JP4465291B2 - Multi-layer analytical element and manufacturing method thereof - Google Patents

Description

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

  In the fields of clinical testing, food testing, and environmental analysis, demands for processing samples quickly and easily are increasing year by year, and dry analytical elements that meet these needs are widely used. Among the dry analytical elements, the spreading layer for receiving, spreading and diffusing blood is disclosed in JP-A-55-164356, JP-A-57-66359, JP-A-60-222769, etc. As represented, 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.

  In a dry analytical element having a non-fibrous porous film represented by Japanese Patent No. 2514087, it is described that a non-fibrous porous material of a development layer contains a hydrophilic polymer. Depending on the polymer agent to be contained and the production method thereof, the performance (particularly the liquid volume dependency) is poor and not substantial.

JP-A-55-164356 JP-A-57-66359 JP 60-222769 A Japanese Patent No. 2514087

  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 having a porous membrane as a spreading layer, and having a stable performance that has a larger signal / noise ratio (S / N ratio) and is not affected by liquid volume. And a method of manufacturing the same.

As a result of diligent studies to solve the above problems, the present inventors have found that in a multilayer analytical element for liquid sample analysis in which a porous liquid sample development layer composed of a functional layer and a non-fibrous porous film is laminated and integrated, It was found that the above-mentioned problems can be solved by including a water-soluble polymer in the porous porous membrane without interacting with the functional layer, and further by setting the coating amount of the water-soluble polymer to 1.5 to 10 g / m ^2. The invention has been completed.

That is, according to the present invention, the porous liquid sample development layer comprising at least one functional layer and at least one non-fibrous porous membrane is laminated and integrated in this order on one surface of the water-impermeable flat support. In the multilayer analytical element for liquid sample analysis, the water-soluble polymer is contained in the non-fibrous porous film without interacting with the functional layer, and the coating amount of the water-soluble polymer is 1.5 to 10 g / m. ^A multilayer analytical element for liquid sample analysis is provided.

Preferably, the coating amount of the water-soluble polymer is 1.5~5.0g / m ^2.
Preferably, the water-soluble polymer is polyvinyl pyrrolidone or a derivative thereof, cellulose ether, alginic acid or an alginic acid derivative, polyvinyl alcohol or a derivative thereof, polyacrylic acid or a derivative thereof, polyethylene glycol, polyethylene oxide, a water-soluble polysaccharide or a derivative thereof, or It is a mixture of these.

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 acylate, saponified cellulose acylate, polyester, polyester carbonate, polyethylene, polyethylene chlorotrifluoroethylene copolymer, polyethylene tetrafluoroethylene copolymer, polyvinyl chloride, polyolefin , Polycarbonate, polytetrafluoroethylene, polyvinylidene difluoride, polyphenylene sulfide, polyphenylene oxide, polyfluorocarbon DOO, polypropylene, polybenzimidazole, poly (methyl methacrylate), styrene - acrylonitrile copolymer, styrene - butadiene copolymer, ethylene - saponified vinyl acetate copolymer, polyvinyl alcohol, or a mixture thereof.
More preferably, the non-fibrous porous membrane is polysulfone, polyethersulfone, cellulose acylate, 6,6-nylon, or 6-nylon.

Preferably, the multilayer analytical element for liquid sample analysis of the present invention is a non-fibrous porous film so that the coating amount is 1.5 to 10 g / m ² after laminating the non-fibrous porous film on the functional layer. It is manufactured by applying to a fibrous porous membrane.

According to another aspect of the present invention, a porous liquid sample development layer comprising at least one functional layer and at least one non-fibrous porous membrane is laminated and integrated in this order on one surface of a water-impermeable flat support. In the method for producing a multilayer analytical element for analyzing a liquid sample, a non-fibrous porous membrane is laminated on a functional layer, and then a water-soluble polymer is applied so that the coating amount becomes 1.5 to 10 g / m ^2. There is provided the above manufacturing method comprising applying to a fibrous porous membrane.

In the multilayer analytical element of the present invention, the water-soluble polymer is contained in the non-fibrous porous membrane without interacting with the functional layer, and the coating amount of the water-soluble polymer is 1.5 to 10 g / m ² . This makes it possible to provide a multilayer analytical element with improved liquid volume dependency.

Hereinafter, embodiments of the present invention will be described in detail.
In the multilayer analytical element for liquid sample analysis of the present invention, a porous liquid sample development layer comprising at least one functional layer and at least one non-fibrous porous membrane is arranged in this order on one side of a water-impermeable flat support. The non-fibrous porous membrane contains a water-soluble polymer without interacting with the functional layer, and the coating amount of the water-soluble polymer is 1.5 to 10 g / m ^2. It is characterized by being.

  In the multilayer analytical element for liquid sample analysis of the present invention, the water-soluble polymer is contained in the non-fibrous porous film used as the porous liquid sample developing layer without interacting with the functional layer. By adopting such a configuration, the diffusion rate of the specimen in the non-fibrous porous membrane is substantially constant. As this means, a non-fibrous porous film uniformly dispersed with a water-soluble polymer is laminated without interacting with the lower layer (functional layer), or the non-fibrous porous film is placed on the functional layer. After laminating, the non-fibrous porous film can be impregnated with the water-soluble polymer without interacting with the lower layer.

The coating amount of the water-soluble polymer is 1.5~10g / m ^2, more preferably from 1.5~5.0g / m ^2. When the application amount of the water-soluble polymer is less than 1.5 g / m ² , the liquid amount dependency becomes large and stable measurement cannot be performed. In addition, when the coating amount of the water-soluble polymer is larger than 10 g / m ^{2, the} measurement speed becomes difficult because the developing speed of the sample becomes slow.

  The polymer dispersed in the spreading layer is not particularly limited as long as it is a water-soluble polymer. Examples include cellulose ethers such as carboxymethyl cellulose, methyl cellulose, hydroxypropyl cellulose, alginic acid and alginic acid derivatives, polyvinyl alcohol and derivatives thereof, polyacrylic acid and derivatives thereof, polyethylene glycol and polyethylene oxide, water-soluble polysaccharides and derivatives thereof, and the like. It is done. Moreover, these copolymers may be used and these may be mixed and used.

  As the water-impermeable flat support, a water-impermeable support used in a conventionally known dry analytical element can be used. Examples of water-impermeable supports include polyethylene terephthalate, bisphenol A polycarbonate, polystyrene, cellulose esters (eg, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) Mention may be made of a film or sheet-like support in the range of 50 μm to about 1 mm, preferably about 80 μm to about 300 μm.

  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 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. Further, the porous liquid sample spreading layer composed of at least one non-fibrous porous membrane is subjected to physical activation treatment disclosed in JP-A-57-66359, preferably glow discharge treatment or corona discharge treatment. It is applied to at least one surface, or hydrophilic treatment such as surfactant impregnation treatment disclosed in JP-A-55-164356, JP-A-57-66359, preferably nonionic surfactant impregnation treatment, hydrophilic polymer impregnation treatment, etc. Treatment, or a combination of two or more of these treatment steps is performed to make the non-fibrous porous membrane hydrophilic, strengthening the adhesive force with the functional layer on the lower side (side closer to the support), or developing area Can be controlled. 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.

  Preferable specific 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, Polyether sulfone, polysulfone, mixture of polyether sulfone and polysulfone, cellulose acylate, saponified cellulose acylate, polyester, polyester carbonate, polyethylene, polyethylene chlorotrifluoroethylene copolymer, polyethylene tetrafluoroethylene copolymer, poly Vinyl chloride, polyolefin, polycarbonate, polytetrafluoroethylene, polyvinylidene difluoride, polyphenylene sulfide, polyphenylene oxide, Examples include trifluorocarbonate, polypropylene, polybenzimidazole, polymethyl methacrylate, styrene-acrylonitrile copolymer, styrene-butadiene copolymer, saponified ethylene-vinyl acetate copolymer, polyvinyl alcohol, or a mixture thereof. It is done.

  Among these, 6,6-nylon, 6-nylon, polyethersulfone, polysulfone, a mixture of polyethersulfone and polysulfone, cellulose acylate, saponified cellulose acylate, polyester, polyethylene, polypropylene, polyolefin, polyacrylonitrile, More preferred are polyvinyl alcohol, polycarbonate, polyester carbonate, polyphenylene oxide, polyamide, polyimide, polyamideimide, or a mixture thereof.

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

There are no particular restrictions on the thickness, pore diameter, porosity, water transmission rate, whiteness, etc. of the porous liquid sample development layer comprising a non-fibrous porous membrane, but the thickness is 50 to 500 μm, preferably 50 to 350 μm, Preferably it is 80-200 micrometers. The pore diameter is preferably 0.001 to 100 [mu] m, more preferably about 0.1 to 50 [mu] m. The porosity is preferably 50 to 95%, more preferably 60 to 90%. The water permeation rate is preferably 1 to 5000 ml per minute per 1 cm ² of non-fibrous porous membrane, more preferably 5 to 2000 ml when water is passed at 25 ° C. at a pressure of 1 kg / cm ^2. It is desirable. Regarding the whiteness, it is desirable that the surface reflection absorbance (ODr) at the time of drying is 0.5 or less. More preferably, it is desirably 0.3 or less. A lower whiteness is advantageous because a dynamic range of ODr can be obtained.

  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 a porous liquid sample development layer is provided on the functional layer. A multilayer analysis element for liquid sample analysis, and (2) a porous liquid in which one or a plurality of functional layers are provided on a transparent support, and a reagent for sample analysis is further provided on the functional layer And a multilayer analytical element for liquid sample analysis provided with a sample development layer. That is, the porous liquid sample development layer in the present invention may or may not contain a sample analysis reagent. However, the porous film as the spreading layer needs to be analyte-free.

  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 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.

  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 30 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.

  The adhesive layer is preferably made of a hydrophilic polymer that can adhere the spreading layer when wetted with water or when swollen with water, thereby allowing the layers to be integrated. 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 and the like are preferable. 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, such as a method of applying an aqueous solution containing a hydrophilic polymer and a surfactant added as necessary onto 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 generated in the presence of a test component diffuses and can be optically detected through a 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.

In order to laminate a porous liquid sample development layer made of a non-fibrous porous film on a functional layer such as a reagent layer, a water absorption layer or an adhesive layer, the method disclosed in JP-A-55-164356, JP-A-57-66359, etc. be able to. That is, the functional layer such as the reagent layer, the water absorbing layer or the adhesive layer is not dried after application, or the functional layer after drying is water, an aqueous solution containing a surfactant, an aqueous solution containing a surfactant and a hydrophilic polymer, An organic solvent containing a surfactant, a surfactant, a hydrophilic polymer, an aqueous solution containing an organic solvent, or the like is supplied substantially uniformly by a wire bar coating device and a coating device using a die. The feed rate is preferably 1.0~50.0g / m ^2, more preferably from 10.0~30.0g / m ^2. The functional layer is substantially swelled or partially dissolved by the supplied aqueous solution or organic solvent, and then the porous liquid sample developing layer composed of a non-fibrous porous membrane is substantially swelled or dissolved on the functional layer. Laminate and apply evenly light pressure. In swelling or dissolving the functional layer, heating by irradiation of radiant heat such as an infrared heater, a method in which a heater is brought into contact with a support, and the like can be used.

  The adhesion force between the porous membrane and the support in the analysis element laminated by the above method is preferably 3.0 g or more as the force for peeling the porous membrane from the 1 cm wide strip-like analysis element. More preferably, it is 10.0 g. If the amount of adhesion is weak, it may cause troubles such as peeling off in the next processing step.

  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. Thus, a container containing one or more pieces can have information such as item names, lot numbers, and calibrations. For example, IC, magnetism, or barcode can be used for these pieces of information, which can be easily read on hardware and useful for automation.

  In the multilayer analytical element of the present invention, for example, an aqueous liquid sample liquid in the range of about 2 μL to about 30 μL, preferably 4 μL to 15 μL is spotted on the porous liquid sample 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. The color development or discoloration in the multilayer analytical element is reflected and photometrically measured from the support side, 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 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 Preparation of Analytical Element Dried at High Temperature An aqueous solution (pH = 7.0) having the following composition was added to a colorless and transparent smooth film of 180 μm polyethylene terephthalate coated with gelatin, and the thickness after drying was 14 μm. It was applied and dried.
Surfactant 11.63 g / m ²
Gelatin 16.34 g / m ²
Here, polyoxy (2-hydroxy) propylene nonylphenyl ether (Surfactant 10G, manufactured by Aurin) was used as the surfactant. Next, water was supplied to the entire surface of the film at a supply rate of about 30 g / m 2 and moistened, and then a polysulfone porous film HS200 (manufactured by “Fuji Photo Film Co., Ltd.”) was laminated.

An aqueous solution having the following composition was applied and dried on the porous film.
Hydroxypropyl cellulose 3.9 g / m ²
Surfactant 0.62 g / m ²

Comparative Example 1: Preparation of analytical element dried at a relatively low temperature The lower layer was applied in exactly the same formulation as in Example 1.
The overcoat aqueous solution was applied and dried at the following prescribed amount.
Hydroxypropylcellulose 1.2 g / m ²
Surfactant 0.62 g / m ²

Comparative Example 2: Preparation of analytical element dried at a relatively low temperature The lower layer was applied in exactly the same formulation as in Example 1.
The overcoat aqueous solution was applied and dried at the following prescribed amount.
Hydroxypropyl cellulose 13.9 g / m ²
Surfactant 0.62 g / m ²

  The integrated multilayer analytical element of Example 1 and Comparative Examples 1 and 2 was cut into 12 mm × 13 mm square chips and housed in a mount member to produce an analytical element.

  In the analytical elements produced in Example 1 and Comparative Examples 1 and 2, three levels (0, 0.2, 0.4, 0.8 mg / ml) of red pigments (Fuji Photo Film Co.) with the following structural formula 10 μL of 7% BSA aqueous solution was dropped, and 540 nm OD was measured.

  The measurement was performed for 6 minutes at the wavelength of 540 nm while incubating at 37 ° C. FIG. 1 shows the state of development at that time. Compared with Example 1, Comparative Example 1 has a high development speed, but the boundary between the development part and the non-development part is unclear. Also, it can be seen that Comparative Example 2 has a much slower development speed than that of Example 1 and Comparative Example 1, and a circular development cannot be obtained.

  Next, using the slides prepared in Example 1 and Comparative Example 1, the liquid volume dependency was examined. The liquid volume was changed from 8 μL to 11 μL, and the rate of change at each liquid volume was plotted with the value at the time of 10 μL spotting as 100. As the specimen, the red pigment 0.4 mg / dL BSA solution was used. The results are shown in FIG. Note that the slide prepared in Comparative Example 2 developed slowly and could not be measured. From the above results, the analytical element created in the example has a variation within ± 5% with respect to the change of 10 μL ± 1 μL, whereas the analytical element created in Comparative Example 1 has a variation of ± 5% or more. I knew it would happen ...

FIG. 1 shows a state of development when a BSA aqueous solution of a red pigment is dropped on analysis elements of Examples and Comparative Examples. FIG. 2 shows the results of examining the liquid volume dependency using the analysis elements created in Example 1 and Comparative Example 1.

Claims (2)

A multilayer analytical element for liquid sample analysis comprising a water-impermeable planar support, a functional layer comprising at least one reagent layer, and a porous liquid sample spreading layer comprising at least one non-fibrous porous membrane, Production of multilayer analytical element for liquid sample analysis in which porous liquid sample development layer comprising at least one functional layer and at least one non-fibrous porous membrane is laminated and integrated in this order on one side of a permeable flat support In the method, the non-fibrous porous membrane is polysulfone, polyether sulfone or a mixture thereof, and after laminating the non-fibrous porous membrane on the functional layer, the coating amount of hydroxypropyl cellulose is 1.5 to 10 g / m. Said manufacturing method including apply | coating to a non-fibrous porous film so that it may become ^two . The method of Claim 1 that the application quantity of this hydroxypropyl cellulose is 1.5-5.0 g / m < ² >.