JPH0675067B2 - Integrated multi-layer analysis element - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a flat plate-shaped or sheet-shaped integrated multi-layer analytical element for quantitatively analyzing a predetermined analyte (component to be analyzed) in an aqueous liquid sample. More particularly, the present invention relates to an integrated multi-layer analytical element in the form of a flat plate or a sheet, which is useful for a clinical test suitable for quantitatively analyzing an analyte in an aqueous liquid sample, particularly a biological fluid such as whole blood. .

[Prior Art] As one form of a dry analytical element, a reagent layer in which a color reaction reagent is dispersed and contained in a hydrophilic polymer binder typified by gelatin on a transparent support and a porous development layer ( Hereinafter, an integrated multi-layer analytical element (hereinafter sometimes referred to as a multi-layer analytical element) having a structure in which a development layer) is closely adhered is disclosed in Japanese Patent Publication No. 53-21677, US Pat.
JP-A-55-90859, JP-A-55-164356, JP-A-57-66359, JP-A-52-3488, US Patent Re30267, JP-A-58-77661, JP-A-60-
222769, JP-A-57-148250, JP-A-57-125847 and the like are proposed and put to practical use. In these multilayer analysis elements, the light-shielding fine particles such as titanium dioxide fine particles are contained in the spreading layer or a light-shielding layer is provided between the spreading layer and the reagent layer in order to adapt to a whole blood sample. Since the multi-layer analytical element can perform accurate quantitative analysis or semi-quantitative analysis of test components by a dry operation by simply spotting about 10 μl of whole blood or plasma aqueous liquid sample on the spreading layer, It is an analytical tool that is becoming increasingly used in.

[Problems of the prior art] In the above-mentioned integrated multilayer analytical element, a hydrophilic polymer binder typified by gelatin is used in a layer other than the developing layer such as a reagent layer or a light-shielding layer, so that it is water-permeable, In general, the diffusion of analytes is slow due to migration, and it is impermeable or difficult to permeate high molecular weight analytes, lipids or hydrophobic analytes, and cannot be applied to these types of analytes. was there. Further, when whole blood is used as the aqueous liquid sample, there is a problem that it cannot be applied to all analytes.

In order to deal with conventional polymer analytes, lipids or hydrophobic analytes, in the multilayer analytical element for quantitative cholesterol analysis described in Japanese Patent Publication No. 56-45599, a color reagent composition containing an enzyme may be contained in the developing layer. Proposed. However, this multilayer analysis element has a problem that the light shielding effect is insufficient for a whole blood sample. Further, in the multilayer analysis element described in JP-A-59-120957, the reagent composition is contained in the voids of the continuous void-containing porous structure layer in which the solid fine particles are held in the hydrophilic polymer binder in contact with the developing layer. Porous reagent layers have been proposed. This multilayer analysis element has a problem that the reagent composition contained in the porous reagent layer is limited.

OBJECT OF THE INVENTION It is an object of the present invention to provide an integrated multi-layer analytical element capable of rapidly diffusing and permeating an analyte in an amount sufficient for a color reaction to a reagent layer.

Another object of the present invention is to have a function of separating red blood cells in a whole blood sample, avoiding interference by red blood cells during reflection optical density measurement for a colorimetric method and performing quantitative analysis of an analyte in a whole blood sample. It is to provide an integrated multi-layer analytical element that enables it.

Another object of the present invention is to provide an integrated multilayer analytical element suitable for quantitative analysis of polymeric analytes, lipids or hydrophobic analytes.

Another object of the present invention is to provide an integrated multi-layer analytical element which contains a sufficient amount of color reagent composition for color reaction with an analyte, has a large calibration curve gradient and high measurement accuracy. Is.

Another object of the present invention is to provide an integrated multi-layer analytical element which is less susceptible to color reagent deterioration over time, has less fog (background coloring), has high measurement sensitivity, and has high measurement accuracy.

Furthermore, the present invention is directed to an integrated multi-layer analytical element described in Japanese Patent Application No. 59-126267 (Japanese Patent Application Laid-Open No. 61-4959), that is, fine through-holes so that uniform passage of a liquid is not hindered between two adjacent surfaces. Integrated multi-layered analysis comprising at least two layers of microporous sheet intimately bonded and integrated (adhesively porous or microporous) with an adhesive partly arranged to form It is also an invention that improves elements.

[Structure of the Invention] The present invention relates to an integrated multilayer analytical element having a plurality of layers including at least one layer containing a color reagent composition, wherein the fibrous porous sheet, the upper non-fibrous porous sheet , And the lower non-fibrous porous sheet are laminated and integrated in this order so that the three layers of porous sheets do not substantially impede the uniform passage of liquid between their two adjacent surfaces. The uppermost non-fibrous porous sheet has an average minimum pore size in the range of 0.8 μm to 30 μm, which are substantially intimately adhered and integrated by an adhesive partly arranged so as to form a slightly penetrating void. Is an integrated multi-layer analytical element.

[Detailed Description of Configuration of the Invention] The fibrous porous sheet is located in the outermost layer (the lower non-fibrous porous sheet or the uppermost layer farthest from the support) and mainly functions as a layer having a spreading action, That is, the upper surface (the lower non-fibrous porous sheet or the surface on the side far from the support) is spotted and supplied to the aqueous liquid sample without substantially unevenly distributing the components contained therein. The received aqueous liquid sample is diffused laterally (in the direction of the sheet surface) so that a substantially constant amount of liquid sample is distributed per unit area,
At the same time, it is a layer having an action (developing action or metering action) of supplying an aqueous liquid sample in which an aqueous liquid sample is received at a substantially constant volume ratio per unit area to an adjacent lower layer. Further, the fibrous porous sheet is a layer that also acts to filter out non-soluble substances (eg, red blood cells in whole blood sample) that interfere with analysis in the aqueous liquid sample.

As the fibrous porous sheet, woven fabric, knitted fabric, paper and filter paper containing organic polymer fiber pulp, paper and filter paper containing glass fiber pulp, fibrous nonwoven fabric and the like can be used.

As the woven fabric, Japanese Patent Laid-Open Nos. 55-164356 and 60-222770 are available.
The plain woven fabric for the spreading layer described in, etc. is preferable, and among the plain woven fabrics, thin cloth fabric, gold cloth fabric, broad fabric, poplin fabric and the like are preferable. A spun yarn (twisted yarn) is preferable as a yarn constituting the woven fabric. The yarn thickness of the woven fabric is represented by a cotton spun yarn count of about 20S to about 150S, preferably about 40S to about 1.
20S range, or about 35D to about 300D, preferably about 45D to about 130D expressed in denier of silk thread, the thickness of woven fabric is about 100μm to about 500μm, preferably about 120μm to about 350μm, woven fabric The porosity of the dough is about 40% to about 9
It is in the range of 0%, preferably about 50% to about 85%.

As knitted fabrics, there are JP-A-60-222769 and JP-A-60-222770.
The warp knitted fabric for the spreading layer described in, etc. is preferable, and among the knitted knitted fabrics, the tricot knitted fabric,
Raschel knitted fabric, Milanese knitted fabric, and double tricot knitted fabric are preferable. A spun yarn (twisted yarn) is preferable as a yarn constituting the knitted fabric. The yarn thickness of the knitted fabric is in the range of about 40S to about 150S, preferably about 60S to about 120S expressed in cotton spun yarn count, or about 35S expressed in silk yarn denier.
The range of D to about 130D, preferably about 45D to about 90D, the gauge number in the knitting process of the knitting fabric is about 20 to about 50, and the thickness of the knitting fabric is about 100 μm to about 600 μm, preferably about 150 μm To about 400 μm, the porosity of the knitted fabric is about 40% to about 90%, preferably about 50% to about 85%.

As a papermaking paper containing organic polymer fiber pulp, Japanese Patent Laid-Open No.
-148250 and the like, a papermaking paper made of a polyethylene fiber-only pulp, and a papermaking paper made of a mixed pulp of polyethylene fibers (30 to 70%) and natural fibers such as cellulose fibers are preferable. The thickness of the paper is in the range of about 80 μm to about 400 μm, preferably about 100 μm to about 250 μm, and the porosity of the paper is in the range of about 20% to about 80%, preferably about 50% to about 70%.

Woven fabrics and knitted fabrics (hereinafter, both may be collectively referred to as cloths) among the fibrous porous sheets are substantially degreased by washing or the like and at least oils or fats supplied or attached at the time of yarn manufacturing or cloth manufacturing Use the removed cloth.
As the papermaking paper and the papermaking filter paper subjected to the calendar treatment, those which are substantially free of fats and oils are used. The fibrous porous sheet is subjected to the physical activation treatment (preferably glow discharge treatment or corona discharge treatment) described in JP-A-57-66359 or the like on one side or both sides, or in JP-A-55-164356, Surfactants described in JP-A-57-66359 and JP-A-57-148250,
Preferably, nonionic surfactant aqueous solution is impregnated, coated or sprayed, hydrophilic polymer aqueous solution is impregnated, coated or sprayed, hydrophilic cellulose derivative described in JP-A-60-222770 and nonionic having HLB value of 10 or more. Of surface active agent by impregnation, coating or spraying, strengthening adhesion with lower layer by porous adhesion by two or more treatments, metering action, metering action corresponding to hematocrit value, etc. Can be controlled.

In the integrated multi-layer analytical element of the present invention, the upper non-fibrous porous sheet of the two layers of non-fibrous porous sheets which are porous-adhered by the fine through-pore structure adhesive layer and are adjacent to each other is mainly used in the aqueous liquid sample. It functions as a layer that has the function of filtering non-lytic substances that interfere with analysis (eg, fine particle components such as red blood cells and white blood cells in whole blood samples), and the function of shielding light. The lower non-fibrous porous sheet functions as a layer having a light-shielding function or a porous reagent layer containing and holding a part or all components of the reagent composition.

As a non-fibrous porous sheet, Japanese Patent Publication Sho 53-21677, U.S. Patent 3
992158, U.S. Pat. No. 1421341 and the like, cellulose esters such as cellulose acetate, cellulose butyrate, cellulose acetate butyrate (mixed ester), cellulose nitrate; cellulose ethers such as ethyl cellulose; carbonate polymers such as bisphenol A polycarbonate. Membrane filter (brush polymer layer) made of polyamide, such as polycapramide (6-nylon), polyhexamethylene adipamide (6,6-nylon), etc. , 198
Polyethylene microporous membrane such as polyethylene microporous membrane and polypropylene microporous membrane described in Membrana, Inc. catalog (published in July 1982), etc.
53-21677, US Pat. No. 3,992,158, etc. Polymer microbeads, glass microbeads, continuous microvoid-containing porous layer in which fine particles such as diatomaceous earth are held in a hydrophilic polymer binder, JP-A-55-90859 Non-fibrous isotropic porous layers such as continuous microvoid-containing porous layers (three-dimensional lattice-like granular structure layers) in which polymer microbeads are adhered in point contact with a polymer adhesive that does not swell with water Can be used.

Instead of a two-layer non-fibrous porous sheet that is porous-bonded with an adhesive having a fine through-hole structure, Japanese Patent Application No.
2-91543), a composite membrane filter (composite brush polymer layer) having a continuous void structure in which two polymer microporous layers formed by the multilayer coating method or the multilayer casting method are closely adhered and integrated at the interface. Can be used.

The void size of the lower non-fibrous porous sheet is about 20 nm to about
30 μm, preferably about 50 nm to about 10 μm, porosity about 20% to about 90%, preferably about 40% to about 85%, thickness about 20 μm to about 500 μm, preferably about 80 μm
To about 350 μm. The void size of the upper non-fibrous porous sheet (mean minimum pore size, that is, the pore size value measured by the well-known mercury intrusion method) is about 0.8 μm to about 3
0 μm, preferably about 1.0 μm to about 10 μm, porosity about 20% to about 90%, preferably about 40% to about 85%, thickness about 20 μm to about 500 μm, preferably about 80 μm
To about 350 μm, the total thickness of the upper and lower two layers of non-fibrous porous sheet is about 40 μm to about 500 μm, preferably about 80
The range from μm to about 350 μm is advantageous for obtaining sufficient measurement sensitivity.

In order to eliminate the optical interference in the measurement of the red reflection optical density of hemoglobin in whole blood in a non-fibrous porous sheet, Japanese Patent Publication No. 53-21677, U.S. Pat. No. 3,992,158, Japanese Patent Publication No. 55-90859, Japanese Patent Application No. 60
-232726 (JP-A-62-91543) and the like, white insoluble titanium dioxide fine particles, barium sulfate fine particles,
It is possible to enhance the function as a light reflecting layer or a light shielding layer by incorporating black hardly soluble carbon black, white aluminum fine particles, or fine particles such as fine flakes in any one or two layers. Alternatively, a dispersion liquid of these fine particles is permeated into the non-fibrous porous sheet, preferably from the side opposite to the side (lower side) to which the fibrous porous sheet is adhered, and is adhered to the inner surface of the void to contain the same. The purpose can be achieved. The fine particle dispersion may contain a binder such as gelatin, polyvinyl alcohol, or polyacrylamide in a content within a range that does not substantially hinder the passage of the polymer analyte. Among these particles, titanium dioxide particles and barium sulfate particles are preferable. When the three-dimensional lattice-like granular structure layer is used, it is possible to use a three-dimensional lattice-like granular structure layer obtained by adhering any of the fine particles in a point contact manner with an adhesive.

A known surfactant is used for the two non-fibrous porous sheet layers,
Preferably, a nonionic surfactant can be impregnated and held. By keeping the surfactant impregnated, the diffusion, permeation and passage of the aqueous liquid sample becomes uniform.

In the multilayer analytical element of the present invention, the color reagent composition contains any one layer or two or more layers (in the embodiment having a support, any one layer other than the support or two or more layers).
Contained in. Examples of the layer containing the color reagent composition include a hydrophilic polymer binder layer, a hydrophilic polymer binder layer and a lower non-fibrous porous sheet layer.
Layer, hydrophilic polymer binder layer and upper non-fibrous porous sheet layer, two layers, two non-fibrous porous sheet layer, hydrophilic polymer binder layer and two non-fibrous porous sheet layer There are three layers, a fibrous porous sheet layer and two non-fibrous porous sheet layers. The color reagent composition can be blended so that all of its components are contained in any two or three layers of these, and at the time of manufacturing a multilayer analytical element of a specific plural component of the color reagent composition or before using the analytical operation. For the purpose of preventing performance deterioration such as increased fog or decreased sensitivity due to the interaction of the specific components of the color reagent composition during storage, each of the specific components is divided into two or more different layers. Can be included. Even in an embodiment in which all the components of the substantially same color reagent composition are contained in two or more layers, the content of the reagent composition in each layer per unit area is substantially the same as the content. There are different modes.

The fibrous porous sheet or the two-layer non-fibrous porous sheet may contain all or a part of the color reagent composition. In this case, in the fibrous porous sheet or the upper non-fibrous porous sheet, a part of the reagent composition containing an enzyme, particularly an enzyme such as an oxidase or a hydrolase and a component that activates or assists its action are added. By keeping the content, it may be preferable because the measurement sensitivity becomes high. Alternatively, JP-A-59-21398, JP-A-60-122348 (JP-A-61-278)
The pretreatment reagent composition such as the endogenous ammonia scavenging reagent composition described in 761) is contained in and retained in the fibrous porous sheet or the upper non-fibrous porous sheet to function as a pretreatment layer or a trap layer. You can also

The color reagent composition is determined by the biochemical or chemical reaction involving (catalyzing) the analyte in the aqueous liquid sample and the enzyme selected to analyze the analyte,
When the selected reaction is a reaction involving two or more kinds of enzymes, all components of the reagent composition containing these enzymes can be contained in the above-mentioned two or three layers, and if necessary, the reagent composition The specific plural components in the product may be contained in two or more different layers.

As an example of a color reagent composition containing at least one enzyme,
U.S. Pat.No. 3,992,158, Japanese Patent Publication No. 53-21677, Japanese Patent Publication No. 54-26793, Japanese Patent Publication No. 55-164356, Japanese Patent Publication No. 57-208997, Japanese Patent Publication No. 59-20853, Japanese Patent Publication No.
9-46854, JP-A-59-54962, JP-B-55-25840 and the like, improved Trinder reagent composition for glucose analysis containing glucose oxidase and peroxidase, JP-B-56-45599, JP-A-59-193352, etc. Cholesterol oxidase, a reagent composition for cholesterol analysis containing peroxidase and optionally cholesterol esterase, JP-A-52-3488, JP-A-58-77661, and JP-A-56-70460
Etc. Reagent composition for blood urea nitrogen (BUN) analysis containing urease, lipoprotein lipase, glycerol kinase, α-glycerol-3-phosphate oxidase, triglyceride containing peroxidase described in JP-A-53-24893 Reagent composition for glycerol analysis, bilirubin analysis reagent composition containing bilirubin-specific oxidase and peroxidase described in JP-A-54-151193 and JP-A-60-78580, JP-A-53-26188, and JP-A-59 -193352 etc., a reagent composition for uric acid analysis containing uricase and peroxidase, a color reagent composition for detecting hydrogen peroxide containing peroxidase described in JP-A-55-124499, JP-A-58-86457, etc. is there.

As an example of a method for holding the enzyme-containing reagent composition in the porous sheet before incorporating the porous sheet into the multilayer analysis element, the porous sheet is immersed in an aqueous solution containing the enzyme-containing reagent composition or an organic solvent-containing solution to form a porous sheet. There is a method in which the functional sheet is taken out of the solution and dried (preferably reduced pressure drying, reduced pressure freeze drying).

As a preferable example of separating and containing each of a plurality of specific components in the color reagent composition in two or more different layers, an electron transfer agent, a dye precursor, an oxidized coenzyme, and the above-mentioned oxidation There is a color reagent composition for measuring an enzyme activity or a protein-bonded low-molecular-weight analyte, which contains an enzyme or an enzyme substrate that can convert a type coenzyme into a reduced type coenzyme, through the reduced type coenzyme. In this case, the electron transfer agent and the dye precursor are separated into two different layers (the other components are separated together with either one of these two components, or are appropriately separated into different layers).
It is often preferable to keep the content of the coloring reagent composition because it can reduce deterioration of the color reagent composition with time, reduce fog, increase measurement sensitivity, and improve measurement accuracy.

As an example of a color reagent composition containing an electron transfer agent, a dye precursor, an oxidized coenzyme, an enzyme capable of converting the oxidized coenzyme to a reduced coenzyme or an enzyme substrate as a main component, Clinica Chimica
Acta, 12 , 210 (1965), JP-A-59-44658, JP-A-59-8809
Coloring reagent composition for measuring lactate dehydrogenase activity described in 7, Clinica Chimica Acta, 28 , 431 (1970), JP-A-50-44894, JP-A-57-208998, JP-A-59-44658, JP-A-59-44658,
59-88097, Color reagent composition for measuring aspartate aminotransferase activity, Color reagent composition for measuring alanine aminotransferase activity, JP-B-46-998
A color reagent composition for measuring creatine kinase activity described in 8, a color reagent composition for measuring creatine phosphokinase activity described in JP-A-49-11395, JP-A-59-44658, and JP-A-59-88097. Color reagent composition for measuring testosterone activity and color reagent composition for measuring androsterone activity described in US Pat. No. 3,791,933, color reagent composition for measuring amylase activity described in Japanese Patent Publication No. 56-39637, and Japanese Patent Publication No. 53-21677. And the color reagent composition for triglyceride analysis described in JP-A-50-126494, JP-A-53-24893, and JP-B-56-38199.

In the present invention, a light-transmitting water-impermeable support can be provided on the lower side of the lower non-fibrous porous sheet layer (the side opposite to the upper non-fibrous porous sheet layer), and A water absorbing layer containing a hydrophilic polymer binder may be provided between the lower non-fibrous porous sheet layers. When the lower non-fibrous porous sheet layer is provided on the support without a water absorbing layer, a thin layer of hydrophilic polymer binder, for example, in the range of about 0.5 μm to about 5 μm is used as an adhesive layer on the surface of the support. When the adhesive layer is not yet dried, or the dried adhesive layer is wetted with water almost uniformly, and the non-fibrous porous sheet is laid on the adhesive layer, and the pressure is lightly and evenly applied to the whole. It can be closely adhered and adhered to be integrated.

When the reagent layer is provided between the lower non-fibrous porous sheet and the water absorbing layer or between the lower non-fibrous porous sheet layer and the support, the reagent layer is the same as the hydrophilic polymer of the water absorbing layer. It is preferable to provide it as a layer in which the color reagent composition is dispersed and contained in a hydrophilic polymer binder.

As an example of a method for holding an enzyme-containing reagent composition in a porous layer incorporated in a multilayer analytical element in a multilayer analytical element having a support or a water-absorbing layer, JP-A-59-171864, JP-A-60-222769 Then, as described in JP-A-60-222770, etc., after providing a porous layer on the adhesive layer or the water absorbing layer, etc., an enzyme-containing reagent composition-containing aqueous solution or organic solvent-containing solution is placed on the porous layer. There is a method of applying and drying (preferably drying under reduced pressure).

The multilayer analytical element of the present invention may be provided with a light-permeable water-impermeable support on the lower side of the lower non-fibrous porous sheet (opposite side of the upper non-fibrous porous sheet). As the support, a light-transmissive (transparent) water-impermeable support which has been used in a conventionally known multilayer analytical element can be used. Specific examples thereof include polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose ester (eg, cellulose diacetate, cellulose triacetate, cellulose acetate pyropionate, etc.), and a thickness of about 50 μm to about 1 mm, preferably Is transparent in the range of about 80 μm to about 300 μm, that is, in the form of a film (sheet) or a flat plate having a smooth surface that transmits electromagnetic radiation in at least a part of the wavelength range of about 200 nm to about 900 nm. Can be used. If necessary, titanium dioxide fine particles, barium sulfate fine particles, carbon black and the like may be dispersed and contained in the support to control the optical performance. If necessary, a publicly known undercoat layer or an adhesive layer may be provided on the surface of the support to strengthen the adhesion between the water absorbing layer or the adhesive layer which may be provided on the support and the support.

The multilayer analytical element of the present invention may be provided with a water absorbing layer between the lower non-fibrous porous sheet and the support. The water-absorbing layer is a layer whose main component is a hydrophilic polymer that absorbs and swells water, and is a layer that can absorb water of an aqueous liquid sample that has reached or penetrated the interface of the water-absorbing layer. It has the function of promoting the permeation of the aqueous liquid component and plasma into the lower non-fibrous porous sheet (porous reagent layer). The hydrophilic polymer used in the water absorption layer has a swelling rate of 30 ° C when absorbing water.
About 150% to about 2000%, preferably about 250% to about 1500
% Polymer. Specific examples of hydrophilic polymers include acid-treated gelatin described in JP-A-59-171864 and JP-A-60-115859, gelatin such as deionized gelatin, phthalated gelatin, gelatin derivatives such as hydroxyacrylate-grafted gelatin, and JP-A- 59-171864, JP-A-60-115859
Agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, etc. These hydrophilic polymers can be used alone or in combination of two or more.
Generally, gelatin or a gelatin derivative is preferable for the water absorbing layer, but when the analyte is albumin or total protein, it is preferable to use a hydrophilic polymer other than gelatin which is a protein derivative, polyacrylamide, polyvinyl alcohol or the like.

The dry thickness of the water absorbing layer is in the range of about 2 μm to about 100 μm, preferably about 5 μm to about 30 μm, and the coating amount is about 2 g / m ²
To about 100 g / m ² , preferably about 5 g / m ² to about 30 g / m ² . A well-known pH buffer, organic carboxylic acid,
It is possible to adjust the pH at the time of use (at the time of carrying out the analytical operation) by adding an acidic polymer, a basic polymer or the like. Further, the water absorbing layer may contain known mordants, polymer mordants and the like. The water absorbing layer is preferably substantially transparent, but if necessary, a small amount of titanium dioxide fine particles, barium sulfate fine particles, carbon black and the like may be dispersed and contained in the water absorbing layer to adjust the optical performance.

In the integrated multilayer analytical element of the present invention, the porosity of the fibrous porous sheet and the two layers of the non-fibrous porous sheet are such that the uniform passage of the aqueous liquid is smoothly performed from the upper side to the lower side. The sheets are substantially intimately adhered to each other by an adhesive layer having a slightly penetrating void structure (or a minute penetrating hole structure) to be integrated (porous adhesion or microporous adhesion). In the present specification, the uniform passage of the aqueous liquid means that the aqueous liquid spotted and supplied to the upper surface of the fibrous porous sheet having continuous micropores is diffused by the metering action of the continuous micropores and the layer below the layer. Aqueous liquid passing through the upper surface of the adjacent upper non-fibrous porous sheet while maintaining the distribution state that has reached the side surface, or the aqueous solution reaching the lower surface of the upper non-fibrous porous sheet It means that the aqueous liquid passes through the upper surface of the adjacent lower non-fibrous porous sheet while substantially maintaining the distribution state of the liquid.

Examples of the adhesive layer having a slightly penetrating void structure include those in which dot-shaped adhesives such as circles, squares, stars, and irregular shapes are arranged in an orderly manner, like parallel dots in printing, parallel thin lines (corrugated or Microscopically, such as straight lines), crossed thin lines (corrugated or straight lines) arranged (in this case, it becomes a fine through-hole structure adhesive layer), screenless dots (grain pattern) The shape, size, and arrangement of dots are random, but an adhesive layer is used which is arranged so as to be one of partial arrangements such as those arranged so that the uniformity can be felt macroscopically. The ratio of the total area occupied by adhesive dots and lines per unit area, that is, the adhesive area ratio is the halftone dot area ratio in printing (edited by the Printing Society of Japan, "Printing Engineering Handbook", published 1983, pages 262-263). By the same definition, about 80
% Or less, preferably about 50% or less, most preferably about 5%
To about 20%. It is desirable that the dot diameter or line thickness of the adhesive is small within the range of about 50 μm to about 1 mm without impairing the adhesive strength. The mutual spacing of the adhesive dots or lines can be determined experimentally to the extent that there is no capillary gap between the two opposing surfaces of the two porous sheets adhered in the range of about 30 μm to about 3 mm. As an example, two 150 μm-thick, smooth-surfaced membrane filters are substantially adhered and integrated with an adhesive arranged in a dot shape with a dot diameter within the above numerical range and a dot interval ( There is no substantial difference in the spreading pattern and the area of the liquid formed when water droplets are spotted on one surface after the porous adhesion). As another example, when adhering a 150 μm thick smooth surface membrane filter to a 200 μm thick tricot knitted fabric, attach the adhesive only to the ridges of the yarn on the latter surface and then attach the two. Can be porous bonded.

As the adhesive, a liquid adhesive, a non-Newtonian liquid adhesive, a hot melt type (heat melting type) adhesive, or the like can be used. As the liquid adhesive, an adhesive having a viscosity of about 1000 cps or more and a low spinnability is preferable because it is localized on the surface of the porous sheet and is less likely to penetrate inward of the porous sheet. As a specific example, a polyvinyl alcohol aqueous solution,
There are dextrin aqueous solution and carboxymethyl cellulose aqueous solution. As the non-Newtonian liquid adhesive, a liquid adhesive in which solid fine particles and the like are appropriately mixed to impart non-Newtonian viscosity is preferable. Specific examples thereof include starch paste, methyl cellulose-containing starch paste, and vinyl acetate-butyl acrylate copolymer aqueous emulsion. As the hot melt adhesive, a solid adhesive at a temperature of about 55 ° C. or lower is preferable. Specific examples thereof include ethylene-vinyl acetate copolymer and ethylene-isobutyl acrylate copolymer. As the hot-melt adhesive, ones such as thread, fine wool, granules, fine particles and powder can be appropriately selected and used.

A printing method as a preferable method for forming a partially existing pattern of the adhesive, that is, a dot pattern, a parallel fine line (corrugated or straight) pattern, a cross fine line (corrugated or straight) pattern, a grain pattern, etc. There is a method by. As a printing method, a method of directly forming an adhesive pattern on the surface of the porous sheet by an intaglio or gravure plate,
By the offset printing method, the adhesive pattern is once transferred onto a rubber roller, release paper, etc., and then this adhesive pattern is transferred to the surface of the porous sheet, directly or by a screen printing method through a polyethylene terephthalate gauze screen or a metal screen. There is a method of forming an adhesive pattern on the surface of the porous sheet by the offset method. When using a hot melt adhesive, these methods can be carried out at a temperature higher than the melting temperature of the adhesive.

As another method of forming a partial presence pattern of the adhesive, solid particles or the like are appropriately mixed on the surface of the temporary support to form a thin layer of the liquid adhesive to which non-Newtonian viscosity is applied, and then formed. There is a method of forming a screenless dot-like adhesive pattern for printing on the surface of the membrane filter or the paper by immediately peeling the membrane filter or the paper after the layers are evenly stacked.

Another method of forming a partial presence pattern of adhesive is to apply a thin layer of adhesive to the surface of a temporary support, on which the porosity of regular fine uneven surfaces such as woven or knitted fabrics is applied. The sheets are evenly stacked and then immediately peeled off to attach the adhesive in spots only to the ridges (thread ridges) of the cloth of the woven or knitted fabric. There is a method of adhering to a smooth surface porous sheet (such as a membrane filter) and closely adhering (porous adhesion).

Another method of creating a partial presence pattern of adhesive is to immerse the entire fibrous porous sheet in an adhesive solution,
Remove the sheet from the solution, squeeze the sheet to squeeze out the adhesive solution so that the adhesive adheres only to the surface of the fiber, and replace the fibrous porous sheet with another smooth surface porous sheet (membrane filter). Etc.) to adhere and integrate (porous adhesion).

In this way, the porous sheet having the partially existing adhesive pattern formed on the surface is overlaid on another porous sheet (having no adhesive pattern formed on the surface) (when a hot-melt adhesive is used). Is applied at a temperature higher than the melting temperature of the adhesive) to bring the facing porous sheets into close contact with each other by applying a substantially uniform light pressure, so that the porous sheets mutually have a slightly through-hole structure (or a slightly through-hole structure). Are substantially intimately adhered and integrated by the adhesive layer of (in this specification, this may be referred to as porous adhesion or microporous adhesion). Porous adhesion can be applied to adhesion between porous sheets, between porous sheets and non-porous sheets, and between non-porous sheets.

In the integrated multi-layer analytical element of the present invention, one or more layers of fibrous porous sheet or non-fibrous porous sheet can be provided by porous adhesion, and if necessary, the above-mentioned Functional layers described in various patent specifications, such as indicator layers, mordant layers or detection layers, semipermeable membrane layers, barrier layers, migration prevention layers, pretreatment layers or trap layers, light shielding layers or light reflection layers. The layer (background layer), the second water absorbing layer, etc. can be appropriately selected and provided.

INDUSTRIAL APPLICABILITY The integrated multilayer analysis element of the present invention is suitable for quantitative analysis of a predetermined analyte (analyte) to be tested in an aqueous liquid sample, for example, total protein in blood, particularly whole blood, albumin,
It is not only particularly useful for high molecular weight analytes such as various enzymes, high molecular weight proteins (proteins) such as bilirubin, hydrophobic analytes such as cholesterol and triglyceride, but also for glucose and urea in blood and urine, It is also useful for low molecular weight analytes such as uric acid and creatinine. Further, it is also possible to carry out the quantitative analysis of the antibody or antigen by an immunological method by allowing the porous layer in the multilayer analysis element to contain and retain the antigen or antibody. The monolithic multilayer analytical element of the present invention is particularly useful for whole blood samples, but is also applicable to other aqueous liquid samples such as plasma, serum, urine, spinal fluid, foods, beverages and the like.

The multilayer analysis element of the present invention is cut into small pieces such as a square or a circle of approximately the same size having a side of about 15 mm to about 30 mm.
-28331, Japanese Utility Model 56-142454, JP 57-63452, Japanese Utility Model 58-323
50, to be used as a chemical analysis slide by putting it in the slide frame described in the special table Sho 58-501144, etc.
It is preferable from various viewpoints such as storage and measurement operation. Depending on the purpose of use, it can be used by storing it in a cassette or magazine in the form of a long tape, or by sticking or storing a small piece on a card with an opening.

The multi-layer analytical element of the present invention can analyze a test component in a liquid sample by the operations described in the above-mentioned patent specifications and the like.
Ie about 5 μl to about 50 μl, preferably 8 μl to 30
A drop of an aqueous liquid sample of whole blood, plasma, serum, etc., in the range of μl is spotted on the spreading layer, and it ranges from about 20 ℃ to about 40 ℃ for 1 to 10 minutes.
At a substantially constant temperature in the range, preferably at a substantially constant temperature near 37 ° C, from the light-transmissive support side to the absorption maximum wavelength of the color, fluorescence, turbidity or ultraviolet ray in the element or its The optical density of the reagent development layer is reflected and measured using light having a wavelength in the vicinity, and the content of the test component in the liquid sample can be determined by the principle of the colorimetric measurement method using a calibration curve prepared in advance. Quantitative analysis of the test components can be performed with high accuracy by keeping the amount of the aqueous liquid sample spotted, the incubation time and the temperature constant. This measurement operation can be performed with a chemical analyzer described in JP-A-56-77746, JP-A-58-21566, JP-A-58-161867 or the like, which is extremely easy and allows highly accurate measurement.

Example 1 Integrated multilayer analytical element for quantitative determination of total bilirubin Daifilin per 100 g of aqueous solution (Chemical Abstracts Reg
istry No.479-18-5) 10 g, sulfosalicylic acid 2 g, 2,4-dichlorobenzenediazonium 0.2 g of a coloring reagent composition aqueous solution for analysis of bilirubin containing 0.2 g of sulfosalicylate was prepared.

This solution has a minimum pore size of 1.2 μm, thickness of 140 μm, and porosity of about 80%.
The cellulose acetate membrane filter (1) was dipped to fill the voids with the aqueous solution, and the membrane filter was taken out from the aqueous solution and dried as it was to impregnate and retain the color reagent composition to form a porous reagent layer.

Next, a starch paste was screen-printed through 100 mesh halftone dots (halftone dot area ratio about 20%) to obtain a minimum pore diameter of 3.0 μm and a thickness of 140 μm.
The cellulose acetate membrane filter with m and porosity of about 80% was attached to the surface of the solid component at a rate of about 3 g / m ² , and immediately the membrane filter impregnated with the above color reagent composition was laminated, and starch glue was applied. After drying, the two membrane filters were adhered and integrated by porous adhesion.

Then, in the same manner, a tricot knitted fabric having a thickness of about 250 μm made of PET spun yarn corresponding to 100S was porous-bonded and tightly integrated on the upper membrane filter. In this way, an integrated multi-layer analytical element for quantitative determination of total bilirubin (Example 1) was prepared in which the three-layered porous sheet was closely adhered and integrated (adhesively bonded) with the adhesive having the fine through-hole structure. did.

Separately, hematocrit value 45%, bilirubin content 0.5 mg /
Whole blood sample that was assayed with dl (test value by Jendrassik-Grof method), and the whole blood was centrifuged to separate plasma and blood clot, and a part of the plasma was a control serum with a total bilirubin content assay value of 21 mg / dl. Replace with an equal volume of Omega Bilirubin (trade name), mix again with blood clot and hematocrit value 45
%, Bilirubin content (test value) 4.1; 8.0; 16.2 mg / dl control whole blood samples were prepared.

Then, in order to evaluate the element of the present invention, 20 μl of each of these whole blood sample and control whole blood sample were spotted on the plain weave fabric layer of the multilayer analysis element, incubated at 37 ° C. for 6 minutes, and The coloration in the element was reflected and measured from the PET support side with visible light having a wavelength of 540 nm, and the results shown in Table 1 were obtained. The multi-layer analytical element of the present invention developed a whole blood sample well.

From the results shown in Table 1, in the monolithic multilayer analytical element for quantifying total bilirubin having the porous reagent layer of the present invention, the reflection optical density is small in the low bilirubin content range and the slope of the calibration curve is large. Is wide and the measurement accuracy is good. This is presumably because the analyte bilirubin diffuses and penetrates to the reagent layer sufficiently, but erythrocytes are separated in the layer above the reagent layer, and therefore do not interfere with the reflection optical density measurement without the light shielding layer.

Example 2 Integrated multilayer analytical element for quantifying total protein A reagent for measuring total protein having the following composition so that the layer thickness after drying is 40 μm on a glow-discharge treated colorless transparent PET support having a thickness of 180 μm. The composition solution was applied and dried.

Reagent composition for total protein measurement Solution composition Water 60g Polyoxydiethylene lauryl sulfate 50% aqueous solution 40g Tartaric acid 100g Copper sulfate pentahydrate 150g Sodium tartrate 15g Lithium hydroxide monohydrate 200g Acrylamide-N-vinylpyrrolidone copolymer (composition Ratio 1: 1) 20% aqueous solution (viscosity about 40cps at 40 ° C) 2000g After wetting the surface of the reagent layer after drying with water at about 25 ° C almost uniformly, the minimum pore size as the lower non-fibrous porous sheet 3.0μ
A cellulose acetate membrane filter with m, a thickness of 140 μm, and a porosity of about 80% was laminated and dried to tightly integrate the reagent layer and the membrane filter.

Next, a starch paste was applied to the surface of the lower membrane filter at a rate of about 3 g / m ² by screen printing through 100 mesh halftone dots (halftone dot area ratio about 20%), and immediately the upper non-fibrous porosity was applied. As a performance sheet, five kinds of cellulose acetate membrane filters having different minimum pore sizes shown in Table 2 were laminated, and the starch paste was dried to porously adhere the two membrane filters to integrate them. Then, in the same way, place a 100S equivalent on the upper membrane filter.
A plain woven fabric made of PET spun yarn and having a thickness of about 140 μm was adhered and integrated by porous adhesion. In this way, 5 types of integrated multilayer analytical elements for quantitative determination of total protein were prepared, in which 3 layers of porous sheets were porous-bonded.

Separately, hematocrit value 43%, plasma total protein content 7.2g / d
The whole blood sample assayed as l and the whole blood were centrifuged to separate plasma and clot, and a part of the plasma was replaced with an equal volume of physiological saline or physiological saline containing 15% human albumin and separated again. A control whole blood sample having a hematocrit value of 43% and a plasma total protein content (assay value) of 1.8 g / dl and 10.0 g / dl was prepared by mixing with a blood clot.

Next, 20 μl of each of the whole blood sample and the control whole blood sample was spotted on a plain woven fabric layer of 5 kinds of multilayer analysis elements, incubated at 37 ° C. for 6 minutes, and the central wavelength was 540 nm.
A standard curve was prepared by measuring the coloration in the element from the PET support side with visible light of the sample, and the total plasma protein content was 10.0 g / dl and 1.8 g / d.
The difference in the reflection optical density value of l was obtained to evaluate the quantitative accuracy of the total protein amount, and the results shown in Table 2 were obtained.

From the results in Table 2, the minimum pore size of the upper non-fibrous porous sheet
At 0.45 μm, the slope of the calibration curve is small and the quantification accuracy is poor;
0.8 μm has a slightly smaller calibration curve gradient than the minimum pore size of 1.2 to 5.0 μm, and the quantification accuracy is slightly inferior, but can be used; 1.2 to 5.0 μm has a large calibration curve gradient and quantification accuracy is good and can be used It is clear. That is, in this multilayer analysis element, the minimum pore size of the upper non-fibrous porous sheet is
It is clear that 0.8 μm or more is necessary, and 1.2 μm or more is preferable.

Example 3 Integrated multi-layer analytical element for quantifying total protein The same procedure as in Example 2 except that a cellulose acetate membrane filter having a minimum pore size of 0.45 μm and 1.2 μm was used as the lower non-fibrous porous sheet. We prepared a total of 10 types of integrated multi-layer analytical elements for quantitative determination of total protein, with 5 types each with different minimum pore sizes of the porous sheet.

When evaluated in the same manner as in Example 2, the same results as in Example 2 were obtained. From this result, it was clarified that the void size of the upper non-fibrous porous sheet controls the quantitative accuracy.

Claims (15)

[Claims] 1. An integrated multi-layer analytical element having a plurality of layers including at least one layer containing a color reagent composition, comprising a fibrous porous sheet, an upper non-fibrous porous sheet, and a lower side. The non-fibrous porous sheets are laminated and integrated in this order, and the three-layer porous sheet has micro-penetrating voids so that the uniform passage of liquid is not substantially hindered between the two adjacent surfaces. The upper part of the upper non-fibrous porous sheet has an average minimum pore size of 0.8 μm.
An integrated multi-layer analytical element, characterized in that it is in the range of m to 30 μm. 2. The partially arranged adhesive is in the form of dots.
The analytical element according to claim 1, which is an adhesive arranged in a crossed linear shape, a parallel linear shape, or a grain shape. 3. The light-permeable, water-impermeable support is adhered to and integrated with the lower non-fibrous porous sheet on the opposite side of the fibrous porous sheet. Analysis element. 4. The analytical element according to claim 3, wherein a water absorbing layer containing a hydrophilic polymer binder is provided between the lower non-fibrous porous sheet and the support. 5. A hydrophilic polymer binder layer provided between the support and the lower non-fibrous porous sheet, wherein the color reagent composition is contained in the hydrophilic polymer binder layer.
Analysis element described in. 6. The analytical element according to claim 3, wherein at least one layer of the three-layered porous sheet contains and holds the coloring reagent composition. 7. The analytical element according to claim 6, wherein the color reagent composition is contained in the lower non-fibrous porous sheet. 8. The analytical element according to claim 3, wherein the color reagent composition is contained in two or more layers excluding the support. 9. The analytical element according to claim 8, wherein the coloring reagent composition is contained and held in the two-layer non-fibrous porous sheet. 10. The analytical element according to claim 8, wherein the color reagent composition is contained in two layers of the fibrous porous sheet and the upper non-fibrous porous sheet. 11. A hydrophilic polymer binder layer provided between the support and the lower non-fibrous porous sheet and the lower non-fibrous porous sheet, wherein the color reagent composition is two layers. The analytical element according to claim 3, which is contained in two layers of the hydrophilic polymer binder layer and the upper non-fibrous porous sheet. 12. A hydrophilic polymer binder layer in which the color reagent composition is provided between the support and the lower non-fibrous porous sheet, and three layers of the two non-fibrous porous sheets. The analysis element according to claim 3, which is contained in. 13. The analytical element according to claim 3, wherein the color reagent composition is contained in the three-layer porous sheet. 14. The color reagent composition containing the color reagent composition in two or more layers, and the color reagent compositions contained in the two or more layers are substantially the same. The analytical element according to claim 3, which is a product. 15. The color reagent composition is contained in two or more layers, and the color reagent composition contained in the two or more layers is a plurality of two or more components thereof. 4. The analytical element according to claim 3, wherein the respective components are contained in different layers.