JPS6239773A - Monolithic multilayered analytical element for analyzing bilirubin - Google Patents

Abstract

PURPOSE:To secure the preservability of a diazonium salt without lowering the developability and color forming property of bilirubin, by using an anionic surfactant as a surfactant. CONSTITUTION:At least one water absorbing layer and a porous reagent layer are laminated to a light pervious and water impermeable support in this order and a diazonium salt capable of forming azobilirubin upon the reaction with bilirubin is contained in the porous reagent layer. By containing an anionic surfactant in this monolithic multilayered analytical element, the storage stability of the diazonium salt is secured without lowering the developability and color forming property of bilirubin.

Description

[Detailed Description of the Invention] [Field of Industrial Application] Bilirubin, which is the main component of bile pigments in biological fluids, is produced in blood serum by the decomposition of heme derived from hemoglobin in red blood cells, and then transferred to the liver. After being taken up, it is excreted into bile as glucuronic acid conjugates. Bilirubin in serum increases with an increase in hemoglobin content and a decline in liver function, so quantitative analysis of bilirubin is considered an essential test item for clinicopathological diagnosis.

The present invention relates to an integrated multilayer analysis element that is used to dryly measure bilirubin simply, quickly, and with high precision.

[Conventional technology]

Regarding bilirubin in serum, there is a method for quantifying bilirubin's inherent yellow color by absorbance measurement, and Van den
Ehrlich was discovered by Bergh.
diazotized sulfanilic acid (
p-sulfobenzenediasonium, Ehrlich's reagent)
A method for colorimetrically quantifying red azobilirubin formed by a coupling reaction between bilirubin and bilirubin (generally called the "diazo method") is known.

Many attempts have been reported to quantify bilirubin by the diazo method using dry analytical elements that can be easily handled as foam. Descriptions regarding them are, for example,
No. 3-28119, JP-A-54-33094, JP-A No. 55
-44992, 56-10255, 56-30
This can be found in various publications such as No. 503 No. 503 and No. 57-23859.

An example of using a surfactant to promote the reaction between bilirubin and diazonium salt in this dry analysis element is as follows.
For example, JP-A-58-134067, JP-A-59-
It is disclosed in JP-A-171864, JP-A-59-230160, JP-A-60-10171, and the like. Nonionic surfactants such as p-octylphenoxypolyethoxyethanol and p-nonylphenoxypolyglycidol have been used exclusively as the surfactant.

[The problem that the invention attempts to solve]

When bilirubin is analyzed by the diazo method using dry analytical elements, storage stability of diazonium salts is generally a problem. Therefore, in order to improve the storage stability of noazonium salts, malic acid, sulfosalicylic acid, etc.
Countermeasures have been taken, such as adding p-styrenesulfonic acid, acrylic acid, etc. (Japanese Unexamined Patent Publication No. 171864/1986) to the layer containing the diazonium salt. As a result of various studies aimed at further enhancing the storage stability of this diazonium salt, the present inventor happened to find that the nonionic surfactant conventionally used inhibits the storage stability of the diazonium salt. This is considered to be one of the reasons why conventionally used nonionic surfactants are water-absorbing, increasing the water content and deteriorating the storage stability of the diazonium salt.

Therefore, an object of the present invention is to ensure the storage stability of diazonium salts without reducing the spreadability and coloring properties of bilirubin.

Another object of the present invention is to further improve the storage stability of diazonium salts.

[Means for solving problems]

These objects of the present invention are achieved by using an anionic surfactant as the surfactant.

That is, the present invention provides at least one water-absorbing layer and a porous reagent layer that are laminated in this order on a light-transparent water-impermeable support, and also includes noazonium that can react with bilirubin to produce azobilirubin. The present invention relates to an integrated multilayer analytical element for bilirubin analysis, characterized in that the integrated multilayer analytical element contains a salt in the porous reagent layer, and contains an anionic surfactant.

As the light-transmitting and water-impermeable support, known supports used in general multilayer analytical elements can be used. Specific examples include polyethylene terephthalate, polycarbinate of bisphenol A, folystyrene, and cellulose ester (cellulose).

50 #L to about 1 m, preferably about 80 μm
About 3004! A range of transparent supports can be used. If necessary, the surface of the support is provided with a well-known T-layer or VC layer according to the above-mentioned product specifications, etc., to strengthen the adhesion with the water-absorbing layer, etc. provided on the support. be able to.

The water absorption layer increases the concentration of the analyte that diffuses into the porous reagent layer by absorbing the solvent of the sample liquid without allowing the analyte bilirupine in the sample liquid to penetrate, thereby increasing the concentration of the analyte that is cypyrirupin and the coloring reagent. This layer has the effect of increasing the reaction efficiency with the diazonium salt.

The water-absorbing layer is composed of a material whose main component is a hydrophilic polymer, and has groups that easily absorb water that has penetrated into the layer through strong hydrogen bonds, has a high swelling rate, and has a high swelling rate. It is preferable to use a polymer that is difficult to dissolve in.

Specific examples of hydrophilic polymers used in the water absorption layer include:
gelatin described in JP-A-59-171864, etc.;
Natural polymers such as dextran, starch, and edible starch, and their crosslinked products; Examples include homoprimers or copolymers made of various monomers, copolymers of acrylamide and the above monomers, and crosslinked products thereof.

Among them, gelatin is particularly preferred.

Further, the water absorption layer can contain a basic polymer or an acidic polymer. The basic polymer or acidic polymer in the water absorption layer works with the acidic polymer contained in the porous reagent layer to adjust the pH of the reagent layer and buffer the reaction environment between the analyte and diazonium salt. Therefore, the high precision quantification achieved by the analytical element of the present invention can be further enhanced. = Basic polymers or acidic polymers that can be used for the above purpose include basic polymers or acidic primers described in JP-A-59-171864, JP-A-59-143959, etc. be able to.

The thickness of the water-absorbing layer is preferably in the range of 3 to 50 μm, particularly preferably in the range of 10 to 30 μm.

Examples of materials constituting the matrix of the porous reagent layer include membrane filters (plush primer single layer) disclosed in Japanese Patent Publication No. 53-21677, etc., imitation particles such as polymer micro♂♂, glass micropeas, and diatomaceous earth. A porous layer containing continuous micropores held in a hydrophilic polymer binder, in which polymer microbeads, glass microbeads, etc. disclosed in JP-A-55-90859 are bonded in point contact with a polymer adhesive that does not swell with water. A non-fibrous isotropic porous layer such as a continuous microvoid-containing porous layer (three-dimensional lattice-like granular structure layer), JP-A-55-164356,! ! f1f Fabric layer disclosed in Japanese Patent Publication No. 57-148250 KM
(f) A fibrous porous layer such as a layer made of paper containing organic $rimer fibers and matrix. The porous layer used as the porous reagent layer mentioned above also has a function as a spreading layer. If the function as a spreading layer is insufficient, a porous layer functioning as a spreading layer can be laminated and integrated on the porous reagent layer.

A method of laminating and integrating a spreading layer on a porous reagent layer is described, for example, in Japanese Patent Application No. 59-120957 and Japanese Patent Application No. 1983.
It can be carried out according to the method described in Japanese Patent No.-126267.

The diazonium salt used as a bilirubin detection indicator in the present invention can react with bilirubin to produce azobilirubin. Publication No. 58-134067, JP 59-145965
No. 59-171864. “Clinical Chemistry Analysis ■Nitrogen-containing Components” 2nd edition, edited by the Japanese Society of Analytical Chemistry (Tokyo, (
Tokyo Kagaku Dojin Co., Ltd., published in 1979) No. 248-279
Examples include various diazonium salts described on page 12.

Preferred compounds among the diazonium salts used in the analytical element of the present invention are p-sulfobenzenediazonium salt and 2,4-dichlorobenzenediazonium! , 2.5
-dichlorobenzenediazonium salt, p-nitrobenzenediazonium salt, etc.! ,) get lost. As salt,
Tetrafluorophosphate, hexafluorophosphate)
, p-1 luenesulfonate, sulfosalicylate, and the like.

The content of diazonium salt is the analytical element L of the present invention.
The range is from about 1 mmol to about 150 mmol per m2, preferably from about 2 mmol to about 100 mmol.

The integrated multilayer analysis element for bilirubin analysis of the present invention is characterized in that it contains an anionic surfactant.

As the anionic surfactant, a sulfonic acid type surfactant is preferable since it has a large effect on stabilizing the noazonium salt. Such sulfonic acid surfactants include alkyl sulfate ester salts, preferably Na, K or Ll salts of alkyl sulfates having 10 to 18 carbon atoms, such as lithium lauryl sulfate, sodium myristyl sulfate. , sodium stearyl sulfate, etc., dialkyl sulfosuccinates, preferably Na + dialkyl sulfosuccinates having from 5 to 18 carbon atoms per alkyl group.
K or Ll salts, such as sodium di(2-ethylhexyl)sulfosuccinate, sodium dioctadecylsulfosuccinate, sodium diisobutylsulfosuccinate, and 4-lyoxyethylene alkyl ether sulfate salts, preferably having 10 carbon atoms -18 alkyl and a polyoxyethylene alkyl ether sulfate salt containing a range of 8 to 15 oxyethylene units, e.g. Examples include sodium lyoxyethylene lauryl ether sulfate. Among these sulfonic acid-based anionic surfactants, alkyl sulfate ester salts and dialkyl sulfosuccinates are preferred.

The anionic surfactant is preferably contained in the same porous reagent layer as the noazonium salt. The content is approximately 0.2 per analytical element 1-z. to about 2, preferably about 0.3? About 1.5? A certain amount within the range of is appropriate.

The analytical elements of the present invention include nonionic interfaces such as polyoxyethylene alkylphenyl ethers typified by octylphenoxypolyethoxyethanol and polyglycitol alkylphenyl ethers typified by nonylphenoxypolyglycidol, which are similar to those conventionally used. An active agent can be used in combination in an amount within a range that does not impair the action of the anionic surfactant.

As other reagents, acidic polymers are included in the reagent layer. The acidic polymer has the effect of stably preserving the diazonium salt in the analytical element, and if a basic polymer is present in the water absorption layer, it works with the basic polymer to stabilize the reagent layer. It also shows the effect of buffering the reaction environment between the analyte and the diazonium salt.

Examples of acidic polymers include p-styrenesulfonic acid,
Acrylic acid, methacrylic acid, maleic acid, maleic acid m
hydrate, maleic acid monoamide, maleic acid monoester, N-(sulfoalkyl)acrylamide [preferably N-(β-sulfo-t-butyl)acrylamide, etc.]
, and N-(sulfoalkyl)methacrylamide [preferably N-(β-sulfo-t-butyl)methacrylamide, etc.], or a polymer of monomers having one or more unsaturated bonds selected from the group consisting of and other unsaturated bond-containing monomers.

Note that the acidic polymer can also be used as a mixture with other water-soluble polymers such as gelatin and polyvinyl alcohol, or as a mixture with an aqueous latex of a hydrophobic polymer.

The reagents contained in the analytical element of the present invention include diazonium salts and acidic reagents. In addition to the reamer, reagents such as a diazo coupling reaction accelerator and other substances can be contained as necessary.

Reaction accelerators are known in the art and are known in the art.
Examples include alcohol (e.g., methanol, ethanol, etc.), caffeine, sodium benzoate, sodium acetate, dyphyllin (7-(2,3-dihydroxypropylthiophylline (479-18-5)), urea,
These include gum arabic, fluorionic surfactants, acid amides, and sulfoxides.

The integrated multilayer analytical element of the present invention may have a water absorption layer or a porous reagent layer divided into two or more layers and may be laminated, or layers other than these may be provided. Examples of such layers include a spreading layer, an analyte (bilirubin, a combination of bilirubin and other substances, etc.) diffusion prevention layer, a light blocking layer, and the like.

Among the various reagents mentioned above, diazonium salts and acidic polymers are contained in the reagent layer, but other reagents may be contained in other layers. For example, a spreading layer may be provided and an anionic surfactant may be added thereto.

The integrated multilayer analytical element of the present invention can be manufactured according to the known methods described in the aforementioned patent specifications and the like. In order to incorporate the above-mentioned diazonium salt, anionic surfactant, acidic polymer, etc. into the porous reagent layer, for example, a solution of these reagents is impregnated or applied to the material of the porous reagent layer, and then dried. method can be used. Alternatively, a water absorption layer and a reagent layer are sequentially laminated on a support, and the impregnating liquid is applied to the top layer of the integrated material,
A drying method can also be used.

The integrated multilayer analytical element of the present invention has a negative side of about 15 to about 3
Cut into small pieces such as 0 square or circular pieces of approximately the same size,
Publication No. 6079, Japanese Utility Model Application Publication No. 56-142454, Publication No. 32350 of Japanese Utility Model Publication No. 58-32350, Special Publication No. 58-50114
It is preferable to use it as an analysis slide by placing it in a slide frame as disclosed in Publication No. 4 or the like from all viewpoints such as manufacturing, packaging, transportation, storage, and measurement operations.

The integrated multilayer analytical element of the present invention can be measured according to the method disclosed in the above-mentioned product specification. That is, from about 5 μl to about 30 μl, preferably from about 8 μl to about 115 μl! A sample of the aqueous liquid is spotted onto the top layer of the porous reagent layer, spreading layer, etc. and inked at a substantially constant temperature ranging from about 20°C to about 45°C. Then, from the light-transmitting support side, the color optical density of azobilirubin produced by the coupling reaction between bilirubin and diazonium salt in the multilayer analytical element is determined by reflection photometry, and the color optical density of the azobilirubin produced in the multilayer analytical element is determined by reflection photometry. Quantify bilirubin.

Bilirubin in serum is classified into direct bilirubin and indirect bilirubin depending on the binding form. In other words, free bilirubin, which is produced by the decomposition of heme derived from hemoglobin in red blood cells, is highly hydrophobic and has poor water solubility, so the reaction rate with diazonium salts is slow, whereas encapsulated or albumin-bound bilirubin is It has high water solubility and the reaction rate with diazonium salts is fast. Therefore, in the determination of bilirubin based on the conventional diazo method, direct bilirubin (inclusive and protein-bound bilirubin) and indirect bilirubin (free bilirubin) are determined by utilizing the above-mentioned reaction rate differences between various types of bilirubin. Each was quantified.

Similarly, in the analytical element of the present invention, direct bilirubin and indirect bilirubin can be respectively quantified based on the same principle.

[Effect]

Anionic surfactants, like nonionic surfactants, enhance the spreadability and reactivity of bilirubin due to their surfactant action. In addition, the sulfonic acid-based anionic surfactant preferably used in the present invention reduces the deterioration of the diazonium salt, which is a coloring reagent, over time, so the shelf life of the bilirubin analysis element can be significantly extended without deterioration of its performance.

〔Example〕

Example 1 Gelatin F-72 was applied onto a 180-piece polyethylene (PET) film with a gelatin undercoat.
67, f! I) An aqueous solution containing 0.031 P of oxyethylene nonylphenyl ether (containing 8 to 15 oxyethylene units) and 30 η of bis(vinylsulfonylmethyl) ether was applied to a thickness of 20 tmbllc after drying and dried to form a water-absorbing layer. Established.

Next, water was supplied as dampening water at a rate of 3097 m'' to moisten the water absorbing layer, and then the PET was made hydrophilic by glow discharge.
Tricot knitting made of spun fiber yarn (average thickness 250 mm) was laminated by pressure bonding.

On top of that, 1 m2 equivalent of an aqueous reagent composition solution with the following composition,
920017 was applied and dried to form a porous reagent layer, thereby preparing an integrated multilayer analytical element for bilirubin quantification.

Daphyllin 15nosulfosalicylic acid 307 water
70116 Acetone 50ml As a comparative example, the same amount of polyoxyethylene nonylphenyl ether (containing an average of 10 oxyethylene units) was added to the sodium lauryl sulfate in the reagent composition (Comparative Example 1), and the same amount was added without sodium lauryl sulfate. (Comparative Example 2) was prepared.

After thoroughly drying these elements, heat at 4℃, 25℃ or 45℃.
Diazonium 1st base (residual amount) after being left at ℃ for 6 days
was measured to determine the residual rate of diazonium salt. For the analysis of diazonium salts, the sample was cut into square chips with 15 mm sides, placed in a 20 m test tube, and mixed with 500 m of disodium chromotrope, 100 M of water, and 1001 r of ethanol.
After the addition of 10 mA', the reagent solution consisting of Ll was left at room temperature for 30 minutes, and the absorption was measured using a spectrophotometer with light having a wavelength of 510 nm for quantification. The value obtained as a result was calculated as a percentage, assuming that the diazonium salt cap before storage was 100%.

Example 2 180 μm thick PET with gelatin undercoat
Vinyl alcohol-itaconic acid copolymer (containing itaconic l'+20.5 to 1.0 t) on the film 30? , sogelinol ether of butanol (crosslinking agent) 14
A coating solution containing 250 ml of water was applied to give a dry film thickness of 20 mm, and dried to form a water-absorbing layer. A developing layer was provided in the same manner as in Example 1, and then the water absorption layer was subjected to a crosslinking reaction at 60° C. for 90 hours.

An aqueous reagent composition solution having the following composition was applied onto the developing layer at a rate of 180 ml per η and dried to prepare an integrated multilayer analytical element for quantifying bilirubin.

Dyphyllin 15? Sulfosalicylic acid 30? water
70m 7 tons
50ml Comparative example: one in which the surfactant in the reagent composition contained the same amount of polyoxyethylene nonylphenyl ether (containing an average of 10 oxyethylene units) (Comparative Example 3), and one in which the surfactant was omitted. (Comparative Example 4) was prepared.

Regarding these elements, 4C125 was prepared in the same manner as in Example 1.
C. or 45.degree. C. for 6 days, and then the content of siazonium salt was measured, and the following results were obtained.

Example 3 The same amount of ethylene as the crosslinking agent for the water absorption layer in Example 2.

An integrated multilayer analytical element for quantifying bilirubin was prepared in the same manner as in Example 2 except that glycol diglycinol ether was used. The diazonium salt 6 was prepared in the same manner as in Example 1.
When the residual amount was measured after 1 day, it was found to be 100 g and 25 g at 4℃.
It was 96 inches at ℃ and 78 inches at 45℃.

〔Effect of the invention〕

The integrated multilayer analytical element of the present invention has good storage stability of diazonium salt, and can be used for bilirubin measurement even after long-term storage. In addition, the spreadability and coloring stability of bilirubin are good, and bilirubin can be measured quickly and with high precision.

Claims (7)

[Claims] (1) At least one water absorption layer and a porous reagent layer are laminated in this order on a light-transparent water-impermeable support, and a diazonium salt capable of reacting with bilirubin to produce azobilirubin is added. An integrated multilayer analytical element for bilirubin analysis, characterized in that the integrated multilayer analytical element contains an anionic surfactant in a porous reagent layer. (2) The analytical element according to claim 1, wherein the anionic surfactant is contained in the same layer as the diazonium salt. (3) The analytical element according to claim 1 or 2, wherein the anionic surfactant is a sulfonic acid surfactant. (4) The analytical element according to claim 3, wherein the anionic surfactant is an alkyl ether sulfate ester salt or a dialkyl sulfosuccinate. (5) The anionic surfactant is a sodium, potassium or lithium salt of an alkyl ether sulfate having 10 to 18 carbon atoms, or a dialkyl sulfosuccinic acid having 5 to 18 carbon atoms per alkyl group. The analytical element according to claim 3, which is a sodium, potassium or lithium salt of (6) The analytical element according to any one of claims 1 to 3, wherein the porous reagent layer is a layer that also serves as a spreading layer. (7) The analytical element according to any one of claims 1 to 3, wherein a spreading layer is further integrally provided on the porous reagent layer.