JPH0599929A - Dry analyzing element for detection of copper ion in urine - Google Patents

Abstract

PURPOSE:To prevent the deterioration of a color former in an analyzing element and to make it possible to preserve the element for a long period from the manufacture of the element to the time of use by incorporating cationic polymer compound. CONSTITUTION:A dry analyzing element for detection of copper ions in urine is provided with water absorbing porous material containing the reagent composition incorporating the N,N-dialkyl derivative of 4-(3,5-dibromo-2-pyridylazo) aniline, ascorbic acid salt, surface-active agent, cationic polymer compound and pH modifier whose pH range is 4.0-8.0.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a dry analytical element containing a reagent composition for detecting copper ions in urine. For more information,
Improving the storage stability of a reagent composition for detecting copper ions containing an N, N-dialkyl derivative of 4- (3,5-dibromo-2-pyridylazo) aniline and ascorbate in a dry analytical element Dry analytical element.

[0002]

BACKGROUND OF THE INVENTION Wilson's disease, an inborn error of metabolism, is a serious disease in which copper is excessively deposited in organs, especially in the liver and brain, resulting in its damage. The patient has a marked decrease in the ceruloplasmin, a copper-binding serum protein. On the other hand, unstable albumin-bound copper is increased, and most of it is excreted in urine. Therefore, if a decrease in serum ceruloplasmin level or an increase in urinary copper level is found early by screening or the like, it can be treated by administration of the copper chelating agent D-penicillamine or the like. From the viewpoint of screening, which requires rapid processing of a large number of specimens, it is easier to collect specimens without any special operation than blood serum ceruloplasmin measurement, which requires blood collection and subsequent centrifugation to obtain specimens. The method for measuring copper ion in urine using a urine sample capable of performing the above is more advantageous. Further, if it can be processed by a dry analysis method, a simpler measurement is possible.

[0003]

2. Description of the Related Art A simple colorimetric method is preferable for a urinary copper ion screening test because the color can be developed by a simple operation and the color change can be visually judged. The most well-known reagent for the colorimetric determination of copper ion is bathocuproine, but it has a drawback in that it has low measurement sensitivity.
The greatest contribution to the detection sensitivity is the extinction coefficient of the color former.From this point, the trace colorimetric method for serum copper using a water-soluble porphine compound that reacts with copper ions and has the largest extinction coefficient is also applicable. Proposed ("Clinical Chemistry" Vol. 8, 266
Page (1979)). However, this method requires a complicated deproteinization operation before the reaction, and thus has difficulty in a screening test for treating a large number of samples in terms of simplicity. Also,
"J.Clin.Chem.Clin.Biochem." 19: 541 (1981)
6-Methylpicolinic acid thioamide described in 1) is a highly sensitive colorimetric reagent proposed for the colorimetric determination of copper ions in urine, but it was measured using a spectrophotometer after solvent extraction of the produced dye. Therefore, the screening test is too complicated.

The N, N-dialkyl derivative of 4- (3,5-dibromo-2-pyridylazoaniline was developed as a reagent for quantitative analysis of silver and copper (Japanese Patent Laid-Open No. 61-87664, Chemic.
al Abstracts, 106: 32842z), of which 4- (3,5-dibromo-2-pyridylazo) -N-ethyl-N- (3-sulfopropyl)
Aniline (hereinafter sometimes abbreviated as DiBr-PAESA)
Is a highly sensitive detection reagent that has a high extinction coefficient when bound to porphin and then copper ion (“Clin. Chem.” 35
Vol. 4, p. 522-554 (1989)). Moreover, the absorption curve of the chelate compound of this compound and copper shifts to the long wavelength side in the presence of an anionic surfactant (“Anal.Chim.Acta”).
176 Vol. 41 (1985)), changes in color tone before and after chelate formation (red to purple) by using anionic surfactant together.
Can be visually discriminated and can be said to be a detection reagent suitable for screening. A colorimetric assay method for copper ions using this compound has already been proposed for serum samples.
For example, in “Anal. Chim. Acta”, Vol. 176, p. 41 (1985), TCA (trichloroacetic acid) was added to a serum sample for protein denaturation to release copper ions bound to serum ceruloplasmin. Is colorimetrically measured with the above compound. On the other hand, "Clin. Chem." Vol. 35, No. 4, 522-554 (1
989), in order to omit the above TCA treatment, ascorbic acid is added to release the copper ion bound to serum ceruloplasmin, and this is colorimetrically measured with the above compound.

Serum ceruloplasmin does not exist in urine, but it is considered that copper ions in urine are also coordinated with proteins in urine. Therefore, even when measuring copper ions in urine, "Clin. Chem." Vol. 35, No. 4, 522-554
Page (1989), same as 4- (3,5-dibromo-2-
It is considered that the use of a pyridylazo) -N, N-diethylaniline derivative in the presence of ascorbic acid and an anionic surfactant is advantageous in terms of sensitivity. However, when the pH at the time of reaction is low and the presence of free ascorbic acid, the generated dye fades very quickly, and it becomes difficult to discriminate after about 30 minutes. In a screening test in which a large number of samples are tested, it may take some time from reaction to determination, and free ascorbic acid cannot be used at a low pH.

The present inventors have already clarified the peculiarities in the case of such a urine sample, and as a solution thereof, use ascorbate in place of ascorbic acid and further adjust the pH during the reaction from 4.5 to 7.5. It was proposed that the range be set to (Japanese Patent Application No. 2-229934). That is, the reagent composition for detecting urinary copper ions proposed herein is a 4- (3,5-dibromo-2-pyridylazo) -N, N-diethylaniline derivative,
Ascorbate, surfactant, and pH at the time of use
It contains a pH adjusting agent or a pH buffering agent in the range of 4.5 to 7.5. Furthermore, the inventors have made it possible to impregnate and hold this reagent composition in a water-absorbing porous material, which is suitable for a screening test for urinary copper ions, is simple in operation, and allows trace amounts of copper ion concentrations to be visually observed by color change or the like. We proposed analytical elements (test pieces) that can be judged.

However, when the dry analysis element disclosed in Japanese Patent Application No. 2-229934 is further examined,
It has become clear that when the storage period after the element is manufactured and used is extended, the color former is deteriorated and the detection sensitivity of copper ions is significantly impaired. The present inventors have completed the present invention as a result of studies to improve the storage stability of such a dry analytical element.

[0008]

That is, the object of the present invention is to provide a reagent dry analytical element for detecting copper ion in urine, which can be stored for a long time after production and is suitable for screening which can be immediately judged by visual inspection. Especially.

[0009]

[Means for Solving the Problems] The above-mentioned problems are 4- (3,5-
Dibromo-2-pyridylazo) aniline N, N-dialkyl derivative, ascorbate, surfactant, cationic polymer compound, water-absorbing water containing a reagent composition containing pH-adjusting agent in the range of pH 4.0 to 8.0 A dry analytical element for the detection of copper ions in urine, characterized in that it comprises a porous material.

That is, the present invention provides 4- (3,5-dibromo-2-
DISCLOSURE OF THE INVENTION The inventors found that a cationic polymer compound is extremely effective for improving the storage stability of a dry analytical element containing an N, N-dialkyl derivative of pyridylazo) aniline (color former) and ascorbate. It is based on the above, and by containing a cationic polymer compound together with these reagents in a water-absorbing porous material, the storage stability after use of the element and before use is improved. The details of how the cationic polymer compound contributes to the storage stability of the reagent are unknown. It is a color former
The N, N-dialkyl derivative of 4- (3,5-dibromo-2-pyridylazo) aniline has a low pK _a2 of 2-3, and dissociates under the pH condition of the analytical element (range 4.0 to 8.0) to give anionic properties. It is a substance. On the other hand, ascorbate is also anionic. Since these anionic substances form a salt with the cationic polymer compound and are immobilized, it is considered that the deterioration of the color former due to the ascorbate is prevented.

[0011]

Description of means for solving the problems is a compound which develops color by binding the color former copper ions (color former) 4-
As the N, N-dialkyl derivative of (3,5-dibromo-2-pyridylazo) aniline, one represented by the following general chemical formula is used.

[0012]

[Chemical 1]

Wherein R is an alkyl group (eg methyl,
And an sulfoalkyl group (eg, sulfomethyl, 2-sulfoethyl, 3-sulfopropyl group, etc.), and R ′ is a lower alkyl group (eg, methyl, ethyl, propyl group, etc.). Of these, a particularly preferred compound is 4- (3,5-dibromo-2-pyridylazo) -N, N-diethylaniline (Chemica
l Abstracts registration number (abbreviated as CASRN) 100743-66-6),

[0014]

[Chemical 2]

4- (3,5-dibromo-2-pyridylazo) -N-ethyl-N- (3-sulfopropyl) aniline represented by the following chemical formula (CASRN: 100743-65-5; abbreviated name: DiBr- PAESA).

[0016]

[Chemical 3]

[0017] Instead of using the ascorbate-free ascorbic acid, using an ascorbate such as free sodium ascorbate or adding an alkali to the ascorbic acid, by a range of pH from 4.0 8.0, copper ions It is possible to substantially eliminate the receding color due to the color formation due to the binding with the color forming agent. As the alkali added to free ascorbic acid, sodium hydroxide, potassium hydroxide and the like are generally used. Sodium as ascorbate,
An alkali metal salt of potassium and an alkaline earth metal salt of calcium or magnesium can be used.

Cationic Polymer Compound The cationic polymer compound is extremely effective for improving the storage stability of the dry analytical element over time. As the cationic polymer compound, a polymer having a repeating unit represented by the following chemical formula is used.

[0019]

[Chemical 4]

Here, L is a linking group connecting Z ⁺ and an atom in the polymer main chain, and Z ⁺ is a linear or heterocyclic ammonium, phosphonium or sulfur-containing group represented by the following structural formula. Is.

[0021]

[Chemical 5]

Where Q ⁺ is N ⁺ or P ⁺ and R
¹ , R ² and R ³ may be the same or different and each is a hydrocarbon having less than 8 carbon atoms selected from alkyl, allyl, aralkyl and aryl, and D is a heterocycle. The necessary atom, X ^−, is an acid anion such as halide ion, nitrate, methosulfate, p-toluenesulfonate.

As the cationic polymer compound, a homopolymer consisting of the above-mentioned monomer and a copolymer containing 25 to 90% by weight of the above-mentioned monomer are used. Preferred cationic polymer compounds include the following compounds described in JP-A-53-89796. Poly (N, N, N-trimethyl-N-vinylbenzylammonium chloride), styrene- [benzyl (dimethyl-p-vinylbenzylammonium chloride] copolymer, styrene-[(vinylbenzyl)-(trihexyl)
Ammonium chloride] copolymer, (N, N, N-trimethyl-N-vinylbenzylammonium chloride) -styrene copolymer, styrene- (N-vinylbenzylammonium chloride) -divinylbenzene copolymer. Also, J.Am.C
hem.Soc., 75, 3326 (1953),
Poly- (4-vinylbenzyl) -3'-carbamoylpyridinium chloride and poly- (4-vinylbenzyl)-(1'-methylpiperidine) chloride can also be used. Also, J.Am.Chem.So
c., Vol. 77, p. 514 (1955), polyvinyldimethylsulfoniumnium bromide can also be used.

On the acidic side where the pH adjuster pH is less than 4.0, the color of the dye formed by the binding of the color former and the copper ion disappears. On the alkaline side above pH 8.0, in the case of a urine sample, the color stability of the dye formed by the combination of the color former and copper is poor. Therefore, the reagent composition contains a pH adjusting agent having a pH in the range of 4.0 to 8.0. As the pH adjuster, an alkaline agent such as sodium hydroxide or an acid agent such as acetic acid, phosphoric acid or citric acid can be appropriately used. It is also possible to use a pH buffering agent as a pH adjusting agent to adjust the pH to a range of 4.0 to 8.0. pH buffers include acetic acid, phosphoric acid, succinic acid, citric acid, phthalic acid, and Good buffers (see “Biochemist
ry ", Vol. 5, pp. 467-477 (1966)," Anal. Biochem. "10
Volume 4, pages 300-310 (1980)).
(N-morpholino) ethanesulfonic acid), BIS-Tri
s, ADA (N- (2-acetamido) iminodiacetic acid), P
IPES (piperazine-N, N'-bis (2-ethanesulfonic acid), ACES (N- (acetamido) -2-aminoethanesulfonic acid), MOPSO (3- (N-morpholino) -2-hydroxypropanesulfonic acid) ), BES (N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid) and the like can be used.

The Surfactant The surfactant increases the color density of the copper chelated color former, further the absorption curve is used to shift to the long wavelength side. As the surfactant for that purpose, not only an anionic surfactant but also a nonionic surfactant can be used. Examples of the anionic surfactant used include alkyl sulfate ester salts, polyoxyethylene alkyl ether sulfate ester salts, polyoxyethylene phenyl ether sulfate ester salts, alkylbenzene sulfonates and alkane sulfonates. Typical examples include sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium polyoxyethylene alkylphenyl ether sulfate, and the like. As the nonionic surfactant, sorbitan fatty acid ester, pentaerythritol fatty acid partial ester, propylene glycol monofatty acid ester, glycerin fatty acid monoester, polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene polyoxypropylene glycol, Polyoxyethylene fatty acid partial ester, polyoxyethylene sorbitol fatty acid partial ester, polyoxyethylene fatty acid ester, fatty acid diethanolamide, fatty acid monoethanolamide, polyoxyethylene fatty acid amide and the like can be used. Specific examples of preferable nonionic surfactants include polyoxyethylene octyl phenyl ether (Tri
ton X-100, etc.) and p-nonylphenoxypolyglycidol.

Aspect 1 of Configuration of Analytical Element As one aspect of the present invention, there is an analytical element for detecting copper ions, which is made of a water-absorbing porous material containing a reagent composition. As the water-absorbing porous material, filter paper, sulfuric acid paper, non-woven fabric, microfilter and the like can be used. The thickness of the material used is about 0.3 mm to about
It is in the range of 2 mm, preferably about 0.5 mm to about 1.2 mm.
When the thickness is small, the amount of the sample sucked in is small, so that the color change due to the reaction is small and it becomes difficult to make a visual judgment.
On the other hand, if the sample is too thick, the sample is likely to be unevenly sucked, and the determination may be erroneous. The method for incorporating the reagent composition into the water-absorbing porous material can be carried out by immersing the water-absorbing porous material in a solution of the reagent composition and drying. In addition, it can be carried out by preparing a material using a composition liquid containing a reagent composition in the porous material preparation stage. Specifically, it can be produced by dissolving the reagent composition in a papermaking solution and making a paper at the step of making a filter paper.

Aspect 2 of configuration of analysis element: Another aspect of the present invention
As an embodiment, a water-impermeable support is coated with an aqueous solution of a natural polymer or a synthetic polymer to form a water-absorbing layer (hydrophilic polymer layer), and a porous material layer is laminated on the water-absorbing layer (one-layer structure) In the element, there is an integrated multi-layer element for detecting copper ion in urine in which a porous layer contains a reagent composition for detecting copper ion.

As the water-impermeable support, a transparent organic polymer film such as polyethylene terephthalate, cellulose ester (cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), polycarbonate, polystyrene, polymethylmethacrylate or the like is used. Can be used. A white opaque support can also be used. In this case, titanium dioxide fine particles and the like can be dispersed and contained when the above organic polymer film is formed.

The water absorbing layer is a layer whose main component is a hydrophilic polymer which absorbs water and swells. Examples of hydrophilic polymers that can be used include gelatin, gelatin derivatives, agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, and polyvinylpyrrolidone. The coating amount of the water absorbing layer when dried is about 3 g / m ² to about 100 g.
/ m ² , preferably in the range of about 5 g / m ² to about 30 g / m ² .

A non-fibrous porous material or a fibrous porous material is used for the porous material layer. As the non-fibrous porous material, a membrane filter, a non-woven fabric or the like is used.
As the fibrous porous material, filter paper, woven fabric, knitted fabric and the like are used.

When the reagent composition is contained in the water-absorbing porous material or the multilayer integrated element, the content thereof is N of 4- (3,5-dibromo-2-pyridylazo) aniline per 1 m ² .
The N-dialkyl derivative is preferably in the range of 25 μmol to 180 μmol. When the amount is less than this amount, the color change due to the binding of copper ions is small and the visual determination is difficult, and when the amount is large, the coloring density of the color former itself is high and the color change due to the copper ion binding is difficult to be visually determined.
Cationic polymer compounds in the range 60 mg to 12 g, ascorbate in the range 3 mmol to 180 mmol, surfactants in the range 5 mmol to 25 mmol, and 24 mmol to 300 mmol with pH modifiers or buffers. It is preferred that the millimolar range is contained per m ² .

As an example of incorporating a reagent composition into a water-absorbing porous material, an aqueous solution of a cationic polymer compound is impregnated at a rate of 1 mL per 15 cm ² of filter paper, dried and then added to an acetic acid-sodium acetate buffer solution. (3,5-dibromo-2
-Pyridylazo) -N-ethyl-N-sulfopropylaniline (DiBr-PAESA), p-nonylphenoxyglycidol and a solution in which sodium ascorbate is dissolved can be impregnated and dried. As an example of incorporating the reagent composition into the monolithic multi-layer element, white cellulose triacetate film (
(Support)) acetic acid-a solution of a cationic polymer compound in a sodium acetate buffer solution in a sodium acetate buffer solution, p-nonylphenoxyglycidol and sodium ascorbate are applied and dried, and a tricot knitted fabric is laminated on it, and then applied. 4- (3,5-dibromo-2-pyridylazo) -N,
It can be prepared by coating and drying a methanol solution of N-dialkylaniline. The coating amount of the water absorption layer when dried is about 3 g
/ m ² to about 100 g / m ² , preferably about 5 g / m ² to about 30 g / m ^2.
In the range, the thickness is in the range of about 3 μm to about 100 μm, preferably in the range of about 5 μm to about 30 μm.

[0033]

Preferred Embodiments The preferred embodiments of the present invention are summarized as follows. (1) 4- (3,5-dibromo-2-pyridylazo) aniline N, N
-Dialkyl derivative, ascorbate, surfactant,
A dry analytical element for detecting copper ions in urine, comprising a water-absorbing porous material containing a cationic polymer compound and a reagent composition containing a pH adjusting agent having a pH in the range of 4.0 to 8.0. (2) The dry analytical element according to (1), which comprises one layer of a water-absorbing porous material containing the reagent composition. (3) The dry analytical element according to (1), wherein a hydrophilic polymer layer and a water-absorbing porous material layer containing the reagent composition are integrally laminated in this order on a water-impermeable support. (4) The N, N-dialkyl derivative of 4- (3,5-dibromo-2-pyridylazo) aniline is 4- (3,5-dibromo-2-pyridylazo) -N-ethyl-N- (3- The dry analytical element according to (1), which is sulfopropyl) aniline or 4- (3,5-dibromo-2-pyridylazo) -N, N-diethylaniline.

[0034]

Example 1 Preparation of Reagent Composition Solution 4- (3,5-Dibromo-2-pyridylazo) -N-ethyl-N-sulfopropylaniline (DiBr-PAESA) 100 μg, p-nonylphenoxypolyglycidol 50 mg, 25 mg of sodium ascorbate was dissolved in 1 mL of 0.2 M MES-sodium hydroxide buffer (pH 6.0).

Preparation of Test Specimen with Filter Paper Impregnated with Composition The blood-collecting filter paper made by Advantech was divided into two parts, and one was treated as a test piece 1 for a comparative example without any treatment. The other was immersed in a 0.9% aqueous solution of polyvinylbenzyl-N-methylpiperidine chloride, which is a cationic polymer compound, and dried to obtain a test piece 2 for preparing an example. Then, dip both test pieces 1 and 2 in the reagent composition, and 1 mL of the composition per 15 cm ^{2 of} filter paper.
Was impregnated in the ratio of. After this was dried, it was cut into pieces of 2 mm × 5 mm and attached to a white stick made of polyethylene terephthalate with a double-sided tape to prepare test pieces of Examples and Comparative Examples.

Urine samples S1, S2, and S3 were included in both test pieces prepared by observing the color change of the test piece by the urine sample, and the color change was observed. The color change occurred within 10 seconds.
The subject S1 was a healthy person, and the amount of copper in urine measured by the atomic absorption method was 0.1 ppm or less. S2 and S3 were urine of Wilson's patients, and the amount of copper in the urine was 1.1 ppm and 2.1 ppm, respectively. The following Table 1 shows the coloration result of each test piece after the sample was spotted.

[0037]

[Table 1]

In all the test pieces, the color change was clearly observed in the samples S2 and S3. After inserting a test piece into a urine sample to contain urine, it is clear that a simple operation of observing the color change makes it possible to easily screen whether or not a further detailed examination is required. is there.

Color change due to urine sample after aging of test piece Each test piece was allowed to stand in a refrigerator at 5 ° C. for 6 months and then taken out, and the same test as above was carried out to observe the color change. The results are shown in Table 2.

[0040]

[Table 2]

With the reagent piece 1 of the comparative example, it was difficult to distinguish between the healthy subject sample S1 and the Wilson disease patient samples S2 and S3 after 6 months. However, in the example of the present invention (test piece 2) impregnated with a polymer containing a diethylaminoethyl group, a clear color change was observed in the subjects S2 and S3, which was clearly distinguishable from a healthy person. Thus, it was shown that the test piece of the present invention can be stored for a long period of time after the test piece is prepared.

[0042]

Example 2 Preparation of test piece using monolithic multi-layer element A water-impermeable polyethylene terephthalate film (support) was coated with 10% by weight aqueous gelatin solution as an undercoat layer, and this layer was knitted as a porous material. A fabric (polyethylene terephthalate spun yarn equivalent to 50 denier, 36 gauge knitted tricot knitted fabric having a thickness of about 250 μm) was applied and dried. On top of this, the reagent composition of Example 1 was knitted on fabric 15
The composition was applied at a ratio of 1 mL per cm ² and dried. this
Cut into 4mm x 4mm pieces, and attach the support side to a white stick made of polyethylene terephthalate with double-sided tape Test piece 3
(Comparative example) was produced. On the other hand, as a knitted fabric to be laminated on the gelatin subbing layer, a cationic polymer of polyvinylbenzyl-N-methylpiperidine chloride was previously prepared.
A knitted fabric impregnated with a 0.9% aqueous solution was used, and otherwise the same as the test piece 3 (comparative example), and a test piece 4 (example) was obtained.

Observation of Color Change by Urine Sample Each test piece was put into a urine sample to be examined, and the urine sample was contained therein. The color change occurred within 10 seconds. The results are shown in Table 3 below.

[0044]

[Table 3]

Color change was clearly observed in the specimens S2 and S3 of each test piece in the urine to be examined. It is possible to easily screen whether or not further detailed examination is required by a simple operation of observing the color change after inserting a multi-layer integrated test piece into a urine sample and including urine. Is clear.

Color change due to urine sample after aging of each test piece Each test piece was allowed to stand in a refrigerator at 5 ° C. for 6 months and then taken out, and the same test as above was carried out to observe the color change. The results are shown in Table 4 below.

[0047]

[Table 4]

With the reagent piece 3 of the comparative example, it was difficult to distinguish the normal human sample S1 from the Wilson disease patient samples S2 and S3 after 6 months. However, in the example of the present invention (test piece 4) impregnated with a polymer containing a diethylaminoethyl group, a clear color change was observed in the subjects S2 and S3, which was clearly distinguishable from a healthy person. Thus, it was shown that the test piece of the present invention can be stored for a long period of time after the test piece is prepared, even as a multilayer analytical element. Even after long-term storage, the convenience was not lost, and the visual judgment could be reliably performed.

[0049]

As described above, the analytical element for detecting copper ions in urine of the present invention can be stored for a long period of time after production,
Used for screening test for the presence of copper ions in urine samples,
This has an excellent effect that the visual determination can be reliably performed on a large number of samples in a short time with a simple operation.

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Claims (1)

[Claims] 1. An N, N-dialkyl derivative of 4- (3,5-dibromo-2-pyridylazo) aniline, an ascorbate, a surfactant, a cationic polymer compound, a pH of 4.0 to 4.0.
A dry analytical element for detecting copper ions in urine, comprising a water-absorbing porous material containing a reagent composition containing a pH adjusting agent in the range of 8.0.