JP2006098102A - Multilayer analysis element - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a multilayer analysis element capable of maintaining a characteristic of a film single body, even in the case where a porous film is laminated, and having no liquid quantity dependency. <P>SOLUTION: Concerning a multilayer analysis element for liquid sample analysis wherein at least one non-fibrous/non-porous polymer layer and a porous liquid sample developing layer comprising at least one non-fibrous porous film are laminated integrally in this order on one surface of a water-impermeable optically-transmissible flat support, in this multilayer analysis element for the liquid sample analysis, the non-fibrous/non-porous polymer layer and the porous liquid sample developing layer comprising the non-fibrous porous film adhere to each other by a water-insoluble polymer. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a dry multilayer analytical element used for clinical inspection, food inspection, environmental analysis, and the like, and a method for producing the same.

  In the fields of clinical testing, food testing, and environmental analysis, demands for processing samples quickly and simply are increasing year by year, and dry analysis elements that meet these needs are widely used. Among the dry analytical elements, the spreading layer for accepting, spreading, and diffusing blood and the like is represented by JP-A-55-164356, JP-A-57-66359, JP-A-60-222769, and the like. Fibrous porous materials have been used.

  This fibrous porous material develops quickly when a liquid sample is spotted, and is excellent in handleability during production. It is also applicable to viscous samples such as whole blood and is widely used.

  However, in this field, measurement with higher accuracy (reproducibility) has been required, and some problems have occurred with fibrous porous materials (cloth spreading layers). One of them is the problem of cloth lot fluctuation. Usually, the fabric spreading layer includes a woven fabric and a knitted fabric. Differences between lots and in-lot differences are found in the weaving and knitting methods. Specifically, the number of stitches per unit area, weight per unit area, thickness, and the like. In addition, there is a material washing step as an intermediate step, but there are lot-to-lot differences and lot-to-lot differences in the hydrophilicity of the cloth depending on the degree of washing. Furthermore, since the cloth spreading layer is not smooth, if it is to be manufactured by securing a sufficient adhesive force by the lamination method, the spreading layer has to be bitten into the lower layer. As a result, the lower layer is disturbed, which is not preferable for measurement with higher accuracy. In addition, when the cloth is bonded to the lower layer, the structure tends to stretch due to its structure, and the void volume tends to change. For this reason, the development area at the time of liquid sample spotting tends to change, which is a cause of in-lot difference and high accuracy measurement not being achieved. In recent years, it has been desired to measure with a smaller number of samples. However, in the cloth spreading layer, when the amount of the sample liquid is reduced, the variation in the amount of reflected light due to the influence of the stitch becomes more significant, and the spreading layer is bonded. In this case, there is a problem that high-precision measurement cannot be performed due to non-uniform disturbance in the lower layer.

  As a technique to replace the cloth spreading layer, a method for producing a porous film by coating has been proposed. Representative inventions are a so-called brush polymer layer (JP-A-49-53888) utilizing a polymer phase transition reaction during coating and drying, and a bead spreading layer (JP-A-55-90859) formed by coating microbeads. Are known. However, these methods have the disadvantage that the film quality of the spreading layer is weak and the spreading layer is fragile and easily peeled when the sheet-like coating is slid (when processed).

  As one of the methods for improving these drawbacks, a method of laminating a non-fibrous porous membrane which is homogeneous and has a high membrane strength as a spreading layer has been proposed (Japanese Patent Application Laid-Open No. S49-53888). Kaisho 56-97872). When applying a porous membrane that is more uniform than the cloth as the spreading layer, when the specimen is spotted on a single membrane, the spreading area changes depending on the liquid volume, so the specimen volume per unit area is constant. Can keep. That is, there is no dependency on the liquid amount. However, it was found that the film was deteriorated when the film was supplied in a conventional manner with dampening water supplied to the polymer layer, laminated, a slide was prepared, and the liquid volume dependency was examined.

JP-A-55-164356 JP-A-57-66359 JP 60-222769 A Japanese Patent Laid-Open No. 49-53888 JP 55-90859 A Japanese Patent Laid-Open No. 56-97872

  The present invention has been made to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a multilayer analytical element that maintains the characteristics of a single membrane and has no liquid volume dependency even when a porous membrane is laminated.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have determined that a dry type for liquid sample analysis in which a porous liquid sample development layer composed of a non-fiber / non-porous polymer layer and a non-fiber porous film is laminated and integrated. In a multi-layer analytical element, the present inventors have found that the above-mentioned problems can be solved by adhering a non-fibrous / non-porous polymer layer and a porous liquid sample development layer comprising a non-fibrous porous film with a water-insoluble polymer, thereby completing the present invention. It came to do.

  That is, according to the present invention, a porous liquid sample development comprising at least one non-fibrous / non-porous polymer layer and at least one non-fibrous porous membrane on one side of a water-impermeable and light-transmissive flat support. In the dry multi-layer analytical element for liquid sample analysis in which the layers are laminated and integrated in this order, the non-fiber / non-porous polymer layer and the porous liquid sample development layer comprising the non-fibrous porous membrane are in close contact with the water-insoluble polymer. A dry multilayer analytical element for analyzing liquid samples is provided.

  Preferably, the non-fibrous porous membrane is 6,6-nylon, 6-nylon, acrylate copolymer, polyacrylate, polyacrylonitrile, polyacrylonitrile copolymer, polyamide, polyimide, polyamideimide, polyurethane, polyethersulfone, Polysulfone, mixture of polyethersulfone and polysulfone, cellulose acetate, cellulose butyrate, cellulose acetate butyrate (mixed ester), cellulose nitrate, polyester, polyester carbonate, polyethylene, polyethylene chlorotrifluoroethylene copolymer, polyethylene tetrafluoroethylene Copolymer, polyvinyl chloride, polyolefin, polycarbonate, polytetrafluoroethylene, polyvinylidene difluoride, polyphenol Ren sulfide, polyphenylene oxide, polyfluorocarbonate, polypropylene, polybenzimidazole, polymethyl methacrylate, styrene-acrylonitrile copolymer, styrene-butadiene copolymer, saponified ethylene-vinyl acetate copolymer, polyvinyl alcohol, or It is a mixture of these. More preferably, the non-fibrous porous membrane is polysulfone, polyethersulfone, cellulose acetate, 6,6-nylon, or 6-nylon.

Preferably, the non-fibrous porous membrane is an asymmetric membrane.
Preferably, the water insoluble polymer is a hot melt adhesive.

  In the dry multi-layer analytical element of the present invention, the non-fiber / non-porous polymer layer and the porous liquid sample development layer comprising the non-fibrous porous membrane are closely attached to each other by the water-insoluble polymer. Can be maintained, and it has become possible to provide a dry multilayer analytical element with improved liquid volume dependency.

Hereinafter, embodiments of the present invention will be described in detail.
The dry multilayer analytical element for liquid sample analysis of the present invention comprises at least one non-fibrous / non-porous polymer layer and at least one non-fibrous porous membrane on one side of a water-impermeable and light-transmissive flat support. The porous liquid sample development layer is laminated and integrated in this order, and the non-fiber / non-porous polymer layer and the porous liquid sample development layer composed of the non-fibrous porous film are adhered to each other by the water-insoluble polymer. It is characterized by being.

  In the conventional method, dampening water is supplied to the non-fiber / non-porous polymer layer and then the non-fiber porous film is laminated to form the porous liquid sample development layer. By disposing a water-insoluble polymer having a non-fibrous / non-porous polymer layer between the non-fibrous porous membrane and the non-fibrous porous membrane, the characteristics of the non-fibrous porous membrane alone are maintained. As a result, the characteristic that the development area changes according to the amount of liquid indicated by the single membrane is maintained even when it is slid, and the dependency on the liquid amount is improved.

  The water-insoluble polymer used in the present invention is not particularly limited as long as it can adhere the non-fiber / non-porous polymer layer and the porous liquid sample developing layer made of the non-fibrous porous film, but preferably a hot melt adhesive. Can be used. The hot-melt adhesive is a type of adhesive that is solid at normal temperature and is used by heating and melting. As the hot melt material, materials described in Industrial Materials Vol. 26, No. 11, P4 to P5 can be used. That is, ethylene-vinyl acetate copolymer, ethylene-vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, ethylene copolymer such as ethylene-acrylic acid copolymer, low molecular weight polyethylene and atactic polypropylene, etc. Polyolefins, polyamides such as nylon, polyester copolymers, thermoplastic rubbers such as styrene block copolymers such as SBS, styrene butadiene rubber, butyl rubber, urethane rubber, rosin, petroleum resin, terpene resin, paraffin, synthetic Various materials such as wax can be used.

The amount of the water-insoluble polymer is about 1 to 10 g / m ^2, and preferably is about 1 to 5 g / m ^2, for example, 3 g / m ^2.

  The dry multilayer analytical element of the present invention is preferably provided with at least one non-fiber / non-porous polymer layer on one side of a water-impermeable and light-transmissive flat support, and then the non-fiber / non-porous It can be produced by laminating a non-fibrous porous film coated with a water-insoluble polymer on the upper surface of the polymer layer.

  As the water-impermeable and light-transmissive planar support, a water-impermeable and light-transmissive support that is used in conventionally known dry analytical elements can be used. Examples of water-impermeable and light-transmitting supports include polymers such as polyethylene terephthalate, bisphenol A polycarbonate, polystyrene, cellulose esters (eg, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.). A film or sheet-like transparent support having a thickness in the range of about 50 μm to about 1 mm, preferably about 80 μm to about 300 μm can be mentioned.

  If necessary, an undercoat layer may be provided on the surface of the support to strengthen the adhesion between the adhesive layer provided on the support and the support. Further, instead of the undercoat layer, the surface of the support may be subjected to a physical or chemical activation treatment to improve the adhesion.

  The dry multilayer analytical element of the present invention includes a porous liquid sample developing layer composed of at least one non-fibrous porous membrane. The porous liquid sample spreading layer is a layer having a function of spreading in a plane without substantially uneven distribution of components contained in an aqueous specimen and supplying the functional layer at a substantially constant rate per unit area. It is.

  The porous liquid sample spreading layer need not be limited to only one layer, and a laminate in which two or more non-fibrous porous membranes are bonded with an adhesive partially disposed can be used. In addition, the porous liquid sample development layer may contain a development control agent such as a hydrophilic polymer for the purpose of controlling the development. Furthermore, a reagent for performing a target detection reaction, a reagent for promoting the detection reaction, various reagents for reducing or preventing interference / interfering reactions, or a part of these reagents may be included. it can.

  The porous liquid sample development layer in the present invention is made of a non-fibrous porous film. Preferably, the non-fibrous porous membrane is a porous membrane made of an organic polymer. As the porous film made of the organic polymer described above, either a symmetric film or an asymmetric film can be used. In the case of an asymmetric porous membrane, the asymmetry rate is preferably 2.0 or more, and in the case of a symmetric porous membrane, the asymmetry rate is preferably less than 2.0. The asymmetric porous membrane referred to in the present specification is a porous membrane in which the average pore diameter of one surface is larger than the average pore diameter of the other surface, and the asymmetry rate is the average of the larger average pore diameter of the smaller one. It is the value divided by the hole diameter.

  Preferred examples of the porous film made of organic polymer include 6,6-nylon, 6-nylon, acrylate copolymer, polyacrylate, polyacrylonitrile, polyacrylonitrile copolymer, polyamide, polyimide, polyamideimide, polyurethane, Polyethersulfone, polysulfone, a mixture of polyethersulfone and polysulfone, cellulose acetate, cellulose butyrate, cellulose acetate butyrate (mixed ester), cellulose nitrate, polyester, polyester carbonate, polyethylene, polyethylene chlorotrifluoroethylene copolymer, Polyethylene tetrafluoroethylene copolymer, polyvinyl chloride, polyolefin, polycarbonate, polytetrafluoroethylene, polyvinylidene diph Olide, polyphenylene sulfide, polyphenylene oxide, polyfluorocarbonate, polypropylene, polybenzimidazole, polymethyl methacrylate, styrene-acrylonitrile copolymer, styrene-butadiene copolymer, saponified ethylene-vinyl acetate copolymer, polyvinyl alcohol Or a mixture thereof.

  Among these, 6,6-nylon, 6-nylon, polyethersulfone, polysulfone, a mixture of polyethersulfone and polysulfone, cellulose acetate, cellulose butyrate, cellulose acetate butyrate (mixed ester), cellulose nitrate, polyester, More preferred are polyethylene, polypropylene, polyolefin, polyacrylonitrile, polyvinyl alcohol, polycarbonate, polyester carbonate, polyphenylene oxide, polyamide, polyimide, polyamideimide, or a mixture thereof.

  More preferred are polysulfone, polyethersulfone, cellulose acetate, 6,6-nylon, or 6-nylon, particularly preferred is polysulfone or polyethersulfone, and most preferred is polysulfone.

The film thickness of the non-fibrous porous film is preferably 80 to 300 μm, more preferably 100 to 200 μm, and particularly preferably 130 to 160 μm.
Moreover, the average pore diameter of the non-fibrous porous membrane is preferably 0.3 to 10 μm, and more preferably 0.45 to 5 μm.

  Examples of the configuration of the dry multilayer analytical element for liquid sample analysis of the present invention include: (1) One or more non-fiber / non-porous polymer layers (functional layers) are provided on a transparent support; Dry multi-layer analytical element for liquid sample analysis in which a porous liquid sample spreading layer is provided on a non-porous polymer layer (functional layer), and (2) one or more non-fibers on a transparent support A liquid in which a non-porous polymer layer (functional layer) is provided and a porous liquid sample developing layer containing a reagent for sample analysis is provided on the non-fiber / non-porous polymer layer (functional layer) A dry multilayer analytical element for sample analysis. That is, the porous liquid sample development layer in the present invention may or may not contain a sample analysis reagent.

  Further, when the porous liquid sample developing layer contains a reagent, the reagent-containing film can be produced by previously immersing the porous film in a reagent solution and drying it. Alternatively, a reagent-containing non-fibrous porous membrane can be produced by applying a reagent solution on the porous membrane and drying it, but the means is not particularly limited.

  The dry multilayer analytical element of the present invention includes at least one non-fiber / non-porous polymer layer (functional layer). The number of non-fiber / non-porous polymer layers (functional layers) is not particularly limited as long as it is 1 or more, and may be one layer or a plurality of layers of two or more.

  Specific examples of the non-fiber / non-porous polymer layer (functional layer) include a water-absorbing layer that absorbs a liquid reagent, a mordant layer that prevents diffusion of a dye generated by a chemical reaction, and a gas-permeable layer that selectively permeates gas. , An intermediate layer that suppresses / promotes mass transfer between layers, a removal layer that removes endogenous substances, a light shielding layer for stable reflection photometry, a color shielding layer that suppresses the effects of endogenous dyes, blood cells and plasma Separation layer, a reagent layer containing a reagent that reacts with the analyte, a coloring layer containing a color former, and the like.

  As an example of the present invention, for example, a hydrophilic polymer layer can be provided as a functional layer on a support, optionally via another layer such as an undercoat layer. The hydrophilic polymer layer is, for example, a non-porous, water-absorbing and water-permeable layer, basically a water-absorbing layer consisting only of a hydrophilic polymer, or a coloring reagent that directly participates in a coloring reaction using a hydrophilic polymer as a binder. A reagent layer including a part or all of it, and a detection layer containing a component (for example, mordant) that fixes and immobilizes the coloring dye in the hydrophilic polymer can be provided.

Hereinafter, the functional layer will be described.
(Reagent layer)
The reagent layer is a water-absorbing material in which at least a portion of the reagent composition that reacts with a test component in an aqueous liquid to produce an optically detectable change is substantially uniformly dispersed in the hydrophilic polymer binder. It is a water-permeable layer. This reagent layer also includes an indicator layer, a coloring layer and the like.

  Hydrophilic polymers that can be used as a binder in the reagent layer generally have natural or synthetic hydrophilic properties with a water swell ratio of from about 150% to about 2000%, preferably from about 250% to about 1500% at 30 ° C. Polymer. Examples of such hydrophilic polymers include gelatin (eg, acid-treated gelatin, deionized gelatin, etc.) and gelatin derivatives (eg, phthalated gelatin, hydroxyacrylate) disclosed in JP-A-60-108753 and the like. Graft gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone and the like.

  The reagent layer can be a layer that is appropriately crosslinked and cured using a crosslinking agent. Examples of cross-linking agents include known vinyl sulfone cross-linking agents such as 1,2-bis (vinylsulfonylacetamido) ethane and bis (vinylsulfonylmethyl) ether for gelatin, aldehydes such as aldehydes for methallyl alcohol copolymers, Examples include glycidyl group-containing epoxy compounds.

  The dry thickness of the reagent layer is preferably in the range of about 1 μm to about 100 μm, more preferably in the range of about 3 μm to about 30 μm. The reagent layer is preferably substantially transparent.

As a reagent included in the reagent layer and other layers of the dry multilayer analytical element of the present invention, a reagent suitable for the detection can be selected according to the test substance.
For example, when analyzing ammonia (when the test substance is ammonia or an ammonia-producing substance), leuco dyes such as leucocyanine dyes, nitro-substituted leuco dyes and leucophthalein dyes (US Reissued Patent No. 30267 or Japanese Patent Publication No. 58-19062): pH indicators such as bromophenol blue, bromocresol green, bromthymol blue, quinoline blue and rosoleic acid (Kyoritsu Shuppan Co., Ltd., Chemical University) Dictionary, Vol. 10, pages 63-65): Triarylmethane dye precursor: Leucobenzylidene dye (described in JP-A-55-379 and JP-A-56-145273): Diazonium salt and azo dye Coupler: A base-bleachable dye or the like can be used. The blending amount of the color developing ammonia indicator with respect to the weight of the binder is preferably in the range of about 1 to 20% by weight.

  The reagent that reacts with the ammonia-producing substance that is the test substance to generate ammonia is preferably an enzyme or a reagent containing an enzyme, and an enzyme suitable for analysis is selected according to the type of the ammonia-producing substance that is the test substance. It can be selected and used. When an enzyme is used as the reagent, the combination of the ammonia-generating substance and the reagent is determined from the specificity of the enzyme. Specific examples of a combination of an ammonia-generating substance and an enzyme as a reagent include urea: urease, creatinine: creatinine deiminase, amino acid: amino acid dehydrogenase, amino acid: amino acid oxidase, amino acid: ammonia lyase, amine: amine oxidase, diamine: amine oxidase Glucose and phosphoamidate: phosphoamidate hexose phosphotransferase, ADP: carbamate kinase and phosphate carbamol, acid amide: amide hydrolase, nucleobase: nucleobase deaminase, nucleoside / nucleoside deaminase, nucleotide: nucleotide deaminase, guanine: guanase Etc. As an alkaline buffer that can be used for the reagent layer in the case of analyzing ammonia, a buffer having a pH in the range of 7.0 to 12.0, preferably 7.5 to 11.5 can be used.

  In the case of analyzing ammonia, the reagent layer includes a reagent that reacts with the ammonia-generating substance to generate ammonia, an alkaline buffering agent, and a hydrophilic polymer binder having film-forming ability. (Curing agent), stabilizer, heavy metal ion trapping agent (complexing agent) and the like can be contained. The heavy metal ion trapping agent is used for masking heavy metal ions that inhibit enzyme activity. Specific examples of the heavy metal ion trapping agent include complexane such as EDTA · 2Na, EDTA · 4Na, nitrilotriacetic acid (NTA), and diethylenetriaminepentaacetic acid.

  Examples of the glucose measuring reagent composition include glucose described in U.S. Pat. No. 3,992,158; JP-A-54-26793; JP-A-59-20853; JP-A-59-46854; There is an improved Trinder reagent composition comprising oxidase, peroxidase, 4-aminoantipyrine or a derivative thereof, 1,7-dihydroxynaphthalene.

(Light shielding layer)
A light shielding layer can be provided on the reagent layer as necessary. The light shielding layer has water permeability or water penetration in which fine particles having a light absorbing property or light reflecting property (collectively referred to as light shielding property) are dispersed and held in a hydrophilic polymer binder having a small amount of film forming ability. Is a sex layer. The light shielding layer is a color of an aqueous liquid spotted and supplied to a developing layer, which will be described later, when a detectable change (color change, color development, etc.) generated in the reagent layer is reflected and measured from the support side having light permeability. Especially, when the sample is whole blood, the red color of hemoglobin is shielded and also functions as a light reflection layer or a background layer.

  Examples of the light-reflecting fine particles include titanium dioxide fine particles (rutile type, anatase type or brookite type fine crystal particles having a particle diameter of about 0.1 μm to about 1.2 μm, etc.), barium sulfate fine particles, aluminum fine particles or Examples of the light-absorbing fine particles include carbon black, gas black, and carbon micro beads. Among these, titanium dioxide fine particles and barium sulfate fine particles are preferable. Particularly preferred are anatase-type titanium dioxide fine particles.

  Examples of the hydrophilic polymer binder having a film-forming ability include weakly hydrophilic regenerated cellulose, cellulose acetate, etc. in addition to the hydrophilic polymer similar to the hydrophilic polymer used in the production of the reagent layer described above. Of these, gelatin, gelatin derivatives, polyacrylamide and the like are preferable. In addition, gelatin and a gelatin derivative can be used by mixing a known hardening agent (crosslinking agent).

  The light shielding layer can be provided by applying an aqueous dispersion of light shielding fine particles and a hydrophilic polymer on the reagent layer by a known method and drying. Further, instead of providing the light shielding layer, light spreading fine particles may be contained in the above-described spreading layer.

(Water absorption layer)
In the dry multilayer analytical element of the present invention, a water absorbing layer can be provided between the support and the reagent layer. The water-absorbing layer is a layer mainly composed of a hydrophilic polymer that swells by absorbing water, and is a layer that can absorb water of an aqueous liquid sample that reaches or penetrates the interface of the water-absorbing layer. It has the effect of promoting the penetration of plasma, which is an aqueous liquid component, into the reagent layer. The hydrophilic polymer used for the water absorbing layer can be selected from those used for the reagent layer. In general, gelatin or a gelatin derivative, polyacrylamide, polyvinyl alcohol, particularly the aforementioned gelatin or deionized gelatin is preferred for the water-absorbing layer, and the aforementioned gelatin as the reagent layer is most preferred. The dry thickness of the water-absorbing layer is about 3 μm to about 100 μm, preferably about 5 μm to about 30 μm, and the coating amount is about 3 g / m ² to about 100 g / m ² , preferably about 5 g / m ² to about 30 g. / M ² range. The water-absorbing layer can contain a pH buffer, which will be described later, a known basic polymer, or the like to adjust the pH during use (when the analysis operation is performed). Further, the water-absorbing layer can contain a known mordant, polymer mordant and the like.

(Detection layer)
The detection layer is generally a layer in which a dye or the like produced in the presence of a test component diffuses and can be optically detected through a light-transmitting support, and can be composed of a hydrophilic polymer. A mordant may be included, for example, a cationic polymer relative to the anionic dye. The water absorption layer generally refers to a layer in which the dye produced in the presence of the test component does not substantially diffuse, and is distinguished from the detection layer in this respect.

  A surfactant can be contained in the reagent layer, the water absorbing 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 including a nonionic surfactant in the reagent layer or the water absorption layer, water in the aqueous liquid sample is easily absorbed into the reagent layer or the water absorption layer substantially uniformly during the analysis operation, and the liquid with the development layer Contact is quick and substantially uniform.

  The test substance targeted by the dry multilayer analytical element of the present invention is not particularly limited, and any liquid sample (for example, body fluid such as whole blood, plasma, serum, lymph, urine, saliva, spinal fluid, vaginal fluid; Specific components in drinking water, liquor, river water, factory wastewater, etc.) can be analyzed. For example, albumin (ALB), glucose, urea, pyrilbin, cholesterol, protein, enzyme (for example, lactate dehydrogenase, CPK (creatine kinase), ALT (alanine aminotransferase), AST (aspartate aminotransferase), GGT (γ -Blood enzymes such as glutamyl transpeptidase) can be analyzed.

  The dry multilayer analytical element of the present invention can be prepared by a known method. The hemolytic reagent may be added in advance to the aqueous reagent solution to be applied or impregnated. As another method, an aqueous solution containing a surfactant or a hydrophilic polymer for controlling the development area, an organic solvent (ethanol, methanol, etc.) solution, or a water-organic solvent mixed solution is applied from above the development layer. It can also be impregnated. Analysis of a test substance using this can also be performed according to a known method.

  For example, the dry multi-layer analytical element of the present invention is cut into small pieces such as a square having a side of about 5 mm to about 30 mm or a circle of substantially the same size. Japanese Utility Model Laid-Open No. 56-142454 (corresponding US Pat. No. 4,387,990), Japanese Patent Laid-Open No. 57-63452, Japanese Utility Model Laid-Open No. 58-32350, Japanese Patent Application Laid-Open No. 58-501144 (corresponding international official: It can be used as a chemical analysis slide by being housed in a slide frame described in WO083 / 00391) and the like, which is preferable from the viewpoint of manufacturing, packaging, transportation, storage, measurement operation, and the like. Depending on the purpose of use, it is used in a long tape form in a cassette or magazine, or a small piece is placed in a container with an opening, or a small piece is affixed or placed in an open card, or a cut piece is used. It can be used as it is.

  In the dry multilayer analytical element of the present invention, an aqueous liquid sample solution in the range of, for example, about 2 μL to about 30 μL, preferably 4 μL to 15 μL is spotted on the porous liquid sample developing layer. The spotted dry multilayer analytical element is incubated at a constant temperature in the range of about 20 ° C to about 45 ° C, preferably at a constant temperature in the range of about 30 ° C to about 40 ° C for 1 to 10 minutes. The color development or discoloration in the dry multilayer analysis element is reflected and photometrically measured from the side of the light-transmitting support, and the amount of the test substance in the sample can be determined by the principle of the colorimetric measurement method using a calibration curve prepared in advance.

  The measuring operation is disclosed in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867 (corresponding to US Pat. No. 4,424,191). With the described chemical analyzer, highly accurate quantitative analysis can be performed with extremely easy operation. Depending on the purpose and required accuracy, semi-quantitative measurement may be performed by visually judging the degree of color development.

Since the dry multilayer analytical element of the present invention is stored and stored in a dry state until analysis is performed, it is not necessary to prepare the reagent at the time of use, and generally the dry state has higher reagent stability. It is simpler and quicker than the so-called wet method in which the reagent solution must be prepared at the time of use. Moreover, it is also excellent as an inspection method capable of quickly performing a high-accuracy inspection with a small amount of liquid sample.
The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1
An aqueous solution having the following composition was coated on a 180 μm polyethylene terephthalate colorless transparent smooth film coated with gelatin so that the thickness after drying was 16 μm and dried.
Gelatin 16.0 g / m ²
Surfactant 0.2 g / m ²
Here, polyoxy (2-hydroxy) propylene nonylphenyl ether (Surfactant 10G, manufactured by Aurin) was used as the surfactant.

  Next, the film and a polysulfone membrane (SE-200: manufactured by Fuji Photo Film Co., Ltd., hereinafter referred to as PS membrane) were partially bonded by a gravure printing method. For partial adhesion, a hot melt material (Nitite H6352-2A; Nitta Gelatin Co., Ltd .; styrene-butadiene copolymer; softening point 76 ° C., viscosity 4100 cps at 140 ° C.) was dissolved at 135 ° C. Using the gravure plate having the pattern shown, the film was transferred to a PS film, faced to the surface of the film, and passed through a laminating roller to be bonded and integrated. The integrated multilayer analytical element was cut into 12 mm × 13 mm square chips and placed in a slide frame (described in JP-A-57-63452) to produce a slide of Example 1.

Comparative Example 1
An aqueous solution having the following composition was coated on a 180 μm polyethylene terephthalate colorless transparent smooth film coated with gelatin so that the thickness after drying was 16 μm and dried.
Gelatin 16.0 g / m ²
Surfactant 0.2 g / m ²
Here, polyoxy (2-hydroxy) propylene nonylphenyl ether (Surfactant 10G, manufactured by Aurin) was used as the surfactant.

Next, water was supplied onto the film at a supply rate of about 15 g / m ² and moistened, and then the polysulfone membrane (SE-200: manufactured by Fuji Photo Film Co., Ltd., hereinafter referred to as PS membrane) was lightly pressurized. Overlaid and dried. The integrated multilayer analytical element was cut into 12 mm × 13 mm square chips and placed in a slide frame (described in JP-A-57-63452) to produce a slide of Comparative Example 1.

Measurement example 1
In order to compare the liquid volume dependency of the slides of Example 1 and Comparative Example 1, each slide was prepared by dissolving a dye having the following structural formula in a 7% BSA (Bovine Serum Albmin) aqueous solution (concentration 0 to 1.5 mg / concentration). 10 mL of mL) was spotted. Thereafter, the mixture was incubated at 37 ° C. for 6 minutes, and the reflection density (ODr) at 5400 nm was measured with Fuji Dry Chem 5000 (manufactured by Fuji Photo Film Co., Ltd.). The relationship between the reflection concentration and the aqueous solution concentration was approximated by a cubic equation to obtain a calibration curve.

  Next, the liquid having a concentration of 0.5 mg / mL was spotted by changing the amount of spotted liquid to 8, 9, 10, 11, 12 μL. Thereafter, the mixture was incubated at 37 ° C. for 6 minutes, and the reflection density (ODr) at 5400 nm was measured with Fuji Dry Chem 5000 (manufactured by Fuji Photo Film Co., Ltd.). The reflection density was substituted into the calibration curve obtained as described above, and the density corresponding to each liquid quantity was obtained. The measurement results are shown in Table 1. In contrast to the comparative example, it is clear that the example has improved liquid volume dependency as shown by the rate of change.

* Here, the rate of change indicates the difference between the maximum value and the minimum value when the measured value at 10 μL is 100%, and when 8 to 12 μL is changed.

FIG. 1 shows a gravure pattern used in the examples.

Claims (5)

A porous liquid sample spreading layer comprising at least one non-fibrous / non-porous polymer layer and at least one non-fibrous porous membrane is laminated and integrated in this order on one surface of a water-impermeable and light-transmissive flat support. In the dry multi-layer analytical element for liquid sample analysis, the non-fiber / non-porous polymer layer and the porous liquid sample developing layer made of a non-fibrous porous membrane are in close contact with the water-insoluble polymer. Multi-layer analytical element. Non-fibrous porous membrane is 6,6-nylon, 6-nylon, acrylate copolymer, polyacrylate, polyacrylonitrile, polyacrylonitrile copolymer, polyamide, polyimide, polyamideimide, polyurethane, polyethersulfone, polysulfone, poly Mixture of ether sulfone and polysulfone, cellulose acetate, cellulose butyrate, cellulose acetate butyrate (mixed ester), cellulose nitrate, polyester, polyester carbonate, polyethylene, polyethylene chlorotrifluoroethylene copolymer, polyethylene tetrafluoroethylene copolymer , Polyvinyl chloride, polyolefin, polycarbonate, polytetrafluoroethylene, polyvinylidene difluoride, polyphenylenesulfur , Polyphenylene oxide, polyfluorocarbonate, polypropylene, polybenzimidazole, polymethyl methacrylate, styrene-acrylonitrile copolymer, styrene-butadiene copolymer, saponified ethylene-vinyl acetate copolymer, polyvinyl alcohol, or these The multilayer analytical element according to claim 1, which is a mixture of The multilayer analytical element according to claim 1 or 2, wherein the non-fibrous porous membrane is polysulfone, polyethersulfone, cellulose acetate, 6,6-nylon, or 6-nylon. The multilayer analytical element according to any one of claims 1 to 3, wherein the non-fibrous porous membrane is an asymmetric membrane. The multilayer analytical element according to any one of claims 1 to 4, wherein the water-insoluble polymer is a hot melt adhesive.

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