JPS63255659A - Color reagent and multilayer analytical element containing the same - Google Patents

Abstract

PURPOSE:To obtain a high sensitivity reagent forming 1mol. of a dye from 1mol. of hydrogen peroxide by the catalytic action or the participation of peroxidase and a multilayer analytical element containing the same, by containing an imidazole derivative represented by a specific formula. CONSTITUTION:An imidazole derivative represented by formula (wherein R<1> is an alkyl group, an alkoxy group or the like, R<2> and R<3> are respectively a phenyl group, a naphthyl group or the like, R<4> is a phenyl group, a naphthyl group or the like and X is an oxygen atom. or a sulfur atom.) is contained in a reagent. By this method, the highly sensitive reagent forming 1mol. of a dye from 1mol. of hydrogen peroxide by the catalytic action or participation of peroxidase and a multilayer analytical element containing the same can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a coloring reagent made of an imidazole derivative and a multilayer analytical element containing the same. More specifically, in the presence of hydrogen peroxide, a substance with an oxidizing effect such as peroxidase acts as a catalyst to develop a new color, as well as hydrogen peroxide containing this or an analyte (analyte) that can produce hydrogen peroxide. The present invention relates to a multilayer analytical element suitable for quantitative analysis of analytes) or beluogysidase activity.

[Conventional technology]

Measuring the generated components and test components (analytes) contained in body fluids such as blood and urine is useful for diagnosing diseases, elucidating pathological conditions,
It is well known that it is useful in determining the progress of treatment. Among these, a method for measuring analytes such as cholesterol, glucose, lactic acid, triglyceride, triglyceride, talleatinine, and free fatty acids in body fluids involves using an oxidase enzyme capable of producing hydrogen peroxide to produce hydrogen peroxide. A method is to develop this into a dye using peroxidase and an oxidizable coloring reagent that is a coloring component, and measure the optical density corresponding to the amount of this dye to determine analyte a using the principle of colorimetry. It is widely used.

As a method for quantifying hydrogen peroxide, for example, a coloring reagent composition combining 4-aminoantipyrine and a soenol compound or an N,N-disubstituted aniline compound, 3-methylbenzothiapurinone hydrazone (MBTH ) and ayuphosphorus compounds, 2,7-diamitufluorene, 0-tolidine derivatives, N,N-dimethyl-p-phenylenediamine, 0-dianisidine, 〇-aminophenol, etc. The methods used are well known.

However, these conventionally used coloring reagents require 2 moles of hydrogen peroxide to form 1 mole of coloring body (dye), and therefore have relatively low sensitivity, making them difficult to quantify trace components. It was a disadvantageous coloring reagent. Also, 4
Except for the combination coloring reagent composition with -aminoantipyridine, all of them have problems such as low stability of the color former (dye).

On the other hand, it is a highly sensitive hydrogen peroxide reagent in which 1 mole of color former (dye) is formed from 1 mole of hydrogen peroxide, and the stability of the color former (pigment) is relatively good, and the coloring wavelength is comparable. Doryarylimidazole derivatives of dye precursors (leuco dyes) on the longer wavelength side have been proposed. (Tokuko Showa 57-551
9, Japanese Patent Publication No. 57-26118, Japanese Patent Publication No. 58-45557
, JP-A-61-229868, U.S. Patent No. 3,297
, No. 710, etc.). Further, diarylimidazole leuco dyes (Japanese Patent Application Laid-Open No. 59-193352) and diarylindolylimidazole leuco dyes (Japanese Patent Application Laid-Open No. 61-4960) have also been proposed.

However, the molecular extinction coefficient (ε value) of these existing triarylimidazole derivatives is approximately 50,000, which is sufficiently satisfactory for quantifying trace components in biological samples such as blood and urine. = t', J: I can't say it.

[Problems to be Solved by the Invention] The object of the present invention is to solve the problems of the prior art mentioned above, and to solve the problems of the prior art as described in (1) above, and to solve the problems of the prior art as described above, and to solve the problems of the prior art described above. It is an object of the present invention to provide a highly sensitive coloring reagent, a coloring reagent composition, and a multilayer analytical element containing the reagent or reagent composition, which can be formed with high sensitivity, that is, having a molecular extinction coefficient of about 60,000 or more.

The present invention provides: (1) a coloring reagent comprising an imidazole derivative represented by the general formula [1]; (2) an imidazole derivative represented by the general formula [1], peroxidase, and an oxidase enzyme capable of producing hydrogen peroxide. and (3) a multilayer analytical element comprising a layer containing a hydrophilic polymer binder and a porous layer laminated in this order on a light-transparent water-impermeable support. The present invention is a multilayer analytical element in which the layer containing the hydrophilic polymer panida or the porous layer contains a coloring reagent composition containing an imidazole derivative represented by the general formula [1].

In general formula [1], R' is an alkyl group, an alkoxy group, a halogen atom, a nido group, a cyan group, a -8031f group, or -5O311I
' group, -CQOI (group or -C00M2 group, -C00M2 group, substituted phenyl group or substituted naphthyl group. ' and ' each represent an alkali metal ion or ammonium ion. As a substituent of the substituted phenyl group or substituted naphthyl group, an alkyl group is used. , an alkoxy group, a halogen atom, a nitro group, a cyan group, an alkylamino group or a (dialkylamine) group, and the number of substituents is 1 or 2, and the substituents may be the same or different from each other. .

R2 and R3 each represent a phenyl group, a naphthyl group, a substituted phenyl group or a substituted naphthyl group. The substituent of the substituted phenyl group or substituted naphthyl group is an alkyl group, an alkoxy group, an alkylamino group, a (dialkylamino) group, an (alkoxyalkyl)amino group or a [di(alkoxyalkyl)]amino group, and the substituted The number of bases is 1
3 to 3 substituents may be the same or different from each other.

Alternatively, R2 and R3 may be interconnected to form a fused 6-membered ring structure including the two carbon atoms of the imidazole ring.

R4 is a phenyl group, a naphthyl group,! Represents a substituted phenyl group or a substituted naphthyl group. Substituents for the substituted phenyl group or substituted naphthyl group include an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, a -303H group, or a -3O group.
ffM' group, -CQOI group or -C00M2 group (M' and M2 each represent an alkali metal ion or an ammonium ion), the number of substituents is 1 or 2, and the substituents may be the same as each other. May be different.

X represents an oxygen atom or a sulfur atom.

[Detailed explanation of the structure of the invention]

In the imidazole derivative (2-pyrazolonyl-4,5-diarylimidazole derivative leuco dye) represented by the general formula [1], the alkyl group represented by R1 is a methyl group, ethyl group, propyl group, butyl group, or hexyl group. , an octyl group, a decyl group, and a straight or branched alkyl group having 1 to about 10 carbon atoms. The alkyl group may further be bonded with a halogen atom (chlorine, bromine, etc.), a cyano group, a nitro group, a carboxy group, a sulfonic acid group, or the like as a substituent. Examples of alkoxy groups include methoxy group, ethoxy group, propoxy group,
Examples include straight-chain or branched alkoxy groups having 1 to about 10 carbon atoms, such as butoxy and bertyloxy groups. Examples of the substituents of the substituted phenyl group or substituted naphthyl group include methyl group, ethyl group, propyl group, butyl group,
Straight chain or branched alkyl groups having 1 to about 10 carbon atoms such as pentyl, hexyl, octyl and decyl groups; 1 to about 10 carbon atoms such as methoxy, ethoxy, propoxy and butoxy groups; 10 linear or branched alkoxy groups, halogen atoms such as chlorine, bromine, fluorine, iodine, nitro group, cyano group, -5O, H group, or S03 group (M+ is an alkali such as sodium, potassium, lithium, etc.) (represents a metal ion or ammonium ion), -C
Examples thereof include an OOH group and a -C00M2 group (M2 represents an alkali metal ion such as sodium, potassium, and lithium, or an ammonium ion). The number of substituents is one or two, and when there are two substituents, the substituents may be the same or different.

R2 and R3 each have a substituent at at least one of the 1.2-position or 1.4-position (ortho-position or para-position in the case of a substituted phenyl group) from the position bonded to the imidazole ring. Represents a phenyl group or a naphthyl group. Substituents for the substituted phenyl group or substituted naphthyl group include linear or branched alkyl groups having 1 to about 5 carbon atoms (e.g., methyl group, ethyl group, propyl group, butyl group),
Straight-chain or branched alkoxy groups having 1 to about 5 carbon atoms (e.g., methoxy, ethoxy, propoxy, butoxy), straight-chain or branched alkyl groups having 1 to about 10 carbon atoms Alkylamino group (e.g., methylamino group, ethylamino group, propylamino group) or dialkylamino group (e.g., dimethylamino group, diethylamino group, dipropylamino group) having one or two of them, total number of carbon atoms (Alkoxyalkylamino) group having one or two linear or branched alkoxyalkyl groups of 2 to about 10 (e.g., methoxymethylamino group, methoxyethylamino group) or [di(alkoxyalkyl)]amino Examples include groups (eg, [di(methoxymethyl)]amino group, [di(methoxyethyl)]amino group). The number of substituents is 1 to 3, and when there are 2 or 3 substituents, the substituents may be the same or different.

When R4 is a substituted phenyl group or a substituted naphthyl group, the substituent includes a linear or branched alkyl group having 1 to about 10 carbon atoms (e.g., methyl group, ethyl'l=, propyl group, butyl group). , pentyl group, isopropyl group), linear or branched alkoxy groups having 1 to about 5 carbon atoms (e.g., methoxy group, ethoxy group, propoxy group, butoxy group), halogen atoms (e.g., chlorine atom, bromine atom) atom, iodine atom, fluorine atom), nitro group, cyano group, -3O3H
group, SO3M' group, -COOH group, or -303M2 group (and M2 each represents an alkali metal ion such as sodium, potassium, lithium, or ammonium ion), etc. The number of substituents is 1 to 3.
When there are two or three substituents, the substitutions may be the same or different from each other.

Preferred specific examples of the inmidazole derivative represented by the general formula (1) are shown below.It goes without saying that the compounds used in the present invention are not limited to these compounds.In the following chemical structural formula: , Me represents a methyl group, and Et represents an ethyl group.

Ngate e2 Me Me U3tl Ndoe2 Ox The imidazole derivative represented by the general formula [1] can be synthesized according to the method described in US Pat. No. 3,297,710. Next, a synthesis example of a compound represented by the general formula [1] will be shown, but compounds not specifically shown can also be easily synthesized according to the following synthesis example.

[Synthesis Example 1] Synthesis of 5-methoxy-3-methyl-1-phenyl-2-pyrazole-4-carbaldehyde 17.4 g of 3-methyl-1-phenyl-2-pyrazolin-5-one was added to dimethylformamide (DMF). 50mL
To? To the flask, 45.9 g of POCl was added over 20 minutes, and the mixture was heated under reflux for 4 hours to react. After cooling, it was poured into ice water, and the product was extracted with methylene chloride. After washing with saturated brine, it was dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The black portion was removed by silica gel column chromatography (eluent: methylene chloride) to obtain 4.53 g of crude crystals of 5-chloro-3-methyl-1-phenyl-2-pyrazole-4-carbaldehyde. This was dissolved in 50 mL of methanol, 5.68 g of sodium carbonate was added, and the mixture was heated under reflux for 2 hours. Methanol was distilled off to about half its volume under reduced pressure, water was added, and the mixture was extracted with methylene chloride. After washing with saturated brine, drying over magnesium sulfate and distilling off the solvent under reduced pressure, the target compound 3 was obtained as an oil.
．． 53g was obtained.

[Synthesis Example 2] Synthesis of 2-(3-methyl-1-phenyl-5-oxo-2-bilaprin-4-yl)-4,5-bis[4-(dimethylamino)phenylciimidazole (Compound 1) (2) 4.46 g of 2-bis[4-(dimethylamino)phenyl]ethane-1,2-dione and 5-methoxy-3-methyl-1-phenyl-2-pyrazole obtained in Synthesis Example 1
4-carbaldehyde 3°26g, ammonium acetate 1
6.5 g was reacted in 90 mL of acetic acid by heating under reflux for 2 hours. After cooling, 120 mρ of water was added, and the mixture was neutralized with ammonium water under ice cooling, and crystals were precipitated. The precipitated crystals were collected by filtration, washed with water, dried, and then recrystallized with ethanol to obtain 1.2 g of a white target compound. λm at melting point 163°C and oxidation coloration
ax 680 nm (ε69000) The reaction (coloring reaction) between the reagent for hydrogen peroxide analysis of the present invention and hydrogen peroxide is represented by the formula [2], for example.

Since the dye (dye A) is produced stoichiometrically by reaction [2], hydrogen peroxide can be quantified based on a calibration curve prepared in advance by measuring the absorbance or reflection spectrophotometry of the produced dye. It can be carried out.

The analysis method of the present invention can be carried out, for example, as follows. The imidazole derivative represented by the general formula [1] and peroxidase as a reaction catalyst are added to the test sample simultaneously or in an appropriate order in the form described above, and the mixture is heated at 5°C to 40°C, preferably 20°C. from 40
at a temperature in the range of 1 to 6 °C and a pH of 3.0 to 9.0, preferably pl+4.5 to 8 °C.
The reaction is allowed to proceed in a time range of 0 minutes, and the absorbance of the reaction mixture containing the produced dye is measured. Or the general formula [1] in a multilayer analytical element (film or sheet)
The porous spreading layer contains an imidazole derivative represented by
μL of test sample solution, or about 10 μL of the porous layer
A fixed amount of test sample solution in the range of μl to about 30 μm is spotted, and the temperature is in the range of 20°C to 40°C, preferably 25°C to 40°C, for 1 minute to 20 minutes, preferably 2 The optical density of the multilayer analytical element is measured by reflection spectrophotometry by inking over a time range of 10 minutes to 10 minutes. The absorbance or reflected optical density can be measured using the end point method and the r
The reaction rate method can be appropriately selected and implemented.

The coloring reagent, coloring reagent composition, and multilayer analytical element of the present invention are suitable for colorimetric analysis of hydrogen peroxide in a sample containing a trace amount of hydrogen peroxide (or an analyte capable of producing hydrogen peroxide) as an analyte. Particularly advantageous.

By further combining an oxidase (oxidase) with the imidazole derivative represented by the general formula [1] and peroxidase as a coloring reagent, a wider range of applications becomes possible.

In other words, when the substance to be finally quantified produces hydrogen peroxide through the action of an oxidase, by combining the oxidase with the reagent of the present invention, the produced hydrogen peroxide can be quantified and used for various purposes. It becomes possible to quantify substances. Specifically, glucose, uric acid, cholesterol, triglycerides in urine and body fluids (e.g. blood),
Lactic acid, taleatinin, free fatty acids, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, cholinesterase, taleatin bosphokinase, lactate dehydrogenase, etc. can be measured. Examples of oxidases used in these measurements include glucose oxidase, uricase, 2-cholesterol oxidase, L-α-glycerophosphate oxidase, and pyruvate oxidase. These oxidases may be of any origin.

When using oxidase together, H
z O2 may be generated in advance, but 11□0□
It is preferable that the production reaction and the color development reaction proceed simultaneously. The amount of the oxidizing enzyme to be used is appropriately determined depending on the type, target substrate, or contaminant.

The peroxidase used in the present invention is a catalyst for the oxidation reaction of a hydrogen donor by hydrogen peroxide.

Peroxidases include peroxidases of plant origin and animal origin (EC1, 11°7) disclosed in Japanese Patent Publication No. 56-45599, Japanese Patent Publication No. 57-5520, etc., and peroxidases of microbial origin (EC1, 11°7), one type or a combination of two or more types can be used as appropriate.

Examples of peroxidases of plant origin include horseradish,
Examples of peroxidases of animal origin include peroxidase extracted from potatoes, fig sap, caps, and radish; lactoperoxidase extracted from milk; and held peroxidase extracted from white blood cells.
Alterna as a peroxidase of microbial origin
ia, Cochliobolus, Curvula
Peroxidases derived from microorganisms that belong to the genus Ria and genus Pellicularia and have the ability to produce non-specific peroxidase can be mentioned.

In addition, special public service No. 56 45599, special public service No. 57-552
Ferric thiocyanate, iron tannate, iron fluorocyanide (all iron (+2) compounds), potassium chromium sulfate, sodium iodide, potassium iodide, ammonium molybdate, potassium molybdate, etc. disclosed in 0 etc. have peroxidase activity. It is also possible to use an inorganic compound having

Among these, non-specific peroxidases of plant or microbial origin are preferred.

As seen in the example of formula [2] above, the imidazole compound represented by general formula [1] and hydrogen peroxide react stoichiometrically under the catalytic action of peroxidase to produce a dye. , a coloring reagent composition that combines an imidazole compound represented by the general formula [1] and HzO□ as a reagent, and a sample containing peroxidase of unknown activity value are added or spotted to a multilayer chemical analysis element containing both. By measuring the optical density of the produced dye, peroxidase activity in a sample can be analyzed or quantified by the principle of colorimetry. This method can be used in enzyme immunoassay using peroxidase as a labeling substance.

10, a bright multilayer analytical element and its preferred embodiments are as follows.

(1) In a multilayer analytical element in which a layer containing a hydrophilic polymer binder and a porous layer are laminated and integrated in this order on a light-transparent water-impermeable support, the layer containing the hydrophilic polymer binder or the A multilayer analytical element in which a porous layer contains a coloring reagent composition containing an imidazole derivative represented by the general formula [1].

(2) The multilayer analytical element according to (1), wherein the coloring reagent composition further contains peroxidase and an oxidase enzyme capable of producing hydrogen peroxide.

(3) The porous layer is a porous spread layer (1) or (
The multilayer analysis element described in 2).

(4) The multilayer analytical element according to (1) or (2), wherein the porous layer is a constant area porous layer.

(5) The multilayer analytical element according to (1) to 4), wherein the coloring reagent composition is contained in a layer containing the hydrophilic polymer binder.

(6) The multilayer analytical element according to (3), wherein the coloring reagent composition is contained in the developing layer.

(7) The multilayer analytical element according to (4), wherein the coloring reagent composition is contained in the constant area porous layer.

The multilayer analytical element of the present invention and the multilayer analytical element of each of the above embodiments are disclosed in Japanese Patent Application Laid-open No. 55-464356 and Japanese Patent Application Laid-Open No. 57-66359.
An integrated multilayer analytical element having a fabric development layer (e.g., plain weave such as broad, boblin, etc.) described in JP-A-60-222
An integrated multilayer analytical element having a fabric development N (e.g., tricot knit, double tricot knit, Mitenese knit, etc.) as described in JP-A-57-148250, and a spread layer made of paper containing organic polymer fiber pulp described in JP-A-57-148250 Integrated multilayer analytical element having
, 992, 158, etc., non-fibrous isotropic porous membrane filters (plush polymer single layer), polymer microbeads, glass microbeads, continuous microporous porous layers comprising diatomaceous earth held in a hydrophilic polymer binder, etc. Integrated multi-layer analysis element with gender development layer, JP-A-1987-
A non-fibrous isotropic porous layer comprising a continuous microvoid-containing porous layer (three-dimensional lattice-like granular structured material layer) in which the polymer microbeads described in 90859 are bonded in point contact with a polymer adhesive that does not swell with water. Using constituent materials and components of an integrated multilayer analytical element having a spread layer and an integrated multilayer analytical element having a constant area porous layer described in Utility Model Application Publication No. 57-42951, etc., the patent specifications of these It can be manufactured according to the method described.

Among these, in an embodiment in which a reagent composition containing at least one enzyme is contained and retained in a spreading layer, textile spreading layers and knitted fabric spreading layers are typical in that it is easy to contain and hold the reagent composition in the spreading layer. An integrated multilayer analytical element having a fibrous spread layer is preferred.

The multilayer analytical element of the present invention has a side of about 15 mm to about 30 mm.
Cut into small pieces such as squares or circles of approximately the same size,
Special Publication No. 57-28331, Utility Publication No. 56-142454,
JP-A-57-63452, Utility Model Publication 58-32350, Japanese Patent Application Publication No. 58-501144, etc., it is possible to use it as a chemical analysis slide by placing it in the slide frame described in Japanese Patent Publication No. 57-63452, Utility Model Application Publication No. 58-32350, Japanese Patent Application Publication No. 58-501144, etc. Preferred from this point of view. Depending on the purpose of use, it can be used in the form of a long tape and stored in a cassette or magazine, or small pieces can be attached or stored in a card with an opening.

The multilayer analytical element of the present invention can analyze analytes in liquid samples by the operations described in the aforementioned patent specifications. In other words, about 5μ! , about 30μ, preferably 8μ
A sample of aqueous liquid such as whole blood, plasma, serum, urine, etc. in a range of 1 to 15 μL is spotted on the developing layer, and the sample part is placed in a range of about 20°C to about 40°C for 1 to 10 minutes. Inkation is carried out at a constant temperature, preferably at a substantially constant temperature around 37°C, and the color development within the element is made optically transparent using light at or near the wavelength of maximum absorption of visible light. The content of the analyte in the liquid sample can be determined by performing reflection photometry from the support side and using a previously prepared calibration curve based on the principle of colorimetric measurement. Quantitative analysis of analytes can be performed with high precision by keeping the amount of liquid sample deposited, incubation time, and temperature constant. The measurement operation was performed using JP-A-60-12-5543, JP-A-60-220862, JP-A-61-294.367, and JP-A-58-161867.
The chemical analyzer described in et al. allows highly accurate quantitative analysis to be performed with extremely easy operation.

The coloring reagent, coloring reagent composition, and multilayer analysis element containing the same of the present invention can be applied to hydrogen peroxide or the like in urine, body fluids (e.g., blood), etc. not only of humans but also of various animals, such as livestock and pet animals. Of course, it can also be used to analyze analytes that produce hydrogen peroxide.

EXAMPLES Hereinafter, the present invention will be explained in more concrete detail with reference to Examples.

Example 1 (1) Preparation of coloring solution: A coloring solution was prepared by dissolving 15 mg of compound (1) in acetone and dissolving 5000 units of peroxidase in an aqueous borate buffer solution of pH 7.5 to make a total volume of 25 mL.

(2) Quantification of hydrogen peroxide Place 3 mL of the two-color solution in a sample cell, add 20 μl of hydrogen peroxide of known concentration, and create a blank (add distilled water instead of the hydrogen peroxide aqueous solution).
As a control, the absorbance was measured at a wavelength of 690 nm after 10 seconds at 37°C, and the following results were obtained.

Example 2 A reagent layer coating solution consisting of the following components was applied onto a 185 μm thick colorless transparent polyethylene terephthalate (PET) film having a gelatin undercoat layer and dried.

Components of reagent layer coating solution (solvent, appropriate amount of water) uricase
1500/bo compound (])
0.2g/bobel oxidase
2500U/bogelatin 10
g/n (pH 8,5 borate buffer) The prepared reagent layer was wetted with water, and a cellulose acetate membrane filter (
A multilayer analytical element for quantifying uric acid was prepared by laminating and integrating the elements (average pore diameter: 3.0 μm, thickness: 180 μm).

In order to evaluate the obtained multilayer analytical element, 10 μL of each uric acid aqueous solution (concentration 6; 10; 12 mg/dL and a uric acid-free blank solution) was spotted on the porous development layer.
Immediately after inking for 2 min at 37°C, PE was measured using a Maches reflectance spectrophotometer equipped with a cyan filter.
Measure the color luminosity density from the illumination side at a wavelength of 690 nm.
Reflection photometry was performed nearby and the following values were obtained.

(Concentration mg/dL) 0 (Blank) 0.43 6 0.80 10 1.77 12 From the results in 1.9, using this multilayer analytical element, the uric acid concentration was determined by colorimetry using reflectance photometry. It became clear that it could be implemented.

Example 3 185 μm thick colorless transparent PE with gelatin subbing layer
A reagent layer coating solution consisting of the following components was applied onto the T film and dried.

Components of reagent layer coating solution (solvent, appropriate amount of water) uricase
200 U/bo compound (1)
0.25g/pogelatin
10g/poperoxidase 3000
U/Pooctylphenoxypolyethoxyethanol (contains 10 oxyethylene units on average) 0.15g/rI'!9
A light reflective layer coating dispersion consisting of the following components was coated on the H8,5 borate buffer reagent layer and dried.

Components of the light-reflecting layer coating dispersion liquid (dispersion medium: appropriate amount of water) Titanium dioxide fine powder 3 g/n (gelatin Ig/booctylphenoxyboriethoxyethanol (contains an average of 10 oxyethylene units) 0.1 g/bright reflection An interfering substance exclusion layer coating solution consisting of the following components was further applied and dried on the layer.

Components of the interference removal surface coating solution (solvent: appropriate amount of water) Ascorbic acid oxidase 4000 U/rfl Gelatin 4 g/rl? Octylphenoxypolyethoxyethanol (contains 10 oxyethylene units on average) 0.1g/PopH8.5
Next, the borate buffer and the interference removal layer are moistened with water, and a broad cloth woven from 100-count cotton yarn (washed with water, degreased, and then dried) is laminated and integrated to form a multilayer for uric acid determination. Analytical elements were prepared.

Next, in the same manner as in Example 2, the color reflective optical density values of the multilayer analytical element for known uric acid concentrations were obtained.

(Concentration mg/d 1.) 0 (Blank) 0.25 6 0.93 10 1.19 12 1.36 [Effects of the Invention] All of the coloring reagents made of the imidazole derivative of the present invention have a concentration of 1 molar excess. One mole of pigment is generated from hydrogen oxide, and its molecular extinction coefficient is over 60,000, making it possible to perform quantitative analysis of components, especially trace components, in biological fluids such as blood and urine with high sensitivity using the principle of colorimetry. Can be implemented.

Claims (9)

[Claims] (1) A color reagent comprising an imidazole derivative represented by the general formula [1]. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [1] In the general formula [1], R^1 is an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, a -SO_3H group, or -SO_3M^
1 group, -COOH group or -COOM^2 group, substituted phenyl group or substituted naphthyl group. M^1 and M^2 each represent an alkali metal ion or an ammonium ion. The substituent of the substituted phenyl group or substituted naphthyl group may be an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, an alkylamino group, or a (dialkylamino) group. R^2 and R^3 are phenyl group, naphthyl group, respectively.
Represents a substituted phenyl group or a substituted naphthyl group. As a substituent of the substituted phenyl group or substituted naphthyl group, an alkyl group,
Alkoxy group, alkylamino group, (dialkylamino) group, (alkoxyalkyl)amino group or [di(alkoxyalkyl)]amino group, the number of substituents is 1 to 3, and the substituents are mutually They may be the same or different. Alternatively, R^2 and R^3 may be linked to each other to form a fused 6-membered ring structure including the two carbon atoms of the imidazole ring. R^4 represents a phenyl group, a naphthyl group, a substituted phenyl group or a substituted naphthyl group. Substituents for the substituted phenyl group or substituted naphthyl group include an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, a -SO_3H group, or a -
SO_3M^1 group, -COOH group or -COOM^2 group (M^1 and M^2 each represent an alkali metal ion or ammonium ion), the number of substituents is 1 or 2, The substituents may be the same or different. X represents an oxygen atom or a sulfur atom. (2) A coloring reagent composition containing an imidazole derivative represented by general formula [1], peroxidase, and an oxidase enzyme capable of producing hydrogen peroxide. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [1] In the general formula [1], R^1 is an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, a -SO_3H group, or -SO_3M^
1 group, -COOH group or -COOM^2 group, substituted phenyl group or substituted naphthyl group. M^1 and M^2 each represent an alkali metal ion or an ammonium ion. The substituent of the substituted phenyl group or substituted naphthyl group is an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, an alkylamino group or a (dialkylamino) group, and the number of substituents is 1 or 2. The substituents may be the same or different from each other. R^2 and R^3 are phenyl group, naphthyl group, respectively.
Represents a substituted phenyl group or a substituted naphthyl group. As a substituent of the substituted phenyl group or substituted naphthyl group, an alkyl group,
Alkoxy group, alkylamino group, (dialkylamino) group, (alkoxyalkyl)amino group or [di(alkoxyalkyl)]amino group, the number of substituents is 1 to 3, and the substituents are mutually They may be the same or different. Alternatively, R^2 and R^3 may be linked to each other to form a fused 6-membered ring structure including the two carbon atoms of the imidazole ring. R^4 represents a phenyl group, a naphthyl group, a substituted phenyl group or a substituted naphthyl group. Substituents for the substituted phenyl group or substituted naphthyl group include an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, a -SO_3H group, or a -
SO_3M^1 group, -COOH group or -COOM^2 group (M^1 and M^2 each represent an alkali metal ion or ammonium ion), the number of substituents is 1 or 2, The substituents may be the same or different. X represents an oxygen atom or a sulfur atom. (3) A multilayer analytical element in which a layer containing a hydrophilic polymer binder and a porous layer are laminated and integrated in this order on a light-transparent water-impermeable support, and the layer containing the hydrophilic polymer binder or the A multilayer analytical element characterized in that a porous layer contains a coloring reagent composition containing an imidazole derivative represented by the general formula [1]. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [1] In the general formula [1], R^1 is an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, a -SO_3H group, or -SO_3M^
1 group, -COOH group or -COOM^2 group, substituted phenyl group or substituted naphthyl group. M^1 and M^2 each represent an alkali metal ion or an ammonium ion. The substituent of the substituted phenyl group or substituted naphthyl group is an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, an alkylamino group or a (dialkylamino) group, and the number of substituents is 1 or 2. The substituents may be the same or different from each other. R^2 and R^3 are phenyl group, naphthyl group, respectively.
Represents a substituted phenyl group or a substituted naphthyl group. As a substituent of the substituted phenyl group or substituted naphthyl group, an alkyl group,
Alkoxy group, alkylamino group, (dialkylamino) group, (alkoxyalkyl)amino group or [di(alkoxyalkyl)]amino group, the number of substituents is 1 to 3, and the substituents are mutually They may be the same or different. Alternatively, R^2 and R^3 may be linked to each other to form a fused 6-membered ring structure including the two carbon atoms of the imidazole ring. R^4 represents a phenyl group, a naphthyl group, a substituted phenyl group or a substituted naphthyl group. Substituents for the substituted phenyl group or substituted naphthyl group include an alkyl group, an alkoxy group, a halogen atom, a nitro group, a cyano group, a -SO_3H group, or a -
SO_3M^1 group, -COOH group or -COOM^2 group (M^1 and M^2 each represent an alkali metal ion or ammonium ion), the number of substituents is 1 or 2, The substituents may be the same or different. X represents an oxygen atom or a sulfur atom. (4) The multilayer analytical element according to claim 3, wherein the coloring reagent composition further contains peroxidase and an oxidase enzyme capable of producing hydrogen peroxide. (5) The multilayer analytical element according to claim 3 or 4, wherein the porous layer is a porous expansion layer. (6) The multilayer analytical element according to claim 3 or 4, wherein the porous layer is a constant area porous layer. (7) Claims 3 to 6, wherein the coloring reagent composition is contained in a layer containing the hydrophilic polymer binder.
Multilayer analysis elements described in. (8) The multilayer analytical element according to claim 5, wherein the coloring reagent composition is contained in the developing layer. (9) The multilayer analytical element according to claim 6, wherein the coloring reagent composition is contained in the constant area porous layer.