JPH0219905B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a multilayer analytical element for analyzing components in an aqueous liquid. More specifically, the present invention relates to a multilayer analytical element with improved storage stability of reagents. BACKGROUND ART Analytical elements that detect specific biochemical substances present in body fluids have been developed and used in clinical trials and the like. These methods utilize the phenomenon in which a reagent impregnated into an analytical element reacts with a substance to be detected and develops a color. Recently, particularly, analytical elements in which filter paper or the like is impregnated with reagents have been widely used. For example, US Patent No. 3050373 or US Patent No. 3061523
As described in No. 1, etc., there are some that are made by soaking a fibrous porous layer such as filter paper in a reagent solution and drying it. Furthermore, attempts have been made to avoid various interferences when performing detection using this method. For example, as described in Japanese Patent Publication No. 50-39558, a semipermeable membrane is coated by impregnating filter paper with a reagent solution and drying it, then immersing it in a solution such as ethyl cellulose and drying it. To suppress the absorption of excess liquid sample, to control the retention of the liquid sample, or to further improve the
As described in Japanese Patent No. 6551, a filter paper impregnated with a reagent is covered with a fine mesh such as fabric or web to protect it from contamination due to contact with hands or the like. Although these analytical elements are easy to handle and provide immediate results, they often have non-uniform coloration, which causes variations in analytical results, resulting in poor quantitative performance and are limited to qualitative or semi-quantitative analysis. It's on. This is because it is difficult to obtain a uniform fibrous porous layer such as a filter paper due to its structure, and even when it is impregnated with a reagent, it is usually not possible to obtain a uniform analysis of the reagent. Furthermore, when using something like a fibrous filter paper, due to the so-called chromatographic phenomenon, excessive non-uniform migration of analysis sample components or reaction reagents occurs in the reagent-containing layer, resulting in local high concentration. It's for a reason. A multilayer analytical element, such as that disclosed in Japanese Patent Publication No. 21677/1983, is known as a device that has improved the drawbacks of these conventional analytical elements and has dramatically improved quantitative performance compared to conventional analytical elements. However, the multilayer analytical elements described above also exhibit significant deterioration of the reagents contained in the carrier during storage, thus significantly penalizing the accuracy and reliability of the analysis. Because the materials that make up the multilayer analytical element are permeable to aqueous fluids, they are greatly affected by air and water, and the peroxide contained in the analytical element oxidizes the indicator composition. This is due to the deterioration of the activity of peroxidase, which exhibits catalytic action. The ability to accurately and consistently proportion the concentrations of the components of an aqueous fluid to produce a detectable product is thus hindered. Various attempts have been made to improve the above storage stability. For example, as in US Pat. No. 3,630,957, the carrier is manufactured from a hydrophobic material, as in US Pat. Nos. 3,212,855 and 3,598,704, a water-soluble polymer or a hydrophilic colloid is added to a water-absorbing carrier, A method of physically isolating reagents is disclosed. However, none of these methods lead to an essential solution. On the other hand, JP-A-54-50393 discloses that the storage stability can be improved by adding a certain type of water-dispersible copolymer to the reagent layer. However, the water-dispersible copolymer described above has the disadvantage that it is difficult to introduce an anionic monomer and aggregation occurs during production of the copolymer, deteriorating the performance as a multilayer analytical element. The present invention aims to eliminate the above-mentioned drawbacks. That is, the first object of the present invention is to provide a multilayer analytical element with improved storage stability of reagents. A second object of the present invention is to provide a multilayer analytical element that has quantitative properties and can be easily measured without requiring skilled operating techniques. Such and the following objects of the present invention are to provide a light-transparent support with at least one reagent layer containing at least one reagent that reacts with a component in a fluid sample and containing a hydrophilic colloid material; In a multilayer analytical element having a porous carrier layer above, at least one of the reagent layers has a polymer interface containing at least one of the compounds represented by the following general formula [], [], or [] as a main component. This is achieved by a multilayer analytical element characterized by containing at least one type of water-dispersible copolymer obtained by emulsion polymerization of a monomer having a vinyl group and a monomer having a vinyl group. General formula [] In the formula, R ₁ is a substituted or unsubstituted divalent organic group,
M ₁ represents a hydrogen atom or a monovalent cation, and n
represents 30 to 95 mol%, and m represents 70 to 5 mol%. General formula [] In the formula, R ₂ and R ₃ each represent a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a cyano group, or
COOR represents ₅ groups (R ₅ represents an alkyl group), and each may be the same or different.
R ₄ represents a hydrogen atom or a lower alkyl group, M ₂ ,
M ₃ , M ₄ , and M ₅ each represent a hydrogen atom or a monovalent cation, a is 30 to 95 mol%, and b is 70 to 95 mol%.
0 mol%, c is 70-0 mol%. However, b+c
is 70-5 mol%. General formula [] In the formula, R ₆ and R ₇ are a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a cyano group, or a COOR ₉ group (R ₉ represents an alkyl group). R ₈ represents a hydrogen atom or a lower alkyl group, and M ₆ , M ₇ , M ₈ , M ₉ are
Each represents a hydrogen atom or a monovalent cation.
x is 30 to 70 mol%, y is 5 to 50 mol%, z is 70 to 70 mol%
It represents 5 mol%, and y+z is 70-30 mol%. Next, typical examples of the polymeric surfactant of the present invention are shown below, but the invention is not limited thereto.

【table】

[Table] Regarding the above-mentioned polymeric surfactant and the synthesis method of water-dispersible copolymer using the same, please refer to JP-A-55-
It is described in detail in Japanese Patent No. 50240, and can be easily synthesized according to those documents. The vinyl monomers used in the present invention may be used alone or in combination as long as they are hydrophobic and capable of radical polymerization.
It is also possible to copolymerize a hydrophilic monomer such as acrylic acid with a hydrophobic vinyl monomer. Preferred vinyl monomers include copolymerizable ethylenically unsaturated nitriles, styrenes, acrylic esters, methacrylic esters, acrylamides, methacrylamides, vinyl heterocyclic compounds, and crosslinkable monomers. polymers, vinyl halides, vinylidene halides, vinyl esters, vinyl ethers, conjugated dienes,
can be mentioned. Examples of copolymerizable ethylenically unsaturated nitriles include acrylonitrile, methacrylonitrile, and α-chloroacrylonitrile. Examples of styrenes include styrene, p-methylstyrene, α-methylstyrene, p-chlorostyrene, and chloromethylstyrene. Examples of acrylic esters include methyl acrylate, ethyl acrylate, and acrylic acid-n.
-butyl, n-propyl acrylate, iso-butyl acrylate, sec-butyl acrylate, 2-hydroxyethyl acrylate, acrylic acid-
Mention may be made of 2-hydroxypropyl.
Examples of methacrylic acid esters include methyl methacrylate, ethyl methacrylate, n-butyl methacrylate, 2-hydroxyethyl methacrylate, and 2-hydroxypropyl methacrylate. Examples of acrylamides include acrylamide, diacetone acrylamide, methylol acrylamide, and methyl acrylamide. Examples of methacrylamides include methacrylamide and benzylmethacrylamide. Examples of vinyl heterocyclic compounds include N-vinylpyrrolidone, N-vinylimidazole, vinylpyridines (e.g., 4-vinylpyridine, 2-vinylpyridine,
vinylpyridine, etc.). Examples of the crosslinkable monomer include divinylbenzene, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, and pentaerythritol trimethacrylate. Examples of vinyl halides include vinyl chloride and vinyl fluoride. Examples of vinylidene halides include vinylidene chloride and vinylidene fluoride. Examples of vinyl esters include vinyl acetate and vinyl butyrate. Examples of vinyl ethers include vinyl methyl ketone, and examples of conjugated dienes include:
Examples include 1,3-butadiene, isoprene, 2,3-dimethyl-1,3-butadiene, and the like. Furthermore, examples of hydrophilic vinyl monomers include carboxylic acid monomers and salts thereof such as acrylic acid, methacrylic acid, and itaconic acid, 3-methacryloyloxypropane-1-sulfonic acid, and 2-acrylamido-2-methyl. Examples include sulfonic acid monomers such as propane sulfonic acid and salts thereof. The above-mentioned polymeric surfactant can be used in an amount of 0.01% to 90% by weight based on the vinyl monomer. Preferably, 0.5% to 80% by weight can be used. As mentioned above, various vinyl monomers can be used alone or in combination, but preferably 50% or more and 99.5% or less of hydrophobic vinyl monomers can be used. Examples of preferred hydrophobic vinyl monomers include acrylic esters, methacrylic esters,
Styrenes can be mentioned. As mentioned above, the water-dispersible copolymer of the present invention can be produced by polymerizing vinyl monomers using the polymeric surfactant of the present invention and a water-soluble radical polymerization initiator using a conventional emulsion polymerization method. However, it is also possible to use a low-molecular-weight surfactant (for example, anionic and nonionic surfactant) in combination, if necessary. For example, put degassed distilled water in a 1-4 neck flask equipped with a stirrer, cooling tube, thermometer, and nitrogen gas inlet tube.
600ml Trax H-45 (manufactured by NOF Corporation, surfactant, active ingredient 30%) 10ml 7.79g of polymeric surfactant of compound example (7), 43g of n-butyl methacrylate
Add 35.21 g of ethyl acrylate, and stir at 200 rpm at room temperature under a nitrogen stream while flowing cooling water. Once the monomers in the flask were uniformly emulsified, the internal temperature was raised to 70°C, and potassium persulfate, 0.48
Simultaneously add an aqueous solution of 0.35 g of sodium metabisulfite dissolved in 20 ml of degassed distilled water,
After polymerization for 6 hours at a rotation speed of 300 rpm and a temperature of 70° C., it is cooled to room temperature. This results in a polymeric surfactant with a polymer solid content of 11.8% and a polymerization rate of 99.9%.
A water-dispersible copolymer of (7)/n-butyl methacrylate/ethyl acrylate=9/50/41 (weight ratio) can be obtained. Other polymeric surfactants can also be synthesized by appropriately selecting monomers according to the same recipe. The light-transmitting support of the present invention (hereinafter referred to as the support of the present invention) may be of any type as long as it is liquid-impermeable. The particular material used as the support is not critical, but various polymeric materials are suitable for this purpose, such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene. The thickness of the support is arbitrary, but typically is about 50 microns to about 250 microns. Although it is possible to coat the reagent layer according to the present invention directly onto the support according to the present invention, in some cases it is possible to use a light-transparent subbing layer to improve the adhesion between the reagent layer and the support. It is also possible to increase it. The purpose of the reagent layer is to contain reagents that cause a reaction with the component to be analyzed to produce a detectable product. Preferred are coatings in which one or more reagents are dispersed or dissolved in a hydrophilic colloid material as a binder. The hydrophilic colloid substances are preferably natural or synthetic substances, more preferably polysaccharides such as gelatin, gelatin derivatives, hydrophilic cellulose derivatives, dextran, gum arabic, agarose, etc. and synthetic hydrophilic substances, such as polyvinyl Included are water-soluble polyvinyl compounds of alcohol and polyvinylpyrrolidone, water-soluble acrylamide polymers, and the like. These choices may depend in part on the optical properties of the detectable product, etc. The water-dispersible copolymer of the present invention can achieve the above-mentioned purpose by adding or partially substituting the hydrophilic colloid substance in the reagent layer. These water-dispersible copolymers of the present invention can contain from about 5 to about 60%, more preferably from about 15 to about 50%, of the total amount of binder in the reagent layer. This achieves the aforementioned objectives and others. Another purpose is to reduce curling of the coating film, and yet another purpose is to improve the dimensional stability of the coating film. The above objective is well known in the photographic industry, but also in the field of multilayer analytical elements, it can provide higher analytical reliability. A further unexpected effect was that the color density of the color reaction increased. It goes without saying that an increase in color density in an analytical reaction leads to an increase in detection sensitivity, which is extremely advantageous. By appropriately selecting the test reagents in the aforementioned reagent layer, glucose, albumin, urea nitrogen, bilirubin, ammonia, uric acid, cholesterol,
It can be easily configured for use in the analysis of blood components such as triglycerides, serum glutamate oxaloacetate transaminase, lactate dehydrogenase, lactate, creatinine, amylase, chloride, calcium, as well as many other components. Although it is effective to add the water-dispersible copolymer of the present invention to improve the storage stability of these test reagents, a particularly preferred example is to add a suitable copolymer that exhibits catalytic action to oxidize a predetermined component. Enzymes and indicator compositions that can react with peroxide in the presence of peroxidase to produce a detectable product, compounds for adjusting PH, and the like. Many examples are given in West German Publication No. 2735690. One such specific example is a reagent for glucose analysis consisting of glucose oxidase, peroxidase, 7-hydroxy-1-naphthol, 4-aminoantipyrine hydrochloride, and a phosphate buffer system with a pH of 6. The porous carrier layer for forming a multilayer analytical element with respect to the reagent layer can be a diffusion layer, a reflective layer, a blocking layer, an undercoat layer, a filtration layer, or a layer as described in U.S. Pat. It can have a registration layer. As described in the above patent specification, these are made by dispersing cellulose diacetate in various white pigments in the form of a slurry in the coexistence of a good solvent and a poor solvent, and coating the slurry on the above-mentioned reagent layer. This is called a non-fibrous porous medium layer in which a layer having a constant pore diameter and high porosity is formed. Needless to say, a microfilter known by the trade name Millipore Filter is also useful. On the other hand, similar to the above-mentioned layer, JP-A-56-
Fibrous porous carriers, such as those described in US Pat. No. 24,576, can also be used. These layers (1) uniformly distribute a fixed volume of fluid sample at a fixed volume per unit area within the reagent layer, (2) remove substances or factors that inhibit analytical reactions in the fluid sample, and (3) It has three functions: it provides a good background effect by reflecting the measurement light that passes through the support during spectrophotometric analysis. These three functions can be performed in separate layers or all in one layer depending on the case. For example, it is also possible to use a combination of the above-mentioned functions, such as using one layer for only the reflective background function and having the other two functions in one layer. The fluid sample used in the present invention may be an aqueous solution, but the present invention is applicable to biological fluid samples such as blood, lymph fluid, urine, and the like. The blood may include whole blood containing blood cell components (red blood cells, white blood cells, platelets, etc.), or plasma or serum excluding these components. The amount of fluid sample used for analysis is selected depending on the purpose, but is usually about 5 to 5
About 50μ is preferred, and more preferably about 8 to about 20μ. As mentioned above, the elements of the invention can be used by bringing them into contact with a fluid sample to be analyzed and measuring the concentration of detectable products formed within said elements by various spectroscopic measurement methods. It is used by calculating the concentration of a predetermined component in the fluid sample. According to the invention, a variety of different devices can be manufactured,
And it can be used not only in the field of clinical chemistry but also in other chemical research fields. Specific embodiments of the present invention and comparisons of conventional analytical elements will be described using examples, but it goes without saying that the present invention is not limited thereto. Example 1 On a 180μ thick transparent polyethylene terephthalate support (1) Deionized gelatin 160mg/dm as reagent layer ² Polymer surfactant (7)/n-butyl methacrylate/ethyl acrylate = 9/50/ Latex consisting of 41 (by weight) 160 mg/dm ² 1,7-dihydroxynaphthalene 6.60 mg/dm ² 4-aminoantipyrine hydrochloride 9.70 mg/dm ² Glucose oxidase 244.1 international units/dm ² Peroxidase 248 AAP units/dm ² Dimedone 2.15 mg/dm ² 3,3-dimethylglutaric acid 19.6 mg/dm ² Reagent layer of approximately 30 μ dry film thickness (2) Cellulose acetate as non-fibrous porous layer 70.4 mg/dm ² Titanium dioxide 505.9 mg/dm ² Octyl Phenoxypolyethoxyethanol (nonionic surfactant; trade name Triton X-
100, manufactured by Rohm and Haas) 13.8mg/dm ² A non-fibrous porous media layer with a dry film thickness of approximately 140μ consisting of a polyurethane polymer 15.5mg/dm ² The above analytical element for glucose analysis of the present invention is an analytical element () In addition, instead of the non-fibrous porous carrier layer, a filter paper (No.
7) was pasted onto the reagent layer to form an analytical element (). On the other hand, as a comparative analysis element (), one in which the water-dispersible copolymer of the present invention was replaced with gelatin;
The water-dispersible copolymer of the present invention is a water-dispersible copolymer (methyl acrylate/
3-Acryloyloxypropanesulfonic acid
The element substituted with Na/2-acetoacetoxyethyl acrylate = 88.75/4.75/6.5) was used as a comparative analytical element (). (However, the diffusion layer is a non-fibrous porous carrier layer.) The above analytical elements (), () and comparative analytical elements (), () were stored at 5°C in the dark and at 25°C in the dark for 20 weeks. After being left for storage, the reflection density was measured at 540 nm, and the degree of deterioration after storage Δ (%) = {(D _R − D _RO )/D _RO } × 100 D _R : 20 weeks from the difference from the measurement position before being left. The reflection density at 540 nm after standing ( _DRO : reflection density at 540 nm before standing) was determined.

[Table] As shown in Table 1 above, it can be seen that the analytical element of the present invention is a good one with less fogging due to neglect than the comparative analytical element. Example 2 Control serum containing glucose of 250 mg/dl, 350 mg/dl, and 500 mg/dl was prepared using the analytical elements for glucose analysis () and () of the present invention used in Example 1 and the comparative analytical element (). After incubation at 37° C. for 7 minutes using 10μ, the reflection density at 540 nm was measured.

[Table] As shown in Table 2, the analytical element of the present invention has a higher color density and a larger slope than the comparative analytical element, indicating that it has high sensitivity.

Claims (1)

[Scope of Claims] 1. A light-transmissive support, at least one reagent layer containing at least one reagent that reacts with a component in a fluid sample and containing a hydrophilic colloid substance, and a porous carrier layer above the reagent layer. In the multilayer analytical element, at least one of the reagent layers contains a polymeric surfactant containing at least one of the compounds represented by the following general formula [], [], or [] as a main component, and a vinyl group. 1. A multilayer analytical element comprising at least one water-dispersible copolymer obtained by emulsion polymerization of a monomer having the following. General formula [] [In the formula, R ₁ is a substituted or unsubstituted divalent organic group,
M ₁ represents a hydrogen atom or a monovalent cation, and n
represents 30 to 95 mol%, and m represents 70 to 5 mol%. ] General formula [ ] [In the formula, R ₂ and R ₃ are each a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a cyano group, or
COOR represents ₅ groups (R ₅ represents an alkyl group), and each may be the same or different.
R ₄ represents a hydrogen atom or a lower alkyl group,
M ₂ , M ₃ , M ₄ , M ₅ are each a hydrogen atom or 1
represents a valent cation, a is 30 to 95 mol%, b is
70-0 mol%, c is 70-0 mol%. However, b
+c is 70-5 mol%. ] General formula [ ] [In the formula, R ₆ and R ₇ are a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a cyano group, or a COOR ₉ group (R ₉ represents an alkyl group). R ₈ represents a hydrogen atom or a lower alkyl group, and M ₆ , _M7 , _M8 , _M9 are
Each represents a hydrogen atom or a monovalent cation.
x is 30 to 70 mol%, y is 5 to 50 mol%, z is 70 to 70 mol%
It represents 5 mol%, and y+z is 70-30 mol%. 2. said reagent is oxidized by said hydrogen peroxide in the presence of an enzyme and peroxidase capable of catalyzing the oxidation of components in said fluid sample to peroxides, producing a detectable product. A multilayer analytical element according to claim 1, comprising an indicator composition capable of producing a product.