JP3323404B2 - Highly accurate substance measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring an analyte in a sample by measuring a detectable substance which is generated with a quantitative correlation based on a chemical reaction of the analyte. And a method for suppressing the decomposition of the detectable substance by a substance coexisting in the reaction system, stabilizing the substance, and facilitating the measurement. The method of the present invention is used for analyzing biological components and environmental samples.

[0002]

2. Description of the Related Art Detection and detection of analytes in samples, such as biological components such as urine and blood in body fluids, foods, medicines, trace substances existing in the natural environment, industrial chemicals, and trace components in waste. As a method of quantification, there is a method of measuring a detectable substance (dye, etc.) that is quantitatively generated by chemically reacting with a specific substance generated in a reaction system involving the analyte, and is included in a body fluid in a clinical test. It is used for quantification of various existing components, environmental analysis, and the like.

For example, hydrogen peroxide produced by a reaction between an analyte in a biological sample and an oxidase or the like is subjected to an oxidation-reduction reaction with an oxidizable color former (dye precursor) in the presence of peroxidase, There is a method of colorimetrically determining a dye compound that is quantitatively generated.

[0004] Such a method of measuring a substance to be analyzed using a reaction system for generating hydrogen peroxide is an important measurement method because there are many reactions in which hydrogen peroxide is generated as a released substance accompanying oxidation. Has become.

However, in the conventional method, accurate measurement has not always been easy. That is, in these measurement methods, the amount or concentration of a detectable substance such as a dye compound needs to have a stoichiometric correlation with a specific substance such as hydrogen peroxide. Oxidizing properties, such as strong reducing properties of ascorbic acid and the like contained in biological samples, affect the redox system in the above colorimetric method, and detectable substances such as the dye compounds are decomposed and measured. It can cause errors.

For example, in these measurement methods, when an excessive amount of hydrogen peroxide is generated from an analyte such as glucose by an oxidase such as glucose oxidase even temporarily, a reaction between the dye precursor and hydrogen peroxide is caused. Besides, a reaction between the produced dye and hydrogen peroxide occurs. As a result, there is a disadvantage in that the dye that has been produced is decomposed by hydrogen peroxide at the same time as the production thereof, resulting in fading.

In addition, if a peroxidase-like enzyme such as peroxidase that generates reactive oxygen species such as superoxide, which is highly reactive from hydrogen peroxide, or a transition metal ion and a complex thereof that exert a similar effect are present in a sample, Reactive oxygen species react with the generated dye, decompose,
Fading occurs. This interference also had a disadvantage in the measurement. Also, if a reaction that produces a detectable substance such as a dye is performed in the state of being exposed to the atmosphere, the generated dye is oxidized by oxygen in the air or dissolved oxygen in the reaction solution, causing decomposition and fading. There was something.

[0008] Accordingly, various attempts have been made to search for a dye precursor that provides a stable substance that is difficult to decompose, and to add various stabilizers, but these methods are still insufficient. Also,
Biological samples contain reducing substances such as ascorbic acid, uric acid, and bilirubin, which have a large effect on the oxidation-reduction reaction, and how accurate measurement is performed in the presence of ascorbic acid is particularly important in clinical practice. It has been a long-standing problem in analysis, and interference suppression means such as selective decomposition by enzymes, decomposition by addition of periodic acid, oxidative decomposition by iron-ethylenediaminetetraacetic acid chelate, and selective separation by semi-permeable membranes are the same as the dye precursors described above. (Yoshihide Ota, Yutaka Ogawa, “Clinical Laboratory” 34 (4), 50)
2-504 (1990): Japanese Patent Publication No. 41223: Japanese Patent Publication No. 2-4861: Japanese Patent Publication No. 4-18630: Japanese Patent Laid-Open No. 5-9
5797: JP-A-7-155196).

In addition to the oxidation-reduction reaction, various known reactions (eg, condensation reaction such as acid-base reaction and coupling reaction of diazonium salt, complex formation reaction, etc.) have a quantitative relationship with a specific analyte. Dye (for example,
Azo dyes, etc.), and there is a method of measuring a specific analyte by optically quantifying the generated dyes. These methods are described in detail in, for example, Handbook of Analytical Chemistry. This is an important measurement method.

[0010] However, some of the dyes formed in this manner include unstable oxygen which is decomposed by environmental oxygen, an oxidizing substance, a peroxidase-like substance or a reducing substance in the sample, hydrogen ions, a base, light or the like of the sample. When measuring such substances, for example, it is necessary to add stabilizers, remove interfering substances, operate quickly, and operate in a nitrogen-substituted environment or a light-shielded environment. Otherwise, the measurement may be in error.

In addition, an electrochemical reaction occurs when an oxidation / reduction reaction is caused between an electron mediator (mediator) and a substance to be analyzed by an enzyme or the like, and the resulting oxidized / reduced electron mediator is reduced / oxidized at an electrode. There is a method of obtaining the amount of the analyte from the response, which is an important measurement method. At this time, the enzymatic reaction is performed for a certain period of time, during which the electron mediator is oxidized / reduced to accumulate the oxidized / reduced form of the electron mediator, and after a certain time, the accumulated oxidized / reduced form of the electron mediator is removed. There is a method of measuring a substance to be analyzed with high sensitivity by reducing / oxidizing at an electrode to generate a large electrochemical response. However, in the conventional method, the accumulated oxidant / reducer of the electron transfer substance may undergo a decomposition reaction such as reduction / oxidation by a coexisting reducing substance or oxidizing substance, thereby causing an error in measurement.

[0012]

If the detectable substance is stable without decomposition, a stoichiometric relationship is assured at the time of measurement, and a better S / B ratio (signal And the ratio to the background), thereby improving the accuracy of the analysis and increasing the sensitivity. Therefore, many efforts have been made to develop a reaction system that produces a detectable substance that is stable and easy to measure. Examples of various reagents that have been developed so far as reactants producing such stable detectable substances are also described, for example, in Analytical Chemistry Handbook.

[0013] However, searching for a reaction system that produces such a stable substance requires a great deal of effort.
Work continues on seeking a reaction system that produces a stable and easily detectable substance. Therefore, even in the presently used measuring methods, there are many cases where a coexisting substance such as pH, moisture, a redox substance, or an unstable substance decomposed by light must be measured as a detectable substance.

The present invention has been made in view of such circumstances, and a method of measuring a substance to be analyzed by measuring a detectable substance which is quantitatively generated from the substance to be analyzed by a chemical reaction is described. It is an object of the present invention to provide a method for improving the accuracy of measurement and achieving high sensitivity by stabilizing a detectable substance.

[0015]

Means for Solving the Problems As a means for solving the above-mentioned problems, the present inventors have found that the presence of a layered inorganic compound in a reaction system including the reaction for producing the detectable substance allows the detection of the detectable substance. To be able to stabilize
The present invention has been completed.

That is, the present invention provides a method for detecting a substance which can be detected using a reaction system including a reaction for producing a substance which is detectable with a quantitative correlation with the substance to be analyzed based on a chemical reaction of the substance to be analyzed. A method of measuring the substance to be analyzed by measuring the amount of the analyte, wherein the decomposition of the detectable substance by a coexisting substance in the reaction system is suppressed by causing a layered inorganic compound to be present in the reaction system. It relates to a method for measuring a substance.

In the present invention, the presence of the layered inorganic compound in the reaction system for producing the detectable substance to be quantified is substantially simultaneous with the production of the detectable substance or before it is decomposed by the coexisting substance. Thus, a complex of the detectable substance and the layered inorganic compound is formed, and as a result, it is possible to prevent the detectable substance from being decomposed by the action of a substance coexisting in the reaction system.

[0018] The measuring method to which the present invention is applied includes a reaction system including a reaction for generating a detectable substance with a quantitative correlation with the analyte based on a chemical reaction of the analyte in the sample. The method is not particularly limited as long as it is a method of measuring the substance to be analyzed by measuring the detectable substance using the method. For example, the present invention is not limited to the case of using a reaction system in which a detectable substance to be quantified by the chemical reaction of the analyte is directly generated, and the chemical reaction of the analyte and the chemical reaction of the detectable substance are generated. Are indirectly linked via another chemical reaction.

Among them, the method of the present invention is preferably applied to a measuring method using a reaction system in which the reaction for producing a detectable substance is an oxidation-reduction reaction, a reaction system for producing an azo compound, or the like. For example, in an oxidation-reduction reaction system, an oxidizing substance, a reducing substance, or a peroxidase-like substance often coexists in a reaction system as a reaction intermediate or a contaminant in a sample. The detectable substance may be degraded, and in such a case, the method of the present invention is useful.

Further, the measuring method of the present invention is suitably used for a method utilizing a substance which can be detected by an optical method or an electrochemical method as a detectable substance. The detectable substance is not particularly limited as long as it is capable of forming a complex by causing an interaction such as adsorption with the layered inorganic compound of the present invention. Such detectable substances are produced by oxidation-reduction reactions, acid-base reactions, condensation reactions, etc., and are widely used in dyes detectable by optical methods and mediators (electron mediators) detectable by electrochemical methods. Can be found.

The term "optically detectable dye" as used herein includes a dye compound, a fluorescent dye, a luminescent substance, and the like, and the term "dye-forming reaction" refers to an optically detectable dye. Any reaction may be used as long as it is a reaction that produces, for example, not only color development but also color change, fluorescence, light emission, and the like.

The measurement method of the present invention is also preferably applied to a method utilizing an oxidation-reduction reaction that produces an oxidized / reduced form of a mediator (electron transfer substance) as a detectable substance. The reaction of generating the oxidized / reduced form of the mediator is a reaction in which the reduced form / oxidized form of the mediator is oxidized / reduced by an oxidoreductase or the like. What is necessary is just to be able to detect by.

The measurement method of the present invention preferably detects and quantifies biological components such as urine and blood in body fluids, foods, medicines, trace substances existing in the natural environment, industrial chemicals, trace components in wastes, and the like. It is used for the method.

In particular, a method of optically measuring a dye or the like quantitatively generated by a redox reaction between hydrogen peroxide generated from a biological component by an oxidase reaction or the like and an oxidizable color forming agent is known as a body fluid in a clinical test. It is used for the quantification of various components and environmental analysis, etc., according to the method of the present invention, such as excess oxidizing substances such as hydrogen peroxide and ascorbic acid, uric acid, bilirubin and the like present in the reaction system. It is possible to solve the problem that a measurement error occurs due to decomposition and fading of a dye or the like due to the action of a reducing substance. Of course, the effectiveness of the present invention is not limited to this example.

[0025]

Embodiments of the present invention will be described below in detail. The method of the present invention can be specifically applied to a measurement method using the following reaction system, but is not limited thereto.

(A) A method utilizing a reaction system including a reaction for producing hydrogen peroxide or an oxidation reaction using hydrogen peroxide as an oxidizing agent. Specifically, a chemical reaction such as an oxidase reaction is performed from a substance to be analyzed. To generate hydrogen peroxide, which is subjected to an oxidation-reduction reaction with an oxidizable color forming agent (dye precursor) in the coexistence of peroxidase (POD). A method for measuring a substance to be analyzed by colorimetric quantification of a dye compound which is a detectable substance generated in association therewith.

(B) a reaction for producing nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH);
Alternatively, a method using a reaction system including a reaction using NADH or NADPH as a reducing agent. Specifically, for example, NADH or NADPH is produced from a substance to be analyzed via a dehydrogenase reaction system or the like. To reduce it by acting on the dye precursor in the presence of the electron transport system,
A measurement method for the colorimetric determination of a dye compound, which is a detectable substance formed thereby.

(C) Under acidic conditions, an aromatic primary amine is reacted with nitrous acid to produce a diazonium salt, the produced diazonium salt is reacted with a coupling reagent, and then the produced azo dye is quantified. Nitrates, diazonium salts,
A method for measuring the amount of a coupling compound.

(D) A fluorescent enzyme substrate such as 4-methylumbelliferone having a phosphate ester releases a phosphate by the action of alkaline phosphatase to generate a fluorescent substance, and the generated fluorescent substance is excited by excitation light. A method of measuring the amount of a substance or alkaline phosphatase labeled with a fluorescent enzyme substrate, which measures the fluorescence emitted upon irradiation.

(E) oxidizing / reducing a mediator such as 1,4-diaminobenzene with an oxidoreductase or the like,
A method for measuring the amount of an oxidoreductase or a substance labeled with an oxidoreductase, which measures a current response when an oxidized / reduced form of a produced mediator is reduced / oxidized by an electrode reaction.

The detectable substance used in these methods is not particularly limited as long as it can be adsorbed on the layered inorganic compound of the present invention. Specific examples of the compounds that can be adsorbed on the layered inorganic compound include amines such as amines and polyamines; imines such as imines and polyimines; polyenes; and aromatics such as aniline derivatives, benzoquinone derivatives and aromatic condensed ring compounds. Compounds; xanthene, azine,
A heterocyclic compound such as thiazine; a complex of an ion and a cyclic ligand such as crown ether or valinomycin; etc., which contains a quaternary nitrogen atom, a phenolic hydroxyl group, a sulfonic acid group, or a carboxyl group in the molecule. Is also good.

Layered inorganic compounds such as smectite are known to adsorb a wide variety of substances.
Smectite, one of them, adsorbs anionic substances, cationic substances and nonionic polar organic compounds, and hydrotalcite adsorbs anionic substances. Substances that can be adsorbed on layered inorganic compounds are described, for example, in H. Van Olphen's book `` An Introduction to Clay Colloid Chemistry, Secon
d Edition ”(Krieger Publishment, Malabar)
It is described in detail in chapter "Interaction of Clays and Organic Compounds". In addition, Tadayoshi Kato, Tokiko 50-8462
(US Pat. No. 3,950,180) introduces a large number of adsorbable compounds. Among them, substances that can be detected by an optical method or an electrochemical method are exemplified.

For example, a substance which can be detected by an optical method such as a dye compound or a fluorescent dye formed from a dye precursor by an oxidation-reduction reaction, an acid-base reaction, a condensation reaction or the like is used. A mediator that can be detected by an electrochemical method is used.

Hereinafter, substances detectable by an optical method will be described. Among substances detectable by an optical method, as the dye compound, a compound having a conjugated system such as an aromatic ring is preferably used. Specifically, 4-amino-1,2-dihydro-1,5- A coupler represented by dimethyl-2-phenyl-3H-pyrazol-3-one (4-aminoantipyrine: abbreviated as 4-AA);
Hydrogen donor (N-ethyl-N- (3-sulfopropyl)
-3,5-dimethylaniline and other Trinder reagents)
Which is formed by oxidative condensation of a compound; oxidized color forming dyes such as ortho-tolidine and benzidines (3,3 ′, 5,5′-tetramethylbenzidine); 2,6-dichloro-4-[(4-hydroxyphenyl ) Imino] -2,
A dye formed by oxidizing a leuco body such as 5-cyclohexadien-1-one; a fluorescent substance formed by oxidizing 4-hydroxyphenylacetic acid; a luminescent substance such as a chemiluminescent substance and its exciter; a reducing dye of a tetrazolium salt A dye formed by reduction of formazan, 1,1'-dimethyl-4,4'-bipyridylium salt or the like; a compound which develops or changes color due to a change in pH, such as bromocresol green; 2-methoxy-4-morpholinobenzenediazonium Azo dyes formed by coupling of diazonium salts such as salts; dyes formed by various known color reactions such as the reaction of aldehydes with 2,3-dimethyl-2,3-bis (hydroxyamino) butane; histamine and ortho Produced by various known reactions such as reaction with phthalaldehyde A fluorescent substance; a dye or a fluorescent substance formed by the reaction of an enzyme substrate such as 4-methylumbelliferyl phosphate with an enzyme; and 2- (5-bromo-2-pyridylazo) -5- [N-propyl-N- (3-sulfopropyl) amino] aniline salt and a dye formed of a metal ion and the like.

Here, the hydrogen donor is 4-amino-1,2- (2-amino-1,2-) by the action of peroxidase in the presence of hydrogen peroxide.
Compounds such as phenol that form quinone dyes by condensation with dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one (4-AA) or 3-methyl-2-benzothiazolinone hydrazone And specifically, dichlorophenol, orthomethoxyphenol, 1,2,2
3-trihydroxybenzene, dimethylaniline, N-
Ethyl-N-sulfopropylmethanisidine, N-ethyl-N-sulfopropylaniline, N-ethyl-N-
(3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3,5
-Dimethylaniline, N-ethyl-N-sulfopropylmethtoluidine, N-ethyl-N- (2-hydroxy-
3-sulfopropyl) metanisidine, N-ethyl-N
-(2-hydroxy-3-sulfopropyl) aniline,
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline, N-ethyl-
N- (2-hydroxy-3-sulfopropyl) meta toluidine, N- (3-sulfopropyl) aniline and the like can be mentioned.

For example, in a method using a reaction system in which 4-AA reacts with a hydrogen donor in the presence of hydrogen peroxide to form a quinone-based dye, the quinone-based dye formed is measured with an absorptiometer or the like. Then, the substance to be analyzed is measured by indirectly quantifying hydrogen peroxide.

The ortho-tolidine and benzidines include ortho-tolidine, dianisidine, 3,3'-diaminobenzidine, 3,3 ', 5,5'-tetramethylbenzidine, N- (3-sulfopropyl) -3,3'. , 5
5′-tetramethylbenzidine and the like.

The leuco compound is a colorless dye precursor which is oxidized to form a dye and develops a color. Dyes in which the leuco body has been oxidized include 2,6-dichloro-4-[(4-hydroxyphenyl) imino] -2,5-cyclohexadien-1-one and 2,6-dichloro-4-[(3 -Chloro-
4-Hydroxyphenyl) imino] -2,5-cyclohexadien-1-one, 7- (diethylamino) -3-
Imino-8-methyl-3H-phenoxazine salt, 3-
(Diethylamino) -7-amino-5-phenylphenazinium salt, 3,7-bis (dimethylamino) phenothiazine-5-ium salt, 1-hydroxy-5-methylphenazinium salt, 7-hydroxy-3H- Phenoxazin-3-one-10-oxide, and leuco forms include 4,4′-benzylidenebis (N, N-dimethylaniline) and 4,4′-bis [N-ethyl-N- (3
-Sulfopropylamino) -2,6-dimethylphenyl] methane, 1- (ethylaminothiocarbonyl) -2
-(3,5-dimethoxy-4-hydroxyphenyl)-
4,5-bis (4-diethylaminophenyl) imidazole, 4,4'-bis (dimethylamino) diphenylamine, N- (carboxymethylaminocarbonyl)-
4,4'-bis (dimethylamino) diphenylamine salt, 10- (carboxymethylaminocarbonyl)-
3,7-bis (dimethylamino) phenothiazine salt and the like.

Dye precursors that are oxidized to form a color include:
In addition, 4-methoxyphenol, 4-ethoxyphenol, 2-ethoxyphenol, 1- (2-hydroxy-5-methoxyphenyl) ethanone, 2-hydroxy-5-methoxybenzoic acid, 2-hydroxy-5-methoxybenzaldehyde Methyl 2-hydroxy-5-methoxybenzoate, 4-methoxy-2-nitrophenol, 2-chloro-4-methoxyphenol, 4-hydroxy-3-methoxybenzaldehyde, 4-hydroxy-3-methoxybenzoic acid and the like. No.

Further, 3- (4-hydroxyphenyl)-
2-propenoic acid, 2-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid,
3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2
-Aminobenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 3,4-diaminobenzoic acid, 3,5-diaminobenzoic acid, 4-amino-2-chlorobenzoic acid, 4-amino-3-methyl Benzoic acid, 4-amino-3-methoxybenzoic acid, 4-aminophthalic acid and the like can be mentioned.

Also, 2,4-diamino-6-hydroxypyrimidine, 4,5-diamino-6-hydroxypyrimidine, 4-amino-2,6-dihydroxypyrimidine,
6-hydroxy-2,4,5-triaminopyrimidine,
4,5-diamino-2,6-dihydroxypyrimidine,
4-amino-6-hydroxy-2-methylpyrimidine,
4-amino-6-hydroxypyrimidine, 4-amino-
6-hydroxy-2-methoxypyrimidine and the like.

Also, 4-hydroxyphenylacetic acid or the like is oxidized to produce a fluorescent substance.
-Hydroxyphenylacetic acid, (4-hydroxy-3-methoxyphenyl) acetic acid, 3- (4-hydroxyphenyl) propionic acid, 4-hydroxy- (2-aminoethyl) phenol, 4-hydroxy-N, N, N- Trimethylbenzenemethaminium, alpha-aminoparahydroxyhydrocinnamic acid, 4-hydroxyphenethylamine, N- (4-hydroxyphenyl) acetanilide,
Fluorescent substances generated by oxidizing 2,7-dichlorofluorescein-diacetate and the like are included.

As a luminescent substance such as a chemiluminescent substance,
Firefly luciferin, Cypridina luciferin, aequorin, lucigenin derivatives, luminol derivatives, acridinium esters, peroxalates and the like.

For example, in a method using a reaction system in which benzidines or leuco bodies are oxidized in the presence of hydrogen peroxide to form a color, the resulting dye is measured with an absorptiometer or the like to indirectly use hydrogen peroxide. The analyte is measured by quantifying

In the above-mentioned method utilizing a reaction system in which a fluorescent substance or a luminescent substance is produced, the substance to be analyzed is measured by indirectly quantifying hydrogen peroxide by measuring with a fluorometer or a luminometer. It is.

Of course, in the oxidation reaction for producing such a dye, the oxidizing agent involved in the oxidation reaction is not limited to hydrogen peroxide, and various known oxidizing agents may be used. An oxidizing enzyme such as peroxidase may be added. Further, prior to the oxidation reaction for producing the dye, a reaction for producing the oxidizing agent may have occurred.

As the tetrazolium salt, 2,3,
5-triphenyltetrazolium salt, 2,5-diphenyl-3- (1-naphthyl) -2H-tetrazolium salt,
3,3 ′-[3,3′-dimethoxy- (1,1′-biphenyl) -4,4′-diyl] -bis [2- (4-nitrophenyl) -5-phenyl-2H-tetrazolium]
Salt, 3,3 ′-[3,3′-dimethoxy- (1,1′-
Biphenyl) -4,4'-diyl] -bis (2,5-diphenyl-2H-tetrazolium) salt, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyl-2H-tetrazolium Salt, 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-
Disulfophenyl) -2H-tetrazolium salt, 2-
(4-iodophenyl) -3- (2,4-dinitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium salt, 3,3 ′-(1,1′-biphenyl-
4,4'-diyl) -bis (2,5-diphenyl-2H
-Tetrazolium) salt, 3- (4,5-dimethyl-2-)
Thiazolyl) -2,5-diphenyl-2H-tetrazolium salt and the like.

The dyes produced by the reduction include:
1,1′-dimethyl-4,4′-bipyridylium salt,
Reductants such as 1,1'-dibenzyl-4,4'-bipyridylium salt are exemplified.

Also, 7-hydroxy-3H-phenoxazin-3-one-10-oxide and the like are reduced to produce a fluorescent substance.
3H-phenoxazin-3-one-10-oxide, 5
-Cyano-2,3-bis (4-methylphenyl) -2H
-Tetrazolium salts, fluorescent substances formed by reduction of 2,3-bis (4-cyanophenyl) -5-cyano-2H-tetrazolium salts, and the like.

For example, in the method using a reaction system in which a tetrazolium salt or a leuco compound undergoes a reduction reaction in the presence of a reducing agent to form a color, the resulting dye is measured indirectly by using an absorptiometer or a fluorometer. The analyte is measured by quantifying the reducing agent. Further, a reaction in which a reducing agent is generated may occur before a reduction reaction in which a dye is generated.

In the reduction reaction for producing such a dye, nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate is preferably used as a reducing agent for the reduction reaction. However, the present invention is not limited to this, and various known reducing agents may be used.

Compounds that develop or change color due to a change in pH include sulfonphthalein dyes such as bromocresol green, bromophenol blue, phenol red, bromopyrogallol red, and pyrogallol red; and triphenylmethane such as malachite green and rosolic acid. Dye, quinaldine red, N
Quinoline dyes such as-(parahydroxyphenyl) -2,6-dichloroparabenzoquinone imine; oxazone dyes such as 7-hydroxy-3H-phenoxazin-3-one 10-oxide; 6,7-dihydroxy-4
Coumarin-based dyes such as methyl coumarin; and conductive polymer compounds such as aniline oligomers.

For example, in a method using a reaction system in which a compound that forms or changes color due to a change in pH is formed or changes color by an acid or base, the resulting dye is measured with an absorptiometer or the like to indirectly determine the acid or base. By doing so, the analyte is measured.

For example, in a method using a reaction system in which a compound that forms or changes color due to a change in pH is formed or changes color by hydrogen ions, the resulting dye is measured with an absorptiometer or the like to determine the hydrogen ion concentration. It measures the analyte.

The azo dyes formed by coupling of diazonium salts include azo dyes formed by coupling of indoxyl and 2-methoxy-4-morpholinobenzenediazonium salt, urobilinogen and 3,3'-dimethoxybiphenyl. Azo dyes formed by coupling of 4,4'-diazonium salt, azo dyes formed by reaction of 4-aminobenzenearsonic acid and N-1-naphthylethylenediamine in the presence of nitrite, also nitrite And azo dyes formed by the reaction of 2,4-dichloroaniline with N, N-diethyl-N'-1-naphthylnaphthylethylenediamineoxalate (Tsuda reagent) in the presence of aniline.

In the method using a reaction system in which an azo dye is formed, the dye to be formed is measured by an absorptiometer or the like, and an analysis target substance (in the above example, indoxyl, urobilinogen, Nitrate) is measured. The reaction for producing the azo dye is not limited to the above-mentioned example, but is preferably applied to reactions for producing various known azo dyes.

Dyes formed by various known color reactions include, but are not limited to, dyes formed by the following known color reactions. Examples of the color reaction include a reaction between hydrogen peroxide and 1,4-diaminobenzene when detecting aldehyde, and a reaction between 2,3-dimethyl- when detecting aldehyde.
Reaction of 2,3-bis (hydroxyamino) butane, reaction of 3-methyl-2-benzothiazolinone hydrazone with oxidizing agent when detecting aldehyde, reaction of 10H-phenothiazine and bromine when detecting secondary amine Reaction, reaction of 2,2′-dithiodipyridine when detecting thiol, and the like.

In the method utilizing the known color reaction,
The resulting dye is measured with an absorptiometer or the like, and the analyte (the aldehyde, secondary amine, or thiol in the above example) that is the starting material of the reaction is measured. The known color reaction that can be used is, of course, not limited to the above examples.

Examples of the fluorescent substance generated by various known reactions include, but are not limited to, fluorescent substances generated in a known detection reaction performed using the reagents described below. . As the reagent used for the detection reaction for producing a fluorescent substance, 2-hydroxy-1,2-acid when detecting a guanidino compound is used.
Examples include diphenylethanone, orthophthalaldehyde for detecting histamine, orthophthalaldehyde for detecting spermidine, and 1,2-diamino-4,5-dimethoxybenzene for detecting alpha keto acid.

In the method using the known detection reaction,
The resulting fluorescent substance is measured by a fluorometer or the like, and the analysis target substance (in the above example, a guanidino compound, histamine, spermidine, or alpha-keto acid) that is a starting substance of the reaction is measured. The known detection reactions that can be used are, of course, not limited to the above examples.

The enzyme substrate which reacts with an enzyme to produce a dye or a fluorescent substance includes N-tosyl-L-phenylalanine-2-amidoacridone which is a substrate of chymotrypsin, and L-alanine-2 which is a substrate of aminopeptidase. -Amidoacridone, 7-acetoxy-N-methylquinolinium salt for measuring esterase, 7-acetoxy-3H-phenoxazin-3-one which is a substrate of esterase, 4-methylun which is a substrate of phosphatase Examples include, but are not limited to, berypheryl phosphate, phosphatase substrate 5,10,15,20-tetrakis (4-phosphonooxyphenyl) porphine, and the like.

For example, in the method utilizing the reaction in which the enzyme substrate is decomposed by the enzyme, the dye or the fluorescent substance to be produced is measured by an absorptiometer or a fluorimeter, and the enzyme is indirectly quantified. The target substance is measured. The enzyme or enzyme substrate may be, for example, chemically bound to the antibody or a fragment thereof.

The complex dye is a dye or a fluorescent substance formed by forming a complex between a metal ion or an anion and a compound such as a ligand by a coordination bond or an ionic bond. Compounds that form a complex with a metal ion to form a color or discolor include compounds known as metal indicators and chromoionophores, as well as compounds that form a complex with a colored transition metal ion to color. Include ethylenediaminetetraacetic acid, 2,2-bipyridine, 1-hydroxy-2- (2-hydroxyphenylazo) benzene, dibenzo-18-crown-6, dicyclohexyl-18-crown-6, cyclic polyamines, calix [ 4] arenes, 3- [N, N-bis (carboxymethyl) aminomethyl] -1,2-dihydroxyanthraquinone, 5 ′, 5 ″ -dibromopyrogallol sulfonephthalein, 2-hydroxy-1- (1-hydroxy -2-
Naphthylazo) -6-nitro-4-naphthalenesulfonate, 2,6-dichloro-4'-hydroxy-3 ',
3 "-dimethylfuchsone-5 ', 5" -dicarboxylate, 3,3'-bis [N, N-bis (carboxymethyl) aminomethyl] fluorescein, 8- [N, N-
Bis (carboxymethyl) aminomethyl] -4-methylumbelliferone, 2,7-bis (2-arsonophenylazo) -1,8-dihydroxy-3,6-naphthalenedisulfonic acid, 5-chloro-2- Hydroxy-3-
(2,4-dihydroxyphenylazo) benzenesulfonic acid, 5-[(hexahydro-2,4,6-trioxo-5-pyrimidinyl) imino] -2,4,6 (1H, 3
H, 5H) -pyrimidinetrione salt, 2- (5-bromo-2-pyridylazo) -5- [N-propyl-N- (3
-Sulfopropyl) amino] aniline salt, 1,8-dihydroxy-2- (2-pyridylazo) -3,6-naphthalenedisulfonic acid salt, 2-nitroso-5- [N-propyl-N- (3-sulfopropyl) ) Amino] phenol and the like.

Particularly, as the compound which forms a colored complex with a monovalent cation, tetrakis [3,5-bis (trifluoromethyl) phenyl] borate salt, tetraphenylphosphonium salt and the like can be mentioned. Particularly, as a compound which forms a fluorescent complex with calcium ions or the like, 1-
[2-Amino-5- (2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl) phenoxy] -2
-(2-amino-5-methylphenoxy) ethane-N,
N, N ', N'-tetraacetate, 1- [2-amino-5-
(2,7-dichloro-6-hydroxy-3-oxy-9
-Xanthenyl) phenoxy] -2- (2-amino-5
-Methylphenoxy) ethane-N, N, N ', N'-tetraacetic acid pentaacetoxymethyl ester, 1- [6-amino-2- (5-carboxy-2-oxazazoyl) -5-
Benzofuranyloxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetate;
1- [6-Amino-2- (5-carboxy-2-ogizazoyl) -5-benzofuranyloxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ',
N'-tetraacetic acid pentaacetoxymethyl ester, 1-
[2-Amino-5- (6-carboxy-2-indolyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ', N'-tetraacetate, 1-
[2-amino-5- (6-carboxy-2-indolyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ', N'-tetraacetic acid pentaacetoxymethyl ester, 8-amino-2-[(2-
Amino-5-methylphenoxy) methyl] -6-methoxyquinoline-N, N, N ', N'-tetraacetate, 8-amino-2-[(2-amino-5-methylphenoxy) methyl] -6 -Methoxyquinoline-N, N, N ', N'-tetraacetic acid pentaacetoxymethyl ester, 3,3'-bis [N, N-bis (carboxymethyl) aminomethyl] fluorescein, 8- [N, N -Bis (carboxymethyl) aminomethyl] -4-methylumbelliferone.

Also, a tetraphenylarsonium salt which forms a colored complex with an anion, a complex with N-ethoxycarbonylmethyl-6-methoxyquinolinium bromide or boron which forms a complex with a chloride ion, which reduces the fluorescence intensity. 8-hydroxy-1- (salicylideneamino)-
3,6-naphthalenedisulfonic acid salt and the like.

In the method utilizing the reaction for forming the complex, the amount of the dye or the fluorescent substance is determined by measuring the dye or the fluorescent substance generated by the ion and the ligand with an absorptiometer or a fluorometer. By doing so, the analyte (often an ion) is measured.

Next, the substance which can be detected electrochemically will be described. Substances that can be detected by the electrochemical method include electron mediators (mediators) and complexes of ionophores and ions.

An electron mediator is a chemical substance that oxidizes / reduces a substance to be analyzed by an enzyme or the like and directly accepts / donates electrons from / to the substance to be analyzed. The substance to be analyzed is measured from the electrochemical response when the / oxidized substance is oxidized / reduced at the electrode.
In addition, the electron mediator and the analyte do not need to directly exchange electrons. The electron mediator oxidizes / reduces the analyte with an enzyme or the like. It may be a chemical substance that accepts / donates electrons indirectly to the substance. The oxidized / reduced form of the electron mediator, which is quantitatively related to the analyte, is reduced at the electrode
The analyte is measured from the electrochemical response during oxidation.

As the electron mediator, the potential within the measurable range of the electrode used (normally -1.2 V to
+1.0 V), specifically, 1,1′-dimethyl-4,4′-bipyridylium salt, 1,1′-dibenzyl-4,4′-bipyridylium salt, 4-diaminobenzene, 2-methyl-1,4-
Naphthoquinone, N-methylphenazinium salt, 1-hydroxy-5-methylphenazinium salt, 1-methoxy-
5-methylphenazinium salt, 9-dimethylaminobenzoalphaphenoxazine-7-ium salt, ferrocene derivative, hexacyanoiron (II) salt, 7-hydroxy-3
H-phenoxazin-3-one 10-oxide, 3,
7-diamino-5-phenylphenazinium salt, 3-
(Diethylamino) -7-amino-5-phenylphenazinium salt, 1,4-benzenediol, 1,4-dihydroxy-2,3,5-trimethylbenzene, N, N,
N ′, N′-tetramethyl-1,4-benzenediamine, Δ2,2′-bi-1,3-dithiol, 2,6-dimethylbenzoquinone, 2,5-dimethylbenzoquinone,
2,3,5,6-tetramethyl-2,5-cyclohexadiene-1,4-dione, 2,6-dichloro-4-
[(4-hydroxyphenyl) imino] -2,5-cyclohexadien-1-one, 2,6-dichloro-4-
[(3-Chloro-4-hydroxyphenyl) imino]-
2,5-cyclohexadien-1-one, 7- (diethylamino) -3-imino-8-methyl-3H-phenoxazine salt, 3,7-bis (dimethylamino) phenothiazine-5-ium salt and the like. .

In this example, the detectable substance is an oxidized / reduced form of the electron transfer substance, and the reaction for producing the detectable substance is an oxidation / reduction reaction of the electron transfer substance. As described above, an electrochemical response such as an oxidation-reduction current when an oxidant / reductant of an electron transfer substance existing in a quantitative relationship with an analyte is reduced / oxidized by an electrode is measured, and the analyte is measured. It is.

An ionophore is a compound such as a ligand which selectively forms a coordination bond or an ionic bond with a specific ion to be analyzed and forms a complex, and which is used in a liquid membrane electrode. Is particularly well known.

Specifically, as ionophores that form complexes with cations, tetrakis [3,5-bis (trifluoromethyl) phenyl] borate, tetraphenylphosphonium, valinomycin, cyclo (N ′, N′-)
Dioctyl-D-asparaginyl-L-prolyl-L-
Alanyl) ₂ , bis (benzo-15-crown-5),
Bis [(benzo-15-crown-5) -4-methyl]
Pimerate, bis (12-crown-4), bis [(1
2-crown-4) methyl] -2-dodecyl-2-methylmalonate, 14-crown-4, dodecyl-methyl-14-crown-4, 6,6-dibenzyl-1,4,
8,11-tetraoxacyclotetradecane, dibenzo-18-crown-6, dicyclohexyl-18-crown-6, 4,16-di-N-octadecylcarbamoyl-3-oxabutyryl-1,7,10,13,19-
Pentaoxa-4,16-diazacyclohexicosan and the like.

Examples of ionophores that form complexes with anions include tetraphenylarsonium salts and 6-methoxy-N- (3-sulfopropyl) quinolinium salts.

A liquid film electrode is a device in which a porous polymer layer or the like is provided on the surface of the electrode, and the polymer layer is impregnated with ionophores, and is bonded to only specific ions in the sample solution to move in the polymer layer. In this method, only a specific ion is selectively separated, and a membrane potential generated at that time is measured to measure a specific ion as a substance to be analyzed. Of course, the use of the ionophore in the electrochemical detection method is not limited to this liquid membrane electrode.

In the bulk liquid, the ionophore is bound to a specific ion, and the unbound ion cannot move.
The ions are selectively separated at an electrode provided with a polymer layer capable of transferring only the complex formed by bonding, and the membrane potential generated at that time can be measured to measure the ions as the analyte.

In this example, the detectable substance is a complex of an ionophore and a specific ion, and the reaction for forming the detectable substance is a complex formation reaction between the ionophore and a specific ion by coordination bond or ionic bond. It is. As described above, the substance to be analyzed is measured by electrochemically measuring the membrane potential generated according to the concentration of a specific ion as the substance to be analyzed.

The reaction system of the present invention may be a solution reaction system or a reaction system using an analytical element prepared through unit operations such as drying, precipitation, and filtration. It may be in a state like dry chemistry after drying and solidification or film formation.

The substances to be analyzed by such a method using a reaction system include biological components such as urine and blood in body fluids, foods, medicines, trace substances existing in the natural environment, industrial chemicals, and waste. Trace substances in a substance.

Next, the layered inorganic compound of the present invention will be described. The layered inorganic compound of the present invention includes Si tetrahedron, Al
It is an inorganic compound having a crystal structure in which a sheet structure in which polyhedrons such as octahedrons are connected in a planar manner is layered, and includes a layered clay mineral and hydrotalcite.

The clay mineral refers to an aluminum silicate mineral which occupies most of clay (fine earth-like inorganic particulate matter which is plastic when wet with water).
Are the smallest constituent units of Si tetrahedron surrounded by four O (oxygen atoms) and Al or Mg octahedron surrounded by six OH groups or O.

The structure of the layered clay mineral is such that the Si tetrahedron shares one face and the remaining vertices O are oriented in the same direction to form a hexagonal mesh sheet (tetrahedral sheet), while Al (or M
g) Octahedrons share a ridge angle to form a sheet (octahedral sheet), which is layered. Minerals formed by stacking many 1: 1 layers of tetrahedral sheets and octahedral sheets one by one are sandwiched between 1: 1 type minerals, one octahedral sheet and two tetrahedral sheets. Minerals made up of many layers of 2: 1 layers are converted into 2: 1 type minerals and 2:
The one in which another octahedral sheet is sandwiched between the layers of type 1 is called a 2: 1: 1 type mineral. The octahedral sheet is made of Mg
(OH) ₂ in which metal ions are present at all octahedral positions is the trioctahedral type, and the octahedral sheet is Al (OH) _{3 in} which 1/3 of the holes are voids.
It is called octahedral. In the present invention, it is preferable to use a 2: 1 type mineral.

The element constituting the layered inorganic compound of the present invention is preferably composed of at least one selected from lithium, sodium, potassium, magnesium, aluminum, silicon, oxygen, hydrogen, fluorine, and carbon. Specifically, a compound represented by any one of the following formulas 1 to 9 can be mentioned. Note that these formulas may include water of crystallization.

Of course, these formulas are formulas as a mineralically or chemically pure compound, and an actual layered inorganic compound may contain impurities such as sodium silicate. Even if chemical formulas are defined, they may not always agree with these formulas, for example, see the literature (D. W, Thompson, JT Butter).
worth, J. Colloid Interf.Sci., 151, 236-243 (199
This is also described in 2)).

[0084]

Embedded image M _x Si ₄ (Al _2-x Mg _x ) O ₁₀ X ₂ (1) (In the formula 1, M is any one of H, Li, Na, and K, and X is OH or F and x
Is a positive number less than 2. )

[0085]

Embedded image M _x (Si _4-x Al _x ) Al ₂ O ₁₀ X ₂ (2) (in the formula 2, M is any one of H, Li, Na, and K, and X is OH or F and x
Is a positive number less than 4. )

[0086]

Embedded image M _x Si ₄ (Mg _3-x Li _x ) O ₁₀ X ₂ (3) (in the formula 3, M is any one of H, Li, Na, and K, and X is OH or F and x
Is a positive number less than 3. )

[0087]

Embedded image M _x (Si _4-x Al _x ) Mg ₃ O ₁₀ X ₂ (4) (in the formula 4, M is any one of H, Li, Na, and K, and X is OH or F and x
Is a positive number less than 4. )

[0088]

Embedded image MSi ₄ Mg _2.5 O ₁₀ X ₂ (5) (In the formula 5, M is any one of Li and Na, preferably Na. X is OH or F And preferably F.)

[0089]

Embedded image M ₂ Si ₄ Mg ₂ O ₁₀ X ₂ (6) (In the formula 6, M is any one of Li and Na, preferably Li. X is any one of OH and F. And preferably F.)

[0090]

Embedded image _{Mg 6 Al 2 (OH) 16} X x ··· (7) (X in Formula 7 is _{halogen, NO 3, SO 4, CO} 3,
Any of OH or an anionic form of an organic acid,
Preferably it is CO ₃ . x is X, halogen, OH, NO
₃ , when it is a monovalent organic acid, X is SO ₄ , C
O ₃ is 1 for a divalent organic acid. )

[0091]

_{Embedded image} Na _0.33 Si ₄ (Mg _2.67 Li _0.33 ) O ₁₀ X ₂ (8) (In the formula 8, X is either OH or F, and is preferably OH.)

[0092]

_{Embedded image} Na _ab (Si _4-a Al _a ) (Mg _3-b Al _b ) O ₁₀ X ₂ (9) (In the formula 9, X is either OH or F, and is preferably Is OH. A is a positive number less than 4, b is a positive number less than 3, and ab> 0.)

Specific examples of the layered inorganic compound of the present invention include 1: 1 type clay minerals such as kaolinite, halloysite, and serpentine; talc, pyrophyllite, smectite,
2: 1 type clay minerals such as vermiculite (in the above formula, represented by formula 2; the same applies hereinafter), mica containing tetrasilicon mica (formula 5) and tenionite (formula 6); 1: 1 type clay minerals; 2: 1 to 2: 1: 1 type intermediate minerals; quasicrystalline clay minerals such as imogolite; amorphous clay minerals such as allophane; hydrotalcite (Formula 7); Can be

Smectite includes two octahedrons such as montmorillonite (formula 1), bentonite containing 40 to 80% of montmorillonite and beidellite (formula 2) depending on the ion species in the isomorphous tetrahedron and octahedral lattice. Hectorite (formula 3, preferably formula 8), saponite (formula 4, preferably formula 9), trioctahedral type such as nontronite; and the like.

The hydrotalcite is represented by the formula in the above formula.
7, specifically Mg₆Al_Two(OH) ₁₆CO_Three・ 4H_TwoIn O
A layered mineral represented by Mg (OH)_Two(Blue rhinoceros
G: Mg in the center²⁺Layers of oxygen octahedra with
Mg with structure²⁺Part of Al³⁺Is also isomorphic to
And have a positive charge, but CO2 between the layers_Three ^2-By
It maintains electrical neutrality and has anion exchange capacity.
is there. Not a silicate mineral, but often a clay mineral
Treated.

Some compositions of the above-mentioned layered inorganic compounds of the present invention are shown in Table 1 below.

[0097]

[Table 1] <Table 1> ──────────────────────────────────── Mineral name Composition ^* ── ────────────────────────────────── Kaolinite Si ₂ Al ₂ O ₅ (OH) ₄ Halloysite _{Si 2 Al 2 O 5 (OH} ) 4 · 2H 2 O serpentine _{^{Si 2 (Mg 2+, Fe 2+}} ) 3 O 5 (OH) 4 talc _{(talc) Si 4 Mg 3 (} OH) 2 O 10 Pairofi Pyrophyllite Si ₄ Al ₂ (OH) ₂ O ₁₀ Montmorillonite MI _x Si ₄ (Al _2-x Mg _x ) O ₁₀ (OH) ₂ · nH ₂ O Beidellite MI _x (Si _{4- x} Al _x ) Al ₂ O ₁₀ (OH) ₂ · nH ₂ O Hectorite MI _x Si ₄ (Mg _3-x Li _x ) O ₁₀ (OH, F) ₂ · nH ₂ O Saponite MI _x (Si _4-x Al _x ) Mg ₃ O ₁₀ (0H) ₂・ nH ₂ O Nontronite MI _x (Si _4-x Al _x ) Fe ₂ O ₁₀ (OH) ₂・ nH ₂ O vermiculite (Vermiculite) MI _x (Si _4-x Al _x ) Al ₂ O ₁₀ (OH) ₂・ NH ₂ O Hydrotalcite Mg ₆ Al ₂ (OH) ₁₆ CO ₃・ 4H ₂ O ────────────────────────── ────────── *: MI Is an exchangeable cation represented by a monovalent cation

The particle size of the layered inorganic compound of the present invention is not particularly limited as long as the particle size is small enough to perform uniform dispersion. In addition, since the layered inorganic compound is generally a plate-like particle and is in a dynamic equilibrium where a plurality of particles repeatedly aggregate and cleave, it is difficult to define the average particle size itself. It is not easy to specify the range, but if it is forcibly mentioned, the value measured by a means such as light scattering method or observation with an electron microscope is 1 nm or more and 20 μm or less in a state of being dispersed in water. Is preferred. More preferably, the thickness is 10 nm or more and 2 μm or less.

It is considered that these compounds have an ion-exchanging ability to perform adsorption depending on the charge and polarity of a dye or the like, and the ion-exchanging ability is derived from the layer charge generated by the substitution of metal ions constituting the layer. I do. Therefore, the absolute value of the layer charge is 0.2 to 0.2 for the atomic group having the composition shown in Table 1.
It preferably has a value of about 1.

Those containing a transition metal ion such as iron as a substitute ion in the structure or as an impurity cause coloration, and exhibit a redox characteristic or the like to cause a side reaction, resulting in transparency and protection. -It is preferable that there is no substitution with a transition metal ion, since the stability is inferior, but the present invention is not limited to this.

These layered inorganic compounds such as clay minerals include 4
Pillars such as quaternary ammonium salts can be provided to adjust the interlayer distance, interlayer charge and polarity in advance. Among the above-mentioned layered inorganic compounds of the present invention, a more preferred one is a 2: 1 type clay mineral, and a particularly preferred one is a swellable clay mineral having ion exchange ability.

Among the swellable clay minerals, more preferred are smectites, especially synthetic smectites such as synthetic hectorite or synthetic saponite, and synthetic mica such as swellable synthetic mica (or Na type mica) represented by synthetic fluorine mica. (Natural mica is usually a non-swelling clay mineral.)
It is. The swelling action is derived from having exchangeable cations or anions, and uses a swellable layered inorganic compound to quickly adsorb a detectable substance on the surface of the layered inorganic compound called an interlayer or a card house structure. Is preferred. Clay minerals adsorb anionic, cationic and nonionic polar organic compounds, and hydrotalcite adsorbs anionic compounds. Compounds that can be adsorbed on layered inorganic compounds are described in, for example, H. Van Olphen's book `` An
Introduction to Clay Colloid Chemistry, Second Ed
ition ”(Krieger Publishment, Malabar), Chapter 11,“ In
teraction of Clays and Organic Compounds ". In the present invention, these may be used alone or in combination of two or more.

The above-mentioned layered inorganic compound of the present invention can be used without limitation to synthetic products and natural products. Preferably, synthetic products are used. Synthetic products, unlike natural products, can quantitatively handle chemically uniform and adsorbed dyes and the like.Moreover, since they do not contain colored metals such as iron between layers and have high transparency, quantitative, This is because optical handling is possible.
Here, "synthesis" means, at least in the case of smectite, those mainly produced by a hydrothermal synthesis method or a melting method. Swellable clay minerals obtained by purifying natural products are also preferably used.

Some of such layered inorganic compounds are commercially available. For example, Lucentite SWN or Lucentite SWF (trade name, manufactured by Corp Chemical Co., Ltd.)
(Synthetic hectorite) or ME (fluoromica), trade name Smecton SA (synthetic saponite) manufactured by Kunimine Industry Co., Ltd., thixopy W manufactured by Kyowa Chemical Industry Co., Ltd.
(Synthetic hectorite) or trade name Kyoword 500
(Synthetic hydrotalcite), product name Laponite (synthetic hectorite) manufactured by Lapo, natural bentonite sold by Nacalai Tesque Co., Ltd., and multi-gel (bentonite) manufactured by Toyoshun Mining Co., Ltd. .

The above-mentioned layered inorganic compounds are known to adsorb organic compounds such as amines, polyenes and various dyes. Conventionally, water treatment agents which adsorb oils, dyes, etc., It has been used as a protein remover, a decolorizing purifier by removing impurities by adsorption, and the like. Further, these layered inorganic compounds are known as materials giving a specific reaction field, for example, causing a phenomenon called metachromism, and more recently, it is also known to improve the photostability of natural dyes.

However, in the present invention, the detectable substance is adsorbed to the layered inorganic compound to form a complex, and the detectable substance is protected from the reaction system and participates in the decomposition reaction. It is thought that there is an effect such that the electronic level changes by adsorption, and an attempt was made to add these layered inorganic compounds to the reaction system. As a result, the detectable substance is quantitatively adsorbed on the layered inorganic compound, and the adsorbed detectable substance can be sufficiently stably present in the presence of excess hydrogen peroxide, ascorbic acid, or the like. Was found.

Surprisingly, even if the layered inorganic compound was added to the reactants in advance, the detection reaction was not hindered. Therefore, by the addition of the layered inorganic compound, for example, the measurement of hydrogen peroxide in a reaction system using 4-AA and a hydrogen donor can be performed more quantitatively. In this way, a layered inorganic compound such as a clay mineral is added to the reaction system for measuring the analyte, thereby stabilizing the detectable substance to be generated, and increasing the sensitivity and accuracy in the measurement of the analyte. No examples have yet been found.

The layered inorganic compound of the present invention is added to the reaction system by any method according to the reaction system to be used. The addition amount of the layered inorganic compound is determined according to the reaction system to be used,
Depending on the layered inorganic compound used, the number of adsorption sites is too small for the detectable substance, the detectable substance is not adsorbed and remains in the solution, etc., or the amount of the adsorption site is too large to adsorb the detectable substance. An amount that does not cause concentration bias is desirable.

The preferred amount of the layered inorganic compound to be added to the reaction system is determined as follows. That is, since the layered inorganic compound mainly adsorbs the dye and the like in an amount corresponding to the above-mentioned layer charge, the total number of adsorption sites for the dye and the like can be obtained for various layered inorganic compounds. If the concentration of the reagent is determined in the detection reaction system, the approximate maximum amount of the generated dye and the like can be calculated, so that the total number of adsorption sites of the layered inorganic compound does not exceed the maximum amount of the dye and the like that can be generated. A suitable amount of a layered inorganic compound can be added.

As a method for adding the layered inorganic compound, it is preferable to add the layered inorganic compound to the reaction system in the form of a dispersion in which the layered inorganic compound is dispersed. This is because an interaction such as adsorption between the layered inorganic compound and a detectable substance is likely to occur. Therefore, the term “dispersion” refers to a state in which the layered inorganic compound is dispersed in a liquid, or a state such as a sol or a gel. Good.

When a solvent is used as the dispersion medium, the type of the solvent is not particularly limited, and conventionally known solvents can be used arbitrarily. Examples thereof include water such as distilled water, alcohol such as ethanol, and acetone and the like. Ketones, ethers such as diethyl ether, esters such as ethyl acetate,
From the halogenated hydrocarbons such as chloroform and the aromatic hydrocarbons such as benzene and toluene, it is possible to select a substance suitable for the substance to be analyzed and its detection reaction system.
It is preferable to add a buffer solution dispersed in the buffer using a buffer described below.

The concentration of the solution or dispersion can be appropriately selected according to the reaction system and the like, and is not particularly limited. Further, a dispersion liquid of the layered inorganic compound may be directly dried to form a film. In that case, the film formation may be performed in the presence of another polymer or a porous substance. Since the film formed by drying in this way swells due to the moisture of the sample to cause a reaction, it can be applied to a dry chemistry test piece as it is, and is convenient for actual use.

The dispersion medium in which the layered compound is dispersed is not necessarily the same as the solvent in which the reaction takes place. The layered inorganic compound may be in a state of being dispersed in a dispersion using a solvent in which the reaction is performed as a dispersion medium, or a sol, a gel, an aggregate, an aggregate or a sintered porous through which the solvent in which the reaction is performed can penetrate. It may be a body condition. It is sufficient that the layered inorganic compound exists in a form that can be uniformly dispersed. In dry chemistry and the like, it is well known that a sample liquid containing a substance to be analyzed, such as blood, saliva, or urine, can be used as a reaction solvent.

The timing of adding the layered inorganic compound may be before or after the production of the detectable substance.
It can be added at any stage of the reaction system. The degree of adsorption is affected by the composition of the buffer (pH, ionic strength, components forming the complex, etc.). For example, smectite dispersed in pure water is the food color blue No. 1 (Brilliant Blue).
Smectite dispersed in a bis-tris buffer [prepared from bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane and hydrochloric acid] having a pH of 6.5 is difficult to adsorb FCF). Adsorb to.

The type of buffer and buffer solution used in the present invention is, for example, the above-mentioned bis-tris buffer, phosphate buffer solution, citrate buffer solution, N- (2-acetamide)
Examples include, but are not limited to, an iminodiacetic acid buffer, and it is preferable to appropriately select an iminodiacetic acid buffer according to the reaction system to be used. Further, it is preferable to select conditions suitable for the reaction system to be used, such as the pH and the concentration of the buffer.

Further, in the dispersion of the layered inorganic compound, translucent colloidal aggregation may occur. This aggregate is uniformly redispersed by stirring the liquid. In particular, when aggregation is inconvenient, the use of a phosphate-based buffer solution improves the dispersibility of the layered inorganic compound, so that the occurrence of aggregation can be suppressed.

The time of addition of the buffering agent is not particularly limited, and may be before or after the addition of the layered inorganic compound. For example, as a buffer solution in which the layered inorganic compound is dispersed, the layered inorganic compound may be used. It is preferably added to the reaction system together with the compound. Alternatively, when the layered inorganic compound is used in the form of a dried gel, aggregate, aggregate, or sintered porous body, it is preferable to dry the buffer solution to prepare a dried buffer.

In the measurement, various surfactants can be added to the reaction system. The addition of the surfactant makes it possible to uniformly disperse the specimen containing the hardly soluble substance and to make the penetration uniform and rapid by improving the wettability of the specimen.
However, surfactants disperse substances that adsorb to the interface.
Since there is an action such as dissolution, there is a possibility that the accuracy of the measurement may be impaired by competing with the adsorption of the generated detectable substance to the layered inorganic compound or by causing the generated detectable substance to be dissolved. Therefore, as the surfactant used in combination with the layered inorganic compound in the present invention, it is preferable to select a surfactant that does not hinder the adsorption between the generated detectable substance and the layered inorganic compound. It is also preferable to use a small amount of the surfactant so as not to cause such interference.

The type of the surfactant which does not hinder the adsorption is such that the molecular weight of the surfactant is not extremely large as compared with the dye to be produced, and the organic value and the inorganic value of the surfactant are represented by the following formulas. Are preferably satisfied.

[0120]

(1) (inorganic value) = (2.37 ± 0.23) × (organic value)
−186.2 ± 117.1

The above formula was obtained by examining the relationship between the adsorption inhibition effect and the inorganic and organic values for various surfactants of known structures. That is, the organic value of one carbon is 20, the inorganic value of the hydroxyl group is 100, the organic value of polyethylene oxide is 30, the inorganic value is 60,
The organic value of the nitro group is 70, the inorganic value is 70, etc., points are assigned to each functional group or atom, and these points are summed up for the functional groups and atoms constituting the compound, and the inorganic value is calculated. Taking the sum of the sum and the organic value, and plotting the inorganic value and the organic value on rectangular coordinates,
Compounds with similar properties are located in the same area of orthogonal coordinates, so it is known as an organic conceptual diagram that common properties independent of the structure of the compound appear (Yoshio Koda, "Organic conceptual diagram-basics and applications- 11th page, Sankyo Publishing (1984)).
The present inventors have studied the relationship between the adsorption inhibition effect and the inorganic value and the organic value for many surfactants having a known structure, and the type of surfactant that does not inhibit adsorption is described above in the organic conceptual diagram. It has been found that the expression is satisfied. In addition, the calculation of the inorganic value and the organic value can use the conversion data in the book concerning the above-mentioned organic conceptual diagram, but the above-mentioned formula (Equation 1) is based on the program “PC Personal Computer” made by Yoshio Honma. Conceptual diagram ”(Chemical Software Society of Japan)
It was obtained using the conversion data in the table.

The selection of the type and the amount of the surfactant which does not hinder the adsorption can be carried out, for example, in the following (a) to (d).

(A) A predetermined amount of smectite, 4-AA,
Hydrogen peroxide is added to a reaction solution containing N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline to develop color. (B) A surfactant is further added to the reaction solution having the same composition as in (a) so as to have a predetermined concentration, and hydrogen peroxide is similarly added to develop a color. (C) Smectite is separated by appropriate means such as spontaneous sedimentation, centrifugation, and filtration, and the color tone of the supernatant or filtrate is measured with a spectrophotometer or the like, and the smectite in each of (a) and (b) is measured. The amount of the dye adsorbed on the dye is compared. Alternatively, when aggregation due to adsorption is observed, the degree of aggregation and precipitation is evaluated. (D) Select the type and amount of surfactant that shows little difference between when no surfactant is added and when it is added.

Preferred types of surfactants selected by such a method include sugar alkyl ethers such as n-octyl-β-D-glucopyranoside, n-octyl-β-D-thioglucopyranoside, n-heptyl. -Β-
Sugar alkylthioethers such as D-thioglucopyranoside, n-octanoyl-N-methylglucamide, n-
Sugar amides such as nonanoyl-N-methylglucamide,
β-D-fructopyranosyl-α-D-glucopyranoside monodecanoate, β-D-fructopyranosyl-α
Sugar esters such as -D-glucopyranoside monodecanoate, N, N-bis (3-D-gluconamidopropyl)
Deoxycollamide and the like can be mentioned.

The amount of addition is not particularly limited, and the ratio of addition to the total amount of the layered inorganic compound is not particularly limited. The amount suitable for the type of the surfactant, the type of the layered inorganic compound, and the reaction system is determined. The amount may be selected, but is preferably an amount sufficient to exhibit the effect as a surfactant, for example, an amount that does not greatly exceed the critical micelle concentration of the surfactant used in an aqueous solution. For example, a 0.3% aqueous solution of n-octyl-β-D-thioglucopyranoside, a 0.3% aqueous solution of β-D-fructopyranosyl-α-D-glucopyranoside monodecanoate, N, N-bis ( A 0.3% aqueous solution of (3-D-gluconamidopropyl) deoxycoramide can be preferably used.

The method of measuring a detectable substance in the method of the present invention includes an absorption measurement method, a fluorescence measurement, a luminescence measurement,
Examples include an electrochemical measurement method, a scattered light measurement method, a reflectance measurement method, and the like, but are not limited thereto.
It can be appropriately selected according to the reaction system and the detectable substance. Preferably, an optical measuring method such as colorimetric quantification represented by light absorption analysis using an absorptiometer or the like is exemplified.
Since the layered inorganic compound used in the present invention has almost no absorption in the visible to near-infrared region, optical measurement can be performed either as a colloidal dispersion or as a gel.

When the measurement is performed, means such as the opal glass method can be used as well as the system. Alternatively, the layered inorganic compound may be separated and concentrated by centrifugation, filtration, or the like, and measured. As a specific example, the filtration of the dispersion liquid of the layered inorganic compound exemplified in the present invention is performed, for example, by a method in which an exclusion limit molecular weight is about 10,000 or a pore size is about 5 nm.
Using an ultrafiltration membrane made of polysulfone.

Preferably, an electrochemical measurement method in which an oxidation-reduction current or a membrane potential is measured at an electrode is also used. By bringing the electrode into contact with the aggregate of the layered inorganic compound and the product, the electrochemical response can be measured with high sensitivity.

[0129]

EXAMPLES The present invention will be specifically described below with reference to examples.

[0130]

Example 1 POD (peroxidase), 4-AA and N-ethyl-N- (2-hydroxy-
3-sulfopropyl) -3,5-dimethylaniline (hereinafter abbreviated as EHSDMeA), bis-tris buffer (pH 6.5) as a buffer, smectite as a layered inorganic compound, and final concentration as shown in Table 2. The sample was placed in a disposable cell (manufactured by methacrylate) having a cell length of 1 cm, and the sample was incubated at 37 ° C. for 180 seconds. As another sample, the same composition and concentration were prepared except that smectite was not added, and incubation was performed in the same manner.

After adjusting the temperature of these samples, hydrogen peroxide was added as shown in Table 2, and the absorbance was measured every 2 seconds for 1800 seconds from 20 seconds after the addition of hydrogen peroxide. Jasco V-550 (manufactured by JASCO Corporation) as an apparatus
And the measurement wavelength was 630 nm. Aggregation occurred when smectite was added, so a 0.1 mL slit was used to measure only the aggregated portion. As a background of the smectite-added system, a sample was prepared in which smectite was added at the same concentration and hydrogen peroxide was not added, and the reaction solution was similarly measured.

[0132]

[Table 2] <Table 2> ─────────────────────────── Reagent final concentration ──────────── ─────────────── POD (peroxidase) 1 U / mL 4-AA ^{* 1} 0.05 mmol / L EHSDMeA ^{* 2} 5 mmol / L Bis-Tris buffer ^{* 3} 100 mmol / L smectite ^{* 4} 0.1% hydrogen peroxide 10 mmol / L (3 mL in total) * 1) 4-aminoantiperine (4 -Amino-1,2-dihydroxy-1,5-dimethyl-2-phenyl-3H-perazol-3-one) * 2) N-ethyl-N- (2-humanperoxy-3-sulfofluoropropyl) -3,5 -Dimethylaniline * 3) Bis (2-human peroxyethyl) iminotris (human peroxymethyl) methane * 4) Lucentite SWN (synthetic smectite: ° Chemical Co., Ltd.)

The reagents used are as shown in Table 3 below.

[0134]

[Table 3] <Table 3> ───────────────────────────────── Reagent Reagent concentration Manufacturer Reagent purity ─── ────────────────────────────── POD 30 U / mL Toyobo Co., Ltd. 4-AA 1.5 mmol / L Wako Pure Chemical ( EHSDMeA 150mmol / L Dojindo Chemical Co., Ltd. Pulse-Tris 0.25mmol / L Nacalai Tesque Co., Ltd. Specially Buffered Smectite 0.3% Corp Chemical Co., Ltd. Hydrogen peroxide 300mmol / L Mitoku Chemical Co., Ltd. ) Reagent grade ─────────────────────────────────

The results are shown in FIG. As can be seen from FIG. 1, a decrease in absorbance was observed about 3 minutes after the start of the reaction in the case where no smectite was added, whereas a decrease in absorbance was not observed in the case where smectite was added. Accordingly, it was confirmed that the present detection reaction proceeds in the same manner as when smectite was not added by the addition of smectite, and the dye formed by the oxidative condensation between 4-AA and EHSDMeA was adsorbed on smectite. It was confirmed that the discoloration was suppressed without undergoing oxidative decomposition by hydrogen peroxide.

[0136]

Example 2 POD, 4-AA, EHSDMeA, bis-Tris buffer (pH 6.5), and smectite were used at a cell length of 1c so that the final concentrations were as shown in Tables 4 and 5.
5 samples (sample numbers 1 to 5) were prepared by disposable cells (made of methacrylate), and each was incubated at 37 ° C. for 180 seconds.

For these samples, after adjusting the temperature, Table 5
The reaction was started by adding the amount of hydrogen peroxide shown in (1). Further, 60 seconds after the addition of hydrogen peroxide, the amount of ascorbic acid shown in Table 5 was added, and the absorbance was measured every second for 300 seconds from 20 seconds after the addition. Jasco V-55 as measuring device
0 (manufactured by JASCO Corporation) and the measurement wavelength was 630 nm. In addition, a slit for 0.1 mL was used for measurement in order to measure only the aggregation portion.

[0138]

[Table 4] <Table 4> 試 薬 Reagent final concentration ──────────── ─────────────── POD (peroxidase) 1 U / mL 4-AA 2 mmol / L EHSDMeA 2 mmol / L Bis-Tris buffer 100 mmol / L smectite ^{* 1} (see Table 5) Hydrogen peroxide (Refer to Table 5) L (+) ascorbic acid (Refer to Table 5) (3mL in total) * 1) Lucentite SWN (synthetic smectite: manufactured by Kofu Chemical Co., Ltd.)

[0139]

[Table 5] <Table 5> ────────────────────────────── Sample smectite ascorbic acid hydrogen peroxide number (%) ( mg / dl) (μmol / l) １ 100 100 100 (284 μmol / L) ) 100 3 0.10 100 4 0.15 (284 μmol / L) 100 5 0.1000 ─────

The reagents used are as shown in Table 6 below.

[0141]

[Table 6] <Table 6> ──────────────────────────────── Reagent Reagent concentration Manufacturer Reagent purity ──── ──────────────────────────── POD 30U / mL Toyobo Co., Ltd. 4-AA 60mmol / L Wako Pure Chemical Co., Ltd. EHSDMeA 60mmol / L Dojindo Chemical Co., Ltd. Pulse-Tris 0.25mmol / L Nacalai Tesque Co., Ltd. Specially Buffered Smectite 0.3% Corp Chemical Co., Ltd. Hydrogen peroxide 3mmol / L Santoku Chemical Industry Co., Ltd. Ascorbin Acid 150mg / dl Nacalai Tesque Co., Ltd. Reagent grade ────────────────────────────────

The results are shown in Table 5 and FIG. FIG. 3 is an enlarged view of 0 to 60 seconds in FIG. Table 7 shows the absorbance (Abs) at 0, 60, and 300 seconds after the start of measurement.
Shown in Further, in order to subtract the influence of the occurrence of aggregation, the difference between Samples 3 and 4 and Sample 5 was
Abs was determined. The results are shown as data in parentheses in Table 7.

As can be seen from FIGS. 2 and 3, in the system containing no smectite, the discoloration occurred immediately after the addition of ascorbic acid. At the start of the measurement (20 seconds after the addition of ascorbic acid), only about 20% of the color development at the time of no addition was observed, and after 60 seconds from the start of the measurement (80 seconds after the addition of ascorbic acid),
It became colorless.

On the other hand, in the system to which smectite was added, at the start of the measurement, the sample was not added (ascorbic acid added 20%).
About 90% after the start of measurement (80 seconds after addition of ascorbic acid), and about 50% even after 300 seconds (320 seconds after addition of ascorbic acid). Color development was observed.

From these results, it was confirmed that the addition of smectite suppressed the reductive decomposition of the produced dye by ascorbic acid.

[0146]

[Table 7] ─────────────────────────────────── Sample 0 seconds later 60 seconds later After 300 seconds No. (ΔAbs) (ΔAbs) (ΔAbs) １ 11 .15 1.14 1.11 2 0.23 0.00 0.003 1.26 (1.16) 1.33 (1.23) 1.63 (1.49) 4 1.16 (1. 06) 1.07 (0.97) 0.84 (0.70) 5 0.10 0.10 0.14 ─────────────

[0147]

According to the present invention, by adding a layered inorganic compound such as a clay mineral to a reaction system, a detectable substance such as a dye and a layered inorganic compound are adsorbed, and the detectable substance is protected. This suppresses decomposition by excessive hydrogen peroxide, reducing ascorbic acid, and the like, and stabilizes a detectable substance. Therefore, in the case of a dye, the fading or the like can be prevented, and stable high-sensitivity and high-precision measurement can be performed.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating a change in absorbance in an experiment showing the effect of adding smectite in a POD color developing system performed in Example 1 of the present invention.

FIG. 2 is a graph showing a change in absorbance in an experiment conducted in Example 2 of the present invention showing the effect of adding smectite in a POD color developing system containing ascorbic acid.

FIG. 3 is an enlarged view of 0 to 60 seconds in FIG. 2;

[Explanation of symbols]

A: When smectite was added B: When smectite was not added C: When smectite was added and hydrogen peroxide was not added 1 ... Sample of sample number 1 2 ... Sample No. 2 sample 3 Sample No. 3 sample 4 Sample No. 5 Sample No. 5 sample

────────────────────────────────────────────────── (5) Continuation of the front page (56) References JP-A-5-99927 (JP, A) JP-A-3-15399 (JP, A) JP-A-6-222061 (JP, A) JP-A-57- 35753 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 31/00-31/22 G01N 21/75 G01N 33/48-33/98

Claims (18)

(57) [Claims] 1. Measuring the detectable substance using a reaction system including a reaction that generates a detectable substance with a quantitative correlation with the analyte based on a chemical reaction of the substance to be analyzed. The method for measuring the analyte, by the presence of a layered inorganic compound in the reaction system, characterized in that the decomposition of the detectable substance is suppressed,
How to measure a substance. 2. The method according to claim 1, wherein the layered inorganic compound is a 2: 1 type clay mineral. 3. The method according to claim 2, wherein the 2: 1 type clay mineral is a swellable layered clay mineral. 4. The method according to claim 3, wherein the swellable layered clay mineral is at least one selected from bentonite, smectite, vermiculite, and synthetic fluoromica. 5. The method according to claim 4, wherein the smectite is a synthetic smectite. 6. The method according to claim 5, wherein the synthetic smectite is at least one selected from the group consisting of hectorite and saponite. 7. The measuring method according to claim 1, wherein the detectable substance is a substance that can be detected by an optical method or an electrochemical method. 8. The method according to claim 7, wherein the detectable substance is a dye detectable by an optical method. 9. The method according to claim 8, wherein the dye is an azo compound.
The measurement method described. 10. The method according to claim 7, wherein the detectable substance is an electron mediator detectable by an electrochemical method. 11. The method according to claim 1, wherein the detectable substance is a compound selected from the group consisting of amines, imines, polyenes, aromatic compounds, heterocyclic compounds, and complexes of ions and cyclic ligands. The measurement method according to any one of claims 10 to 10. 12. The method according to claim 1, wherein the reaction for producing the detectable substance is an oxidation-reduction reaction. 13. The reaction for producing a detectable substance is an oxidation-reduction reaction, and the reaction system including the reaction for producing the detectable substance is a reaction for producing hydrogen peroxide or reacting hydrogen peroxide with an oxidizing agent. 13. The method according to claim 12, comprising an oxidation reaction. 14. A colorimetric determination of a dye produced by a redox reaction between hydrogen peroxide generated by a chemical reaction from a substance to be analyzed and an oxidizable color former with a quantitative correlation with the substance to be analyzed. 14. The method according to claim 13, wherein in the method for measuring the substance to be analyzed, a layered inorganic compound is present in the oxidation-reduction reaction system to suppress at least decomposition of the dye by hydrogen peroxide. 15. The reaction for producing the detectable substance is a redox reaction, and the reaction system including the reaction for producing the detectable substance produces nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. 13. The method according to claim 12, which comprises a reaction using nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a reducing agent. 16. The analysis by quantifying an azo compound produced with a quantitative correlation with the analyte by a coupling reaction between a diazonium salt produced by a chemical reaction from the analyte and the reagent to be coupled. 10. The measuring method according to claim 9, wherein in the method for measuring the target substance, a decomposition of the azo compound is suppressed by allowing a layered inorganic compound to be present in the coupling reaction system. 17. The measuring method according to claim 1, wherein the decomposition of the detectable substance includes the decomposition of the detectable substance by ascorbic acid. 18. The method according to claim 1, wherein the reaction system contains a buffer or a dried product thereof.