination of substances in a wide variety of sample materials. Here, an oxidizing enzyme acts on a corresponding enzyme substrate in the presence of an acceptor that receives the electrons of the oxidation reaction. The reduction in the electron acceptor indicates the presence of the enzyme substrate. It has so far proven to be particularly advantageous if the reduced electron acceptor can be detected by color formation, since this is not necessarily only possible using expensive measuring equipment, but can also be done visually if necessary.

Known methods for the colorimetric determination of substances using oxidizing enzymes use oxidases or dehydrogenases. Both enzyme groups belong to the main group of oxidoreductases (Römpps Chemie-Lexikon, Franckh-sche Verlagshandlung, Stuttgart, 8th edition, 1985, volume 4, page 2952; Lexikon Biochemie, ed. HD Jakubke, Verlag Chemie, Weinheim, 2nd edition, 1981 , Page 194), whose members can be differentiated according to their natural electron acceptors.

The natural electron acceptor for oxidases is molecular oxygen (Römpps Chemie-Lexikon, Franckh-sche Verlagshandlung, Stuttgart, 8th edition, 1985, volume 4, page 2946). A representative of the state of the art in the use of oxidases for the colorimetric determination of analytes is A. Kunst et al. in "Methods in enzymatic analysis", ed. HU Bergmeyer, Verlag Chemie, Weinheim, 3rd edition, 1984, volume 6, pages 178-185. There, glucose is detected in serum, plasma or deproteinized blood by reaction with glucose oxidase and atmospheric oxygen in aqueous solution by oxidizing the hydrogen peroxide formed in this reaction by reducing the oxygen in the presence of peroxidase and thus color-forming on phenol and also present in the reaction mixture 4-aminophenazone works. In this reference, the presence of reducing substances such as ascorbic acid, uric acid or glutathione is mentioned as a source of error. Transition metal ions or heme and heme proteins, which can easily occur in blood-derived samples because they decompose hydrogen peroxide, also have a disruptive effect. Also sample ingredients containing hydrogen peroxide and peroxidase and possibly other substances contained in the detection reagent, such as. B. Phenol or 4-aminophenazone lead to color formation, can lead to incorrect results. B. bilirubin or drugs, such as. B. -Methyldopa, which can occur in blood-derived samples or in urine.

In J. Siedel et al. in "Methods in enzymatic analysis", published by HU Bergmeyer, Verlag Chemie, Weinheim, 3rd edition, 1984, volume 8, pages 139-148, the colorimetric determination of total cholesterol in serum or plasma is described in such a way that initially ester-bound cholesterol is also present Cholesterol esterase is released. Then cholesterol is determined by reaction with cholesterol oxidase and atmospheric oxygen in aqueous solution, the hydrogen peroxide formed in this reaction having an oxidizing effect in the presence of peroxidase and thus color-forming action on phenol and 4-aminoantipyrine likewise present in the reaction mixture. The color formation is a measure of the amount of total cholesterol in the sample.

All of the disadvantages mentioned for the above-mentioned method for the determination of glucose by means of oxidase apply equally to the described cholesterol determination method. These disadvantages are independent of whether the detection reaction z. B. in a cuvette or on a dry reagent, as z. B. from EP-A-0 016 387, DE-A-32 47 608, EP-A-0 262 445 or EP-A-0 256 806 are carried out. In particular when carrying out the aforementioned determination methods on solid supports, in so-called dry tests, the oxygen requirement has additionally proven to be disadvantageous. Especially when a lot of oxygen is needed to oxidize high concentrations of enzyme substrate, the diffusion of oxygen from the air into the reaction medium can become a rate-determining step and lead to long reaction times or incorrect results, especially in the case of kinetic determination methods.

Dehydrogenases can generally be divided into those which require nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as natural direct electron acceptors for the oxidation of enzyme substrates and those which are not NAD or NADP- are dependent and therefore use substances other than natural direct electron acceptors in enzymatic oxidation reactions.

The use of dehydrogenases for colorimetric measurements is e.g. B. from DE-A-21 47 466 known. There it is described that lactate is converted to pyruvate and reduced nicotinamide adenine dinucleotide by catalysis of lactate dehydrogenase with nicotinamide adenine dinucleotide. The NADH formed then reacts, for example in the presence of the enzyme diaphorase, with tetrazolium salts to form NAD and colored formazans, the concentration of which can be determined photometrically. Instead of diaphorase, N-methylphenazinium methosulfate is also mentioned as a reduction catalyst for the transfer of the electrons from NADH to the tetrazolium salt.

Disadvantages of this method can be seen in the fact that instead of NADH also other possible biological samples, such as. B. blood, serum, plasma or urine occurring, reducing acting substances, such as glutathione or drugs, such as methyl dopa or dobesylate in the presence of reduction catalysts, such as diaphorase or N-methylphenazinium methosulfate, convert tetrazolium salts into corresponding formazans and thus lead to false-positive results .

The necessity of reduction catalysts, such as diaphorase or phenazinium ethosulfate, for the transfer of the electrons released by the oxidation of an analyte from the analyte / enzyme system to an electron acceptor serving for color formation also has the disadvantage that other sample accompanying substances can be reduced which are otherwise the same Conditions did not result in a reduction, but lead to false negative results.

The invention seeks to remedy the aforementioned disadvantages of the prior art. The invention was specifically based on the object of providing a method for the colorimetric determination of an analyte by means of enzymatic oxidation of the analyte in the presence of an electron acceptor and determination of the electron acceptor by color formation as a measure of the amount of analyte for the analysis, in particular of biological fluids, of which reducing effect , in particular accompanying substances which decompose hydrogen peroxide in samples, in particular biological samples, as liquids derived from blood, e.g. B. do not interfere with serum or urine, the use of reduction catalysts, such as diaphorase or N-methylphenazinium methosulfate, for which no oxygen is required and which thereby enables fast final color values ​​even at high substrate concentrations.

In addition, a means for the colorimetric determination of an analyte by enzymatic oxidation of the analyte containing an oxidoreductase and a color-forming electron acceptor should be provided, which is suitable for carrying out the above-mentioned method.

In particular, the task extends to the selection of those substances which can be used as color-forming electron acceptors for an improved process and agent as indicated above.

The object is achieved by the invention as characterized in the claims.

Accordingly, a method for the colorimetric determination of an analyte by means of enzymatic oxidation of the analyte in the presence of an electron acceptor and determination of the reduced electron acceptor by color formation as a measure of the amount of the analyte was found, which is characterized in that that the analyte is oxidized with a corresponding oxidoreductase in the presence of a substance from the group of compounds with nitrogen in an oxidation state between +1 and -1 as a direct electron acceptor.

In addition, a means for the colorimetric determination of an analyte by enzymatic oxidation of the analyte containing an oxidoreductase and a color-forming electron acceptor was found, which is characterized in that the color-forming electron acceptor is a substance which reacts directly with the analyte / enzyme system from the group of compounds Nitrogen is in an oxidation state between +1 and -1.

In particular, it has been found that the use of a substance from the group of compounds with nitrogen in an oxidation state between +1 and -1 is particularly suitable as a direct electron acceptor of an analyte / oxidase system or an analyte / NAD (P) -independent dehydrogenase system .

According to the invention, “analyte” is understood to mean such a substance that is enzymatically oxidized. In many cases, the analyte will be the substance that is to be directly detected or quantified in the sample to be examined. For example, glucose can be oxidized directly with glucose oxidase and determined colorimetrically. However, it is also possible that the analyte is first formed from another substance by one or more upstream reactions, and the presence and concentration of the analyte can thus be concluded indirectly from the presence and concentration of the starting substance. So z. B. Glycerol are detected and determined in such a way that in a first reaction glycerol is converted into glycerol 3-phosphate and adenosine diphosphate by means of glycerol kinase and adenosine triphosphate and then in a second reaction glycerol 3-phosphate is oxidized with glycerol 3-phosphate oxidase becomes. In this case, glycerol-3-phosphate is the analyte whose concentration corresponds to that of the substance to be determined, namely glycerol. Here too, however, the analyte is the compound that is determined colorimetrically.

In the present invention, the analyte is the substance that is accepted as the substrate of the respective oxidizing enzyme. In order for the analyte to be oxidized, an electron acceptor must also be present, which accepts the electrons from the analyte with the help of the enzyme.

Surprisingly, it has now been found that substances from the group of compounds with nitrogen in an oxidation state between +1 and -1, preferably those with nitrogen in an oxidation state of +1 or -1, can be color-forming electron acceptors for oxidizing enzymes.

In this context, "color-forming" is understood to mean that the electron acceptor after reduction is either itself immediately in a different color than before the enzymatic oxidation of the analyte or that the reduced acceptor itself does not lead directly to color generation, but it does cause it in a subsequent reaction leads to a color change in the reaction mixture. Color changes include changing from colorless to colored as well as from one color to another. The skilled worker is familiar with many possibilities for color formation from subsequent reactions, from which he can choose depending on the circumstances. For example, reactions in which the reduced electron acceptor itself becomes part of a colored compound, such as oxidative coupling reactions, may be mentioned here. Coloring secondary reactions can, however, also be those in which the reduced electron acceptor leads to a color change of the same due to its reducing effect on another substance.

The oxidation state is understood to be a numerical value which indicates the oxidation state of a specific atom in a compound, which can be a neutral molecule or a charged complex ion. The determination of oxidation levels is known to the person skilled in the art. Instructions for determining this numerical value are basic chemistry textbooks, such as B. "Inorganic Chemistry", Hofmann & Rüdorff, F. Vieweg & Sohn, Braunschweig, 20. Edition, 1969, pages 216-218; "Textbook of inorganic chemistry", Hollemann-Wiberg, publishing house Walter deGruyter & Co., Berlin, 71st - 80th edition, 1971, pages 197 - 199 or "Anorganikum", ed. L. Kolditz, VEB German publishing house of the sciences, Berlin, 4th edition, 1972, page 446.

Preferred electron acceptors according to the invention are N-oxides, nitroso compounds, hydroxylamines and oximes. N-oxides, nitroso compounds and oximes are particularly preferred.

In particular, those N-oxides can be used advantageously in which the nitrogen atom of the N-oxide carrying the oxygen atom is part of an aromatic ring system. Examples of these are resazurin or resazurin derivatives, as are described, for example, in EP-A-0 156 347. Here the nitrogen atom of the N-oxide has the oxidation number - 1.

Resazurin and resazurin derivatives are reduced as electron acceptors in the case of enzymatic oxidation to resorufins and resorufin derivatives. This is associated with a clear color change. So z. B. Resazurin colored blue and Resorufin red.

Aromatic N-oxides which can advantageously be used are those of benzfuroxane or benzfuroxane derivatives whose carbon-aromatic partial structure can be substituted by low molecular weight ring substituents. Low molecular weight ring substituents in this context can be those with a molecular weight of up to about 400 daltons.

Particularly preferred benzfuroxanes are represented, for example, by general formula I:<chemistry id="chem0001" num="0001"><img file="EP0354441A2_D0001.tif" /></chemistry> in the R¹ and R² may be the same or different and represent hydrogen, lower alkyl, lower alkoxy, lower alkylcarbonyl or formyl.

In these compounds, the nitrogen atom of the N-oxide has the oxidation state + 1.

Lower alkyl and lower alkoxy are residues with 1-5 carbon atoms. Methyl or methoxy are particularly preferred.

Lower alkylcarbonyl is understood to mean those radicals which contain 1-5 carbon atoms in the alkyl radical. Acetyl is particularly preferred.

When the aforementioned benzfuroxanes of the general formula I are used as electron acceptors in enzymatic oxidations, the colorless compounds are reduced to colored compounds. Essentially, the formation of orange-colored compounds takes place.

For the process according to the invention, the aromatic N-oxides mentioned above can be used as direct color indicators for the enzymatic oxidation of an analyte. For this purpose, the oxidizing enzyme together with the aromatic N-oxide is brought into contact with the sample to be examined. If the sample contains an analyte that can be oxidized by the enzyme, the N-oxide serves as an electron acceptor, which changes its color by reduction and thus indicates the presence of the analyte in the sample. The concentration of the analyte in the sample can be inferred from the intensity of the newly formed color.

Suitable nitroso compounds which can be used for the method according to the invention for the colorimetric determination of an analyte are preferably those which contain the nitroso residue bound to an aromatic group.

Carbon-aromatic nitroso compounds, such as nitrosobenzene and nitrosobenzene derivatives, are particularly preferred. As nitrosobenzene derivatives, all those which are converted by reduction into compounds which can be used for color formation in the sense of the explanation given above are very outstandingly suitable for the process according to the invention.

Nitrosobenzene and nitrosobenzene derivatives are usually weakly colored, yellow, green or brown colored substances that are not colored in the reduced state. In order to be able to detect an analyte by colorimetry, they must therefore lead to color formation in a subsequent reaction. This can be achieved, for example, by reacting the reduced nitroso compound with another substance in such a way that a colored substance is obtained which contains the reduced nitroso compound as a partial structure.

Oxidative coupling reactions are one possibility for this type of color formation. For this reason, nitrosobenzene derivatives are particularly suitable for the process according to the invention which not only act as electron acceptors for oxidizing enzymes, but which can also be used in the reduced state for oxidative coupling reactions.

Nitrosobenzene derivatives of the general formula II are very particularly preferred<chemistry id="chem0002" num="0002"><img file="EP0354441A2_D0002.tif" /></chemistry> in the R represents a hydroxyl group or an amino group, wherein the amino group can be mono- or disubstituted by lower alkyl and the lower alkyl group can in turn be substituted by a hydroxy group, an amino group substituted once or more by lower alkyl, PO₃H₂, SO₃H or CO₂H.

Those nitrosobenzene derivatives of the general formula II which are not particularly volatile are particularly suitable.

Lower alkyl in the above definition means an alkyl radical with 1-5 carbon atoms. Methyl and ethyl are particularly preferred.

The acid residues PO₃H₂, SO₃H and CO₂H can be present as such or in salt form as ammonium, alkali or alkaline earth metal salts. Ammonium salts are those which contain the unsubstituted ammonium cation NH₄⁺ or those which contain ammonium cations substituted one or more times by lower alkyl, aryl and / or lower alkylaryl. Lower alkyl in each case means an alkyl radical containing 1-5 carbon atoms. The aryl radical is an aromatic ring system with 6-10 carbon atoms.

Methyl and ethyl are preferred as lower alkyl. The preferred aryl radical is phenyl, with benzyl being a preferred lower alkylaryl radical.

P-Hydroxy-nitrosobenzene, p-dimethylamino-nitrosobenzene, p-diethylamino-nitrosobenzene and p-dihydroxyethylamino-nitrosobenzene are very suitable as electron acceptors for the process according to the invention.

As stated above, for the method according to the invention, nitrosobenzene or nitrosobenzene derivatives in which nitrogen is in the oxidation state +1 are preferably brought into contact with the sample to be examined and an oxidizing enzyme. In the presence of an analyte in the sample that is accepted as an enzyme substrate, nitrosobenzene or a nitrosobenzene derivative acts as an electron acceptor and is thereby reduced to the corresponding amine. As a rule, this reduction of the nitroso compounds alone is not sufficient for a colorimetric determination of the analyte, since either only color decreases occur or color changes are barely perceptible. The reduced electron acceptor can, however, serve as the starting material for an oxidative coupling reaction, with the most varied of colors being able to be obtained depending on the choice of the coupling partner.

Oxidative coupling reactions are known to the person skilled in the art and are described, for example, in EP-A-0 175 250 or in H. Hünig et al., Angewandte Chemie <u style="single">70</u>, 215-222 (1958). In general, oxidative couplings are often used to produce dyes. In this connection, they can be understood as a reaction between an electron-rich aromatic compound and an oxidizable coupling component, which in the presence of oxidizing agents, such as. B. Sodium or potassium hexacyanoferrate (III), copper salts, mercury salts, iron (III) chloride, lead dioxide, hydrogen peroxide, lead tetraacetate, sodium or potassium salts of persulfuric acid, peracetic acid, periodic acid or chloramine T. According to DE-A 33 31 588, oxidases can also be used as oxidizing agents. In their reduced state, the nitrosobenzenes described above, after having acted as electron acceptors in enzymatic oxidations, are compounds which are available as oxidizable coupling components for oxidative coupling reactions. For this purpose, the amines formed by the reduction can be oxidized by oxidizing agents, as mentioned above by way of example, in order to form a dye with electron-rich coupling components which are simultaneously present in the reaction mixture.

A large number of electron-rich coupling components are available to the person skilled in the art. These coupling components can be selected depending on the desired color of the coupling product. Examples are aromatic amines, phenols or methylene-active compounds. Particularly preferred compounds can be selected from the group of anilines, e.g. B. N-methylanthranilic acid and the anilinophosphonic acids mentioned in EP-A-0 175 250 can be selected.

A further possibility for determining the reduced electron acceptor by color formation in the case of the nitroso compounds is to oxidize the reduced nitroso compounds by other substances, which in turn form a color due to this transition from the oxidized to the reduced form, here too, with color formation, both the transitions from one color to another as well as from the colorless to the colored state. Examples of such color formation reactions are e.g. B. the oxidation of the reduced electron acceptor by means of metal salts, which are reduced to colored metal salts with the metal in a lower oxidation state or possibly completely down to the neutral metal. Copper (II) salts can, for example, be converted into red copper (I) salts and silver salts into metallic silver. It is also possible to reduce phosphorus molybdate to blue molybdenum.

Instead of aromatic nitroso compounds, aromatic oximes can also be used as electron acceptors for enzymatic oxidation for the process according to the invention. Nitroso compounds can be in a tautomeric equilibrium with oximes. This is particularly the case if a hydrogen atom is available in the α position to the nitroso group<chemistry id="chem0003" num="0003"><img file="EP0354441A2_D0003.tif" /></chemistry> or if a corresponding delocalization of the electrons and protons is possible.

It has been found that oximes which can be in a tautomeric nitroso / oxime equilibrium with nitroso compounds which function as electron acceptors in enzymatic oxidation reactions can also be used in processes according to the invention. This applies in particular to oximes of the general formula III<chemistry id="chem0004" num="0004"><img file="EP0354441A2_D0004.tif" /></chemistry> in the R 'is oxygen, a further oxime group or a positively charged amino group, where the amino group can be mono- or disubstituted by lower alkyl and the lower alkyl group can in turn be substituted by a hydroxyl group by one or more amino groups substituted by lower alkyl, PO₃H₂, CO₂H or SO₃H .

Lower alkyl in the above definition means an alkyl radical with 1-5 carbon atoms. Methyl and ethyl are particularly preferred.

The acid residues PO₃H₂, SO₃H and CO₂H can be present as such or in salt form as ammonium, alkali or alkaline earth metal salts.

Ammonium salts are those which contain the unsubstituted ammonium cation NH₄⁺ or those which contain ammonium cations substituted one or more times by lower alkyl, aryl and / or lower alkylaryl. Lower alkyl in each case means an alkyl radical containing 1 -5 carbon atoms. The aryl radical is an aromatic ring system with 6-10 carbon atoms.

Methyl and ethyl are preferred as lower alkyl. The preferred aryl radical is phenyl, with benzyl being a preferred lower alkylaryl radical.

In such oximes, nitrogen has the oxidation state - 1.

Just like nitrosobenzene, the oximes of the general formula III are colorless or colored substances which are not colored in the reduced state. As described above for nitrosobenzenes as electron acceptors in the process according to the invention, aromatic oximes can also be brought to color formation according to their function as electron acceptors. Both nitroso compounds and oximes give amines when exhaustively reduced. For oximes as electron acceptors, basically the same subsequent reactions for color formation can be used in the process according to the invention as for nitroso compounds.

Hydroxylamines which can be used as electron acceptors for the process according to the invention are preferably also aromatic compounds. Derivatives of phenylhydroxylamine substituted in the phenyl ring are particularly suitable as electron acceptors for the process according to the invention. In these compounds the nitrogen atom has the oxidation number - 1.

Very particularly preferred phenylhydroxylamines are those of the general formula IV<chemistry id="chem0005" num="0005"><img file="EP0354441A2_D0005.tif" /></chemistry> in the R˝ has the same meaning as R in the general formula II.

Hydroxylamines are also converted by reduction into the corresponding amines, which are not significantly colored. As described for nitroso compounds and oximes, the oxidation of the analyte and the associated reduction in the electron acceptor by the process according to the invention must therefore be followed by subsequent reactions which, starting from the reduced electron acceptor, lead to color formation. Basically, the same applies here as for the use of nitroso compounds or oximes as electron acceptors in the process according to the invention. The starting compound for all of these subsequent reactions are the electron acceptors of the enzymatic oxidation reduced to the amine stage.

Surprisingly, it was found that compounds with nitrogen in an oxidation state between + 1 and - 1, preferably those with an oxidation state of + 1 or - 1, in particular the compounds from the group of N-oxides, nitroso compounds, oximes and hydroxylamines and very particularly those Compounds from the group of N-oxides, nitroso compounds and oximes, as described above, can serve as color-forming electron acceptors for many enzymatic oxidations that are catalyzed by oxidoreductases. They can preferably be used where no reduction catalysts, such as diaphorase or N-methylphenazinium methosulfate, are desired for the electron transfer from analytes to be oxidized to the color-forming electron acceptor. Surprisingly, it has been found that these substances are advantageous when oxidases or non-NAD (P) -dependent dehydrogenases are used as oxidizing enzymes. If such enzymes are used for the oxidation of an analyte, the electron transfer can take place directly, ie without the participation of a reduction catalyst from the analyte / enzyme system to the aforementioned color-forming electron acceptors. In this context, the analyte / enzyme system is understood to mean the combination of analyte and oxidizing enzyme necessary for the oxidation reaction and, if appropriate, together with coenzymes and / or cofactors, such as metal salts, which naturally interact with the enzyme and are necessary for the oxidation.

Flavin-dependent oxidases are particularly preferred for the process according to the invention. Examples are L- and D-amino acid oxidases, cholesterol oxidase, glucose oxidase, glycerol-3-phosphate oxidase, lactate oxidase and pyruvate oxidase. Oxidases which are very particularly suitable according to the invention are glucose oxidase, glycerol-3-phosphate oxidase, lactate oxidase and pyruvate oxidase.

Of the non-NAD (P) -dependent dehydrogenases, pyrroloquinoline-quinone (PQQ) -dependent dehydrogenases can be used particularly advantageously for the process according to the invention. Glucose dehydrogenase in particular is well suited for the colorimetric determination of glucose in the presence of compounds with nitrogen in an oxidation state between +1 and -1 as color-forming electron acceptors. Likewise, non-NAD (P) -dependent alcohol dehydrogenase can be used for the determination of alcohol, such as ethanol.

The method according to the invention is carried out in such a way that the sample to be examined is contacted with a suitable oxidoreductase and one or more of the color-forming electron acceptors described above. If the sample contains an analyte that is oxidized by the oxidoreductase, the color-forming electron acceptor is reduced. If the reduced electron acceptor has a different color than the original electron acceptor in its oxidized form or if the electron acceptor changed from the colorless state to a colored state due to the enzymatic oxidation process, the intensity of the color formed can be directly visual, possibly by means of comparison colors or photometrically with the concentration of Analytes in the sample are correlated. If the color of the reduced electron acceptor is essentially the same as that of the electron acceptor originally used, or if there is color bleaching or complete decolorization, then a subsequent reaction is connected to the enzymatic oxidation reaction, which leads to a color whose intensity also visually or photometrically indicates the concentration of the analyte can be determined in the sample.

The method can be carried out in a so-called wet test, e.g. B. in a cuvette or as a so-called dry test on an appropriate reagent carrier, the necessary test reagents being on a solid carrier, in particular an absorbent or swellable material. Such test carriers are e.g. B. from EP-A-0 016 387, DE-A-32 47 608, EP-A-0 262 445 or EP-A-0 256 806 known.

Agents for the colorimetric determination of an analyte for carrying out the method according to the invention, as characterized in the claims, are also the subject of the invention. In addition to the oxidoreductase necessary for the enzymatic oxidation of the analyte to be determined, such an agent contains at least one color-forming electron acceptor which takes over the electrons released during the oxidation directly from the analyte / enzyme system. The substances described above for the process according to the invention are used as oxidizing enzymes and color-forming electron acceptors.

The agent according to the invention contains a buffer system in order to maintain a pH value which is suitable for carrying out the method and which depends in particular on the enzymes to be used. It can also contain other suitable additives commonly used for such agents, such as wetting agents, stabilizers, etc. If the oxidation of the electron acceptor does not lead to a measurable color change, the agent according to the invention for the colorimetric determination of an analyte naturally also includes the reagents required for a subsequent reaction.

The agent according to the invention can be in the form of a solution or applied to an absorbent or swellable carrier. In the form of a solution, the agent preferably contains all the reagents required for the process according to the invention. Water, water-soluble organic solvents, such as. B. methanol, ethanol, acetone or dimethylformamide or mixtures of water with such organic solvents in question. For reasons of durability, it can be advantageous to distribute the reagents required for the test over two or more solutions, which are only mixed during the actual test. The latter can apply in particular if, after oxidation of the analyte, the reduced electron acceptor in a subsequent reaction, e.g. B. an oxidative coupling reaction is further implemented. Typical concentrations for the electron acceptors used in the agent according to the invention are: 0.01-100 mmol / l, preferably 0.1-25 mmol / l. Reagents for subsequent reactions are used at least in a stoichiometric ratio to the electron acceptors, preferably in an excess, in particular in a 2-10 fold excess.

The agent according to the invention can also be in the form of a test strip. Such test strips are known in many types, for example from EP-A-0 016 387, DE-A-32 47 608, EP-A-0 262 445 or EP-A-0 256 806. What they all have in common is that they are used for the implementation reagents required for the determination procedure are present on solid support layers. Particularly suitable carrier layers are absorbent and / or swellable materials which are wetted by the sample liquid to be examined. Examples are gelatin, cellulose or synthetic fiber fleeces. The reagents are in solid form in or on these carrier materials.

When the sample liquid is applied to the test strip or the test strip is immersed in the sample liquid, a liquid medium forms in the strip, within which the detection reaction takes place. The color formation caused by the reaction can be visual or photometric e.g. B. be evaluated by reflection photometry.

A special subject of the invention relates to the use of a substance from the group of compounds with nitrogen in an oxidation state between +1 and -1 as a direct electron acceptor of an analyte / oxidoreductase system. In this context, the analyte / oxidoreductase system used is the combination of analyte and oxidizing enzyme necessary for an enzymatic oxidation reaction and, if appropriate, together with coenzymes which naturally interact with the enzyme and are necessary for the oxidation, such as flavin or PQQ or / and cofactors, such as metal salts. Roger that. It has been found that substances from the group of nitroso compounds, N-oxides, hydroxylamines and oximes, preferably nitroso compounds, N-oxides and oximes, can be used quite generally as color-forming electron acceptors in the oxidation of an analyte by means of an oxidoreductase.

The present invention offers the advantage that no reduction catalysts, such as diaphorase or N-methylphenazinium methosulphate, are necessary for the reduction of a color-forming electron acceptor. Its reduction can now be carried out directly by the analyte / enzyme system. Any disruptive side reactions can be avoided.

In particular when oxidases are used for the enzymatic oxidation of analytes, the use of the compounds according to the invention avoids the formation of hydrogen peroxide as a preliminary stage of a colorimetric determination method. This eliminates or reduces the interfering influence of reducing compounds.

Finally, the compounds used according to the invention offer a real alternative wherever the access of atmospheric oxygen is limited or undesirable. Oxygen can be replaced by these compounds as an electron acceptor in enzymatic oxidations. This offers very particular advantages in the case of agents according to the invention in the form of test strips. While these previously had to be constructed in such a way, especially at high analyte concentrations, that in the case of enzymatic oxidations by means of oxidase, air has access to the reagent mixture applied to the test strips, test strips can now be constructed which work particularly quickly and reliably. While so far the film test strips often had to be wiped off after being placed on the test strips so that oxygen could diffuse into the test strips at all, this measure is not necessary when using the electron acceptors according to the invention. By avoiding the time-dependent diffusion of oxygen in the test strips, reaction rates dependent on the analyte concentration are obtained in the case of kinetic measurements and reaction rates which are independent of the analyte concentration in the case of endpoint measurements, which allow faster, more reliable and simpler determination methods than was previously possible.

The following examples show some of the possible process variants for the colorimetric determination of an analyte by means of enzymatic oxidation.

example 1

Reduction of N-oxides by oxidases or non-NAD (P) -dependent dehydrogenases and their substrates

A) 40g polyvinyl propionate (e.g. Propiofan 70 F, BASF AG, Ludwigshafen, Federal Republic of Germany) 45 g sodium alginate (e.g. Algipon from Kelco, Division of Merck &amp; Co., Clark, New Jersey, USA), 1.7% by weight in water 2.5 g protein hydrolyzate (e.g. Crotein C, Croda GmbH, Nettetal, Federal Republic of Germany) 10 ml Tris buffer 0.1 M, pH 7.5 750 IU glucose oxidase (EC 1.1.3.4) and 1 ml resazurin 0.1 M in water were mixed to a homogeneous mass, on one side of matt polycarbonate film (e.g. Pokalon, Lonza, Rheinfelden, Federal Republic of Germany) doctored 140 µm thick (doctor blade gap 200 µm) and at 50 ^OC dried for 30 minutes.

When a solution containing glucose (40 mg / dl) was applied to such a film, the reagent layer changed color from blue to red within 1 minute. The presence of 20 mg / dl uric acid and 20 mg / dl glutathione did not interfere with the color reaction. When solutions containing no glucose were applied, the reagent layer retained the color that it had before the sample was applied. It stayed blue.

Instead of using glucose and glucose oxidase, the same color result was also used Pyruvate and pyruvate oxidase (EC 1.2.3.3.) Lactate and lactate oxidase (EC 1.1.3.2.) Glycerin-3-phosphate and glycerin-3-phosphate oxidase (EC 1.1.3.21) achieved. B) The following benzfuroxanes were used instead of resazurin in the preparation of the film described above<chemistry id="chem0006" num="0006"><img file="EP0354441A2_D0006.tif" /></chemistry><ul id="ul0001" list-style="none"><li>a) R¹ = R²: H</li><li>b) R1: H; R²: CO-CH₃ (production according to NR Ayyangov, Synthesis<u style="single">1987</u>, 616)</li><li>c) R1: H; R²: CHO (manufactured according to ML Edwards, J. Het. Chemistry<u style="single">13</u>, 653 (1976))</li><li>d) R¹ = R²: CH₃ (preparation according to JA Usta et al., J. Het. Chemistry <u style="single">18</u>, 655 (1981))</li></ul> used in a concentration of 250 mg per film batch, then the color changed from colorless to orange in the presence of glucose in the sample examined. The color intensity increased with increasing glucose concentration.

The presence of 20 mg / dl uric acid did not interfere with the color reaction. The substrates / enzyme pairs: Pyruvate / Pyruvate Oxidase (EC 1.2.3.3.) Lactate / lactate oxidase (EC 1.1.3.2.) Glycerin-3-phosphate / glycerin-3-phosphate oxidase EC 1.1.3.21) could also be detected with the aforementioned benzfuroxanes.

Example 2

Reduction of nitroso compounds by glucose oxidase and glucose

<ul id="ul0002" list-style="none"><li>A) 2,000 µl 0.1 M citric acid / NaOH buffer, pH 6.0 200 µl p-nitroso-N, N-dimethylaniline, 0.1 M in ethanol 100 µl glucose oxidase (EC 1.1.3.4) (2500 IU / ml) or glucose dehydrogenase (EC1.1.99.10) 200 µl sample with known glucose content a) 5 mM b) 10 mM c) 15 mM d) 20 mM e) 50 mM in water were mixed and 2 minutes at 25 ^OC incubated. Then were 250 µl N-methylanthranilic acid, 0.1 M in ethanol, 125 µl potassium hexacyanoferrate (II), 0.2 M in water and 125 µl potassium hexacyanoferrate (III), 0.2 M in water added. After a further minute, the mixture was diluted 25 times and the absorption of the reaction mixture, which turns green in the presence of glucose, was measured at 710 nm against a blank value (above reaction mixture without glucose). The results obtained gave a straight line. The extinction coefficient of ε₇₁₀ = 24 000 M⁻¹ cm⁻¹ can be used to determine unknown glucose concentrations in solutions.</li><li>B) In the above test under A), p-nitrose-dimethylaniline was replaced by a) p-nitroso-phenol b) p-nitroso-N, N-diethylaniline c) p-Nitroso-N, N-diethanolaniline (preparation according to D'Amico et al., J. Amer. Chem. Soc. <u style="single">81</u>, 5957 (1959)) thus, in the presence of glucose, a color change of by coupling with N-methylanthranilic acid a) brown to blue b) yellow to green c) yellow to green observed.</li><li>C) In the above test under A), N-methylanthranilic acid was replaced by a) N-methyl-N-methylenephosphonic acid aniline b) 1-hydroxy-naphthalene-2-carboxylic acid c) aniline-2-sulfonic acid in the presence of glucose, a color was obtained by coupling with p-nitroso-N, N-dimethylaniline a) λ_Max = 735 nm b) λ_Max = 590 nm c) λ_Max = 640 nm observed.</li></ul>

When p-nitroso-N, N-dimethylaniline and N-methyl-N-methylenephosphonic acid aniline were used, the dependence of the change in light absorption at 735 nm on the glucose concentration shown in FIG. 1 was found. For this purpose, the absorbance was measured after (1 + 24) dilution in citrate buffer, pH 6 and plotted against the glucose concentration in the test mixture.

Example 3

Detection of glucose by the formation of metallic silver

The approach from citrate buffer, p-nitroso-N, N-dimethylaniline. Glucose oxidase and sample as in Example 2A was at 25 for 2 minutes^OC incubated and mixed with 250 ul 100 mM silver nitrate solution in water and 250 ul gold sol in water.

(Goldsol was prepared according to the following instructions: 0.4 mg HAuCl dest in 0.4 ml water; 0.2 ml 0.1 M NaSCN in water and 0.5 ml 0.1 M in succession to 100 ml boiling distilled water K₂CO₃ added to water and allowed to cool after 10 minutes).

The curves shown in FIG. 2 were obtained with the results obtained at 700, 850 and 1300 nm without intermediate dilution. They can serve as calibration curves for determining the unknown glucose content in solutions.

Example 4

Detection of glucose by molybdenum blue formation

To a solution of 200 mg of 2,18-phosphoromolybdic acid (production possible, for example, according to G. Brauer, "Handbook of Preparative Inorganic Chemistry", Enke-Verlag, Stuttgart, p. 1278 (1954) or A. Rosenheim, A. Traube, Z. Anorg. Chemistry <u style="single">65</u>, 99 (1910) in 920 -x µl 0.1 M citric acid / NaOH buffer pH 5.5, 40 µl 0.1 M p-nitroso-N, N-dimethylaniline (in ethanol) and 40 µl glucose oxidase ( 6250 IU / ml water) added. 1 minute after the addition of x μl (x = 0, 1, 2, 3, 5, 7, 10) of a glucose-containing sample of known glucose content (1M), the mixture was diluted to 50 ml and the change in absorption (ΔE) at 820 nm was measured. As a result, the curve shown in Fig. 3 was obtained. It can serve as a curve for determining the unknown glucose content of solutions, where C represents the concentration in the sample before the dilution carried out for the measurement.

Example 5

Kinetic determination of glucose using non-NAD (P) -dependent glucose dehydrogenase

The following solutions were made: Test buffer: 0.1 M Tris / hydrochloric acid, pH 7.5 containing 1% bovine serum albumin Electron acceptor: 0.1 M p-nitroso-N, N-dimethylaniline in ethanol Indicator: 2,18-phosphoromolybdic acid, 100 mg / ml water Enzyme: glucose dehydrogenase (EC 1.1.99.17), 50 IU / ml test buffer Glucose solution: a) 36 mg glucose / dl human plasma b) 72 mg glucose / dl human plasma c) 144 mg glucose / dl human plasma d) 360 mg glucose / dl human plasma e) 720 mg glucose / dl human plasma f) 1440 mg glucose / dl human plasma g) 3600 mg glucose / dl human plasma In a 1 cm cuvette 1740 µl buffer 250 µl electron acceptor 250 µl indicator and 10 ul glucose dehydrogenase given the mixture to 25 ^OC thermostatted and then added 250 ul glucose solution. With the addition of the glucose solution as the starting point, the change in absorption per minute (ΔE / min) was registered at 820 nm. The following values ​​were obtained:<tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" colsep="0" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" align="center">Glucose concentration (final concentration in the test approach)</entry><entry namest="col2" nameend="col2" align="center">ΔE / min</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">3.6 mg / dl</entry><entry namest="col2" nameend="col2" align="char" char=",">0,20</entry></row><row><entry namest="col1" nameend="col1" align="right">7.2 mg / dl</entry><entry namest="col2" nameend="col2" align="char" char=",">0,38</entry></row><row><entry namest="col1" nameend="col1" align="right">14.4 mg / dl</entry><entry namest="col2" nameend="col2" align="char" char=",">0,57</entry></row><row><entry namest="col1" nameend="col1" align="right">36 mg / dl</entry><entry namest="col2" nameend="col2" align="char" char=",">1,00</entry></row><row><entry namest="col1" nameend="col1" align="right">72 mg / dl</entry><entry namest="col2" nameend="col2" align="char" char=",">1,60</entry></row><row><entry namest="col1" nameend="col1" align="right">144 mg / dl</entry><entry namest="col2" nameend="col2" align="char" char=",">2,24</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">360 mg / dl</entry><entry namest="col2" nameend="col2" align="char" char=",">3,13</entry></row></tbody></tgroup></table></tables>

Example 6

Test strips for glucose detection through the formation of molybdenum blue

1 g Sodium alginate (e.g. Algipon from Kelco, Division of Merck &amp; Co., Clark, New Jersey, USA) 45 g polyvinyl propionate (e.g. Propiophan 70D from BASF, Ludwigshafen, Federal Republic of Germany) 0.75 g sodium nonyl sulfate 10, 15 g of potassium dihydrogen phosphate and 58.5g distilled water 4 g Aerosil COK 84 (Degussa, Hanau, Germany / FRG) are stirred to a homogeneous mass and adjusted to pH 5.5 with 10 N sodium hydroxide solution.

Then then 65 mg glucose oxidase (250 IU / mg), 260 mg of p-nitroso-N, N-dimethylaniline 1.3 g of 2,18-phosphoromolybdic acid added.

The mass was knife-coated onto a 1 mm thick polystyrene film in a layer thickness of 320 μm and at 60 for 1 hour ^OC dried. When a drop of a solution containing glucose is dropped, a clear green color appears within one minute. The intensity of the coloring increases with increasing glucose concentration and can be evaluated visually using a comparison scale or by reflection photometry.

Instead of the electron acceptor p-nitroso-N, N-dimethylaniline, p-benzoquinone-dioxime or p-nitroso-N, N-diethanolaniline could also be used.

When stored in the dark at room temperature, the discoloration is stable for several weeks.

Replacing p-nitroso-N, N-dimethylaniline with peroxidase (100 mg; 200 IU / mg) and phosphomolybdic acid with 3,3 ′, 5,5′-tetramethylbenzidine (300 mg) in the above example gives an oxygen-dependent glucose test , as it is known in principle from the prior art. In contrast to the example above, however, the sample solution must be wiped onto the test strip after application so that oxygen can diffuse in and coloring occurs at all. In addition to this disadvantage, the final coloring with a higher glucose concentration is achieved more slowly and the color produced is less stable in storage. The advantages of replacing oxygen with electron acceptors according to the invention are thus: - no need to wipe the sample after placing it on the test strip - faster response - In the case of kinetic measurement, the reaction rate depends on the analyte concentration - When measuring the end point at the end point, the reaction speed is independent of the analyte concentration - more stable dye

Example 7

Detection of glucose with non-NAD (P) -dependent glucose dehydrogenase and resazurin

To 2050 µl test buffer (0.2 M citrate, 1% by weight albumin, pH 7.0) and 100 µl electron acceptor solution (10 mM resazurin in water), 250 µl sample solution (C_glucose = 0 to 0.5 mM) and 100 µl enzyme solution (glucose dehydrogenase (EC 1.1.99.17) 200 U / ml in test buffer) added. After 2 minutes, the absorbance at 530 nm was measured against a blank value treated in the same way using 100 μl of test buffer instead of enzyme solution. The following results were obtained:<tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="3" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><thead valign="top"><row><entry namest="col1" nameend="col2" align="center">Glucose concentration in</entry><entry namest="col3" nameend="col3" align="center">Change in absorption ΔE₅₃₀</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="center">(mM) cuvette</entry><entry namest="col2" nameend="col2" align="center">(mM) sample</entry><entry namest="col3" nameend="col3" /></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="char" char=",">0</entry><entry namest="col2" nameend="col2" align="char" char=",">0</entry><entry namest="col3" nameend="col3" align="char" char=",">0</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,002</entry><entry namest="col2" nameend="col2" align="char" char=",">0,02</entry><entry namest="col3" nameend="col3" align="char" char=",">0,009</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,004</entry><entry namest="col2" nameend="col2" align="char" char=",">0,04</entry><entry namest="col3" nameend="col3" align="char" char=",">0,018</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,008</entry><entry namest="col2" nameend="col2" align="char" char=",">0,08</entry><entry namest="col3" nameend="col3" align="char" char=",">0,035</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,012</entry><entry namest="col2" nameend="col2" align="char" char=",">0,12</entry><entry namest="col3" nameend="col3" align="char" char=",">0,051</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,016</entry><entry namest="col2" nameend="col2" align="char" char=",">0,16</entry><entry namest="col3" nameend="col3" align="char" char=",">0,068</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,020</entry><entry namest="col2" nameend="col2" align="char" char=",">0,20</entry><entry namest="col3" nameend="col3" align="char" char=",">0,087</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,030</entry><entry namest="col2" nameend="col2" align="char" char=",">0,30</entry><entry namest="col3" nameend="col3" align="char" char=",">0,130</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,040</entry><entry namest="col2" nameend="col2" align="char" char=",">0,40</entry><entry namest="col3" nameend="col3" align="char" char=",">0,174</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="char" char=",">0,050</entry><entry namest="col2" nameend="col2" align="char" char=",">0,50</entry><entry namest="col3" nameend="col3" align="char" char=",">0,217</entry></row></tbody></tgroup></table></tables>

The test is linear up to sample concentrations of 0.5 mM. In this range, the concentration in the cuvette can be calculated from the extinction coefficient ε₅₃₀ = 4350 M⁻¹ cm⁻¹. At higher sample concentrations, an intermediate dilution or a smaller sample volume can be used. Lower sample concentrations can be determined more sensitively using the fluorescence of the Resorufin product.

The test could be carried out in an analogous manner with benzfuroxane and the benzfuroxane derivatives named in Example 1 B and with nitroso compounds according to Example 2.

With non-NAD (P) -dependent alcohol dehydrogenase (EC 1.1.99.8), ethanol was detected in the same way.

Example 8

Determination of lactate with lactate oxidase and nitroso compounds as electron acceptors

In a cuvette were 2240 ul test buffer (0.2 M citric acid / sodium hydroxide, pH 6.35), 5 ul electron acceptor (0.1 MN, N-dimethyl-p-nitroso-aniline in ethanol) and 250 ul sample with known lactate -Concentrations mixed and on 25 ^OC thermostatted. The test was started with 5 μl enzyme solution (lactate oxidase (pediococcus sp.) 200 U / ml test buffer) and the change in extinction ΔE / min at 390 nm was recorded. The following results were obtained:<tables id="tabl0003" num="0003"><table frame="all"><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" colsep="0" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row rowsep="1"><entry namest="col1" nameend="col1" align="center">C._Lactate: mM</entry><entry namest="col2" nameend="col2" align="center">ΔE / min</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="char" char=",">0</entry><entry namest="col2" nameend="col2" align="char" char=",">0</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,1</entry><entry namest="col2" nameend="col2" align="char" char=",">0,093</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,2</entry><entry namest="col2" nameend="col2" align="char" char=",">0,182</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,3</entry><entry namest="col2" nameend="col2" align="char" char=",">0,250</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0,5</entry><entry namest="col2" nameend="col2" align="char" char=",">0,343</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">1,0</entry><entry namest="col2" nameend="col2" align="char" char=",">0,443</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="char" char=",">3,0</entry><entry namest="col2" nameend="col2" align="char" char=",">0,508</entry></row></tbody></tgroup></table></tables> C._Lactate is the lactate concentration that is in the cuvette when the measurement is carried out.

The test could be accelerated or slowed down by changes in the electron acceptor concentration, enzyme concentration, observation wavelength and temperature. N, N-diethyl-p-nitrosoaniline, N, N-bis (2-hydroxyethyl) -p-nitrosoaniline, benzfuroxane and resazurin could also be used as electron acceptors.