JP4243255B2 - Integrated multilayer analytical element for creatinine determination - Google Patents

Description

  The present invention relates to an integrated multilayer analytical element for quantitative determination of creatinine, and more particularly to an integrated multilayer analytical element for quantitative determination of creatinine for analyzing creatinine in a liquid sample such as blood and urine.

  Conventionally, various methods called dry methods have been proposed as methods for measuring urea nitrogen in body fluids in order to simplify and speed up the measurement and eliminate individual differences among measurers. A typical one includes a reagent layer containing urease and an alkaline buffer, and an indicator layer for detecting gaseous ammonia, and a selectively permeable layer that selectively allows only gaseous ammonia to pass between them. It uses an integrated multi-layer analytical element that is integrated between the two. For example, Patent Document 1 (Japanese Patent Laid-Open No. 52-3488) basically discloses an integrated analytical element having the multilayer structure. In this analytical element, a thin hydrophobic polymer layer is used as a selective permeation layer for ammonia gas.

  In Patent Document 2 (Japanese Patent Laid-Open No. 58-77661), an indicator layer for gaseous ammonia, a liquid blocking layer, and an alkaline buffer are contained on a transparent support and, if necessary, react with a substrate to produce ammonia. In the integrated multilayer analytical material for analyzing ammonia or ammonia-producing substrate in a liquid sample, in which a reagent layer having a reagent capable of being dispersed and a spreading layer are integrally bonded and laminated in this order, the liquid blocking layer is made of a porous substance. Disclosed is an integrated multilayer analytical material for analysis of ammonia or ammonia-producing substrate in a liquid sample, characterized in that it comprises an air hole that substantially blocks the liquid sample and allows gaseous ammonia to permeate under operating conditions. Yes. In this multilayer analysis material, an integrated multilayer analysis element is disclosed in which a membrane filter is used as a permselective layer to improve the adhesion to the indicator layer and to increase the sensitivity.

  In addition, Patent Document 3 (Japanese Patent Laid-Open No. 4-157363) uses polyvinyl alkyl ether or the like substantially free of ammonia and ammonium ions for the undercoat layer of the support, or polyvinyl alkyl ether for the binder of the indicator layer. Is used to disclose an integrated multilayer analytical element for analyzing ammonia or ammonia-producing substances with higher color development optical density, lower background color development optical density, and higher measurement accuracy. In Patent Document 4 (Japanese Patent Laid-Open No. 4-157364), poly (N-vinylpyrrolidone) is contained in the porous development layer, ammonia is not substantially contained in the ammonia generation reaction reagent layer, and the pH is about 9. Integral multilayer for analysis of ammonia or ammonia-generating substances with higher measurement optical density, lower background color optical density, and higher measurement accuracy, using a binder that does not generate ammonia at 0 or more and does not change the binder performance Analytical elements are disclosed. Furthermore, Patent Document 5 (Japanese Patent Laid-Open No. 2002-122585) discloses an indicator layer containing an indicator that causes a change detectable by gaseous ammonia on a transparent support, a liquid blocking layer that allows gaseous ammonia to pass through, A reagent layer containing an alkaline buffer and optionally having a reagent capable of reacting with a substrate to generate ammonia, and an analytical element in which a spreading layer is integrally bonded and laminated in this order, and at least two liquid blocking layers An integrated multilayer analytical element for analyzing ammonia or an ammonia-producing substance in a liquid sample characterized by comprising the above porous membrane is disclosed.

JP-A-52-3488 JP 58-77661 A JP-A-4-157363 JP-A-4-157364 JP 2002-122585 A

  However, when trying to quantify creatinine using the analytical element of the prior art, a low molecular amine such as methylamine contained in the sample is gasified in the analytical element to become a gas, which can be detected as an indicator together with gaseous ammonia. It has been clarified by the present inventors that the measurement value of creatinine is undesirably increased and an undesirable increase in the measured value of creatinine is caused. For example, a blood sample of a healthy person does not contain so much low-molecular amine, but a blood sample of a patient with a specific disease state such as kidney disease contains a non-negligible amount of low-molecular amine. Is required.

  Therefore, an object of the present invention is to provide an integrated multilayer analytical element for quantification of creatinine that suppresses an undesirable increase in the measured value of creatinine even if a low molecular amine is contained in the sample.

The above problem can be solved by the following means.
1) An integrated multilayer analytical element for quantification of creatinine, which contains a substance that generates an insoluble salt with an amine.
2) The integrated multilayer analytical element for quantification of creatinine includes an indicator layer containing an indicator that causes a change detectable by gaseous ammonia on a transparent support, a liquid blocking layer that allows gaseous ammonia to pass through, and an alkaline buffer. 1) The integrated type for quantification of creatinine according to 1) above, wherein the reagent layer containing an agent and capable of producing ammonia by reacting with creatinine and the development layer are integrally laminated in this order. Multi-layer analytical element.
3) The integrated multilayer analytical element for quantification of creatinine according to 2) above, wherein a substance that generates an insoluble salt with the amine is contained in the spreading layer.
4) The integrated type for quantification of creatinine according to any one of 1) to 3) above, wherein the substance that generates an insoluble salt with the amine is tetraphenylborate or a tetraphenylborate derivative. Multi-layer analytical element.

  The integrated multilayer analytical element for quantification of creatinine of the present invention contains a substance that generates an insoluble salt with amine, so that even if a low molecular amine is contained in the sample, the measured value of creatinine is not desired. High price is suppressed.

The layer structure of the integrated multilayer analytical element for quantification of creatinine of the present invention can be the same as that of the conventional integrated multilayer analytical element for quantification of creatinine as long as it contains a substance that generates an insoluble salt with amine. Although not particularly limited, it contains at least an indicator layer containing an indicator that causes a change detectable by gaseous ammonia on a transparent support, a liquid blocking layer that allows gaseous ammonia to pass through, and an alkaline buffer, and if necessary, a substrate. It is preferable that the reagent layer capable of reacting to generate ammonia and the developing layer are analytical elements in which they are integrally laminated in this order.
In addition, at least one layer that exhibits a desired function is provided in any place on the transparent support-indicator layer, indicator layer-liquid blocking layer, liquid blocking layer-reagent layer, reagent layer-developing layer, and developing layer. You can also.
Hereinafter, each layer will be described.

(Transparent support)
As the transparent support, a hydrophobic transparent support generally used in such an analytical element, for example, a transparent support made of a polymer such as polyethylene terephthalate, polycarbonate or polyvinyl compound is used. The thickness of the transparent support is about 50 to 1000 μm, usually about 80 to 300 μm.

(Indicator layer)
The indicator layer includes at least an indicator that produces a detectable change with gaseous ammonia. An indicator that produces a change that can be detected by gaseous ammonia means a substance that changes its structure as a result of reaction or interaction with gaseous ammonia and changes its spectroscopic properties such as absorption wavelength. To do. Here, the change in structure includes a chemical structure, for example, a change in atomic composition, a change in bond relationship such as a covalent bond and a hydrogen bond, and a change in steric structure.
As the indicator that produces a change that can be detected by gaseous ammonia, for example, a compound that reacts with gaseous ammonia to cause a change in absorption wavelength (hereinafter referred to as a color-forming precursor) is suitable. Examples of chromogenic precursors that can be used in the analysis element of the present invention include leuco dyes such as leuco cyanine dyes, nitro-substituted leuco dyes, and leucophthalein dyes (Japanese Patent Laid-Open No. 52-3488, US Pat. No. RE 30 267). PH indicators such as bromophenol blue, bromocresol green, bromothymol blue, quinoline blue and rosoleic acid (described in Kyoritsu Shuppan, Chemical Dictionary, Vol. 10, pages 63-65); Triarylmethane dye precursors Leucobenzylidene dyes (described in JP-A No. 56-145273); diazonium salts and azo dye couplers; base-bleachable dyes, and the like.

An indicator layer can be formed by mixing at least one of these coloring precursors with a binder polymer and applying the mixture on a transparent support. The binder polymer preferably contains an adhesive polymer and a water-insoluble vinyl polymer. The adhesive polymer preferably has a Tg of 0 ° C. or lower (more preferably −150 to −5 ° C., particularly preferably −100 to −10 ° C.). The adhesive polymer preferably has a mass average molecular weight of 10,000 to 1,000,000 (more preferably 10,000 to 500,000, particularly preferably 20,000 to 200,000).
The adhesive polymer is mainly composed of polyvinyl alkyl ether (eg, polyvinyl methyl ether, polyvinyl ethyl ether, polyvinyl isobutyl ether, etc.), natural rubber, chloroprene, styrene butadiene rubber, acrylic ester of aliphatic alcohol having 2 to 16 carbon atoms. , A polymer obtained by copolymerizing a small amount of monomers having polar groups such as acrylic acid, acrylamide, etc., a silicone adhesive composed of a combination of silicone rubber and silicone resin, and styrene / isoprene / styrene block polymer Adhesives, rosin resins, terpene resins, hydrogenated petroleum resins, polyisobutylene, indene, dammers, copal, coumarone, picopale, alkyd resins, cellulose esters, neoprene It is. Of these, polyvinyl alkyl ether is preferable.
The addition amount of the adhesive polymer is 1 to 500 times, preferably 3 to 300 times, and more preferably 5 to 100 times the indicator amount of the indicator layer.

The water-insoluble vinyl polymer needs to be insoluble in water, but is preferably soluble in the indicator layer coating solvent, and is compatible with the adhesive polymer and other polymers that can be used in combination. Preferably there is. Here, in this specification, being insoluble in water means having a solubility of 1 g or less in 100 g of water at 20 ° C. The indicator layer coating solvent is generally an organic solvent or a mixed solvent of an organic solvent and water. The water-insoluble vinyl polymer is preferably soluble in alcohol, and particularly preferably ethanol-soluble.
The water-insoluble vinyl polymer preferably has a Tg of 10 ° C. or higher (more preferably 20 to 150 ° C., particularly preferably 30 to 120 ° C.). The water-insoluble vinyl polymer preferably has an average degree of polymerization of 30 to 5000 (more preferably 50 to 3000, particularly preferably 100 to 2500).
The water-insoluble vinyl polymer preferably has an acetal group. Examples of the acetal group include a butyral group, an acetal group, and a formal group, and the water-insoluble vinyl polymer is particularly preferably polyvinyl butyral.
The addition amount of the water-insoluble vinyl polymer is 1 to 50% by mass, preferably 1 to 30% by mass, and more preferably 1 to 20% by mass with respect to the adhesive polymer.

  In addition, polymers other than those exemplified above can also be used as the binder polymer. Other binder polymers include acid-treated gelatin, alkali-treated gelatin, deionized gelatin, and other gelatins; cellulose esters such as cellulose acetate, cellulose acetate butyrate, and cellulose acetate propionate; alkyls such as methylcellulose, ethylcellulose, and propylcellulose. Celluloses; etc. are used.

  The used amount of the color developing precursor is about 0.1 to 50% by mass, preferably about 0.5% to 20% by mass with respect to the mass of the binder polymer. For the purpose of adjusting the sensitivity, the pH can be adjusted by adding various buffering agents, organic acids, inorganic acids and the like. The buffering agent can be selected from those described later. Examples of the organic acid and inorganic acid include ethanesulfonic acid, aspartic acid, azelaic acid, glutaric acid, succinic acid, glutaconic acid, tartaric acid, pimelic acid, malonic acid, apple Acid, 3,3-dimethylglutaric acid, citric acid, P-toluenesulfonic acid, perchloric acid, hydrochloric acid and the like can be used. Furthermore, alkalis such as sodium hydroxide, potassium hydroxide, disodium carbonate, and sodium bicarbonate can be added to the indicator layer. The solvent used to form the coating solution is suitably an organic solvent such as acetone, 2-methoxyethanol, methyl ethyl ketone, methanol, ethanol, or water, or a mixed solvent thereof. These solvents include a color precursor and a binder polymer. Etc. are added so that the solid content concentration is about 1 to 30% by mass, preferably about 3 to 20% by mass. This is coated on a transparent support and dried to form an indicator layer so that the dry film thickness is about 1 to 30 μm, preferably 2 to 20 μm.

  The liquid blocking layer provided on the indicator layer is a liquid such as a coating solution and a sample solution and an interference component dissolved in these liquids (for example, alkaline Preferably, it is made of a microporous material having through-air holes that are substantially impermeable to components and pass gaseous ammonia.

  The liquid blocking layer in the present invention is composed of one or more porous films. In the case of two or more porous membranes, the pore diameter of the uppermost porous membrane in contact with the reagent layer described later is preferably the same as or smaller than the pore diameter of the porous membrane immediately below. Specifically, the pore diameter of the uppermost porous membrane is 0.01 to 1 μm, preferably 0.04 to 0.2 μm, and the pore diameter of the porous membrane immediately below it is 0.2 to 20 μm, preferably 0.5. The ratio of the average pore diameter of the uppermost porous membrane / the pore diameter of the porous membrane immediately below it is 0.001 to 1.0, preferably 0.01 to 0.5. The pore diameter in this specification is an average pore diameter unless otherwise specified. The material of the porous film is not particularly limited, and examples thereof include fluorine-containing polymers such as polyethylene, polypropylene, and polytetrafluoroethylene, cellulose acetate, polysulfone, polyamide (nylons), and mixtures thereof. Preferred is a combination of a polyethylene porous membrane and a polypropylene porous membrane. Each porous film has a thickness of 3 to 40 μm, preferably 5 to 20 μm, and a liquid blocking layer is formed by combining two or more layers, usually 2 to 3 layers. The porosity of the entire liquid blocking layer is 25 to 90%, preferably 35% to 90, and the total layer thickness is 10 to 150 μm, preferably 10 to 50 μm.

  The liquid barrier layer adheres to the indicator layer described above with practical strength. For adhesion, the indicator layer surface may be wetted and pasted and dried. Here, the wet state means that the solvent in which the binder is dissolved remains, or the dried film is wetted with the soluble solvent and the binder is in a swollen state, a dispersed state or a solution state.

  Bonding between the porous films is performed by physical and / or chemical means such as bonding in a point contact manner using an adhesive such as thermocompression bonding or hot melt. These may be sequentially laminated, or may be adhered to the indicator layer after the porous films are first laminated.

  A reagent layer can be provided on the liquid blocking layer. The reagent layer includes, for example, a reagent that reacts with creatinine to generate ammonia (generally an enzyme or a reagent containing an enzyme), an alkaline buffer and a film-forming ability for efficiently releasing ammonia generated by the reaction as gaseous ammonia. It is a layer usually containing a hydrophilic polymer binder having Examples of the reagent for generating ammonia include creatinine deiminase, for example, creatinine iminohydrase (EC3.5.4.21).

  As an alkaline buffer that can be used in the reagent layer, a buffer having a pH in the range of 7.0 to 10.5, preferably 7.5 to 10.0 can be used. Specific examples of the buffer include ethylenediaminetetraacetic acid (EDTA), tris (hydroxymethyl) aminomethane (Tris), phosphate buffer, N, N-bis (2-hydroxyethyl) glycine (Bicine), N-2 -Good's buffer such as hydroxyethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid (HEPPSO), N-hydroxyethylpiperazine-N'-ethanesulfonic acid (HEPES), borate buffer, etc. be able to.

  Examples of the hydrophilic polymer binder having film-forming ability that can be used in the reagent layer include gelatin, agarose, polyvinyl alcohol, polyacrylamide, hydroxymethyl cellulose, hydroxyethyl cellulose, and polyvinyl pyrrolidone.

  In addition to the above components, the reagent layer can contain a wetting agent, a binder cross-linking agent (curing agent), a stabilizer, a heavy metal ion trapping agent (complexing agent), and the like as necessary.

  The reagent layer is a coating liquid obtained by mixing a reagent that reacts with creatinine to generate ammonia, an alkaline buffer, and the above-described reagents added as necessary with a hydrophilic polymer binder such as gelatin having a film forming ability, It can be formed by applying and drying on the liquid blocking layer.

  The amount of the reagent that reacts with creatinine contained in the reagent layer to generate ammonia is usually in the range of about 0.1 to about 50% by weight, preferably about 0.2 to about 20%, based on the weight of the binder. is there. Suitably, the amount of alkaline buffer is in the range of about 0.1 to about 60 weight percent based on the weight of the binder. The dry film thickness of this reagent layer is usually in the range of about 1 to about 40 μm, preferably about 2 to about 20 μm.

  A development layer is provided on the reagent layer. As the spreading layer, a textile fabric spreading layer (for example, broad) described in JP-A-55-164356 (corresponding US Pat. No. 4,292,272), JP-A-57-66359 (corresponding US Pat. No. 4,783,315), etc. Flat knitted fabrics such as poplin), knitted fabric unfolding layer described in JP-A-60-222769 (corresponding EP Patent 0 162 302A) and the like (eg, tricot knitted fabric, double tricot knitted fabric, Miranese knitted fabric), An organic polymer fiber pulp-containing papermaking paper development layer described in JP-A-57-148250, a development layer formed by applying a dispersion of a fiber and a hydrophilic polymer described in JP-A-57-125847, etc. A membrane filter layer described in JP-B-53-21777 (corresponding to US Pat. No. 3,992,158) Rush polymer layer), isotropic microporous development layer containing continuous microvoids in which fine particles such as polymer microbeads are adhered in a point contact manner with a hydrophilic polymer binder, Japanese Patent Laid-Open No. 55-90859 (corresponding US Patent) 4 258 001), etc., and continuous microvoid-containing isotropic microporous layer (three-dimensional lattice-like granular structure layer) developed by adhering the polymer microbeads in a point contact manner with a polymer adhesive that does not swell with water Non-fiber isotropic microporous development layer such as a layer; Japanese Patent Laid-Open No. 61-4959 (corresponding US Pat. No. 5,019,347), Japanese Patent Laid-Open No. 62-138756, Japanese Patent Laid-Open No. 62-138757, Japanese Patent Laid-Open No. 62-138758 (Corresponding EP Patent No. 0 226 465A) a plurality of microporous layers (for example, two layers of woven fabric or knitted fabric and membrane filter, woven fabric or knitted fabric) 3 layer of membrane filter and woven fabric or knitted fabric) with a fine discontinuous or island-like (halftone dot in the printing field) adhesive layer on the surface and a spread layer with excellent blood cell separation ability There is.

  A woven fabric or a knitted fabric used for the spreading layer is subjected to a physical activation treatment typified by a glow discharge treatment or a corona discharge treatment described in JP-A-57-66359 on at least one side of the cloth fabric, or a special feature. Cloth fabric by performing water washing degreasing treatment, hydrophilic treatment such as impregnation with hydrophilic polymer described in JP-A-55-164356, JP-A-57-66359, etc., or by appropriately combining these treatment steps. Can be made hydrophilic to increase the adhesive force with the lower layer (side closer to the support). Further, as described in JP-A-59-171864, JP-A-60-222769, JP-A-60-222770, etc., a polymer-containing aqueous solution or a polymer-containing water-organic solvent mixed solution is formed on the spreading layer. Can be applied to control the development area or spread of the liquid sample.

  In the analysis element of the present invention, an ammonia diffusion preventing layer and an endogenous ammonia supplementing layer can be provided in this order between the reagent layer and the spreading layer. The ammonia diffusion prevention layer and the endogenous ammonia supplementation layer are described in, for example, Japanese Patent Application Laid-Open No. 4-157364, etc., and for example, the ammonia diffusion prevention layer is substantially subjected to ammonia supplementation and ammonia generation reaction. Layer, which can be formed using a hydrophilic polymer such as hydroxypropylcellulose. The thickness of the ammonia diffusion preventing 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 to change it to a state where it cannot substantially reach the reagent layer. Examples of the reagent include a composition containing an enzyme having a catalytic ability of changing ammonia into another substance using ammonia as a substrate. A specific example of the composition is a composition containing α-ketoglutarate, NADPH, and glutamate dehydrogenase using a hydrophilic polymer such as hydroxyethyl cellulose as a binder polymer. The thickness of the endogenous ammonia supplement layer is, for example, 1 to 30 μm.

  Furthermore, a color shielding layer or a light reflection layer can be provided between the reagent layer and the spreading layer. In the color shielding layer or the light reflecting layer, white particles such as titanium dioxide fine particles and barium sulfate fine particles having light shielding properties or light shielding properties and light reflecting properties are dispersed almost uniformly in a hydrophilic polymer binder such as gelatin. The dry layer has a thickness ranging from about 2 μm to about 20 μm.

  Further, a known adhesive layer made of a hydrophilic polymer may be provided on the reagent layer, the ammonia diffusion preventing layer, the endogenous ammonia supplementing layer, the color shielding layer or the light reflecting layer for the purpose of firmly bonding and integrating the spreading layer. it can. The dry thickness of the adhesive layer ranges from about 0.5 μm to about 5 μm.

  A surfactant can be contained in the reagent layer, the ammonia diffusion preventing layer, the endogenous ammonia supplementing layer, the color shielding layer or the light reflecting layer, the adhesive layer, the spreading layer and the like. An example is a nonionic surfactant. Specific examples of nonionic surfactants include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, p-nonylphenoxypolyglycidol, octyl glucoside, etc. There is. By incorporating the nonionic surfactant in the spreading layer, the spreading action (metering action) of the aqueous liquid sample becomes better. By containing a nonionic surfactant in the reagent layer, ammonia diffusion preventing layer, endogenous ammonia supplemental layer, color shielding layer or light reflecting layer, adhesive layer, etc., water in the aqueous liquid sample is substantially contained in the reagent layer during the analysis operation. The liquid contact with the spreading layer becomes quick and substantially uniform.

  As described above, the integrated multilayer analytical element for quantification of creatinine of the present invention is characterized by containing a substance that generates an insoluble salt with an amine. The substance is not particularly limited as long as it is a substance that forms an insoluble salt with an amine, and examples thereof include tetraphenylborate and tetraphenylborate derivatives. Among them, tetraphenylborate (for example, sodium tetraphenylborate) ), Tetraphenylborate derivatives (for example, tetrakis (potassium 4-chlorophenylborate), tetrakis [3,5-bis (1,1,1,3,3,3-hexafluoro-2-methoxy-2) -Propyl) phenyl] sodium borate trihydrate, tetrakis [3,5-bis (trifluoromethyl) phenyl] sodium borate dihydrate, sodium tetrakis (4-fluorophenyl) borate.2 water Japanese) is preferred. The amine contained in the sample (for example, a blood sample) is a low molecular amine such as methylamine.

The substance that generates an insoluble salt with an amine can be contained, for example, in the ammonia diffusion prevention layer, the endogenous ammonia supplementing layer, the developing layer, etc., but the developing layer contains a substance that generates an amine and an insoluble salt. Is preferred. In order to introduce a substance that generates an amine and an insoluble salt into the development layer, a coating liquid (overcoat liquid) containing a substance that generates an amine and an insoluble salt may be applied on the formed development layer and dried. In order to introduce a substance capable of forming an amine and an insoluble salt into the developing layer into another layer, a substance capable of forming an amine and an insoluble salt may be contained in the coating solution for forming the other layer.
The coating amount of the substance that generates an insoluble salt with amine is, for example, 0.1 to 10 g / m ² , preferably 0.5 to 8.0 g / m ² , more preferably 1.0 to 6.0 g / m ² . is there.

  The overcoat solution contains a substance that generates an amine and an insoluble salt, a polymer, and a solvent thereof, completes the analytical element, and the pH of the spreading layer when the specimen is spotted is pH 2-9, preferably pH 3 It is preferable to add an acid such as hydrochloric acid so as to be ˜7. Examples of the polymer include polyvinyl pyrrolidone, polyacrylamide, polyvinyl alcohol, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose and the like, and the mass average molecular weight is preferably 5,000 to 2,000,000. Examples of the solvent include ethanol, 2-methoxyethanol, methanol, propanol, isopropanol, water, or a mixed solvent thereof.

  In order to measure creatinine in a sample using the analytical element of the present invention, an aqueous liquid sample drop such as whole blood, plasma, serum, urine or the like in the range of 3 to 30 μL, preferably 6 to 15 μL, is placed on the spreading layer. After spotting and incubating at a substantially constant temperature in the range of about 20 to 40 ° C. in the range of 1 to 10 minutes, the degree of color change (color development or fading) of the indicator layer from the transparent support side is determined. Reflection metering or visual comparison with standard colors.

EXAMPLES Hereinafter, although an Example and a comparative example further demonstrate this invention, this invention is not limited to the following example.
[Example 1]
On the transparent polyethylene terephthalate (PET) film having a thickness of 180 μm, an indicator layer was applied and dried with an ethanol solution so as to have the following coating amount.
Indicator layer Bromophenol blue 110mg / m ²
Polyvinyl ethyl ether 1.8 g / m ² Mass average molecular weight: about 40,000 Polyvinyl butyral 0.18 g / m ² Average degree of polymerization: about 300
Sodium hydroxide 6.8 mg / m ²

  Next, a polyethylene membrane filter having an average pore size of 0.2 μm, a porosity of 75%, and a thickness of 100 μm was uniformly pressed on the indicator layer to provide a liquid blocking layer. On this liquid blocking layer, the reagent layer was applied from an aqueous solution and dried so as to have the following coating amount.

Reagent layer (pH 9.0)
Hydroxyethyl cellulose 3.0 g / m ²
Average molecular weight: about 40,000 Hydroxyethyl group average substitution degree: DS = 1.0 to 1.3
Average number of moles: MS = 1.8 to 2.5
Sodium tetraborate 1g / m ²
Creatinine iminohydrase 1800 U / m ²
(EC 3.5.4.21)

Further, an ammonia diffusion preventing layer was applied on the reagent layer so as to have the following coating amount and dried.
Ammonia diffusion prevention layer Hydroxypropylcellulose 20.0 g / m ²
Methoxy group 28-30%
Hydroxypropoxy group 7-12%
Viscosity of 2% aqueous solution at 20 ° C. 0.05 Pa. s (50 cps)

Further, an endogenous ammonia supplement layer was applied on the ammonia diffusion preventing layer so as to have the following coating amount and dried.
Endogenous ammonia supplementary layer (pH 8.2)
Hydroxyethyl cellulose 5g / m ²
Ampholite 1.2g / m ²
α-ketoglutaric acid 1.2 g / m ²
NADPH 0.4 g / m ²
Glutamate dehydrogenase 10000 U / m ²
(EC 1.4.1.4)

  The endogenous ammonia supplement layer was moistened almost uniformly with a 0.2% aqueous solution of P-nonylphenoxypolyglycidol, and a polyester knitted fabric (gauge number 40) was immediately crimped uniformly. Further, this laminate was impregnated with an ethanol solution (overcoat solution) of polyvinylpyrrolidone so as to have the following coating amount, dried, slitted to a width of 12 mm, and an integrated multilayer analytical element for creatinine determination was prepared.

Overcoat solution Polyvinylpyrrolidone 7.5 g / m ²
Average molecular weight: about 1,200,000 1N hydrochloric acid 0.04 g / m ²
Sodium tetraphenylborate 1.5 g / m ²

[Comparative Example 1]
An integrated multilayer analytical element for the determination of creatinine was prepared in the same manner as in Example 1 except that sodium tetraphenylborate was removed from the overcoat solution.

  Using the integrated multilayer analytical element for quantification of creatinine obtained above, 10 μl of creatinine having a concentration of 0.58 mM in water as a solvent was spotted on the spreading layer, and the optical density after 5 minutes at a measurement wavelength of 600 nm (OD) was examined. Separately, using the integrated multilayer analytical element for quantification of creatinine obtained above, 10 μl of methylamine at a concentration of 0.58 mM with water as the solvent was spotted on the spreading layer, and 5 at a measurement wavelength of 600 nm was obtained. The color development optical density (OD) after a minute was examined. The results are shown in the table below. In the results of the following table, ΔOD (methylamine) / ΔOD (creatinine) represents the ratio of the former to the latter (the former / the latter) by subtracting the OD of water from the OD of methylamine and the OD of creatinine, respectively. Is.

  From the results of Table 1, in the analytical element of Example 1, the OD increase due to methylamine can be suppressed by about 40% compared to the conventional analytical element that does not use sodium tetraphenylborate (Comparative Example 1). I was able to confirm.

Claims (4)

  An integrated multilayer analytical element for determination of creatinine, comprising a substance that generates an insoluble salt with an amine.   The integrated multilayer analytical element for quantitative determination of creatinine comprises, on a transparent support, at least an indicator layer containing an indicator that causes a change detectable by gaseous ammonia, a liquid blocking layer that allows gaseous ammonia to pass through, and an alkaline buffer. The integrated multilayer analysis for creatinine quantification according to claim 1, wherein the reagent layer that contains and reacts with creatinine to generate ammonia, and the development layer are integrally laminated in this order. element.   The integrated multilayer analytical element for quantification of creatinine according to claim 2, wherein a substance that generates an insoluble salt with the amine is contained in the spreading layer.   The integrated multilayer analytical element for quantification of creatinine according to any one of claims 1 to 3, wherein the substance that generates an insoluble salt with the amine is tetraphenylborate or a tetraphenylborate derivative. .