JP2002350437A - Manufacturing method of dry analysis element - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method of a dry analysis element capable of preventing coloring of a leuco pigment even if an antifogging agent is not used in manufacture of the dry analysis element, and also preventing coloring thereafter before being used. SOLUTION: This manufacturing method of the dry analysis element is characterized by integrating the fading leuco pigment as a layer having pH of 5-12 into the dry analysis element, and executing aging there after.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a dry analytical element for quantitatively determining a leuco dye by coloring it with hydrogen peroxide or peroxidase using the same.

[0002]

2. Description of the Related Art In recent years, enzymological analysis methods in clinical examinations have been highly evaluated for their specificity and are rapidly spreading. Among them, glucose in urine and body fluids, uric acid, cholesterol, triglycerides, lactic acid, creatinine, free fatty acids, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, cholinesterase, creatine phosphokinase,
As a method of measuring lactate dehydrogenase, etc., a method of quantifying a target substance by quantifying hydrogen peroxide generated by the action of an oxidase of a substance to be finally determined in each system is often used. I have.

[0003] These methods involve the preparation of o-tolidine, 2,7-diaminofluorene, N,
A method for converting a chromogen such as N-dimethyl-p-phenylenediamine, o-dianisidine, o-aminophenol or the like to an oxidized color former and performing colorimetric determination, 4-aminoantivirin and phenol or N, N A combination with -dialkylaniline or N, N-dialkyltoluidine,
3-methyl-2-benzothiazolinone hydrazone and o-
Trizine or N, N-dimethylaniline or N, N
A method in which a chromogen such as 4-methoxy-1-naphthol or a derivative thereof which forms a dimer is oxidatively condensed and colorimetrically determined as a color former by a combination with -diethylaniline. Further, a dry analytical element in which a fading leuco dye is incorporated as a color former is also known (JP-A-5959 / 1984).
193352).

[0004]

When a dry analytical element is manufactured by incorporating the above-mentioned leuco dye, the leuco dye sometimes develops color and fog occurs during the formation of multiple layers by repeated coating. For this reason, an antifoggant has conventionally been used. However, antifoggants have a problem of inhibiting oxygen activity. In addition, variations in raw material lots and variations in application are liable to occur, and fog is not stable. Further, there is also a problem that the leuco dye develops color due to natural aging and the accuracy varies.

[0005] An object of the present invention is to provide a dry analysis method in which the leuco dye does not develop color even when an antifoggant is not used in the production of a dry analysis element, and no color develops before use. An object of the present invention is to provide a device manufacturing method.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that leuco dyes have a specific pH.
It has been found that fogging can be eliminated by applying as a solution of the above, followed by aging by heating, and furthermore, it can be stored without causing fogging.

The present invention has been made based on this finding, and incorporates a fading leuco dye into a dry analytical element as a solution having a pH of 5 to 12 or adjusts the pH of the leuco dye layer from the upper layer. The pH of the leuco layer may be adjusted by lamination from the upper layer. Then 30-70 ° C
The present invention relates to a method for manufacturing a dry analytical element, characterized by performing aging.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The leuco dye to which the present invention is applied is one in which the 4-position and 5-position are asymmetric, and there is no nitrogen-containing group in one or both of the 4- and 5-positions.

This leuco dye is an imidazole derivative represented by the following general formula [1].

Embedded image

In the formula [1], R ¹ is an aryl group in which the ortho or para position is substituted with a hydroxyl group,
² represents an aryl group or a substituted aryl group, and R ³ represents an alkyl group, a substituted alkyl group or an alkenyl group, respectively.

The hydroxyaryl group represented by R ¹ in the general formula [1] includes, in addition to a hydroxyl group, a halogen atom (such as a chlorine atom or a bromine atom), a cyano group, an alkoxy group, a monoalkylamino group, a dialkylamino group, or the like. May have one substituent, or two or more of the same or different substituents. The substituted aryl group represented by R ² includes a halogen atom (such as a chlorine atom or a bromine atom), a hydroxyl group, a cyano group, an alkoxy group, a monoalkylamino group,
An aryl group having one substituent such as a dialkylamino group or two or more of the same or different substituents.

The substituted alkyl group represented by R ³ is a hydroxyl group, an alkoxy group, an aryloxy group, a cyano group, a monoalkylamino group, a dialkylamino group, a carboxyl group, an alkoxycarbonyl group, a carbamoyl group, an N-alkylcarbamoyl. Group, N, N-dialkylcarbamoyl group, alkylsulfonyl group, sulfamoyl group, N-alkylsulfamoyl group, N, N-dialkylsulfamoyl group, aryl group, etc.
Or an alkyl group having two or more of the same or different substituents, of which an aryl group and an aryloxy group are preferable.

When R ³ is alkyl, it may have any number of carbon atoms, but preferably has 2 or more, most preferably 6 or more. When R ³ has 3 or more carbon atoms,
The carbon chain may be linear, branched or cyclic. R ³
Is alkenyl, the number of carbon atoms may be any number as long as it is 2 or more, but is preferably 6 or more. When the number of carbon atoms is 3 or more, the carbon chain may be linear or branched. And when the number of carbon atoms is 5 or more, it may be cyclic.

Preferred specific examples of the substituted aryl group represented by R ¹ are 2-hydroxyphenyl group, 4-hydroxyphenyl group, 3,5-dibromo-4-hydroxyphenyl group, 3-bromo-4-hydroxy- 5-methoxyphenyl group, 3-methoxy-4-hydroxyphenyl group,
And a 3,5-dimethoxy-4-hydroxyphenyl group.

Preferred specific examples of the substituent represented by R ² include a phenyl group, a 4-methoxyphenyl group, a 4-ethoxyphenyl group, a 4- (dimethylamino) phenyl group,
-(Diethylamino) phenyl group, etc .;
Preferred examples of the substituent represented by ¹ include the groups mentioned above.

When R ³ is an alkyl group, preferred examples thereof are ethyl, propyl, butyl, hexyl, octyl, decyl, isopropyl, isoamyl, cyclohexyl, cyclohexylmethyl, cyclohexylethyl. And the most preferred of these are octyl, decyl, cyclohexylethyl and the like. When R ³ is a substituted alkyl group,
Preferred specific examples thereof include benzyl group, phenethyl group, 3
-Phenylpropyl group, phenoxymethyl group, 2-phenoxyethyl group, 3-phenoxypropyl group and the like, and among these, phenethyl group, 3-phenylpropyl group, 2-phenoxyethyl group, 3-phenoxypropyl group and the like. It is most preferred in that it gives a compound that is easily soluble in water and has low interlayer diffusion when incorporated into a multilayer chemical analysis element.

When R ³ is an alkenyl group, preferred specific examples thereof include a 3-butynyl group, a 4-ventenyl group and a 3-hexenyl group.

Next, preferred specific examples of the compound represented by the formula (1) are shown, but the compounds used in the present invention are not limited to these compounds. In the following Mc is methyl, Et is ethyl, Ph is a phenyl group, _{B 2} is a benzyl group.

Embedded image

Embedded image

Embedded image

Embedded image

Embedded image

Embedded image

Embedded image

Embedded image

Of these compounds, the compound (7) is preferable because it has a high solubility in water and has a low interlayer diffusion when incorporated into a multilayer chemical analysis element.

Leuco dyes, which are water-soluble, are dissolved in water as they are, and those which are not, are dissolved in a water-soluble organic solvent such as acetone, dimethylformamide, methanol, ethanol, etc., and then added to water to form an aqueous solution or suspension. I do.
The concentration of the leuco dye is about 0.05% to 2%, preferably about 0.1 to 1%. This leuco dye solution has a pH of about 5 to 12, preferably about 6 to 11, and particularly preferably about 7 to 9.5.

For this purpose, it is preferable to use a pH buffer. Examples of the pH buffer include phosphate buffer, imidazole buffer, pyrophosphoric acid, Tris, 2-amino methane propane 1.3-diol buffer, boric acid, glycine, sodium carbonate, and the like. .

Since the leuco dye reacts with hydrogen peroxide under the action of peroxidase to form a color, it is necessary to incorporate the peroxidase into the dry analytical element. Also,
When the layer containing the leuco dye is a coating layer, a hydrophilic polymer binder such as gelatin is also required. These may be contained in the same solution as the leuco dye, or may be separate solutions. In the case of the same solution, the concentration of peroxidase is about 5 to 200 KU / L, preferably about 50 to 100 KU / L, and the concentration of the hydrophilic polymer binder is about 1 to 30%, preferably 5 to 25%.
The degree is appropriate.

In addition, an oxidase corresponding to the object to be analyzed is also added. In other words, when the substance to be finally quantified produces hydrogen peroxide by the action of the oxidase, by combining the oxidase with the reagent system of the present invention, the produced hydrogen peroxide is quantified to obtain various target substances. Can be quantified. Specifically, glucose, uric acid, cholesterol, triglycerides, lactic acid, creatinine, free fatty acids, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, cholinesterase, creatine phosphokinase in urine and fluids (eg, blood), Lactate dehydrogenase and the like can be measured. Examples of the oxidase used in these measurements include glucose oxidase, uricase, cholesterol oxidase, L-α-glycerophosphate oxidase, and pyruvate oxidase. Also,
It is also preferable to use a surfactant (preferably, a nonionic surfactant (eg, polyoxyethylene alkylphenyl ether)).

The basic constitution of the analytical element of the present invention is such that a water-permeable layer and a porous spreading layer are laminated on a water-impermeable transparent support in this order.

The porous developing layer has a function of spreading the components contained in the aqueous sample in a plane without substantially unevenly distributing the components, and supplying the components to the water-permeable layer at a substantially constant rate per unit area. All known non-fibrous and fibrous porous materials can be used as the spreading layer which has been used in the dry analysis element. Specifically, a non-fibrous isotropic microporous medium layer represented by a membrane filter (brushed polymer) disclosed in JP-A-49-53888, a polymer disclosed in JP-A-55-90859, etc. Non-fibrous porous layer typified by a continuous void-containing three-dimensional lattice granular structure layer in which microbeads are adhered in a point contact manner with a water-swellable adhesive;
164356, a porous layer composed of a woven fabric disclosed in 57-66359 and the like, a layer composed of a knitted fabric disclosed in 60-22769 and the like, and the like.
It is not limited to these.

For example, cellulose derivatives (DAC, TA
C, NC, HMC (hydroxymethylcellulose), H
EC (hydroxyethyl cellulose) porous membrane, porous membrane made of ethylene polymer or copolymer such as polyethylene, polypropylene, polystyrene, vinyl chloride, etc., porous membrane made of polyethylene terephthalate, polycarbonate, polysulfone, etc. Acrylic acid, methacrylic acid, a porous film of a vinyl polymer or copolymer of these esters, a porous film of a condensation polymer such as nylon, polyamide, polyurethane, glass particles, and inorganic material particles such as diatomaceous earth. There are porous membranes made by bonding a small amount of polymer, porous membranes made of polytetrafluoroethylene, filter paper, glass fiber filter paper, and the like.

It is not necessary to limit the spread layer to only one layer. As disclosed in JP-A-61-4959, JP-A-62-138756, JP-A-62-135757, and JP-A-62-138758, two or more layers are provided. Can be used in layers.

The spreading layer may contain a nonionic, anionic, cationic or amphoteric surfactant in order to promote the spreading of the sample.

Further, for the purpose of controlling the expandability,
A development controlling agent such as a hydrophilic polymer can be included.

Further, various reagents for promoting the target detection reaction, or for reducing or preventing interference or interference reaction, or a part of the reagents can be included.

The thickness of the spread layer is 20 to 300 μm, preferably 50 to 270 μm, and more preferably 80 to 250 μm.
μm.

The hydrophilic polymer layer, which is a typical layer of the water-permeable layer, usually contains at least a part of a reagent necessary for analysis, in which case this layer is called a reagent layer. For this layer, various known water-soluble, swellable and hydrophilic polymers used in dry analysis elements can be used. A natural or synthetic hydrophilic polymer having a swelling ratio at the time of absorption of water at 30 ° C. of about 150% to about 2000%, preferably about 250% to about 1500% can be used. 59-171864, 60-
Gelatin (eg, acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (eg, phthalated gelatin, hydroxyacrylate-grafted gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinyl Examples include, but are not limited to, pyrrolidone.

In place of the hydrophilic polymer layer, a polymer porous membrane or the like can be used.

The thickness of the hydrophilic polymer layer is about 1 when dried.
μm to about 100 μm, preferably about 3 μm to about 50 μm
m, particularly preferably from about 5 μm to about 30 μm, preferably substantially transparent.

The hydrophilic polymer layer may contain various reagents or a part of the reagents for promoting a desired reaction or preventing or reducing interference or interference.

As the water-impermeable transparent support, there can be used a known water-impermeable transparent support conventionally used in a dry analytical element. Specifically, polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose ester (for example, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) and the like have a thickness of about 50 μm to 1 mm, preferably about 80 μm to about 300μ
m transparent films can be used.

If necessary, a known undercoat layer or adhesive layer may be provided on the surface of the support to enhance the adhesion to the hydrophilic polymer layer.

Various layers are further incorporated in the dry analysis element according to the analysis items and the like. For example, a detection layer, a water absorption layer, a light reflection layer, a light shielding layer, and the like.

The leuco dye is contained in the above-mentioned hydrophilic polymer layer, but may be contained in the spread layer.

When the lamination of each layer is completed, aging is performed next. This aging is performed under normal pressure or reduced pressure atmosphere.
The reaction is carried out at about 30 to 70 ° C, preferably about 35 to 60 ° C, particularly preferably about 45 to 55 ° C, for about 1 to 72 hours, preferably for about 6 to 24 hours.

[0041]

EXAMPLES Example 1 Gelatin-undercoated 18
0μm polyethylene terephthalate colorless transparent smooth filter
An aqueous solution of the following composition is applied to the
And dried under reduced pressure at room temperature. Reaction layer Gelatin 20.0 g / m²  Peroxidase 15.0 KU / m²  Pyruvate oxidase 3.0 KU / m²  Leuco dye 0.3g / m²  pH 8.0

On this reaction layer, polyethylene terephthalate
Laminate a 250μm-thick development cloth made of sheet knit
Then, an aqueous solution having the following composition was applied and dried under reduced pressure at room temperature. Buffer (pH 8.0) 1.2 g / m²  Surfactant 1.0g / m²

Here, the surfactant is polyoxy (2-
Hydroxy) propylene nonylphenyl ether (“S
urfactant 10G, manufactured by Orin Co.), and leuco dye is 2- (3,5-dimethoxy-4-hydroxyphenyl) -4- (4-dimethylaminophenyl)-
5-Phenethylimidazole acetate was used, and trishydroxymethylaminomethane was used as a buffer.

The structural formula of the above leuco dye is shown below.

Embedded image

The above-mentioned integrated multilayer analytical element was aged in a constant temperature room at a temperature of 40 ° C. for 6 hours.

The above-mentioned integrated multi-layer analysis element is 12 mm × 1
Cut into 3 mm square chips, slide frame (Japanese
No.-63452).
A dry slide (1) for YR analysis was prepared.

Comparative Example 1 An integrated multilayer analytical element was prepared in the same manner as in Example 1 except that the leuco dye was changed to the following.

Embedded image

Example 2 An integrated multilayer analytical element was manufactured in the same manner as in Example 1 except that the aging temperature was changed.

Example 3 An integrated multilayer analytical element was manufactured in the same manner as in Example 1 except that the pH of the leuco dye solution was changed.

[Measurement Example 1] The following measurement was performed using the above slide. (1) Aging temperature and fog strength Table 1 shows the results of measuring the fog and the measurement range of the integrated multilayer analytical element manufactured in the same manner as in Example 1 by changing the aging temperature. (2) Table 2 shows the results of measuring the fog strength of the integrated multilayer analytical element manufactured in the same manner as in Example 1 by changing the pH of the aqueous leuco dye solution. When a fading leuco dye is used, fog is stabilized at a low pH at 5 to 12. (3) Table 3 shows the results of measuring the fog and the measurement range of the integrated multilayer analytical element of Comparative Example 1.

[0051]

[Table 1]

[0052]

[Table 2]

[0053]

[Table 3]

[0054]

According to the present invention, fog of a dry analytical element using a leuco dye can be removed without using an antifoggant, thereby improving the analysis accuracy. In addition, stability during storage can be improved.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G042 AA01 BD19 CA10 CB03 DA10 FA11 FB07 FC03 2G045 AA16 AA25 BA11 BB51 BB60 CB03 DA31 FB01 FB11 GC12 HA10 2G054 AA07 AB02 AB05 BA01 BB13 CA21 CE01 GE06

Claims (3)

[Claims] 1. A method for producing a dry analytical element, comprising incorporating a fading leuco dye into a dry analytical element, and then performing aging by heating. 2. The method for producing a dry analytical element according to claim 1, wherein the pH of the leuco dye layer is 5 to 12. 3. The method for producing a dry analytical element according to claim 1, wherein the aging temperature is 30 ° C. to 70 ° C.