Multilayer analysis element for liquid sample analysis

Abstract

Provided is a multilayer analysis element for liquid sample analysis having excellent adhesion and sensitivity. The multilayer analysis element for liquid sample analysis of the present invention comprises, on a transparent support, at least: a detection layer containing a substance that undergoes a detectable change caused by a gaseous substance; a liquid-blocking layer that selectively allows the gaseous substance to permeate therethrough; and a spreading layer, in this order, wherein the detection layer contains a water-soluble polymer having a glass transition temperature of -50°C to -30°C and a cloud point of 30°C or higher.

Description

Multilayer analytical element for liquid sample analysis 【0001】 This invention relates to a multilayer analytical element for liquid sample analysis. 【0002】 Various methods known as dry chemistry have been proposed for measuring urea nitrogen and creatinine in bodily fluids such as blood. For example, Patent Document 1 describes "a multilayer analytical element for liquid sample analysis comprising, in this order, a detection layer containing a substance that causes a detectable change by a gaseous substance, a liquid barrier layer that selectively allows gaseous substances to pass through, and a developing layer, on a transparent support, wherein the detection layer contains polyvinyl methyl ether and polyvinyl butyral" (claims, etc.). 【0003】 Patent No. 4505296 【0004】 Such multilayer analytical elements for liquid sample analysis (hereinafter also simply referred to as "elements") are used in animal testing because they allow for short-time measurements using a dry method and are simple analytical elements. Since animals are small and blood is difficult to collect from their fur-covered skin, there is a desire for testing with smaller blood samples. Furthermore, improved accuracy in low-concentration samples is desired, and to explore the possibility of these measurements, improved measurement sensitivity is desirable. 【0005】 In this context, when the present inventors examined the element described in the example of Patent Document 1, it became clear that the measurement sensitivity (hereinafter also simply referred to as "sensitivity") may be insufficient in some cases. 【0006】 Furthermore, multilayer analytical elements for liquid sample analysis also require adhesion (for example, the adhesion between the transparent support and the detection layer). 【0007】 Therefore, in view of the above circumstances, the present invention aims to provide a multilayer analytical element for liquid sample analysis that is excellent in terms of adhesion and sensitivity. 【0008】As a result of diligent research into the above-mentioned problems, the inventors discovered that the above-mentioned problems can be solved by using a water-soluble polymer with a glass transition temperature and cloud point within a specific range as the detection layer, leading to the present invention. Specifically, the inventors found that the above-mentioned problems can be solved with the following configuration. 【0009】 (1) A multilayer analytical element for liquid sample analysis comprising, in this order, a detection layer containing a substance that causes a change detectable by a gaseous substance, a liquid barrier layer that selectively allows gaseous substances to pass through, and a developing layer on a transparent support, wherein the detection layer contains a water-soluble polymer having a glass transition temperature of -50°C to -30°C and a cloud point of 30°C or higher. (2) The multilayer analytical element for liquid sample analysis according to (1), wherein the water-soluble polymer is a polymer obtained by polymerizing a monomer represented by general formula (A) described later and a monomer represented by general formula (B) described later. (3) The multilayer analytical element for liquid sample analysis according to (1) or (2), wherein the water-soluble polymer is a polymer obtained by polymerizing a monomer represented by general formula (A) described later, a monomer represented by general formula (B) described later and a monomer represented by general formula (C) described later. (4) The above R ２ A multilayer analytical element for liquid sample analysis according to (2) or (3) above, wherein m is a methyl group or an ethyl group. (5) A multilayer analytical element for liquid sample analysis according to any one of (2) to (4) above, wherein m is 3 to 10. 【0010】 As shown below, the present invention provides a multilayer analytical element for liquid sample analysis that has excellent adhesion and sensitivity. Furthermore, by maintaining sufficient adhesion while increasing sensitivity, the present invention provides a multilayer analytical element for liquid sample analysis that enables improved accuracy in low-concentration samples and measurement with smaller sample volumes. 【0011】The multilayer analytical element for liquid sample analysis of the present invention is described below. In this specification, numerical ranges expressed using "~" mean a range that includes the values ​​written before and after "~" as the lower and upper limits. Each component may be used alone or in combination of two or more. When two or more components are used in combination, the content of each component refers to the total content unless otherwise specified. The average means the arithmetic mean. (Meth)acrylic means acrylic or methacrylic. 【0012】 The multilayer analytical element for liquid sample analysis of the present invention (hereinafter also simply referred to as "the element of the present invention") is a multilayer analytical element for liquid sample analysis comprising, in this order, a detection layer containing a substance that undergoes a change detectable by a gaseous substance, a liquid barrier layer that selectively allows gaseous substances to pass through, and a developing layer on a transparent support, wherein the detection layer contains a water-soluble polymer (hereinafter also referred to as "specific polymer") having a glass transition temperature (Tg) of -50°C to -30°C and a cloud point of 30°C or higher. 【0013】 The device of the present invention is thought to solve the above-mentioned problems by adopting such a configuration. The reason is not clear, but it is presumed to be as follows. From the inventors' studies, it has been found that the adhesion strength correlates with the amount of the indicator layer (detection layer) seeping into the liquid barrier layer. It has also been found that the amount of seeping correlates with the Tg of the polymer in the detection layer. Tg can be said to be an indicator of the elastic modulus. In the device of the present invention, since a polymer with a Tg of a certain value or higher (high elastic modulus) is used in the detection layer, it is thought that it does not seep easily into the liquid barrier layer and exhibits excellent adhesion. In addition, the cloud point is determined by how much affinity the polymer has with water, and the higher the affinity with water, the higher the cloud point. Here, the chemical structures of water molecules and ammonia molecules are very similar. In the device of the present invention, since a polymer with a cloud point of a certain value or higher is used in the detection layer, it is thought that it has a high affinity with ammonia molecules and exhibits excellent sensitivity. 【0014】 The following describes each layer of the element of the present invention. 【0015】 [Transparent Support] Examples of transparent supports include those commonly used in analytical elements (especially hydrophobic transparent supports), and specific examples include transparent supports made of polymers such as polyethylene terephthalate, polycarbonate, and polyvinyl compounds. The thickness of the transparent support is approximately 50 to 1000 μm, and usually approximately 80 to 300 μm. In this specification, a so-called semi-transparent state, where one side can be seen from the other, is also considered transparent. 【0016】 [Detection Layer] The detection layer contains at least a substance that undergoes a detectable change due to a gaseous substance, and a specific polymer. 【0017】 [Substances that undergo detectable changes in response to gaseous substances] Substances that undergo detectable changes in response to gaseous substances (hereinafter also referred to as "indicators") refer to substances that, as a result of reacting or interacting with gaseous substances (especially ammonia), undergo a change in structure, which in turn causes a change in spectroscopic properties such as absorption wavelength. Here, structural changes include changes in chemical structure, such as changes in atomic composition, changes in bonding relationships such as covalent bonds and hydrogen bonds, and changes in stereochemistry. Specific examples of substances that undergo detectable changes in response to gaseous substances include compounds that react with gaseous ammonia to produce a change in absorption wavelength (hereinafter also referred to as "chromogenic precursors"). Examples of color precursors include leuco dyes such as leucocyanin dyes, nitro-substituted leuco dyes, and leucophthalein dyes (described in Japanese Patent Publication No. 52-3488 and U.S. Reissued Patent Publication No. 30267); pH indicators such as bromophenol blue, bromo cresol green, bromothymol blue, quinoline blue, and rosolic acid (described in Kyoritsu Shuppan, Encyclopedia of Chemistry, Vol. 10, pp. 63-65); triarylmethane-based dye precursors; leucobenzylidene dyes (described in Japanese Patent Publication No. 56-145273); diazonium salt and azo dye couplers; and basic bleachable dyes. 【0018】<Content> In the detection layer, the content of the indicator (especially the chromogenic precursor) is preferably 0.1 to 50% by mass, more preferably 0.5 to 20% by mass, based on the content of the polymer (specific polymer, binder polymer) described below, because the effects of the present invention are more excellent. 【0019】 <Specific Polymer> The detection layer contains a water-soluble polymer (specific polymer) having a glass transition temperature of -50°C to -30°C and a cloud point of 30°C or higher. Here, water-soluble means that the solubility in 100 g of water at pH 7.0 at 22°C is 0.1 g or more. 【0020】 <Glass Transition Temperature> As described above, the glass transition temperature of the specific polymer is -50°C to -30°C. Among them, it is preferably -45°C to -30°C, more preferably -40°C to -30°C, because the effects of the present invention are more excellent. 【0021】 In this specification, the glass transition temperature (hereinafter also referred to as "Tg") means the extrapolated glass transition start temperature (hereinafter also referred to as "Tig") measured using differential scanning calorimetry (DSC) in accordance with the methods described in JIS K 7121 (1987) or JIS K 6240 (2011). 【0022】 Hereinafter, the method for measuring Tg will be described more specifically. It is measured using a differential thermal thermogravimetric simultaneous measurement device STA7200 manufactured by Hitachi High-Tech Sciences Corporation. After holding at -80°C until the device is stable, it is heated to 80°C at a heating rate of 10°C / min. It is cooled to -80°C at a heating rate of 10°C / min and held until the device is stable, and then heated to 80°C at a heating rate of 10°C / min. In the second heating measurement, a differential thermal analysis (DTA) curve or a DSC curve is created. The temperature at the intersection of a straight line obtained by extending the baseline on the low-temperature side in the DTA curve or DSC curve to the high-temperature side and a tangent line drawn at the point where the slope of the curve of the stepwise change part of the glass transition is maximized is defined as Tg. 【0023】<Cloud point> As described above, the cloud point of the specific polymer is 30°C or higher. Among these, from the reason that the effects of the present invention are more excellent, it is preferably 33 to 60°C, and more preferably 36 to 50°C. 【0024】 In the present specification, the cloud point refers to the lower critical solution temperature (LCST) in an aqueous solution, and means the temperature that causes a phenomenon in which the polymer chain is hydrated and dissolved at low temperatures, but dehydration occurs at high temperatures, causing aggregation and insolubility. 【0025】 Hereinafter, the method for measuring the cloud point will be specifically described. Add 4 mL of ion-exchanged water and 40 mg of the polymer to a 4 mL glass sample container, and dissolve it in a water bath at 20°C. Then, raise the water temperature at a rate of 3°C / min, and take the temperature at which it becomes white and turbid as the cloud point. 【0026】 <Specific examples> The specific polymer is not particularly limited as long as it is a water-soluble polymer having a Tg and a cloud point within the above-described ranges. Specific examples thereof include poly(meth)acrylic, polyvinyl alcohol, polyalkylene oxide, and the like. Among these, from the reason that the effects of the present invention are more excellent, it is preferably poly(meth)acrylic. 【0027】 <Polymer α> From the reason that the effects of the present invention are more excellent, the specific polymer is preferably a polymer (hereinafter also referred to as "polymer α") obtained by polymerizing a monomer represented by the following general formula (A) and a monomer represented by the following general formula (B). Polymer α may be a polymer obtained by further polymerizing another monomer in addition to the above monomers. 【0028】 【0029】 In general formulas (A) and (B), R １ represents a hydrogen atom or a methyl group, R ２ represents an alkyl group having 1 to 4 carbon atoms or an aryl group, l and m represent the average number of repeating units of oxyethylene, l is 1 to 2, and m is 3 or more. 【0030】 As described above, in general formulas (A) to (B), R １represents a hydrogen atom or a methyl group. Of these, a methyl group is preferred because it provides superior effects for the present invention. 【0031】 As described above, in general formulas (A) to (B), R ２ This represents an alkyl group having 1 to 4 carbon atoms, or an aryl group. The alkyl group may be linear, branched, or cyclic, but it is preferably linear, and more preferably a methyl group, for the sake of superior effects of the present invention. Examples of the aryl group (aromatic hydrocarbon group) include aromatic hydrocarbon groups having 6 to 18 carbon atoms, such as a phenyl group, a tolyl group, a xylyl group, and a naphthyl group. 【0032】 As described above, in general formulas (A) to (B), l and m represent the average number of repeating units of oxyethylene, where l is 1 to 2 and m is 3 or more. For the reasons that the effects of the present invention are superior, l is preferably 1 or 2, and more preferably 2. For the reasons that the effects of the present invention are superior, the lower limit of m is preferably 3 or more, and more preferably 4 or more. For the reasons that the effects of the present invention are superior, the upper limit of m is preferably 45 or less, more preferably 23 or less, even more preferably 13 or less, and particularly preferably 9 or less. 【0033】 In polymer α, the ratio of repeating units derived from the monomer represented by general formula (A) (hereinafter also referred to as "repeating unit A") to the total repeating units is preferably 10 to 90% by mass, and more preferably 40 to 85% by mass, for the reasons that the effects of the present invention are superior. In polymer α, the ratio of repeating units derived from the monomer represented by general formula (B) (hereinafter also referred to as "repeating unit B") to the total repeating units is preferably 10 to 90% by mass, and more preferably 15 to 60% by mass, for the reasons that the effects of the present invention are superior. 【0034】<Polymer β> Polymer α is preferably a polymer (hereinafter also referred to as "polymer β") obtained by polymerizing a monomer represented by the following general formula (A), a monomer represented by the following general formula (B), and a monomer represented by the following general formula (C) because the effects of the present invention are more excellent. 【0035】 【0036】 In general formulas (A) to (C), R １ represents a hydrogen atom or a methyl group, R ２ represents an alkyl group having 1 to 4 carbon atoms or an aryl group, and l, m, and n represent the average number of repeating units of oxyethylene. l is 1 to 2, m is 3 or more, and n is 1 to 10. 【0037】 The definitions, specific examples, and preferred embodiments of R １ , R ２ , l, and m are as described above. 【0038】 As described above, in general formula (C), R １ represents a hydrogen atom or a methyl group. Among them, a methyl group is preferable because the effects of the present invention are more excellent. 【0039】 As described above, in general formula (C), n is 1 to 10. Among them, it is preferably 1 to 5, and more preferably 1 to 2 because the effects of the present invention are more excellent. 【0040】 In polymer β, the proportion of the repeating unit derived from the monomer represented by general formula (C) (hereinafter also referred to as "repeating unit C") with respect to all repeating units is preferably 1 to 30% by mass, and more preferably 5 to 20% by mass because the effects of the present invention are more excellent. The preferred embodiments of the proportions of repeating unit A and repeating unit B are as described above. 【0041】<Content> The content of the specific polymer in the detection layer is preferably 10% by mass or more, more preferably 30% by mass or more, even more preferably 50% by mass or more, and particularly preferably 70% by mass or more, for reasons that the effects of the present invention are superior. There is no particular upper limit, and it is 100% by mass. 【0042】 In the detection layer, the content of the specific polymer is preferably 1 to 500 times, more preferably 3 to 300 times, and even more preferably 5 to 100 times, by mass ratio, of the content of the indicator mentioned above, for the reason that the effects of the present invention are superior. 【0043】 [Binder Polymer] The detection layer may contain a binder polymer other than the specified polymer. Examples of such binder polymers include water-insoluble vinyl polymers; gelatins such as acid-treated gelatin, alkali-treated gelatin, and deionized gelatin; cellulose esters such as cellulose acetate, cellulose acetate butyrate, and cellulose acetate propionate; alkylcelluloses such as methylcellulose, ethylcellulose, and propylcellulose; and so on. Among these, water-insoluble vinyl polymers are preferred because they provide superior effects for which the present invention is most effective. 【0044】 <Water-Insoluble Vinyl Polymer> The water-insoluble vinyl polymer preferably has an acetal group for better performance of the present invention. Examples of acetal groups include butyral groups, formal groups, etc. The water-insoluble vinyl polymer preferably is polyvinyl butyral for better performance of the present invention. 【0045】 (Content) If the detection layer contains a water-insoluble vinyl polymer, its content is preferably 1 to 50% by mass, more preferably 1 to 30% by mass, and even more preferably 1 to 20% by mass, relative to the content of the specified polymer described above, for the reasons that the effects of the present invention are superior. 【0046】[Other Components] The detection layer may contain components other than those described above. Examples of such components (other components) include buffers, organic acids, and inorganic acids for pH adjustment. Specific examples of buffers include the alkaline buffer described in the reagent layer below. Specific examples of organic and inorganic acids include ethanesulfonic acid, aspartic acid, azelaic acid, glutaric acid, succinic acid, glutaconic acid, tartaric acid, pimelic acid, malonic acid, malic acid, 3,3-dimethylglutaric acid, citric acid, p-toluenesulfonic acid, perchloric acid, and hydrochloric acid. It may also contain alkalis such as sodium hydroxide, potassium hydroxide, disodium carbonate, and sodium bicarbonate. 【0047】 [Method of Formation] As a method for forming the detection layer, for example, one can mix the above-mentioned components in a solvent and apply the resulting coating solution to the above-mentioned transparent support. As the solvent, organic solvents such as acetone, 2-methoxyethanol, methyl ethyl ketone, methanol, ethanol, or water, or a mixture thereof, are preferred for the above-mentioned solvent, for the reason that the effects of the present invention are superior. The solid content concentration of the coating solution is preferably 1 to 30% by mass, and more preferably 3 to 20% by mass, for the reason that the effects of the present invention are superior. 【0048】 [Thickness] The thickness of the detection layer is preferably 1 to 30 μm, and more preferably 2 to 20 μm, for the sake of achieving superior effects in the present invention. 【0049】 [Liquid Barrier Layer] The liquid barrier layer is preferably composed of a microporous material having through-holes that substantially prevents the permeation of liquids such as coating solutions and sample solutions, and interfering components (e.g., alkaline components) dissolved in these liquids, while selectively allowing the permeation of gaseous substances such as gaseous ammonia, during the manufacturing and / or analytical operations of the device. In this specification, barriers are defined as being partially blocked. 【0050】The liquid barrier layer is preferably composed of one or more porous membranes. In the case of two or more porous membranes, it is preferable that the pore diameter of the uppermost porous membrane in contact with the reagent layer described later is the same as or smaller than the pore diameter of the porous membrane directly below it. Specifically, the pore diameter of the uppermost porous membrane is 0.01 to 1 μm, preferably 0.04 to 0.2 μm, the pore diameter of the porous membrane directly below it is 0.2 to 20 μm, preferably 0.5 to 10 μm, and the ratio of the average pore diameter of the uppermost porous membrane to the pore diameter of the porous membrane directly below it is 0.001 to 1.0, preferably 0.01 to 0.5. In this specification, pore diameter refers to the average pore diameter unless otherwise specified. The material of the porous membrane is not particularly limited, but examples include polyethylene, polypropylene, fluorine-containing polymers such as polytetrafluoroethylene, cellulose acetate, polysulfone, polyamide (nylons), or mixtures thereof. A preferred combination is a porous polyethylene membrane and a porous polypropylene membrane. Each porous membrane has a thickness of 3 to 40 μm, preferably 5 to 20 μm, and two or more of these, usually two to three layers, are combined to form a liquid barrier layer. The porosity of the entire liquid barrier layer is 25 to 90%, preferably 35 to 90%, and the total thickness of the layer is 10 to 120 μm, preferably 10 to 110 μm. 【0051】 The liquid barrier layer is preferably adhered to the detection layer described above with practical strength. Methods of adhesion include, for example, applying the liquid barrier layer to a wet surface and then drying it. Here, "wet state" means that the solvent dissolving the binder remains, or that the dried film is wetted with a soluble solvent, causing the binder to be in a swollen, dispersed, or solution state. 【0052】 [Reagent Layer] A reagent layer may be provided on top of the liquid barrier layer. The reagent layer is typically a layer containing, for example, a reagent that reacts with an ammonia-producing substance to produce ammonia (generally an enzyme or a reagent containing an enzyme) (hereinafter also simply referred to as "reagent"), an alkaline buffer for efficiently releasing the ammonia produced by the reaction as gaseous ammonia, and a hydrophilic polymer binder having film-forming ability. 【0053】[Reagents] Examples of combinations of ammonia-producing substrates and reagents include urea / urease, creatinine / creatinine deiminase, amino acid / amino acid dehydrogenase, amino acid / amino acid oxidase, amino acid / amino acid dehydratase, amino acid / ammonia lyase, amine / amine oxidase, diamine / amine oxidase, glucose and phosphate amidate / phosphate amidate hexose phosphate transferase, ADP / carbamate kinase and phosphate carbamol, acid amide / amide hydrolase, nucleobase / deaminase, nucleoside / deaminase, nucleotide / deaminase, guanine / guanase, etc. 【0054】 [Alkaline buffering agent] For the sake of superior effectiveness of the present invention, the alkaline buffering agent is preferably a buffering agent with a pH in the range of 7.0 to 10.5, and more preferably a buffering agent in the range of 7.5 to 10.0. Specific examples of alkaline buffering agents include ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane (Tris), phosphate buffering agents, Good buffering agents such as N,N-bis(2-hydroxyethyl)glycine (Bicine), N-2-hydroxyethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid (HEPPSO), and N-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), as well as borate buffering agents. 【0055】 [Hydrophilic Polymer Binders] Examples of hydrophilic polymer binders include gelatin, agarose, polyvinyl alcohol, polyacrylamide, hydroxymethylcellulose, hydroxyethylcellulose, and polyvinylpyrrolidone. 【0056】 [Other Components] The reagent layer may contain components other than those described above. Examples of such components (other components) include wetting agents, binder crosslinking agents (curing agents), stabilizers, heavy metal ion trapping agents (complexing agents), etc. 【0057】[Content] In the reagent layer, the content of the hydrophilic polymer binder is preferably 10 to 99% by mass, and more preferably 60 to 90% by mass, for the reason that the effects of the present invention are superior. Also, in the reagent layer, the content of the reagent is preferably 0.1 to 50% by mass, and more preferably 0.2 to 20% by mass, relative to the content of the hydrophilic polymer binder, for the reason that the effects of the present invention are superior. Also, in the reagent layer, the content of the alkaline buffer is preferably 0.1 to 60% by mass, relative to the content of the hydrophilic polymer binder, for the reason that the effects of the present invention are superior. 【0058】 [Method of Formation] Methods for forming the reagent layer include, for example, mixing the above-mentioned components, applying the resulting coating solution to the above-mentioned liquid barrier layer, and drying it. 【0059】 [Thickness] The thickness of the reagent layer is preferably 1 to 40 μm, and more preferably 2 to 20 μm, for the sake of achieving superior effects in the present invention. 【0060】[Expanding Layer] Examples of the expanding layer include woven fabric expanding layers described in Japanese Patent Publication No. 55-164356 (corresponding to U.S. Patent No. 4,292,272) and Japanese Patent Publication No. 57-66359 (corresponding to U.S. Patent No. 4,783,315) (e.g., plain knit fabrics such as broadcloth and poplin), and knitted fabric expanding layers described in Japanese Patent Publication No. 60-222,769 (corresponding to European Patent Publication No. 0162,302) (e.g., tricot knitted fabric, double tricot knitted fabric, Milanese knit). Fibrous microporous developing layers such as: a developing layer of paper containing organic polymer fiber pulp (material fabric), a developing layer formed by coating a dispersion of fibers and hydrophilic polymer (as described in Japanese Patent Publication No. 57-148250), a developing layer formed by coating a dispersion of fibers and hydrophilic polymer (as described in Japanese Patent Publication No. 57-125847), etc.; a membrane filter layer (brush polymer layer) (as described in Japanese Patent Publication No. 53-21677, U.S. Patent No. 3,992158, etc.), and fine particles such as polymer microbeads in point contact with a hydrophilic polymer binder. Non-fibrous isotropic microporous unfolding layers such as a continuous microvoid-containing isotropic microporous unfolding layer (three-dimensional lattice-like granular structure layer) unfolding layer in which polymer microbeads described in Japanese Patent Publication No. 55-90859 (corresponding U.S. Patent No. 4258001), etc., are bonded in a point-contact manner with a polymer adhesive that does not swell with water; Japanese Patent Publication No. 61-4959 (corresponding U.S. Patent No. 5019347), Japanese Patent Publication No. 62-13875 6. Examples include a developing layer with excellent blood cell separation ability, which is formed by laminating and bonding multiple microporous layers (e.g., two layers of woven or knitted fabric and a membrane filter, or three layers of woven or knitted fabric, a membrane filter, and woven or knitted fabric) on their surfaces with fine discontinuous dots or island-like adhesives (halftone dots in the printing field), as described in Japanese Patent Publication No. 62-138757 and Japanese Patent Publication No. 62-138758 (corresponding to European Patent Publication No. 0226465). 【0061】The woven or knitted fabric used for the development layer can be made hydrophilic by applying a physical activation treatment, such as the glow discharge treatment or corona discharge treatment described in Japanese Patent Publication No. 57-66359, to at least one side of the fabric, or by performing a water washing and degreasing treatment, a hydrophilic polymer impregnation treatment, or other hydrophilic treatment described in Japanese Patent Publication No. 55-164356, Japanese Patent Publication No. 57-66359, etc., or by sequentially performing a combination of these treatment steps as appropriate, thereby increasing the adhesion strength with the lower layer (the side closer to the support). Furthermore, as described in Japanese Patent Publication Nos. 59-171864, Japanese Patent Publication No. 60-222769, Japanese Patent Publication No. 60-222770, etc., the development area or spread of the liquid sample can be controlled by applying a polymer-containing aqueous solution or a polymer-containing water-organic solvent mixed solution from above the development layer. 【0062】 [Other Layers] The element of the present invention may have a configuration that includes at least a detection layer, a liquid barrier layer, and a deployment layer on a transparent support in this order, and may have one or more additional layers at any of the following positions: between the transparent support and the detection layer, between the detection layer and the liquid barrier layer, between the liquid barrier layer and the deployment layer, or on the deployment layer. 【0063】When the element of the present invention is used for creatinine quantification, an ammonia diffusion prevention layer and an endogenous ammonia capture layer may be provided between the reagent layer and the developing layer in that order. The ammonia diffusion prevention layer and the endogenous ammonia capture layer are known, as described in, for example, Japanese Patent Application Publication No. 4-157364. For example, the ammonia diffusion prevention layer is a layer in which ammonia capture and ammonia production reactions are substantially prevented, and can be formed using a hydrophilic polymer such as hydroxypropyl cellulose. The thickness of the ammonia diffusion prevention layer is, for example, 2 to 50 μm. The endogenous ammonia capture layer is a layer containing a reagent that acts on endogenous ammonia already present in the liquid sample and changes it to a state in which it is substantially unable to reach the reagent layer. Examples of reagents include compositions containing enzymes that have catalytic activity to convert ammonia into other substances using ammonia as a substrate. Specific examples of such compositions include compositions using a hydrophilic polymer such as hydroxyethyl cellulose as a binder polymer, and containing α-ketoglutaric acid, NADPH, and glutamate dehydrogenase. The thickness of the endogenous ammonia capture layer is, for example, 1 to 30 μm. 【0064】 A color-shielding layer or a light-reflecting layer can be provided between the reagent layer and the developing layer. The color-shielding layer or light-reflecting layer is a layer with a dry thickness ranging from approximately 2 μm to approximately 20 μm, in which white fine particles such as titanium dioxide fine particles or barium sulfate fine particles, which have light-shielding properties or possess both light-shielding and light-reflecting properties, are dispersed almost uniformly in a hydrophilic polymer binder such as gelatin. 【0065】 Furthermore, a known adhesive layer made of a hydrophilic polymer can be provided on top of the reagent layer, ammonia diffusion prevention layer, endogenous ammonia capture layer, color shielding layer, or light reflection layer for the purpose of firmly bonding and integrating the development layer. The thickness of the adhesive layer when dry is in the range of approximately 0.5 μm to approximately 5 μm. 【0066】[Preferred Embodiments] Surfactants can be included in the reagent layer, ammonia diffusion prevention layer, endogenous ammonia capture layer, color shielding layer or light reflection layer, adhesive layer, developing layer, etc. Nonionic surfactants are an example. Specific examples of nonionic surfactants include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, p-nonylphenoxypolyglycidol, octyl glucoside, etc. Including a nonionic surfactant in the developing layer improves the developing action (metering action) of aqueous liquid samples. Including a nonionic surfactant in the reagent layer, ammonia diffusion prevention layer, endogenous ammonia capture layer, color shielding layer or light reflection layer, adhesive layer, etc., makes it easier for water in the aqueous liquid sample to be absorbed substantially uniformly into the reagent layer during analytical operations, and also makes liquid contact with the developing layer rapid and substantially uniform. In the element of the present invention, it is preferable that the above-mentioned layers are sequentially bonded and laminated together as a single unit, for the sake of achieving superior effects of the present invention. 【0067】 [Method of Use] A method for measuring, for example, ammonia or an ammonia-producing substrate in a liquid sample using the element of the present invention includes, for example, placing droplets of an aqueous liquid sample such as whole blood, plasma, serum, or urine in the range of 3 to 30 μL, preferably 6 to 15 μL, onto the developing layer, incubating for 1 to 10 minutes at a substantially constant temperature in the range of about 20 to 40°C, and then measuring the degree of color change (color development or fading) of the detection layer from the transparent support side by reflectance photometry, or visually comparing it with a standard color. 【0068】 The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. 【0069】[Polymer Synthesis] The following polymers (synthetic polymers A-F, X-Y) were synthesized. All synthetic polymers A-F and X-Y were water-soluble polymers. As described below, synthetic polymers A-F all have a Tg of -50°C to -30°C and a cloud point of 30°C or higher, and therefore fall under the category of specific polymers mentioned above. On the other hand, as described below, synthetic polymer X does not fall under the category of specific polymers mentioned above because its Tg is less than -50°C. Also, as described below, synthetic polymer Y does not fall under the category of specific polymers mentioned above because its cloud point is less than 30°C. 【0070】 [Synthetic Polymer A] 618 mL of ethanol was weighed into a flask with a condenser, and after raising the temperature to reflux, the system was thoroughly purged with nitrogen. A solution consisting of 200 g of ethylene glycol monomethyl ether methacrylate, 300 g of polyethylene glycol monomethyl ether methacrylate (average repeating units of oxyethylene: 4), 2.335 g of 2,2'-azobis(isobutyrate)dimethyl, and 63 mL of ethanol was added dropwise over 60 minutes. After the addition was complete, polymerization was carried out under reflux for 90 minutes. Then, a solution consisting of 1.480 g of 2,2'-azobis(isobutyrate)dimethyl and 13 mL of ethanol was added, and polymerization was carried out under reflux for 180 minutes. After polymerization was complete, the mixture was cooled to below 50°C, and then 222 mL of methanol was added to obtain a polymer solution with a concentration of 40%. The obtained polymer is also called synthetic polymer A. 【0071】 Synthetic polymer A is a monomer represented by general formula (A) (where R is in general formula (A)). １ and R ２ (where is a methyl group and l is 1) and a monomer represented by general formula (B) (where R is in general formula (B) １ and R ２ It is a polymer obtained by polymerizing (where is a methyl group and m is 4) and 【0072】 For synthetic polymer A, the Tg was -47°C and the cloud point was 33°C. 【0073】[Synthetic Polymer B] 618 mL of ethanol was weighed into a flask with a condenser, heated to reflux, and then the system was thoroughly purged with nitrogen. A solution consisting of 250 g of ethylene glycol monomethyl ether methacrylate, 250 g of polyethylene glycol monomethyl ether methacrylate (average repeating units of oxyethylene: 9), 2.345 g of 2,2'-azobis(isobutyrate)dimethyl, and 63 mL of ethanol was added dropwise over 60 minutes. After the addition was complete, polymerization was carried out under reflux for 90 minutes. Then, a solution consisting of 1.487 g of 2,2'-azobis(isobutyrate)dimethyl and 13 mL of ethanol was added, and polymerization was carried out under reflux for 180 minutes. After polymerization was complete, the mixture was cooled to below 50°C, and then 222 mL of methanol was added to obtain a polymer solution with a concentration of 40%. The obtained polymer is also called synthetic polymer B. 【0074】 Synthetic polymer B is a monomer represented by general formula (A) (where R is present in general formula (A)). １ and R ２ (where is a methyl group and l is 1) and a monomer represented by general formula (B) (where R is in general formula (B) １ and R ２ It is a polymer obtained by polymerizing (where is a methyl group and m is 9) and 【0075】 For synthetic polymer B, the Tg was -44°C and the cloud point was 46°C. 【0076】[Synthetic Polymer C] 618 mL of ethanol was weighed into a flask with a condenser, and after raising the temperature to reflux, the system was thoroughly purged with nitrogen. A solution consisting of 350 g of diethylene glycol monomethyl ether methacrylate, 150 g of polyethylene glycol monomethyl ether methacrylate (average repeating units of oxyethylene: 4), 2.268 g of 2,2'-azobis(isobutyrate)dimethyl, and 63 mL of ethanol was added dropwise over 60 minutes. After the addition was complete, polymerization was carried out under reflux for 90 minutes. Then, a solution consisting of 1.438 g of 2,2'-azobis(isobutyrate)dimethyl and 13 mL of ethanol was added, and polymerization was carried out under reflux for 180 minutes. After polymerization was complete, the mixture was cooled to below 50°C, and 222 mL of methanol was added to obtain a polymer solution with a concentration of 40%. The obtained polymer is also called synthetic polymer C. 【0077】 Synthetic polymer C is a monomer represented by general formula (A) (where R is present in general formula (A)). １ and R ２ (where is a methyl group and l is 2) and a monomer represented by general formula (B) (where R is in general formula (B) １ and R ２ It is a polymer obtained by polymerizing (where is a methyl group and m is 4) and 【0078】 For synthetic polymer C, the Tg was -43°C and the cloud point was 36°C. 【0079】[Synthetic Polymer D] 618 mL of ethanol was weighed into a flask with a condenser, and after raising the temperature to reflux, the system was thoroughly purged with nitrogen. A solution consisting of 425 g of diethylene glycol monomethyl ether methacrylate, 75 g of polyethylene glycol monomethyl ether methacrylate (average repeating units of oxyethylene: 9), 1.792 g of 2,2'-azobis(isobutyrate)dimethyl, and 63 mL of ethanol was added dropwise over 60 minutes. After the addition was complete, polymerization was carried out under reflux for 90 minutes. Then, a solution consisting of 0.896 g of 2,2'-azobis(isobutyrate)dimethyl and 13 mL of ethanol was added, and polymerization was carried out under reflux for 180 minutes. After polymerization was complete, the mixture was cooled to below 50°C, and 222 mL of methanol was added to obtain a polymer solution with a concentration of 40%. The obtained polymer is also called synthetic polymer D. 【0080】 Synthetic polymer D is a monomer represented by general formula (A) (where R is present in general formula (A)). １ and R ２ (where is a methyl group and l is 2) and a monomer represented by general formula (B) (where R is in general formula (B) １ and R ２ It is a polymer obtained by polymerizing (where is a methyl group and m is 9) and 【0081】 For synthetic polymer D, the Tg was -39°C and the cloud point was 35°C. 【0082】[Synthetic Polymer E] 618 mL of ethanol was weighed into a flask with a condenser, and after raising the temperature to reflux, the system was thoroughly purged with nitrogen. A solution consisting of 300 g of diethylene glycol monomethyl ether methacrylate, 150 g of polyethylene glycol monomethyl ether methacrylate (average repeating units of oxyethylene: 4), 50 g of 2-hydroxyethyl methacrylate, 2.380 g of 2,2'-azobis(isobutyrate)dimethyl, and 63 mL of ethanol was added dropwise over 60 minutes. After the addition was complete, polymerization was carried out under reflux for 90 minutes. Then, a solution consisting of 1.509 g of 2,2'-azobis(isobutyrate)dimethyl and 13 mL of ethanol was added, and polymerization was carried out under reflux for 180 minutes. After polymerization was complete, the system was cooled to below 50°C, and 222 mL of methanol was added to obtain a polymer solution with a polymer concentration of 40%. The obtained polymer is also called synthetic polymer E. 【0083】 Synthetic polymer E is a monomer represented by general formula (A) (wherein general formula (A), R １ and R ２ (where is a methyl group and l is 2) and a monomer represented by general formula (B) (where R is in general formula (B) １ and R ２ (where is a methyl group and m is 4) and a monomer represented by general formula (C) (where R is in general formula (C) １ This polymer is obtained by polymerizing (where is a methyl group and n is 1) and 【0084】 For synthetic polymer E, the Tg was -37°C and the cloud point was 36°C. 【0085】[Synthetic Polymer F] 618 mL of ethanol was weighed into a flask with a condenser, and after raising the temperature to reflux, the system was thoroughly purged with nitrogen. A solution consisting of 350 g of diethylene glycol monomethyl ether methacrylate, 100 g of polyethylene glycol monomethyl ether methacrylate (average repeating units of oxyethylene: 9), 50 g of 2-hydroxyethyl methacrylate, 2.576 g of 2,2'-azobis(isobutyrate)dimethyl, and 63 mL of ethanol was added dropwise over 60 minutes. After the addition was complete, polymerization was carried out under reflux for 90 minutes. Then, a solution consisting of 1.634 g of 2,2'-azobis(isobutyrate)dimethyl and 13 mL of ethanol was added, and polymerization was carried out under reflux for 180 minutes. After polymerization was complete, the system was cooled to below 50°C, and 222 mL of methanol was added to obtain a polymer solution with a concentration of 40%. The obtained polymer is also called synthetic polymer F. 【0086】 The synthetic polymer F is a monomer represented by general formula (A) (wherein general formula (A), R １ and R ２ (where is a methyl group and l is 2) and a monomer represented by general formula (B) (where R is in general formula (B) １ and R ２ (where is a methyl group and m is 9) and a monomer represented by general formula (C) (where R is in general formula (C) １ This polymer is obtained by polymerizing (where is a methyl group and n is 1) and 【0087】 For synthetic polymer F, the Tg was -34°C and the cloud point was 38°C. 【0088】[Synthetic Polymer X] 618 mL of ethanol was weighed into a flask with a condenser, and after raising the temperature to reflux, the system was thoroughly purged with nitrogen. A solution consisting of 150 g of diethylene glycol monomethyl ether methacrylate, 350 g of polyethylene glycol monomethyl ether methacrylate (average repeating units of oxyethylene: 9), 1.792 g of 2,2'-azobis(isobutyrate)dimethyl, and 63 mL of ethanol was added dropwise over 60 minutes. After the addition was complete, polymerization was carried out under reflux for 90 minutes. Then, a solution consisting of 0.896 g of 2,2'-azobis(isobutyrate)dimethyl and 13 mL of ethanol was added, and polymerization was carried out under reflux for 180 minutes. After polymerization was complete, the mixture was cooled to below 50°C, and 222 mL of methanol was added to obtain a polymer solution with a polymer concentration of 40%. The obtained polymer is also called synthetic polymer X. 【0089】 Synthetic polymer X is a monomer represented by general formula (A) (where R is present in general formula (A)). １ and R ２ (where is a methyl group and l is 2) and a monomer represented by general formula (B) (where R is in general formula (B) １ and R ２ It is a polymer obtained by polymerizing (where is a methyl group and m is 9) and 【0090】 For synthetic polymer X, the Tg was -53°C and the cloud point was 44°C. 【0091】 [Synthetic Polymer Y] 618 mL of ethanol was weighed into a flask with a condenser, and after raising the temperature to reflux, the system was thoroughly purged with nitrogen. A solution consisting of 500 g of diethylene glycol monomethyl ether methacrylate, 1.792 g of 2,2'-azobis(isobutyrate)dimethyl, and 63 mL of ethanol was added dropwise over 60 minutes. After the addition was complete, polymerization was carried out under reflux for 90 minutes. Then, a solution consisting of 0.896 g of 2,2'-azobis(isobutyrate)dimethyl and 13 mL of ethanol was added, and polymerization was carried out under reflux for 180 minutes. After polymerization was complete, the mixture was cooled to below 50°C, and 222 mL of methanol was added to obtain a polymer solution with a concentration of 40%. The obtained polymer is also called synthetic polymer Y. 【0092】The synthetic polymer Y is a monomer represented by general formula (A) (where R is present in general formula (A)). １ and R ２ It is a polymer obtained by polymerizing (where is a methyl group and l is 2). 【0093】 For synthetic polymer Y, the Tg was -35°C and the cloud point was 21°C. 【0094】 [Fabrication of the elements] Each element was fabricated as follows. 【0095】 [Example 1] The element of Example 1 was fabricated as follows. 【0096】 <Detection Layer> A detection layer was formed by applying and drying a detection layer-forming composition (ethanol solution) to a transparent polyethylene terephthalate (PET) film (transparent support) with a thickness of 180 μm, in the following coverage amounts. 【0097】 (Coating amount) Bromphenol blue 110 mg / m² ２ Synthetic polymer A 1.8 g / m ２ Polyvinyl butyral (average degree of polymerization: approximately 300) 0.18 g / m ２ Sodium hydroxide 6.8 mg / m² ２ 【0098】 <Liquid Barrier Layer> Next, a liquid barrier layer was created by uniformly pressing a polyethylene membrane filter with an average pore size of 0.2 μm, a porosity of 75%, and a thickness of 100 μm onto the detection layer. 【0099】 <Reagent Layer> Furthermore, a reagent layer was formed by applying and drying a reagent layer-forming composition (aqueous solution) on top of this liquid barrier layer to the following coverage amount. 【0100】 (Covering amount) Hydroxyethylcellulose 3.0 g / m ２ (Average molecular weight: approximately 40,000, average degree of hydroxyethyl group substitution: DS = 1.0 to 1.3, average number of moles: MS = 1.8 to 2.5) Sodium tetraborate 1 g / m ２Creatinine iminohydrase 1800 U / m³ ２ 【0101】 <Development Layer> Furthermore, a development layer was created by uniformly pressing a polyester knitted fabric (gauge number 40) onto this reagent layer. 【0102】 In this way, a multilayer analytical element for liquid sample analysis (the element of Example 1) was obtained in which a detection layer, a liquid barrier layer, and a deployment layer were integrally bonded and laminated on a transparent support in that order. 【0103】 [Example 2] A multilayer analytical element for liquid sample analysis (element of Example 2) was obtained by following the same procedure as in Example 1, except that polyvinyl butyral was not added to the detection layer. 【0104】 [Examples 3-7] Except for using the polymers listed in Table 1 instead of synthetic polymer A, multilayer analytical elements for liquid sample analysis (elements of Examples 3-7) were obtained by following the same procedure as in Example 2. 【0105】 [Comparative Example 1] A multilayer analytical element for liquid sample analysis (element of Comparative Example 1) was obtained by following the same procedure as in Example 1, except that polyvinyl methyl ether (mass-average molecular weight: approximately 40,000) was used instead of synthetic polymer A. The above polyvinyl methyl ether is a water-soluble polymer with a Tg of -25°C and a cloud point of 32°C. Since the above polyvinyl methyl ether has a Tg greater than -30°C, it does not fall under the category of the specific polymer described above. 【0106】 [Comparative Example 2] A multilayer analytical element for liquid sample analysis (element of Comparative Example 2) was obtained by following the same procedure as in Comparative Example 1, except that polyvinyl butyral was not added to the detection layer. 【0107】 [Comparative Example 3] A multilayer analytical element for liquid sample analysis (element of Comparative Example 3) was obtained by following the same procedure as in Example 1, except that synthetic polymer X was used instead of synthetic polymer A. 【0108】 [Comparative Example 4] A multilayer analytical element for liquid sample analysis (element of Comparative Example 4) was obtained by following the same procedure as in Comparative Example 3, except that polyvinyl butyral was not added to the detection layer. 【0109】[Comparative Example 5] A multilayer analytical element for liquid sample analysis (element of Comparative Example 5) was obtained by following the same procedure as in Example 2, except that synthetic polymer Y was used instead of synthetic polymer A. 【0110】 [Comparative Example 6] A multilayer analytical element for liquid sample analysis (the element of Comparative Example 5) was obtained by following the same procedure as in Example 2, except that S-1511 modified (acrylic polymer) manufactured by Toagosei Co., Ltd. was used instead of synthetic polymer A. Since S-1511 modified by Toagosei Co., Ltd. is not water-soluble, it does not fall under the category of the specific polymer described above. 【0111】 [Evaluation] The following evaluations were performed on each obtained element. 【0112】 [Adhesion] A TENSILON UTM-II-20 manufactured by Orientec Co., Ltd. was used as the adhesion force measuring device. The element was cut into strips 10 mm wide and 150 mm long, and the PET film (transparent support) side was attached to the stand with double-sided tape. The PET film and the detection layer were manually peeled off partway, the peeled detection layer and the unfolded layer were clipped together, connected to the measuring device, and measurement was started. The peel force (adhesion force) was recorded as a graph by the recorder, and the peel force could be read from the graph. The adhesion force was evaluated according to the following criteria. The results are shown in Table 1. A score of C or higher is preferable, a score of B or higher is more preferable, and a score of A is even preferable. A: Extremely strong adhesion (80gf or more) B: Strong adhesion (60gf or more, less than 80gf) C: Adhesion is slightly weaker but acceptable (40gf or more, less than 60gf) D: Poor adhesion, occasional peeling (20gf or more, less than 40gf) E: Frequent peeling, impractical (less than 20gf) 【0113】[Sensitivity] A solution containing creatinine at a concentration of 20.0 mg / dL was spot-applied at a rate of 10 μL onto the developing layer, and the chromogenic optical density (OD) after 5 minutes was measured at a measurement wavelength of 600 nm. The sensitivity was then evaluated according to the following criteria. The results are shown in Table 1. A value of C or higher is preferable, a value of B or higher is more preferable, and an A is even preferable. A OD 1.7 or higher B OD 1.6 or higher and less than 1.7 C OD 1.5 or higher and less than 1.6 D OD 1.4 or higher and less than 1.5 E OD less than 1.4 【0114】 【0115】 In Table 1, the "wt%" column for the detection layer polymer represents the percentage (mass%) of repeating units derived from each monomer represented by its general formula relative to the total number of repeating units. 【0116】 As can be seen from Table 1, Examples 1 to 7, in which the detection layer contained the specific polymer, showed excellent adhesion and sensitivity. Among these, Examples 3 to 7, in which the Tg of the specific polymer was -45°C or higher, showed even better adhesion. Among these, Examples 5 to 7, in which the Tg of the specific polymer was -40°C or higher, showed even better adhesion. Among these, Examples 6 to 7, in which the specific polymer was obtained by polymerizing monomers represented by general formula (A), monomers represented by general formula (B), and monomers represented by general formula (C), showed even better sensitivity. 【0117】 On the other hand, Comparative Examples 1 to 6, in which the detection layer did not contain the specific polymer, showed insufficient adhesion and sensitivity in at least one of the two aspects.

Claims

1. A multilayer analytical element for liquid sample analysis comprising, in this order, a detection layer containing a substance that undergoes a change detectable by a gaseous substance, a liquid barrier layer that selectively allows gaseous substances to pass through, and a developing layer, on a transparent support, wherein the detection layer contains a water-soluble polymer having a glass transition temperature of -50°C to -30°C and a cloud point of 30°C or higher.

2. The multilayer analytical element for liquid sample analysis according to claim 1, wherein the water-soluble polymer is a polymer obtained by polymerizing a monomer represented by the following general formula (A) and a monomer represented by the following general formula (B). In general formulas (A) and (B), R １ R represents a hydrogen atom or a methyl group. ２ represents an alkyl group or aryl group having 1 to 4 carbon atoms, and l and m represent the average number of repeating units of oxyethylene, where l is 1 to 2 and m is 3 or more.

3. The multilayer analytical element for liquid sample analysis according to claim 1, wherein the water-soluble polymer is a polymer obtained by polymerizing a monomer represented by the following general formula (A), a monomer represented by the following general formula (B), and a monomer represented by the following general formula (C). In general formulas (A) to (C), R １ R represents a hydrogen atom or a methyl group. ２ represents an alkyl group or aryl group having 1 to 4 carbon atoms, and l, m, and n represent the average number of repeating units of oxyethylene, where l is 1 to 2, m is 3 or more, and n is 1 to 10.

4. The aforementioned R ２ A multilayer analytical element for liquid sample analysis according to claim 2 or 3, wherein the group is a methyl group or an ethyl group.

5. The multilayer analytical element for liquid sample analysis according to claim 2 or 3, wherein m is 3 to 10.

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