JP3359822B2 - Highly sensitive substance measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for quantifying a substance to be analyzed in a sample by measuring the detectable substance using a reaction system that produces a detectable substance such as a dye based on a chemical reaction. The present invention relates to a highly sensitive measurement method for measuring the above-mentioned detectable substance by adsorbing it on a layered inorganic compound. 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 substances to be analyzed in a sample, for example, biological components such as urine and blood in body fluids, foods, medicines, trace substances existing in the natural environment, industrial chemicals, and trace substances in waste. As a method for quantification, there is a method in which a reaction system involving the substance to be analyzed is used, and the amount of a detectable substance such as a dye which is quantitatively generated thereby is measured.

As such a method, for example, hydrogen peroxide produced by a chemical reaction of an analyte is subjected to an oxidation-reduction reaction with an oxidizable color forming agent (dye precursor) in the presence of peroxidase, There is a method of colorimetrically determining the generated dye compound.

In a clinical test, hydrogen peroxide is generated from such a substance to be analyzed, and then the amount of the hydrogen peroxide is measured by quantification of a dye compound which forms the substance, thereby obtaining the amount of the substance to be analyzed. Methods are often used because of their simplicity. However, in the conventional method,
When the amount of the analyte was very small, sufficient measurement sensitivity could not be obtained, and a highly accurate measurement result could not be obtained.

[0005] Further, an oxidation / reduction reaction is caused by an enzyme or the like between an electron mediator (mediator) and a substance to be analyzed, and an electrochemical reaction is performed when the resulting oxidized / reduced electron mediator is reduced / oxidized at an electrode. There is a method to determine the amount of the analyte from the response. However, in the conventional method,
When the amount of the analyte was very small, sufficient measurement sensitivity could not be obtained.

[0006]

SUMMARY OF THE INVENTION The present invention provides a method for quantifying a detectable substance such as a dye, which is generated with a quantitative correlation based on a chemical reaction of a substance to be analyzed.
It is an object to provide a highly sensitive measurement method for measuring a substance to be analyzed.

[0007]

Means for Solving the Problems The present inventors have found that the above problems can be solved by the presence of a layered inorganic compound in a reaction system including a reaction for producing a detectable substance, and have completed the present invention. It is.

That is, the present invention relates to a method for measuring the amount of a detectable substance by measuring the amount of the detectable substance using a reaction system including a reaction for producing a detectable substance based on a chemical reaction of a substance to be analyzed in a sample. A method for measuring a target substance, comprising a step of adding a layered inorganic compound to the reaction system and causing the layered inorganic compound to adsorb the detectable substance.

In the present invention, highly sensitive measurement can be performed by adsorbing a detectable substance to be formed on a layered inorganic compound. That is, for example, as a result of the detectable substance adsorbing to the layered inorganic compound and settling, the measurement sensitivity in optical or electrochemical detection is increased. In this case, the detectable substance may be adsorbed to the layered inorganic compound to form a colloidal aggregate and precipitate, but it is not always necessary to aggregate.

[0010] The measuring method to which the present invention is applied includes 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 analyte in the sample. The method is not particularly limited as long as the method is used to quantitatively measure the target substance by quantifying the detectable substance. Of course, the detectable substance may be the substance to be analyzed itself. Further, for example, the present invention is not limited to the case of using a reaction system in which a detectable substance is directly generated by a chemical reaction of the analyte,
The case where the chemical reaction of the analysis target substance and the formation reaction of the detectable substance are indirectly linked via another chemical reaction is also included. Among them, the method of the present invention is a measurement method using a reaction system in which a detectable substance is a dye or an electron transfer substance generated by a redox reaction, and the detectable substance generated is a dye such as an azo dye or an ionophore and an analyte. It is preferably applied to a measurement method using a reaction system that is a complex with a substance or the like.

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 chemical substances, trace substances in waste, and the like. It is used for the method.

In particular, a method of optically measuring a dye quantitatively generated by a redox reaction between hydrogen peroxide generated by an oxidase reaction from a biological component and an oxidizable color forming agent has been proposed. It is used for quantification of various components, environmental analysis, and the like. By using the method of the present invention for such an analysis / detection method, highly sensitive measurement becomes possible. Of course, the method of the present invention is not limited to this example.

[0013]

Embodiments of the present invention will be described below in detail. The measurement method of the present invention is preferably applied specifically to a measurement method using a reaction system that produces a detectable substance as described below.

The detectable substance 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; heterocyclic compounds such as xanthene, azine, and thiazine;
A complex of an ion with a cyclic ligand such as crown ether or valinomycin; and the like, which may contain a quaternary nitrogen atom, a phenolic hydroxyl group, a sulfonic acid group, or a carboxyl group in the molecule.

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, Second Edition "(Krieger Publi
shment, Malabar), Chapter 11, "Interaction of Clays an
d Organic Compounds ”. Also,
Kato Tadayoshi, Japanese Patent Publication No. 50-8462 (USP3,950,180)
Numerous adsorbable compounds have been introduced. this house,
Substances that can be detected by an optical method and an electrochemical method are included.

Substances that can be detected by an optical method include dyes. Here, the dye includes a fluorescent dye, a luminescent substance, and the like, and the reaction for generating the dye may be any reaction that generates an optically detectable substance. It may provide fluorescence, light emission, or the like.

The dye is preferably a dye compound produced from a dye precursor by an oxidation-reduction reaction, an acid-base reaction, a condensation reaction, or the like. Dyes generated in connection with the oxidation-reduction reaction include 4-amino-1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-one (4-aminoantipyrine: hereinafter, 4-aminoantipyrine). A
Abbreviated as A. ) And a hydrogen donor (N
Dyes formed by oxidative condensation of -ethyl-N- (3-sulfopropyl) -3,5-dimethylaniline or the like; oxidative condensation; ortho-tolidine, benzidines (3,3 ', 5,5'- Oxidized chromogenic dyes such as tetramethylbenzidine; 2,6-dichloro-4-
[(4-Hydroxyphenyl) imino] -2,5-cyclohexadien-1-one, a dye formed by oxidizing a leuco body; a fluorescent substance formed by oxidizing 4-hydroxyphenylacetic acid; a chemiluminescent substance; Luminescent substances and exciters thereof; formazan which is a reducing dye of tetrazolium salt; dye formed by reduction of 1,1′-dimethyl-4,4′-bipyridylium salt; and the like.

Examples of the dye formed by the acid-base reaction include compounds that develop or change color due to a change in pH, such as bromocresol green. In addition, as a dye formed by various reactions known as a color reaction or the like, 2
Azo dyes formed by coupling of diazonium salts such as -methoxy-4-morpholinobenzenediazonium salt; various known color reactions such as a reaction between aldehyde and 2,3-dimethyl-2,3-bis (hydroxyamino) butane A fluorescent substance generated by various known reactions such as a reaction between histamine and orthophthalaldehyde; a dye or a fluorescent substance generated by an enzyme substrate reacting with an enzyme substrate such as 4-methylumbelliferyl phosphate. No.

As a dye formed by a coordination bond or an ionic bond, 2- (5-bromo-2-pyridylazo) -5
And a dye that is a complex formed from-[N-propyl-N- (3-sulfopropyl) amino] aniline salt and a metal ion.

Here, the hydrogen donor means 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 the 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 analyte is quantified by indirectly quantifying hydrogen peroxide.

The ortho-tolidine and benzidine include ortho-tolidine, dianisidine, 3,3'-diaminobenzidine, 3,3 ', 5,5'-tetramethylbenzidine, and 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. Examples of the dye in which the leuco body is oxidized include 2,6-dichloro-4-[(4-hydroxyphenyl) imino] -2,5-cyclohexadiene-1-.
On, 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-phenoxazine-3-
On-10-oxide is mentioned, and as a leuco body,
4,4′-benzylidenebis (N, N-dimethylaniline), 4,4′-bis [N-ethyl-N- (3-sulfopropylamino) -2,6-dimethylphenyl] methane, 1- (ethyl Aminothiocarbonyl) -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.
4-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,
2,7-dichlorofluorescein diacetate and the like,
Fluorescent substances generated by oxidation are exemplified.

Examples of the luminescent substance such as a chemiluminescent substance include firefly luciferin, Cypridina luciferin, aequorin, lucigenin derivative, luminol derivative, acridinium ester, peroxalate 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. By quantifying the target substance, the substance to be analyzed is quantified.

In the method using a reaction system in which a fluorescent substance or a luminescent substance is generated, the substance to be analyzed is quantified by indirectly quantifying hydrogen peroxide by measuring with a fluorimeter or a luminometer. is there.

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-4,4′-biphenylene) -bis [2- (paranitrophenyl) -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 formed by reduction include 1,1
1′-dimethyl-4,4′-bipyridylium salt, 1,
Reductants such as 1'-dibenzyl-4,4'-bipyridylium salt are exemplified. Further, 7-hydroxy-3H-phenoxazin-3-one-10-oxide and the like are reduced to generate a fluorescent substance. As the fluorescent substance, 7-hydroxy-3H-phenoxazin-3-one-10-oxide is used. , 5-cyano-2,3-bis (4-methylphenyl)
And a fluorescent substance formed by reduction of -2H-tetrazolium salt, 2,3-bis (4-cyanophenyl) -5-cyano-2H-tetrazolium salt, and the like.

For example, in a method using a reaction system in which a tetrazolium salt or a leuco body 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 quantified 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.

Examples of the compound which develops or changes 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 quantified.

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. Quantify the analyte.

The azo dyes formed by the coupling of diazonium salts include azo dyes formed by the 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 above-mentioned 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 material (in the above example, indoxyl, urobilinogen, Nitrate). The reaction for generating an azo dye is not limited to the above example, and is preferably applied to reactions for generating various known azo dyes.

The 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 quantified. 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 carried out using the following reagents. . 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 with a fluorometer or the like, and the analyte (the guanidino compound, histamine, spermidine, or alpha keto acid in the above example), which is the starting substance of the reaction, is quantified. 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 generate a dye or a fluorescent substance includes N-tosyl-L-phenylalanine-2-amidoacridone which is a substrate of chymotrypsin and L-alanine- which is a substrate of aminopeptidase. 2-amidoacridone, 7-acetoxy-N-methylquinolinium salt for measuring esterase, 7-acetoxy-3H-phenoxazin-3-one which is a substrate of esterase, 4-methyl which is a substrate of phosphatase Examples include umbelliferyl phosphate, but are not limited thereto.

For example, in a method utilizing a reaction in which an enzyme substrate is decomposed by an enzyme, a dye or a fluorescent substance to be produced is measured by an absorptiometer or a fluorometer, and the enzyme is indirectly quantified. The substance is quantified. 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-1
8-crown-6, cyclic polyamines, calix [4] arene, 3- [N, N-bis (carboxymethyl) aminomethyl] -1,2-dihydroxyanthraquinone, 5 ′, 5 ″ -dibromopyrogallol sulfone phthalate Rain, 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.

In particular, as a compound capable of forming a fluorescent complex with calcium ions and 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-oxazoyl) -5-benzofuranyloxy] -2- (2 -Amino-5-methylphenoxy) ethane-N, N, N ', N'-tetraacetic acid salt, 1- [6-amino-2- (5-carboxy-2-oxazazoyl) -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'-pentaacetoxymethyl tetraacetate Ester, 3,3′-bis [N, N-bis (carboxymethyl) aminomethyl] fluorescein, 8-
[N, N-bis (carboxymethyl) aminomethyl]-
4-methylumbelliferone and the like.

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

In the method utilizing the reaction in which the complex is formed, 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 quantified.

The substances which can be detected optically have been described so far. Next, the substances 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 then directly accepts / donates electrons from / to the substance to be analyzed, and is a reduced form of the electron mediator. The analyte is quantified from the electrochemical response when the oxidized / oxidized product is oxidized / reduced at the electrode.
Also, the electron mediator and the analyte need not directly exchange electrons, and the electron mediator oxidizes / reduces the analyte with an enzyme or the like, and in this case, from or to the analyte. Alternatively, a chemical substance that indirectly accepts / donates electrons may be used. The analyte is quantified from the electrochemical response when the oxidant / reducer of the electron mediator, which has a quantitative relationship with the analyte, is reduced / oxidized at the electrode.

Specifically, 1,1 ′ is used as an electron mediator.
-Dimethyl-4,4'-bipyridylium salt, 1,1'-
Dibenzyl-4,4′-bipyridylium salt, 1,4-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, hexacyano Iron (II) salt, 7-hydroxy-3H-phenoxazin-3-one 10-oxide, 3,7-diamino-5-phenylphenazinium salt, 3- (diethylamino) -7-amino-5-phenylphena Dinium 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 to quantify the analyte. It is. For example, measuring the electrochemical response when oxidized / reduced on an electrode as an electron donor / acceptor, such as ascorbic acid or hydrogen peroxide,
The analyte can be indirectly quantified from the response result.

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

Specifically, tetrakis [3,5-bis (trifluoromethyl) phenyl] borate, tetraphenylphosphonium, valinomycin, cyclo (N ′, N′-) are ionophores which form complexes with cations.
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 method in which a porous polymer layer or the like is provided on the surface of an electrode, and an ionophore is impregnated into the polymer layer, and is bonded to only specific ions in a 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 quantify the 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 quantify 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 quantified by electrochemically measuring the membrane potential generated according to the concentration of the specific ion as the substance to be analyzed.

The measurement method of the present invention is preferably applied to a method using a reaction system that produces such a detectable substance, and preferably includes the following methods.

(A) This is 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, for example, a reaction is carried out from a substance to be analyzed via an oxidase reaction system. A hydrogen peroxide is produced in the presence of a peroxidase, and is subjected to an oxidation-reduction reaction with an oxidizable colorant (dye precursor) in the presence of a peroxidase, and the amount of a dye compound produced by this reaction is measured.

(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 generated from a substance to be analyzed via a dehydrogenase reaction system. A method in which this is caused to act on a dye precursor in the presence of an electron transfer system to reduce the dye precursor, and the amount of the dye compound generated thereby is measured.

(C) Aromatic primary amine is reacted with nitrous acid under acidic conditions to generate a diazonium salt, the generated diazonium salt is reacted with the reagent to be coupled, and the generated 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 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.

Of course, the detectable substance may be the substance to be analyzed itself, for example, by measuring the electrochemical response when glucose dissolved in water is oxidized on the electrode surface, Some substances quantify substances.

The analytes that can be measured in such a reaction system include biological components such as urine and blood in body fluids, foods, medicines, trace substances present in the natural environment, industrial chemicals, and traces in waste. Substances and the like.

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 that occupies most of clay (fine earth-like inorganic particulate matter that 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. As the layered inorganic compound used in the present invention, a 2: 1 type mineral is preferable.

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 mineralically or chemically pure compounds.Since actual layered inorganic compounds may contain impurities such as sodium silicate, the chemical formulas are determined by elemental analysis or the like. May not always agree with these formulas, as described in the literature (D. W, Thompson,
JT Butterworth, J. Colloid Interf. Sci., 151, 2
36-243 (1992)).

[0074]

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. )

[0075]

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. )

[0076]

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. )

[0077]

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. )

[0078]

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.)

[0079]

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.)

[0080]

Embedded image _{Mg 6 Al 2 (OH) 16} X x ··· (7) (X in Formula 7 is _{halogen, N0 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. )

[0081]

_{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.)

[0082]

_{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 montmorillonite (formula 1), bentonite and beidellite (formula 2) which are natural products containing 40 to 80% of montmorillonite depending on the ion species in the tetrahedral and octahedral lattices isomorphously substituted. Dioctahedral type; 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
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.

[0087]

[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 average particle size of the layered inorganic compound of the present invention is not particularly limited as long as it is small enough to allow 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. Also, by having ion exchange ability,
It is considered that adsorption is performed according to the charge or polarity of the dye or the like, and the ion exchange ability is derived from the layer charge generated by the replacement of metal ions constituting the layer. 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.

Further, those containing a transition metal ion such as iron as a substitution ion in the structure or as an impurity cause coloring, and exhibit a redox characteristic or the like to cause a side reaction. Although it is inferior, it is preferable that there is no substitution with a transition metal ion, 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 bentonite, smectite, vermiculite and synthetic fluoromica, particularly preferably synthetic smectite such as synthetic hectorite or synthetic saponite, or swellable clay mica represented by synthetic fluoromica. Synthetic mica (natural mica is usually a non-swelling clay mineral) such as synthetic mica (or Na-type mica). The swelling action is derived from having exchangeable cations or anions, and uses a swellable layered inorganic compound to quickly adsorb a detectable substance to the surface of an interlayer or a clay mineral called a card house structure. Is preferred. Clay minerals include anionic, cationic and nonionic polar organic compounds.
Hydrotalcite adsorbs anionic compounds. Compounds that can be adsorbed on the layered inorganic compound include, for example, H. Van Ol
Phen's book `` An Introduction to Clay Colloid Chemi
stry, Second Edition "(Krieger Publishment, Malab
ar) Chapter 11 “Interaction of Clays and Organic Com
pounds ". 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 being limited to a synthetic product and a natural product, but a synthetic product is preferably used. Unlike natural products, synthetic products can quantitatively handle detectable substances that are chemically uniform and adsorbed.Furthermore, they do not contain colored metals such as iron between layers and have high transparency. This is because it is possible to perform optical and optical handling. 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. In addition, these layered inorganic compounds are known as materials that give a specific reaction field, such as causing a phenomenon called metachromism, and more recently, it is also known to improve the light stability of natural dyes, In the present invention, it has been found that the detection sensitivity can be increased by adsorbing a detectable substance to the layered inorganic compound. There has not yet been found any example in which such a layered inorganic compound such as a clay mineral is used for measuring a substance to increase the sensitivity.

Even more surprisingly, the detection reaction was not hindered even when the layered inorganic compound was previously added to the reaction system. 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.

The method of adding the layered inorganic compound in the method of the present invention depends on the reaction system to be used, but it is preferable to disperse the compound in a solvent and add it to the reaction system in the form of a dispersion.
The solvent is not particularly limited, and conventionally known ones can be arbitrarily used.Examples include water such as distilled water, alcohols such as ethanol, ketones such as acetone, ethers such as diethyl ether, and ethyl acetate. Esters, halogenated hydrocarbons such as chloroform,
From aromatic hydrocarbons such as benzene and toluene,
A substance suitable for the substance to be analyzed and its detection reaction system can be selected. It is preferable to add a buffer solution dispersed in the buffer using a buffer described below. In addition, the use of a sample liquid such as blood, saliva, urine or the like containing a substance to be analyzed as a reaction solvent is known as dry chemistry.

The amount of the layered inorganic compound to be added is determined according to the reaction system to be used, and although it depends on the layered inorganic compound used, the amount of the adsorption site is too small for the detectable substance and the detectable substance is adsorbed. It is desirable that the amount is such that it does not remain in a solution or the like without being detected or that the concentration of a detectable substance is not biased due to too many adsorption sites.

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. Once the concentration of the reagent is determined in the detection reaction system, the approximate maximum amount of the dye and the like to be generated can be calculated, and the total amount of adsorption sites of the layered inorganic compound can be calculated so that the maximum amount of the dye and the like that can be generated does not exceed the maximum amount. Can be added.

The timing of adding the layered inorganic compound is not particularly limited, and may be before or after the reaction for producing the detectable substance. It is preferable to add in advance and to disperse in the reaction system.

Here, what is dispersed in the reaction system is as follows.
This is because an interaction such as adsorption between the layered inorganic compound and a reaction starting material, a reaction intermediate, or a reaction product, which is involved in a production reaction of a detectable substance, is likely to occur. This is also because a state in which there is no variation in concentration that is uniformly dispersed is suitable for detection. Therefore, the term “dispersion” as used herein refers to a state in which the layered inorganic compound is dispersed in a liquid, a sol, a gel, or the like, as long as the above-described interaction is likely to occur and is in a state suitable for detection. Good.

The dispersion medium in which the layered inorganic compound is dispersed is not necessarily the same as the reaction solvent in which the reaction is performed. The layered inorganic compound may be in a state of being dispersed in a dispersion liquid containing the reaction solvent as a dispersion medium, or in a state of a sol, a gel, an aggregate, an aggregate or a sintered porous body through which the reaction solvent can penetrate. There is no particular limitation as long as the layered inorganic compound is present in a form capable of being uniformly dispersed.

The layered inorganic compound having an exchangeable cation or an exchangeable anion, when dispersed in water by stirring, irradiation of ultrasonic waves, or the like, becomes an almost uniform dispersion at an appropriate concentration. However, due to the addition of an electrolyte, the addition of an organic compound, or a long-term standing or temperature change, the particles of the layered inorganic compound aggregate or coagulate,
Occasionally gelation or precipitation occurs. These agglomerations are generally gently interacting particles and can be easily redispersed by stirring.

The dispersion, aggregation and redispersion of such a layered inorganic compound are described, for example, in H. Van Olphen's book “An Introduction to Clay Colloid Chemistry, Secon.
d Edition ”(Krieger Publishment, Malabar), Chapter 3,“ The Theory of Stability of Hydrophobic Sols. ”
Chapter 4, "Successes of the Theory of Stability-Furth
er Theories and Refinement ", Chapter 7," Electric Doubl
e-Layer Structure and Stability of Clay Suspension
s "and Chapter 8" Peptization of Clay Suspensions ".

The degree of adsorption is affected by the composition of the buffer (pH, ionic strength, components forming a complex, etc.). For example, smectite dispersed in pure water hardly adsorbs food color blue No. 1 (Brilliant Blue FCF),
Smectite dispersed in H6.5 bis-tris buffer [prepared from bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane and hydrochloric acid] rapidly adsorbs this dye.

The type of buffer and buffer solution used in the method of the present invention is, for example, the above-mentioned bis-tris buffer, phosphate buffer solution, citrate buffer solution, N- (2-acetamido) iminodiacetic acid buffer. Examples thereof include, but are not limited to, liquids and the like, and it is preferable to appropriately select them 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.

The timing of addition of the buffering agent is not particularly limited, and may be before or after the addition of the layered inorganic compound. However, the buffer solution in which the layered inorganic compound is dispersed is reacted with the layered inorganic compound together with the layered inorganic compound. It is preferably added to the system.

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 inorganic value of the surfactant are represented by the following formulas. Are preferably satisfied.

[0109]

(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 that 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 the 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.

In a preferred embodiment of the present invention, a layered inorganic compound is previously added to the reaction system and dispersed. At this time, translucent colloidal aggregation may occur, but in the present invention, it is not always necessary to cause aggregation. This aggregation can be considered as a complex in which a layered inorganic compound and a detectable substance are adsorbed. This aggregation 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.

It is also possible to further increase the measurement sensitivity by adsorbing the detectable substance finally used for detection on the layered inorganic compound to precipitate the substance, and separating and concentrating the detectable substance from the reaction system. is there. Here, the method for separating the layered inorganic compound to which the detectable substance is adsorbed is not particularly limited, and examples thereof include a method by spontaneous sedimentation, centrifugation, filtration, chromatography, electrophoresis, and solvent evaporation. As a specific example, filtration of the dispersion liquid of the layered inorganic compound exemplified in the present invention is performed using, for example, an ultrafiltration membrane made of polysulfone having an exclusion limit molecular weight of about 10,000 or a pore size of about 5 nm. be able to.

In the present invention, the amount of a detectable substance adsorbed on the layered inorganic compound is measured. Examples of the measurement method include an absorption measurement method, a fluorescence measurement, a luminescence measurement, an electrochemical measurement method, a scattered light measurement method, and a reflectance measurement method. 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, an optical measurement can be performed even in a colloidal dispersion or a gel. When the measurement is carried out in the form of a solution, not only the system but also a means such as the opal glass method can be selected.

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 an electrode into contact with a layered inorganic compound to which a detectable substance is adsorbed, an electrochemical response can be measured with high sensitivity.

[0119]

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

[0120]

Example 1 POD (peroxidase), 4-AA and N-ethyl-N- (2-hydroxy-
3-sulfopropyl) -3,5-dimethoxyaniline (hereinafter abbreviated as EHSDA), bis-tris buffer (pH 6.5) as a buffer, smectite as a layered inorganic compound, and final concentration as shown in Table 2. So that the final concentration of hydrogen peroxide is 120 μmol / l
And reacted to obtain a reaction solution. The absorption spectrum of the agglomerated portion of the obtained reaction solution was 450 to 75
It was measured between 0 nm.

Further, POD, 4-AA, EHSDA, and Bis-Tris buffer were taken so that the final concentration was as shown in Table 3, and hydrogen peroxide was added thereto at a final concentration of 120 μmo.
The reaction was performed by adding 1 / l to obtain a reaction solution. The absorption spectrum of the obtained reaction solution was
It was measured between 0 and 750 nm.

The absorbance was measured using Jasco V-55.
0 (manufactured by JASCO Corporation) at intervals of 0.5 nm. The scanning speed is 200 nm / min and the bandwidth is 1.
0 nm. A 1 cm cell length disposable cell (manufactured by polymethyl methacrylate) was used as the cell, and a 0.1 ml slit was used to measure only the aggregated portion. FIG. 1 shows the results of the measurement.

[0123]

[Table 2] <Table 2> ─────────────────────────── Reagent final concentration ─────────── ＰＯ POD (peroxidase) 1 U / ml 4-AA ^{* 1} 2 mmol / l EHSDA ^{* 2} 2 mmol / l Bis-Tris buffer ^{* 3} 100 mmol / l smectite ^{* 40} 1% (3 ml in total volume) * 1) 4-aminoantihydrin (4-amino-1,2- (Hydroxy-1,5-dimethyl-2-phenyl-3H-perazol-3-one) * 2) N-ethyl-N- (2-humanoxy-3-sulfofluorohyl) -3,5-dimethoxyaniline * 3) Hice (2-human peroxyethyl) imino tris (human peroxymethyl) methane * 4) Lucentite SWN (synthetic smectite: manufactured by Kofu Chemical Co., Ltd.)

[0124]

[Table 3] <Table 3> ─────────────────────────── Reagent final concentration ─────────── POD (peroxidase) 1 U / ml 4-AA 2 mmol / l EHSDA 2 mmol / l Bis-Tris buffer 100 mmol / l Smectite 0% (total volume 3 ml) ────────────────────────

The reagents used are as shown in Table 4 below.

[0126]

[Table 4] <Table 4> ─────────────────────────────── Reagent Reagent concentration Manufacturer Reagent purity ───── ＰＯ POD (III) 30 U / mL Toyobo Co., Ltd. 4-AA 60 mmol / L Wako Pure Chemical Co., Ltd. Reagent Special grade EHSDA 60 mmol / L SIGMA gas-tris 0.25 mmol / L Nacalai Tesque Co., Ltd. Specially Buffered Smectite 0.3% Corp Chemical Co., Ltd. Hydrogen peroxide Santoku Chemical Industry Co., Ltd. ────────────────────────

As can be seen from the results shown in FIG. 1, it was confirmed that the reaction proceeded under the condition that the dye was adsorbed on smectite in the same manner as under the condition where no dye was added. The absorption maximum when smectite was not added was about 593 nm, and the absorption maximum when smectite was added was about 578 nm.

[0128]

Example 2 POD, 4-AA, EHSDA, and bis-Tris buffer (pH 6.5) were added to the final concentrations shown in Table 3 above.
Take a 1 cm cell length quartz cell so that
Incubation was performed at 7 ° C. for 3 minutes. After the temperature was adjusted, hydrogen peroxide having a concentration shown in Table 5 was added to start the reaction, and the absorbance was measured 3 minutes after the start of the reaction. After 3 minutes, the reaction had reached its end point.

The equipment used was Jasco V-550 (manufactured by JASCO Corporation), and the measurement wavelength was 593 nm (wavelength near the absorption maximum). From this result, a calibration curve of hydrogen peroxide when smectite was not added could be obtained.

[0130]

[Table 5]

[0131]

Example 3 [Experimental method] POD, 4-AA, EHSDA, bis-tris buffer (pH 6.5) and synthetic smectite were mixed with a cell length of 1c so that the final concentration was as shown in Table 2 above.
m of a disposable cell (manufactured by polymethyl methacrylate), and the temperature was adjusted at 37 ° C. for 180 seconds. After the temperature adjustment, hydrogen peroxide was added so that the final concentration was as shown in Table 6, and the absorbance was measured every 2 seconds for 1800 seconds from 10 seconds after the addition of hydrogen peroxide. Jasco V- as measuring device
550 (manufactured by JASCO Corporation) using a measurement wavelength of 577 nm
(Wavelength near the maximum absorption wavelength). In order to measure only the aggregation portion, a slit for 0.1 ml was used. Using the measurement result of the hydrogen peroxide concentration of 0 μmol / l as a blank, the difference in absorbance (ΔAbs) 1800 seconds after the start of the measurement was determined, and the calibration curve of hydrogen peroxide when smectite was added was determined.

[Results] The results are shown in FIG. 2 together with the results of Example 2 when no smectite was added. FIG. 3 shows a calibration curve obtained by taking the logarithm of the vertical axis and the horizontal axis for the results of FIG.
Tables 5 and 6, and FIGS. 2 and 3 show that there is a correlation between the absorbance of the dye adsorbed and the hydrogen peroxide.
[When smectite is added, the concentration of hydrogen peroxide is 0 to 200.
A calibration curve of r = 0.999 was obtained between μmol / l. r: Correlation coefficient] From FIGS. 2 and 3, the sensitivity was improved to about 3 μmol / l when the smectite was not added, whereas the minimum detection limit was about 6 μmol / l when smectite was not added. In addition, the slope of the calibration curve was approximately doubled.

[0133]

[Table 6]

[0134]

Example 4 [Experimental method] POD, 4-AA, EHSDA, bis-tris buffer (pH 6.5) and synthetic smectite were mixed with a cell length of 1c so that the final concentration was as shown in Table 2 above.
m of a disposable cell (manufactured by polymethyl methacrylate), and the temperature was adjusted at 37 ° C. for 180 seconds. After the temperature adjustment, hydrogen peroxide was added so that the final concentration was 100 μmol / l, and 60 seconds every 2 seconds from 20 seconds after the addition of hydrogen peroxide.
The absorbance was measured for 0 seconds. Jasc as measuring device
Using oV-550 (manufactured by JASCO Corporation), the measurement wavelength was 57
7 nm (wavelength near the maximum absorption wavelength). In order to measure only the aggregation portion, a slit for 0.1 ml was used. In addition, the measurement result of the hydrogen peroxide concentration of 0 μmol / l was measured as a blank.

Further, POD, 4-AA, EHSDA, and bis-Tris buffer (pH 6.5) were added to a disposable cell (polymethyl methacrylate) having a cell length of 1 cm so that the final concentration was as shown in Table 3 above. Take 1 at 37 ° C
The temperature was adjusted for 80 seconds. After the temperature adjustment, hydrogen peroxide was added to a final concentration of 100 μmol / l, and the absorbance was measured every 2 seconds for 600 seconds from 20 seconds after the addition of the hydrogen peroxide. Jasco V-550 (manufactured by JASCO Corporation) was used as a measurement device, and the measurement wavelength was 593 nm (wavelength near the maximum absorption wavelength). A slit for 0.1 ml was used.

[Results] The results are shown in FIG. FIG. 4 shows that the addition of smectite has a sensitizing effect. It was also confirmed that the color reaction reached the end point about 30 seconds after the addition of hydrogen peroxide. Furthermore, even if smectite was added after color development without adding smectite, dye adsorption and aggregation were observed.

[0137]

Example 5 As a tetrazolium salt, 3,3 ′-(3,3
3'-dimethoxy-4,4'-biphenylene) -bis [2- (paranitrophenyl) -5-phenyl-2H-
Tetrazolium chloride (hereinafter abbreviated as "tetrazolium salt"), phosphate buffer (adjusted to pH 8.5 by mixing disodium hydrogen phosphate and sodium dihydrogen phosphate) as a buffer, L-ascorbic acid , And smectite (trade name Lucentite SWN:
(Synthetic smectite, manufactured by Corp Chemical Co., Ltd.) was reacted in a disposable tube such that the final concentration was as shown in Table 7, and the color was developed. Dilute the obtained coloring solution 10 times,
The absorption spectrum was measured between 400 and 800 nm.

For comparison, a tetrazolium salt, a phosphate buffer and L-ascorbic acid were placed in a disposable tube in the same manner as described above except that smectite was not added so that the final concentration was as shown in Table 7, and the color was developed. . The absorption spectrum of the obtained coloring solution was measured between 400 and 800 nm.

The absorbance was measured using a spectrophotometer (Jasco V-550) manufactured by JASCO Corporation. A disposable cell (made of polymethyl methacrylate) having a cell length of 1 cm was used for the cell. FIG. 5 shows the results of the measurement.

[0140]

[Table 7] <Table 7> ─────────────────────────── Reagent final concentration ───────────テ ト ラ tetrazolium salt 1 mmol / l phosphate buffer (pH 8.5) 100 mmol / l L (+)-ascorbic acid 333 μmol / l smectite 0.1% or 0 % (Total amount 3ml) ───────────────────────────

In the system without smectite, blue color was formed and the absorption maximum wavelength was about 633 nm. The smectite-added system develops a purple-red color and has an absorption maximum wavelength of about 535.
nm. Thus, it was confirmed that the reaction proceeded under the condition in which smectite was added in the same manner as in the condition without smectite addition. In addition, since the absorption maximum is shifted to the shorter wavelength side, when the same color reaction system as in this example is used, the absorbance is measured at the wavelengths near the respective absorption maximum wavelengths of the smectite-added system and the non-added system. 63
It can be seen that it is better to perform at 3 nm (smectite-free system) and 535 nm (smectite-added system).

[0142]

Example 6 The same tetrazolium salt, phosphate buffer (pH 8.5) and smectite as used in Example 5 were placed in a disposable tube such that the final concentration was as shown in Table 8, and the solution was added at 30 ° C. for 3 hours. Incubated for minutes. After the incubation, L-ascorbic acid is added at 0-333 μmo.
1 / l in the range shown in Table 9
The reaction was performed at 30 ° C. for 30 minutes, and the absorbance was measured (measurement wavelength: 535 nm). No ascorbic acid added (0 μmo
1 / l) as a blank, a calibration curve was prepared from the measurement results.

For comparison, a tetrazolium salt and a phosphate buffer were placed in a disposable tube in the same manner as above except that smectite was not added, and the mixture was incubated at 30 ° C. for 3 minutes. After incubation, ascorbic acid (0-333 μmol /
l) was added to each of the concentrations shown in Table 10 at 30 ° C.
The reaction was carried out for 30 minutes and the absorbance was measured (measurement wavelength:
633 nm). A calibration curve was prepared from the measurement results using a sample without ascorbic acid as a blank.

FIG. 6 shows the obtained calibration curve. In the smectite-free system, the final concentration of ascorbic acid was 41.7 to 33.
R (correlation function) = 0.99 in the range of 3.3 μmol / l
72 calibration curves were obtained. In the smectite-added system, a calibration curve of r = 0.9985 was obtained in the range of ascorbic acid final concentration of 5.2 to 133.3 μmol / l. The slope of the obtained calibration curve is about 2.5 times that of the smectite-added system as compared to the smectite-free system, and it can be seen that the sensitizing effect can be obtained by adding smectite.

The absorbance was measured using a spectrophotometer (Jasco V-550) manufactured by JASCO Corporation. A disposable cell (made of polymethyl methacrylate) having a cell length of 1 cm was used for the cell.

[0146]

[Table 8] <Table 8> ─────────────────────────── Final concentration of reagent ─────────── ──────────────── tetrazolium salt 800 μmol / l phosphate buffer (pH 8.5) 100 mmol / l L (+)-ascorbic acid 0-333.3 μmol / l smectite 0.1 % Or 0% (3 ml in total) ───────────────────────────

[0147]

[Table 9]

[0148]

[Table 10]

[0149]

Example 7 Hydrochloric acid, smectite (Lucentite SWN, manufactured by Corp Chemical), 2,4-dichloroaniline, and sodium nitrite were taken in this order so that the final concentration of each reagent was as shown in Table 11, and mixed. Thereafter, a Tsuda reagent (N, N-diethyl-N'-1-naphthylnaphthylethylenediamineoxalate) was added and reacted to form an azo-based dye and develop color. The absorption spectrum of this was measured between 400 and 800 nm. In addition, sodium nitrite has four concentrations (0, 8, 16, and 33 μmo).
1 / l). FIG. 7 shows the results.

For comparison, hydrochloric acid, 2,4-dichloroaniline, and sodium nitrite were added in the same manner as described above except that smectite was not added, so that the final concentrations were as shown in Table 11, and then Tsuda The reagent was added to develop color. The absorption spectrum of this was measured between 400 and 800 nm. In addition, sodium nitrite has four concentrations (0, 8,
16, and 33 μmol / l). Fig. 8 shows the results.
Shown in For the smectite-added system and the smectite-free system, each had a sodium nitrite concentration of 33 μmol.
FIG. 9 shows the absorption spectra for the case of / l. still,
The absorbance was measured using a spectrophotometer (Jasco, manufactured by JASCO Corporation).
V-550) was used. A disposable cell (made of polymethyl methacrylate) having a cell length of 1 cm was used for the cell.

[0151]

[Table 11]

The smectite-free system developed a reddish purple color and had an absorption maximum wavelength of about 540 nm. The smectite-added system develops purple color and has a maximum absorption wavelength of about 555n.
m. Thus, it was confirmed that the reaction proceeded under the condition in which smectite was added in the same manner as in the condition without smectite addition. In addition, since the absorption maximum is shifted to the longer wavelength side, when using the same color reaction system as in this example, the absorbance is measured at a wavelength near the absorption maximum wavelength of each of the smectite-added system and the non-added system. 540
It can be seen that it is better to perform the measurement at nm (smectite-free system) and 555 nm (smectite-added system).

[0153]

Example 8 The same hydrochloric acid as used in Example 7, 2,4
-Dichloroaniline, sodium nitrite and Tsuda reagent were placed in a disposable cell (manufactured by polymethyl methacrylate) so that the final concentration was as shown in Table 12, and the solution was placed at 30 ° C for 10 hours.
After the reaction was carried out for a minute to sufficiently develop the color, smectite was added to form aggregates and sedimentation, and after 30 seconds from the addition of smectite, the absorbance of the aggregates was measured every second for 20 minutes (measuring wavelength: 555 nm). A 0.1 ml slit was used to measure the absorbance of only aggregation. In addition, sodium nitrite was added to each concentration shown in Table 13 in the range of 0 to 50 μmol / l. Sodium nitrite final concentration 0μmo
Start measurement 2 with the result of 1 / l sample as blank
The difference (ΔAbs) from the absorbance at 0 minutes was determined, and a calibration curve was created.

In addition, as a smectite-free system, hydrochloric acid, 2, 2 were used in the same manner as described above so that the final concentration was as shown in Table 12.
After taking 4-dichloroaniline and sodium nitrite into a disposable cell and incubating at 30 ° C. for 3 minutes,
The Tsuda reagent was added, and the absorbance was measured every 10 seconds for 10 minutes 10 seconds after the addition (measurement wavelength: 540 nm). In addition, sodium nitrite was added to each concentration shown in Table 14 in the range of 0 to 50 μmol / l. The absorbance (Abs) 10 minutes after the start of the measurement was determined, and a calibration curve was created. The result of the sample having a final concentration of sodium nitrite of 0 μmol / l was measured as a blank. The reaction system used in this experiment was 10
You have reached the end point in minutes.

FIG. 10 shows the calibration curve obtained. In the smectite-free system, the final concentration of sodium nitrite was 1.6 to 5
R (correlation function) = 0.99 in the range of 0.0 μmol / l
91 calibration curves were obtained. In the smectite-added system, a calibration curve of r = 0.9940 was obtained in the final sodium nitrite concentration range of 0.4 to 25.0 μmol / l. The slope of the obtained calibration curve is about 2.5 times that of the smectite-added system as compared to the smectite-free system, and it can be seen that the sensitizing effect can be obtained by adding smectite.

The absorbance was measured using a spectrophotometer (Jasco V-550) manufactured by JASCO Corporation. A disposable cell (made of polymethyl methacrylate) having a cell length of 1 cm was used for the cell.

[0157]

[Table 12]

[0158]

[Table 13]

[0159]

[Table 14]

[0160]

According to the present invention, a highly sensitive measurement is made possible by adding a layered inorganic compound such as a clay mineral to a reaction system and adsorbing a detectable substance and then measuring the substance. be able to.

The measuring method of the present invention is applicable to the detection and quantification of biological components such as urine and blood in body fluids, foods, medicines, trace substances existing in the natural environment, industrial chemical substances, trace substances in waste, and the like. Can be used.

[Brief description of the drawings]

FIG. 1 is an absorption spectrum diagram measured in Example 1.

FIG. 2 is a diagram showing a calibration curve of hydrogen peroxide obtained in Example 2 and Example 3.

FIG. 3 is a diagram showing a calibration curve obtained by taking a logarithm of a vertical axis and a horizontal axis of the calibration curve of FIG. 2;

FIG. 4 is a graph showing a change over time in absorbance after addition of hydrogen peroxide in Example 4.

FIG. 5 is an absorption spectrum diagram measured in Example 5.

FIG. 6 is a diagram showing a calibration curve of ascorbic acid concentration obtained in Example 6.

FIG. 7 is an absorption spectrum diagram (smectite added system) measured in Example 7.

FIG. 8 is an absorption spectrum diagram (smectite-free system) measured in Example 7.

FIG. 9: Sodium nitrite concentration 3 measured in Example 7
FIG. 4 is an absorption spectrum diagram at 3 μmol / l (smectite added system and non-smectite added system).

FIG. 10 is a diagram showing a calibration curve of sodium nitrite concentration obtained in Example 8.

[Explanation of symbols]

1 ... when smectite is added 2 ... when smectite is not added 3 ... when smectite is added and hydrogen peroxide is not added 4 ... when nitrous acid concentration is 33 μmol / l 5 ... when the nitrite concentration is 16 μmol / l 6 ... when the nitrite concentration is 8 μmol / l 7 ... when the nitrite concentration is 0 μmol / l

──────────────────────────────────────────────────続 き Continuation of front page (51) Int.Cl. ⁷ Identification symbol FI G01N 30/00 G01N 27/46 311K 336B (56) References JP-A-5-99927 (JP, A) JP-A-57-35753 (JP, A) JP-A-6-222061 (JP, A) JP-A-3-15399 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 31/00-31 / 22 G01N 21/75 G01N 33/48-33/98

Claims (16)

(57) [Claims] 1. The method according to claim 1, wherein the amount of the detectable substance is measured by using an aqueous reaction system including a reaction for producing a detectable substance based on a chemical reaction of the analyte in the sample. In the measuring method, the layered inorganic compound is dispersed in the aqueous reaction system, the detectable substance is adsorbed to the layered inorganic compound, and the detectable substance is
Characterized in that it comprises a step of Ru increases the detection sensitivity, the measuring method of the 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 the group consisting of 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 a compound that can be detected by an electrochemical method selected from the group consisting of an electron transfer substance, an electron donor, and an electron acceptor. 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. 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. 15. The method according to claim 1, wherein the reaction system contains a buffer or a dried product thereof. 16. The method according to claim 1, wherein the layered inorganic compound in the reaction system is added to the reaction system in the form of a dispersion of the layered inorganic compound. .