JP2006090859A - Multilayer analysis element - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a multilayer analysis element with lessened inter-lot difference and intra-lot difference, lessened liquid volume dependency, and enhanced measuring accuracy. <P>SOLUTION: This multilayer analysis element for liquid specimen analysis is a multilayer analysis material for liquid specimen analysis made by layering/unifying at least one functional layer and at least one granular structure developing layer in this order on one side of a water-impermeable and light-transmissive planar support body and is characterized in that the functional layer has a film swelling property of a water swelling rate of 300% or less. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a multilayer analysis element used for clinical examination, food inspection, environmental analysis and the like.

  In the fields of clinical testing, food testing, and environmental analysis, demands for processing samples quickly and simply are increasing year by year, and analytical elements that meet these needs are widely used. Among the analytical elements, the development layer for accepting, spreading, and diffusing blood is represented by JP-A-55-164356, JP-A-57-66359, JP-A-60-222769, and the like. As such, 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 addition, the spreading layer is originally required to have a function to supply a constant volume of liquid sample per unit area to the lower layer regardless of the volume of the applied liquid sample. However, since the cloth spreading layer has a strong capillary force due to the cloth filament, this function is required. May be difficult to achieve.

  As a means for solving the drawbacks of the cloth spreading layer as described above, an analytical element using a granular structure composed of inorganic or organic polymer particles held together by an adhesive as a spreading layer has been studied. Since the granular structure development layer is formed by coating, the uniformity thereof is very high, and the difference between lots and the difference between lots are sufficiently small. In addition, unlike the cloth development layer, these granular structure development layers can be bonded to the lower layer without biting into the lower layer. Furthermore, since the strong capillary force does not work like the cloth spreading layer, it is considered that the liquid amount dependency is also improved. From such a viewpoint, Japanese Examined Patent Publication No. 53-21777 discloses a granular structure development layer made of glass beads and gelatin, and Japanese Patent Publication No. 6-4039 discloses a granular structure development layer composed of organic polymer beads and an organic polymer. Further, Japanese Patent Publication No. 63-43701 and Japanese Patent Publication No. 57-182166 disclose a granular structure developing layer made of a water-insoluble organic polymer and an organic polymer latex, and Japanese Patent Publication No. 57-101760, Japanese Patent Application Laid-Open No. Sho. Japanese Patent No. 57-101761 discloses a granular structure development layer that chemically bonds between organic polymer beads during coating. However, as a result of repeated studies, it has been found that the dependency on the liquid amount greatly changes not only due to the sample spreading action of the granular structure spreading layer but also depending on the film quality of the spreading layer lower layer. In addition, the deterioration of the liquid volume dependency has a problem that the measurement accuracy is deteriorated.

JP-A-55-164356 JP-A-57-66359 JP 60-222769 A Japanese Examined Patent Publication No. 53-21777 Japanese Patent Publication No. 6-4039 Japanese Patent Publication No. 63-43701 JP-A-57-182166 JP-A-57-101760 Japanese Patent Laid-Open No. 57-101761

  An object of the present invention is to solve the above-described problems of the prior art. That is, an object of the present invention is to provide a multilayer analysis element that has a small difference between lots and a difference between lots, a small liquid volume dependency, and high measurement accuracy. Furthermore, the present invention has made it a problem to be solved to provide a multilayer analytical element that enables stable production.

  As a result of intensive studies to solve the above problems, the present inventors have laminated at least one functional layer and at least one granular structure development layer in this order on one surface of a water-impermeable and light-transmissive flat support. In the integrated multilayer analysis material for liquid sample analysis, it is found that the above problem can be solved by providing a functional layer having a film swelling property with a water swelling ratio of 300% or less as the functional layer, and to complete the present invention. It came.

  That is, according to the present invention, a multilayer for liquid sample analysis in which at least one functional layer and at least one granular structure development layer are laminated and integrated in this order on one surface of a water-impermeable and light-transmissive flat support. In the analytical material, there is provided a multilayer analytical element for liquid sample analysis, wherein the functional layer has a film swelling property with a water swelling ratio of 300% or less.

Preferably, the granular structure developing layer is composed of organic polymer particles and an organic polymer adhesive.
More preferably, the granular structure spreading layer is formed of organic polymer particles having an average particle diameter of 1 μm to 200 μm made of a non-self-adhesive organic polymer that is impermeable to water, non-swelled in water. It is formed by bonding and is configured to have continuous voids.

  According to the present invention, a multi-layer analytical element that enables stable production by using a granular structure development layer, has small lot-to-lot differences and within-lot differences, improves liquid volume dependency, and has high measurement accuracy. Can be provided. Furthermore, in the present invention, when the water swelling rate of the functional layer is 300% or less, it is possible to provide a multilayer analytical element with improved liquid volume dependency and further improved measurement accuracy.

Hereinafter, embodiments of the present invention will be described in detail.
The multilayer analytical element for liquid sample analysis of the present invention is obtained by laminating and integrating at least one functional layer and at least one granular structure development layer in this order on one side of a water-impermeable and light-transmissive flat support. The functional layer has a film swelling property with a water swelling ratio of 300% or less.

  In the present invention, by providing a functional layer having a film swellability with a water swelling ratio of 300% or less, it is possible to provide a multilayer analytical element having improved liquid volume dependency and further improved measurement accuracy. Furthermore, in the present invention, by providing the granular structure development layer, it is possible to provide a multilayer analytical element with improved liquid volume dependency and further improved measurement accuracy.

  The “water swell ratio” in the “film swellability of water swell ratio of 300% or less” in the present invention means that the functional layer is exposed by removing the granular structure development layer from the multilayer analytical element of the present invention. Is used to refer to the water swell rate obtained by measuring the percent increase in functional layer thickness resulting from immersion in a 37 ° C. water bath for 5 minutes.

  Examples of the configuration of the multilayer analysis element for liquid sample analysis of the present invention include: (1) one or more functional layers are provided on a transparent support, and further a granular structure development layer is provided on the functional layers. A multilayer analytical element for analyzing a liquid sample, and (2) a granular structure containing one or more functional layers on a transparent support, and further containing a reagent for sample analysis on the functional layer And a multilayer analytical element for analyzing liquid samples provided with a development layer. That is, the granular structure development layer in the present invention may or may not contain a reagent for sample analysis.

  As the water-impermeable and light-transmitting flat support, a water-impermeable and light-transmitting support that is used in conventionally known analysis elements can be used. Examples of water-impermeable and light-transmitting supports include polymers such as polyethylene terephthalate, bisphenol A polycarbonate, polystyrene, and 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 functional 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 multilayer analytical element of the present invention includes at least one granular structure development layer. The granular structure development 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. is there.

  The granular structure development layer need not be limited to only one layer, and may be a laminate in which two or more granular structure development layers are bonded with an adhesive partially disposed. The granular structure development layer may contain a development control agent such as a hydrophilic polymer for the purpose of controlling the development. Furthermore, as already described above, a reagent for performing the 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 Can be included.

  The thickness of the granular structure development layer is 50 to 1000 μm, preferably 50 to 500 μm, and more preferably 100 to 350 μm.

  The granular structure developing layer is preferably composed of organic polymer particles and an organic polymer adhesive. The granular structure developing layer is more preferably water-impermeable, non-swelling in water, and non-self-adhesive organic polymer particles having an average particle diameter of 1 μm to 200 μm by an organic polymer adhesive. It is formed by bonding and is configured to have continuous voids.

  The type of the organic polymer constituting the granular structure developing layer is not particularly limited, but it is preferable to use a non-self-adhesive organic polymer that is impermeable to water, does not swell in water. Specific examples of such organic polymers include polyvinyl chloride, copoly (vinyl chloride-vinylidene chloride), polyvinyl acetate, copoly (ethylene-vinyl acetate), polyethylene, polypropylene, copoly (ethylene-propylene), polystyrene, poly Methyl methacrylate, copoly (acrylonitrile-styrene), copoly (acrylonitrile-butadiene-styrene), polycarbonate of bisphenol A, polyoxymethylene, polyphenylene oxide, cellulose acetate butyrate, cellulose acetate propionate, etc. , Poly (styrene-co-vinylbenzyl chloride-co-methacrylic acid) (78/20/2), poly (styrene-co-N, N, N-trimethyl-N-vinylbenzyl-ammonium chloride-co (Methacrylic acid) (88/10/2), poly (styrene-co-divinylbenzene) (98/2), poly (styrene-co-butylacrylate-co-methacrylic acid) (88/10/2), poly ( Styrene-co-methacrylic acid-co-divinylbenzene) (70/25/5 and 98/1/1), poly (vinyl chloride-co-methacrylic acid-co-divinylbenzene) (93/2/5), Poly (styrene-co-2-hydroxyethyl methacrylate-co-methacrylic acid) (88/10/2), poly (styrene-co-methacrylic acid) (98/2), poly (vinyltoluene-co-pt -Butylstyrene-co-methacrylic acid) (61/37/2), poly (vinyltoluene-co-pt-butylstyrene-co-methacrylic acid-co-divinylbenzene) (6 / 37/2/1), poly (methyl methacrylate-co-butyl acrylate) (70/30), poly (styrene-co-acrylonitrile) (70/30), poly (methyl methacrylate-co-N- (m and p-vinylbenzyl) -N, N-dimethylamine hydrochloride-co-ethylene dimethacrylate) (70/20/10), poly (methyl methacrylate-co-2- (N, N-diethylamino) -ethyl methacrylate hydrochloride -Co-ethylene dimethacrylate) (70/20/10). These organic polymers may be used alone or in combination of two or more.

  In the present invention, it is preferable to use organic polymer particles composed of the above-described organic polymer. The average particle diameter of the organic polymer particles is preferably 1 μm to 200 μm, more preferably 5 μm to 100 μm.

  Examples of the organic polymer adhesive constituting the granular structure developing layer include a hydrophilic organic polymer or a hydrophobic organic polymer. Examples of the hydrophilic organic polymer that can be used as an adhesive include gelatin, agarose, gum arabic, carboxymethyl cellulose, polyvinyl alcohol, polyacrylamide, polyvinyl pyrrolidone, and hydroxypropyl cellulose. Examples of the hydrophobic organic polymer that can be used as an adhesive include polyvinyl chloride, polyvinyl acetate, polystyrene, polyisobutylene, polymethyl acrylate, polyethyl acrylate, polymethyl methacrylate, polyvinyl benzyl alcohol, and poly-N—. In addition to known polymers used as adhesives such as isopropylacrylamide, nitrile rubber, chloroprene rubber, phenol resin, melanin resin, and epoxy resin, poly (butyl acrylate-co-styrene-co-2-acrylamido-2-methylpropanesulfonic acid) ) (70/20/10), poly (butyl acrylate-co-styrene-co-2-acrylamido-2-methylpropanesulfonic acid) (76/21/3), poly (ethyl acrylate-co-acrylic acid) 2-acetoacetoxyethyl methacrylate-co-2-acrylamido-2-methylpropanesulfonic acid) (71/24/4/1) and (67/16/16/1), poly (2-hydroxyethyl methacrylate-co -2-acetoacetoxyethyl methacrylate) (15/85) (Tg = −20 ° C.), poly (butyl acrylate-co-acrylic acid) (75/25), poly (butyl acrylate-co-acrylic acid-co-methacrylic) Acid-co-ethylacryloyl acetate) (70/5/15/10), poly (butyl acrylate-co-acrylic acid-co-ethyl acryloyl acetate) (75/15/10), poly (butyl acrylate-co-methacrylic) Acid-co-2-acetoacetoxyethyl methacrylate) (56/34/10), poly Butyl acrylate-co-styrene) (70/30), poly (butyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid-co-acetoacetoxyethyl methacrylate) (85/10/5), poly (butyl acrylate) -Co-acrylic acid-co-2-acetoacetoxyethyl methacrylate-co-2-acrylamido-2-methylpropanesulfonic acid) (67/16/16/1), poly (butyl methacrylate-co-styrene) (90 / 10), poly (ethyl acrylate-co-styrene) (70/30), poly (butyl acrylate-co-2-acrylamido-2-methylpropanesulfonic acid) (90/10), poly (butyl acrylate-co-styrene) ) (50/50), poly (2-ethylhexyl acrylate-co -Acrylic acid-co-2-acetoacetoxyethyl methacrylate-co-2-acrylamido-2-methylpropanesulfonic acid) (67/16/16/1), poly (butyl methacrylate-co-methacrylic acid) (70/30 ), Poly (ethyl acrylate-co-acrylic acid) (80/20) or (70/30), poly (butyl acrylate-co-styrene-co-2-acetoacetoxyethyl methacrylate-co-2-acrylamide-2-) Methylpropanesulfonic acid) (70/22/6/2), poly (butyl acrylate-co-styrene-co-2-acrylamido-2-methylpropanesulfonic acid-co-divinylbenzene) (69/20/10/1) ), Poly (acrylamide-co-2-acetoacetoxyethyl methacrylate) (20/80) It is like (15/85).

  Moreover, the granular structure development layer containing a reagent can be produced by previously containing a reagent in a coating solution for forming the granular structure development layer, and applying and drying. Alternatively, it can be prepared by applying a reagent solution on the granular structure development layer and drying it, but the means is not particularly limited.

  The multilayer analytical element of the present invention comprises at least one functional layer. The number of 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 layers.

  The functional layer in the multilayer analytical element of the present invention is characterized in that it has a film swelling property with a water swelling ratio of 300% or less. The lower limit of the water swelling rate is not particularly limited, but can be, for example, 50% or more, or 100% or more. When there are two or more functional layers, the water swelling ratio of the entire functional layer may be 300% or less, or at least one functional layer (for example, The water swelling rate of the reagent layer and the water absorbing layer to be described may be 300% or less.

Film swellability with a water swell ratio of 300% or less can be achieved by appropriately setting the amount of a crosslinking polymer (for example, a hydrophilic polymer) and a crosslinking agent used when forming a functional layer. For example, in Example 1 herein (results shown in Table 1), 0.04 g / m ² of 1,2-bis (vinylsulfonylacetamido) ethane relative to 16.6 g / m ² of gelatin. When (crosslinking agent) is used, the water swelling rate is 928%, and when 0.2 g / m ² crosslinking agent is used, the water swelling rate is 325% and 0.8 g / m ² crosslinking agent. When used, the water swelling rate is 233%. That is, when the amount of the crosslinking agent used is increased, the water swelling rate decreases. Therefore, a film swelling property with a water swelling ratio of 300% or less can be achieved by using a certain amount or more of the crosslinking agent with respect to a predetermined amount of the crosslinked polymer.

  Specific examples of the functional layer include an adhesive layer that bonds the spreading layer and the functional layer, a water absorbing layer that absorbs the liquid reagent, a mordant layer that prevents the diffusion of the dye generated by the chemical reaction, and a gas permeation that selectively permeates the gas. Layer, an intermediate layer that suppresses and promotes mass transfer between layers, a removal layer that removes endogenous substances, a light shielding layer that stably performs reflection photometry, a color shielding layer that suppresses the influence of endogenous dyes, blood cells and Examples include a separation layer for separating plasma, a reagent layer containing a reagent that reacts with an analyte, and a coloring layer containing a color former.

  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.

(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 50 μm. The reagent layer is preferably substantially transparent.

As a reagent to be included in the reagent layer and other layers of the multilayer analytical element of the present invention, a reagent suitable for detection can be selected according to the test substance.
For example, when analyzing ammonia (when the test substance is ammonia or an ammonia-generating 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 US 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.

(Adhesive layer)
An adhesive layer may be provided on the reagent layer in order to adhere and laminate the spreading layer, optionally via a layer such as a light shielding layer.

  Hydrophilic polymer or hydrophobic polymer that can adhere the spreading layer when the adhesive layer is wet with water or an organic solvent, or when swollen with water, thereby allowing the layers to be integrated Preferably it consists of. Examples of the hydrophilic polymer that can be used for the production of the adhesive layer include hydrophilic polymers similar to the hydrophilic polymer used for the production of the reagent layer. Of these, gelatin, gelatin derivatives, polyacrylamide, polyvinylpyrrolidone and the like are preferable. Examples of the hydrophobic polymer include hydrophobic polymers similar to the hydrophobic polymers that can be used as the adhesive layer constituting the granular structure development layer. Of these, poly-N-isopropylacrylamide is preferred. The dry film thickness of the adhesive layer is generally in the range of about 0.5 μm to about 20 μm, preferably about 1 μm to about 10 μm.

  In addition to the reagent layer, the adhesive layer may be provided on a desired layer in order to improve the adhesive strength between other layers. The adhesive layer can be provided by a known method of applying an aqueous solution or an organic solvent containing a hydrophilic polymer or a hydrophobic polymer and a surfactant or the like to be added as necessary on a support or a reagent layer. .

(Water absorption layer)
The multilayer analytical element of the present invention can be provided with a water absorbing layer 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 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 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 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; or beverage) Specific components in water, alcoholic beverages, 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 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 multilayer 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 having substantially the same size, and disclosed in Japanese Examined Patent Publication No. 57-283331 (corresponding US Pat. No. 4,169,751), Japanese Unexamined Patent Publication No. 56-142454 (corresponding US Pat. No. 4,387,990), Japanese Unexamined Patent Publication No. 57-63452, Japanese Utility Model Publication No. 58-32350, Japanese Patent Publication No. 58-501144 (corresponding international publication: WO083). / 00391) and the like and can be used as a chemical analysis slide, 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 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 granular structure developing layer. The spotted 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. Color development or discoloration in the multilayer analytical 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 based on 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 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 easier and quicker than the so-called wet method in which the 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: Relationship between swelling ratio of functional layer and liquid volume dependency (1) Production of multilayer analytical element having functional layer having different granular structure development layer and swelling ratio 180 μm polyethylene terephthalate coated with gelatin An aqueous solution (pH = 5.1) having the following composition was applied to a colorless transparent smooth film so as to have the following coating amount, dried, and then hardened at 45 ° C. for 67 hours to obtain a water absorbing layer 1.
<Water absorption layer 1>
Gelatin (16.6g / m ² )
Polyoxy (2-hydroxy) propylene nonylphenyl ether (0.2g / m ² )
1,2-bis (vinylsulfonylacetamide) ethane (0.04g / m ² )

In the same manner, the following water absorption layers 2 and 3 were produced.
<Water absorption layer 2>
Gelatin (16.6g / m ² )
Polyoxy (2-hydroxy) propylene nonylphenyl ether (0.2g / m ² )
1,2-bis (vinylsulfonylacetamide) ethane (0.2 g / m ² )
<Water absorption layer 3>
Gelatin (16.6g / m ² )
Polyoxy (2-hydroxy) propylene nonylphenyl ether (0.2g / m ² )
1,2-bis (vinylsulfonylacetamide) ethane (0.8 g / m ² )

Next, a granular structure development layer was applied on the above water-absorbing layer by coating and drying so as to have the following coating amount.
<Granular structure development layer>
Polymethyl methacrylate particles (average particle size 38μm) (197g / m ² )
Polyvinylpyrrolidone K90 (Molecular weight 1 million) (3.6g / m ² )
Polyoxy (2-hydroxy) propylene nonylphenyl ether (2.2 g / m ² )
Glycerin (0.7g / m ² )

As described above, a multilayer analytical element having functional layers having different swelling ratios using a granular structure as a spreading layer was produced.
The multilayer analysis element is cut into a 12 mm × 13 mm square chip, placed in a slide frame (described in JP-A-57-63452), and slides 1, 2 and 3 for measuring the amount of non-adsorbing dye solution dependency. Was made.

(2) Comparison of swelling rate and liquid volume dependency of functional layer The granular structure development layer is removed from each of slides 1, 2 and 3 for measuring the non-adsorptive dye liquid volume dependency, and using a swelling film thickness meter, 37 The swelling rate was measured by measuring the percent increase in water-absorbing layer thickness resulting from soaking in a water bath at 5 ° C for 5 minutes. The measurement results are shown in Table 1.

  In addition, red pigment of the following structural formula (manufactured by Fuji Photo Film Co., Ltd.) is dissolved in 7% HSA (Human Serum Albumin) solution at 0, 0.1, 0.3, 0.5, 0.8 mg / mL and used for liquid volume dependency. It was set as the evaluation liquid.

  This evaluation solution was spotted in 10 μL each on slides 1, 2 and 3 for measuring the amount of non-adsorbing dye solution dependency. Thereafter, each slide was incubated at 37 ° C., the reflection optical density at 540 nm was measured from the support side, and the reflection optical density 5 minutes after the spotting was determined. This produced a calibration curve for the red pigment of each slide. Next, 0.3 mg / mL of the evaluation solution was spotted on slides 1, 2, and 3 for measurement of the amount of non-adsorbing dye solution dependency, respectively 6, 8, 10, and 12 μL. Thereafter, each slide was incubated at 37 ° C., the reflection optical density at 540 nm was measured from the support side, the reflection optical density after 5 minutes from the spotting was obtained, and converted to the red pigment concentration using the calibration curve created earlier. The liquid volume dependency was compared. The measurement results are shown in Table 1.

  As can be seen from the results in Table 1, slides 1 and 2 have a swelling ratio of 300% or more, and a change in the dye concentration depending on the liquid amount is 20 to 30%, whereas slide 3 has a swelling ratio of 300%. The change in the pigment concentration depending on the liquid amount was improved to about 10%.

Example 2: Comparison of sensitivity, accuracy and liquid volume dependency of Ca analysis multilayer analysis element using various development layer materials (1) Preparation method of Ca analysis analysis element using granular structure as development layer Gelatin primer An aqueous solution (pH = 5.6) having the following composition was applied to a 180 μm polyethylene terephthalate colorless and transparent smooth film and dried to obtain the water absorption layer.

Gelatin (11.7g / m ² )
1N potassium hydroxide (1.2g / m ² )
Titanium oxide (0.8g / m ² )
Polyoxy (2-hydroxy) propylene nonylphenyl ether (0.3g / m ² )
1,2-bis (vinylsulfonylacetamide) ethane (0.8 g / m ² )

Next, an aqueous solution (pH = 5.3) having the following composition was applied onto the water-absorbing layer so as to have the following coating amount and dried to form a reagent layer.
Gelatin (11.9g / m ² )
2-morpholinoethanesulfonic acid monohydrate (6.4g / m ² )
1N potassium hydroxide (7.0g / m ² )
CPA (III) (0.4g / m ² )
Polyoxy (2-hydroxy) propylene nonylphenyl ether (0.4 g / m ² )
Di-iso-octylsodium sulfosuccinate (0.1 g / m ² )
1,2-bis (vinylsulfonylacetamide) ethane (0.8 g / m ² )

  Next, a granular structure development layer was applied and dried on the reagent layer so as to have the following coating amount. Thereafter, hardening was performed at 45 ° C. for 67 hours.

<Granular structure development layer>
Polymethyl methacrylate particles (average particle size 38μm) (197g / m ² )
Polyvinylpyrrolidone K90 (3.6g / m ² )
Polyoxy (2-hydroxy) propylene nonylphenyl ether (2.2 g / m ² )
Glycerin (0.7g / m ² )

As described above, a multilayer analysis element for Ca analysis using a granular structure as a development layer was produced.
The multilayer analysis element was cut into a 12 mm × 13 mm square chip and placed in a slide frame (described in JP-A-57-63452) to prepare a slide 1 for Ca analysis.

(2) Preparation of a multilayer analysis element for Ca analysis using a knitted fabric as a spreading layer Up to the reagent layer (1) A reagent layer is prepared in the same manner as the preparation of an analysis element for Ca analysis using a granular structure as a spreading layer. Apply water to the entire surface at a supply rate of about 30 g / m ² and moisten it, and then apply light pressure to a tricot knitted fabric (hereinafter referred to as knitted fabric) knitted with 36 gauge polyethylene terephthalate spun yarn equivalent to 50 denier. And then laminated and dried. Thereafter, hardening was performed at 45 ° C. for 67 hours.

  On the above fabric, an ethanol solution having the following composition was applied and dried so as to have the following coating amount, and a multilayer analysis element for Ca analysis using a knitted fabric as a spreading layer was produced.

Polyvinylpyrrolidone (3.3g / m ² )
Polyoxyethylene (10) octyl phenyl ether (0.4 g / m ² )

  The multilayer analysis element was cut into a 12 mm × 13 mm square chip and placed in a slide frame (described in JP-A-57-63452) to prepare a slide 2 for Ca analysis.

(3) Comparison of Liquid Volume Dependent Serums with water and Ca concentrations of 6.1, 9.7 and 15.2 mg / dl were spotted on the above slides 1 and 2 for Ca analysis, respectively. Thereafter, each slide was incubated at 37 ° C., the reflection optical density at 625 nm was measured from the support side, the reflection optical density 4 minutes after the spotting was obtained, and a calibration curve of Ca concentration and reflection optical density was prepared. Next, changes in Ca concentration according to the liquid volume were compared on slides 1 and 2 when 8, 10, and 12 μL of control serum having a Ca concentration of 9.7 mg / dl was applied. Table 2 shows the comparison results of the liquid amount dependency.

As can be seen from the results in Table 2, the multilayer analysis element 1 for Ca analysis using a granular structure as a spreading layer is more liquid-dependent than the multilayer analysis element 2 for Ca analysis using a knitted fabric as a spreading layer. To do.

Claims (3)

In a multilayer analytical material for liquid sample analysis, in which at least one functional layer and at least one granular structure development layer are laminated and integrated in this order on one surface of a water-impermeable and light-transmissive planar support, A multilayer analytical element for liquid sample analysis, characterized in that the film has a water swell ratio of 300% or less. The multilayer analytical element for liquid sample analysis according to claim 1, wherein the granular structure developing layer is composed of organic polymer particles and an organic polymer adhesive. By adhering organic polymer particles having an average particle diameter of 1 μm to 200 μm composed of a non-self-adhesive organic polymer that is impermeable to water, non-swelled in water, with an organic polymer adhesive The multilayer analytical element for liquid sample analysis according to claim 1 or 2, wherein the multilayer analytical element is formed so as to have continuous voids.