JPS62138758A - Integral type multi-layered analyzing element - Google Patents

Abstract

PURPOSE:To quickly diffuse and penetrate an analyte of the amt. enough for coloration reaction to a reagent layer by tightly adhering 3 layers of porous sheets by an adhesive agent constituting fine through-holes and incorporating a coloring reagent compsn. into >=2 plural layers. CONSTITUTION:This analyzing element consists of a fibrous porous sheet, upper non-fibrous porous sheet, lower non-fibrous porous sheet and hydrophilic polymer binder layer. Three layers of the sheets are adhered by the adhesive agent constituting the fine through-gaps so as not to disturb the uniform passage of liquid. The coloring reagent compsn. is incorporated into >=2 plural layers except a base. A preferable example of incorporating and holding the coloring reagent compsn. separately into the plural layers includes the coloring reagent compsn. for measurement via an electron carrier, etc. The decrease in the deterioration of the coloring reagent compsn. with age, etc. are attained by separately incorporating the electron carrier and dye precursor into separate two layers in this case.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention provides a method for analyzing predetermined analytes (
Concerning an integrated multilayer analysis element in the form of a flat plate or sheet for quantitative analysis of components to be analyzed.

In particular, the present invention relates to an integrated multilayer analytical element in the form of a plate or sheet useful in clinical tests suitable for the quantitative analysis of analytes in aqueous liquid samples, especially biological body fluids 8, such as whole blood.

[Prior Art] As a form of dry analysis element, a reagent layer in which a color-forming reaction reagent is dispersed and contained in a hydrophilic polymer binder represented by gelatin on a transparent support, and a porous development layer ( An integrated multilayer analytical element (hereinafter sometimes referred to as a multilayer analytical element) having a structure in which a developing layer (hereinafter sometimes referred to as a developing layer) is closely adhered is disclosed in Japanese Patent Publication No. 53-21677 and US Pat. No. 3,992,158. JP 55-90859, JP 55-164356, JP 57-66359, JP 52-3488, U.S. Patent Re30267, JP 58-77661, JP 60-222769,
JP-A-57-+48250, JP-A-57-12584
7 etc., and have been put into practical use. In order to adapt these multilayer analytical elements to whole blood samples, do they contain light-shielding particles such as titanium dioxide particles in the developing layer?

A light shielding layer is provided between the developing layer and the reagent layer. The multilayer analytical element is suitable for the clinical testing field because it allows accurate quantitative or semi-quantitative analysis of test components by simply spotting an aqueous liquid sample such as whole blood or plasma at approximately +OU+ on the developed layer. It is an analytical tool that is increasingly used in

[Problems with the prior art] The above-mentioned integrated multilayer analytical element uses a hydrophilic polymer binder such as gelatin in layers other than the development layer, such as the reagent layer or the light shielding layer, so it is water permeable. In general, the penetration of analytes due to diffusion is slow, and they are impermeable or poorly permeable to polymeric analytes, lipids, or hydrophobic analytes, and cannot be applied to these types of analytes. There was a point. Furthermore, when whole blood is used as the aqueous liquid sample, there is a problem in that it cannot be applied to all analytes.

Conventionally, in order to deal with polymer analytes, lipids, or hydrophobic analytes, the multilayer analytical element for quantitative cholesterol analysis described in Japanese Patent Publication No. 56-45599 has been designed to contain a coloring reagent composition containing an enzyme in the developing layer. Proposed. However, this multilayer analysis element had a problem in that the light shielding effect was insufficient for whole blood samples. Also, JP-A-59-
The multilayer analytical element described in 120957 includes a porous reagent layer in which a reagent composition is contained in the pores of a continuous pore-containing porous structure layer in which solid fine particles are held in a hydrophilic polymer binder in contact with a spreading layer. is proposed. This multilayer analytical element has a problem in that the reagent composition that can be contained in the porous reagent layer is limited.

[Object of the Invention] An object of the present invention is to provide an integrated multilayer analytical element in which a sufficient amount of analyte for a color reaction can rapidly diffuse into the reagent layer.

Another object of the present invention is to have the function of separating red blood cells in a whole blood sample, and quantifying analytes in a whole blood sample by avoiding interference by red blood cells during reflection optical density measurement for colorimetric methods. The purpose of the present invention is to provide an integrated multi-layer analysis element that enables analysis.

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

Another object of the present invention is to contain a sufficient amount of a color-forming reagent composition for a color-forming reaction with an analyte.

An object of the present invention is to provide an integrated multilayer analytical element with a large calibration curve slope and high measurement accuracy.

Another object of the present invention is to provide a color reagent composition with little deterioration over time, little fog, and high measurement sensitivity.

The object of the present invention is to provide an integrated multilayer analysis element with high measurement accuracy.

Furthermore, the present invention provides an integrated multilayer analysis element described in Japanese Patent Application No. 59-126267, that is, a part of the integrated multilayer analytical element that forms finely penetrating voids between two adjoining surfaces so that the uniform passage of liquid is not obstructed. The present invention also improves integrated multilayer analytical elements that include at least two layers of microporous sheets that are intimately bonded together (porously bonded or microporously bonded) by a disposed adhesive.

(The following is a blank space) [Constitution of the Invention The present invention consists of a plurality of 7g
In an integrated multilayer analysis element with.

Fibrous porous sheets in order from the side farthest from the special holiday.

an upper non-fibrous porous sheet, a lower non-fibrous porous sheet, and a hydrophilic polymer binder fFj2, each of the three layers of porous sheets having its UA j7? The two surfaces are bonded and integrated in substantially intimate contact with an adhesive that is partially arranged to form micro-penetration gaps so that uniform passage of liquid is substantially unobstructed between the two surfaces; The present invention is an integrated multilayer analytical element in which a coloring reagent composition is contained in two or more layers excluding the support.

[Detailed Description of the Structure of the Invention] The fibrous porous sheet is located as the outermost layer (the uppermost layer furthest from the support) and mainly functions as a layer having a spreading function. i.e. its upper surface (the surface far from the support)
The received aqueous liquid is distributed in an approximately constant amount per unit area without substantially unevenly distributing the aqueous liquid sample supplied to the aqueous liquid sample. The action of spreading the sample laterally (in the direction of the plane of the sheet) and simultaneously supplying the adjacent lower layer with an aqueous liquid sample that receives an approximately constant volume of aqueous liquid sample per unit area (spreading action or metering) This layer has a ring effect). In addition, the fibrous porous sheet contains non-soluble substances that may interfere with analysis in aqueous/alpha samples (e.g.

This layer also acts to filter out red blood cells (red blood cells, etc.) in whole blood samples.

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

As for the woven fabric, JP-A-55-164356. The plain woven fabric for the spreading layer described in JP-A-60-222770 and the like is preferred.

Among plain woven fabrics, there are narrow fabrics, all-medium fabrics, broad fabrics,
Boblin fabric etc. are preferred. Spun yarn (twisted yarn) is preferable as the yarn constituting the woven fabric. The thickness of the thread of the woven fabric is expressed in terms of cotton spun yarn count, from about 2O8 to about 1505. Preferably in the range from about 40S to about 120S equivalent, or from about 35D to about 300D in silk denier, preferably from about /15D to about 130D.

The thickness of the woven fabric is about 100 LIITI to about 500 um.
The porosity of the woven fabric ranges preferably from about 40% to about 90%, preferably from about 50% to about 85%.

As knitted fabrics, warp for developing layers described in JP-A-60-222769, JP-A-60-222770, etc.
Stockinette knitted fabrics are preferred, and among warp stockinette knitted fabrics, tricot knitted fabrics, Raschel knitted fabrics, Milanese knitted fabrics, and double tricot knitted fabrics are preferred.

Spun yarn (twisted yarn) is preferable as the yarn constituting the knitted fabric. The thickness of the yarn of knitted fabric is approximately 4 in terms of cotton spun yarn count.
About 150s from O9, preferably about 60s.

in the range of about 1203 equivalents, or from about 35D to about 130D, preferably from about 45D to about 90 in silk thread denier.
Range D: 1. The gauge number during the knitting process of the knitted fabric is approximately 20 to 50. 2. The thickness of the knitted fabric is approximately 1100I.
The porosity of the vX fabric ranges from about 40% to about 90%, preferably from about 50% to about 85%.

As paper made with organic polymer fiber valves.

Paper making from a valve made only of polyethylene fibers and polyethylene fibers (
Preferably, paper is made from a mixing valve of 30-70%) and natural fibers such as cellulose fibers. The thickness of the paper is approximately 80tnn
The porosity of the paper ranges from about 20% to about 80%, preferably from about 50% to about 70%.

Fibrous porous sea!・Of which woven fabrics and knitted fabrics (
(hereinafter both may be collectively referred to as cloth) is a cloth that has been subjected to a degreasing treatment such as washing with water to substantially remove at least the oils and fats supplied or attached during yarn manufacturing or cloth manufacturing. Paper-made paper and calender-treated paper-made filter paper are used that are substantially free of oils and fats. The fibrous porous sheet may be subjected to a physical activation treatment (preferably glow discharge treatment or corona discharge treatment) described in JP-A-57-66359, etc., on one or both sides, or JP-A-57-66359.
5-164356, JP-A-57-66359, JP-A-57-148250, etc., impregnation treatment, coating or spray treatment with a surfactant (preferably a nonionic surfactant (aqueous soluble)), impregnation with an aqueous hydrophilic polymer solution Treatment, coating or spray treatment, impregnation treatment with an aqueous solution of a hydrophilic cellulose derivative and a nonionic W surfactant with an HLB value of 10 or more as described in JP-A-60-222770, coating or spray treatment, two or more of these By processing, etc., it is possible to control the strengthening of the adhesion with the lower layer due to porous adhesion, the metering effect, the metering effect corresponding to the hematocrit value, etc.

In the integrated multilayer analytical element of the present invention, the upper layer (the side far from the support) of the two adjacent non-fibrous porous sheets that are porously bonded by the microporous adhesive layer is mainly made of an aqueous liquid. Non-dissolved substances in the sample that interfere with the analysis (e.g.
Red blood cells in a whole blood sample.

It functions as a layer that has the effect of filtering fine particle components such as white blood cells) and the light shielding effect. The lower layer functions as a layer having a light-shielding effect or a porous reagent layer that contains and retains some or all of the components of the reagent composition.

Special Publication No. 53-21677 as a non-fibrous porous sheet
.

U.S. Patent No. 3,992,158. Cellulose esters such as cellulose acetate, cellulose butyrate, cellulose acetate butyrate (mixed esters), cellulose nitrate; cellulose ethers such as ethyl cellulose; carbonate ester polymers such as polycarbonates of bisphenol A; polyamides; , for example, membrane filters (plush polymer single layer) made of polycabramide (6-nylon), polyhexamethyleneaziboamide (6,6-nylon), etc., collection of lecture abstracts of the 9th Plastic Film Research Group (The Society of Polymer Science, Japan) Published February 22, 1984), Me
Polyolefin microporous membranes such as polyethylene microporous membranes and polypropylene microporous membranes described in mbrana, Inc. catalog (issued July 1982), polymers described in Japanese Patent Publication No. 53-21677, U.S. Patent No. 3,992,158, etc. A porous layer containing continuous micropores in which microparticles such as microbeads, glass microbeads, and diatomaceous earth are held in a hydrophilic polymer binder, and a polymer adhesive in which the polymer microbeads do not swell with water, as described in JP-A-55-90859. A continuous microvoid-containing porous layer bonded in point contact (a non-fibrous isotropic porous layer such as a three-dimensional lattice-like granular structure rf!), etc. can be used.

Patent application No. 60-232726 instead of a two-layer non-fibrous porous sheet porously bonded with a micro-through hole structure adhesive
Use of a composite membrane filter (single layer of composite plush polymer) with a continuous pore structure in which two polymeric microporous layers formed by the multilayer coating method or multilayer casting method described in 2 are adhered and integrated at the interface. I can do it.

The pore size of the lower non-fibrous porous sheet is from about 20 nm to about 30 Lffr+, preferably from about 50 nm to about 10
um, the porosity ranges from about 20% to about 90%, preferably about 40% to about 85%, and the thickness ranges from about 20 um to about 5001.um. on, preferably about 80um to about 350um
This is the range of LLIrl. The pore size (average minimum pore diameter) of the upper non-fibrous porous sheet is about 0.8 layer m to about 30 m
1 nn, preferably about 1. Ol, Im in the range of about 10 um, porosity from about 20% to about 90%, preferably about 4
Range from 0% to about 85%, thickness from about 201+m to about 5
00 fields, preferably in the range of about 80 to about 350..., the total thickness of the upper and lower two layers of non-fibrous porous sheets is about 40 um
from about 500 pm, preferably from about 80 um to about 350 pm
A range of um is advantageous in order to obtain sufficient measurement sensitivity.

In the non-fibrous porous sheet, Japanese Patent Publication No. 53-21677, U.S. Pat. According to the method described in 60-232726 etc., fine particles such as white slightly soluble titanium dioxide fine particles, barium sulfate fine particles, black hardly soluble carbon black, white aluminum fine particles or fine flakes are contained in one or two layers. The function as a light reflecting layer or a light shielding layer can be enhanced. Alternatively, a dispersion of these fine particles is applied to a non-fibrous porous sheet, preferably on the side opposite to the side to be adhered to the fibrous porous sheet (
The same objective can be achieved by a method in which the material is infiltrated from the bottom (lower side) and attached to the inner surface of the void. The fine particle dispersion may contain a binder such as gelatin, polyvinyl alcohol, or polyacrylamide in an amount that does not substantially impede passage of the polymeric analyte. Among these fine particles, titanium dioxide fine particles and barium sulfate fine particles are preferred. When using a three-dimensional lattice-like granular structure layer, a three-dimensional lattice-like granular structure layer in which any of the fine particles are bonded in point contact with an adhesive can be used.

The two non-fibrous porous sheet layers contain a known surfactant,
Preferably, a nonionic surfactant can be impregnated and retained. By impregnating and retaining the surfactant, the diffusion and penetration of the aqueous liquid sample becomes uniform.

The multilayer analytical element of the present invention is characterized in that the coloring reagent composition is contained in two or more layers other than the support. Examples of layers containing the color reagent composition include two layers: a hydrophilic polymer binder layer and a lower non-fibrous porous sheet layer; 2Pi, a hydrophilic polymer binder layer and an upper non-fibrous porous sheet layer; 2 layers of non-Wi, fibrous porous sheet layer, 3N of hydrophilic polymer binder layer and 2 layers of non-fibrous porous sheet layer, 3N of fibrous porous sheet layer and 2 layers of non-fibrous porous sheet layer. There is 3N. The coloring reagent composition can be mixed so that all of its components are contained in any of these 2N or 3 layers, or it can be used during the production of multilayer analytical elements or analytical operations of specific multiple components of the coloring reagent composition. In order to prevent performance deterioration such as increase in fog or decrease in sensitivity due to interactions between specific components of the coloring reagent composition during previous storage, each of the specific components is divided into two or more different layers. can be contained. Even in an embodiment in which substantially all components of the same coloring reagent composition are contained in multiple layers of 2N or more, the content per unit area of the reagent composition in each layer is substantially the same. There are different aspects.

All or part of the components of the coloring reagent composition can be contained and retained in the fibrous porous sheet or the two-layer non-fibrous porous sheet. in this case.

An enzyme-containing component of the reagent composition in the fibrous porous sheet or the upper non-fibrous porous sheet, especially an oxidase,
It may be preferable to contain and retain an enzyme such as a hydrolase and a component that activates or assists its action, as this increases the measurement sensitivity. Or JP-A-59-21398.
A pretreatment reagent composition such as the endogenous ammonia capturing reagent composition described in Japanese Patent Application No. 60-122348 is contained and held in a fibrous porous sheet or an upper non-fibrous porous sheet to form a pretreatment layer or trap. It can also function as a layer.

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 this analyte; In the case of reactions involving the above enzymes, all components of the reagent composition containing those enzymes are added to the
It can be contained in one layer or three layers, or, if necessary, a plurality of specific components in the reagent composition can be divided and contained in two or more different layers.

As an example of a coloring reagent composition containing at least one enzyme,
U.S. Patent No. 3,992,158. Special Publication No. 53-21677.

JP-A-54-26793, JP-A-55-164356
, JP-A-57-208997, JP-A-59-208
53, JP-A-59-46854, JP-A-59-54
9 [32, Improved Trinder reagent silk composition for glucose analysis containing glucose oxidase and peroxidase, described in Japanese Patent Publication No. 55-25840, etc., Japanese Patent Publication No. 56-45
A reagent for cholesterol analysis containing cholesterol oxidase, peroxidase, and cholesterol esterase, which is optionally blended with cholesterol oxidase and peroxidase described in JP-A No. 599-11-3352 and the like! JP-A-52-3488.

Blood urea nitrogen (BUN) analysis reagent containing urease described in JP-A-58-77661, JP-A-56-70460, etc. Acid compounds, bobrotiine lipase, glycerol kinase, described in JP-A-53-24893, etc. , Reagent composition for triglyceride or glycerol analysis containing α-glyceride-corro-3-phosphate oxidase peroxidase, JP-A-5'1-151193, JP-A-Go-78
A reagent composition for analyzing bilirubin containing the bilirubin-specific oxidase and peroxidase described in 580, JP-A-53
A reagent composition for uric acid analysis containing uricase and peroxidase described in JP-A-26188 and JP-A-59-193352.

Japanese Patent Publication No. 55-124499. There are compositions of coloring reagents for detecting hydrogen peroxide containing peroxidase as described in JP-A-58-86457 and the like.

As an example of a method for retaining an enzyme-containing reagent composition in a porous sheet before incorporating the porous sheet into a multilayer analytical element, a porous sheet is mixed with an enzyme-containing reagent (1) in an aqueous solution or an organic solvent-containing solution. The porous sheet is immersed in the solution, removed from the solution, and dried (preferably vacuum drying or vacuum freeze drying).

An example of a method for retaining an enzyme-containing reagent composition in a porous layer embedded in a multilayer analytical element.

Japanese Patent Publication No. 59-171864. Japanese Patent Publication No. 60-222769
, JP-A-60-222770, etc., after providing a porous layer on a hydrophilic polymer binder layer (water absorption layer or reagent layer), an aqueous solution containing an element-containing reagent composition is applied from above the porous layer. Alternatively, there is a method of applying an organic solvent-containing solution and drying (preferably drying under reduced pressure).

Each of the specific components in the coloring reagent composition is treated with a different 2ra
An example of an electron transfer agent and two dye precursors that are preferably contained and retained in a plurality of layers of i or more.

The main components are an oxidized coenzyme, an enzyme or an enzyme substrate that can convert the oxidized coenzyme into a reduced coenzyme.

There are color reagent compositions for measuring enzyme activity or protein-bound small molecule analytes via reduced coenzymes. In this case, the electron transfer agent and the dye precursor are separated into two separate layers (other components are contained and retained together with one of these two components, or separately separated as appropriate). By doing so, it is often preferable to achieve a reduction in deterioration of the coloring reagent composition over time, a reduction in fog, an increase in measurement sensitivity, and an improvement in measurement accuracy.

An example of a coloring reagent composition containing an electron transfer agent 2 dye precursor, an oxidized coenzyme, and an enzyme or enzyme substrate capable of converting the oxidized coenzyme into a reduced coenzyme is Clnic.
a Chimica Acta, i3, 210 (196
5).

Color reagent composition for measuring lagtate dehydrogenase activity described in JP-A-59-44658 and JP-A-59-88097, C11nica Chimica Acta,
28,431 (+970), JP-A-50-44894
, JP-A-57-208998, JP-A-59-4465
8.

Color reagent composition for measuring 7 subalternate aminotransferase activity, color reagent composition for measuring alanine aminotransferase activity, described in JP-A-59-88097, creatine kinase activity measurement as described in JP-A-46-9988 A coloring reagent composition for measuring creatine phosphokinase activity described in JP-A-49-11395, JP-A-59-44658, and JP-A-59-88097, and a coloring reagent composition for measuring creatine phosphokinase activity described in U.S. Pat. No. 3,791,933. Color reagent composition for measurement, color reagent composition for measurement of anl-rosterone activity, color reagent layer I composition for measurement of amylase activity described in Japanese Patent Publication No. 56-39637, Japanese Patent Publication No. 53 Sho.
Color reagent composition for glycerol analysis described in JP-A-21677, JP-A-50-126494, JP-A-53-248
93, and a coloring reagent composition for triglyceride analysis described in Japanese Patent Publication No. 5G'-38199.

If the reagent layer is placed between the lower non-fibrous porous sheet and the hydrophilic polymer binder layer (in which case the hydrophilic polymer binder layer functions as a water absorption layer),
The reagent layer is preferably provided as a layer in which all or part of the components of the coloring reagent composition are dispersed and contained in a hydrophilic polymer binder similar to the hydrophilic polymer of the hydrophilic polymer binder layer.

The hydrophilic polymer binder layer is a layer mainly composed of a hydrophilic polymer that absorbs water and swells, and is a layer that can absorb water from an aqueous liquid sample that has reached or penetrated the interface of this layer.
When using a whole blood sample, it has the effect of promoting permeation of the aqueous liquid component and plasma into the non-fibrous porous sheet (porous reagent layer).

That is, the hydrophilic polymer binder layer has the function of a water absorption layer. Hydrophilic polymer The hydrophilic polymer used in the binder layer has a swelling rate of approximately 150% at 30°C upon water absorption.
from about 2000%, preferably from about 250% to about 150%
Polymer in the range of 0%. Hydrophilic polymer〇Specific examples include JP-A-59-171864, JP-A-60=11
Acid-treated gelatin described in No. 5859, gelatin such as deionized gelatin, phthalated gelatin, gelatin derivatives such as hydroxyacrylate grafted gelatin, JP-A-59-
Examples include agarose, pullulan, pullulan derivatives, polyacrylamide', polyvinyl alcohol, polyvinylpyrrolidone, and the like, which are described in JP-A No. 171864 and JP-A No. 60-115859. These hydrophilic polymers can be used alone or in combination of two or more. Gelatin or gelatin derivatives are generally preferred for the hydrophilic polymer binder layer, but when the analyte is albumin or total protein, hydrophilic polymers other than protein derivative gelatin, polyacrylamide, polyvinyl alcohol, etc. are used. is preferable.

The dry thickness of the hydrophilic polymer binder layer is approximately 2um.
from about 1100u, preferably from about 5LLII+ to about 3
0μm range, coating amount from about 2g/m2 to about 100g
/+t+2. Preferably about 5g/1T12 to about 30g
/m2 range. The shallow water polymer binder layer contains known pHl buffer agents, 0-organic carboxylic acids, acidic polymers, basic polymers, etc. when used (when performing analysis operations).
(JH can be adjusted. Furthermore, the hydrophilic polymer binder layer can contain a known mordant, polymer mordant, etc. Hydrophilic polymer binder q
It is preferable that the layer be substantially transparent, but if necessary, the optical performance can be adjusted by containing a small amount of titanium dioxide fine particles, barium chain acid fine particles, carbon black, etc. dispersed in the layer.

As the light-transparent water-impermeable support of the integrated multilayer analytical element of the present invention, any light-transparent (transparent) water-impermeable support used in conventionally known multilayer analytical elements can be used. Specific examples include polyethylene terephthalate,
polycarbonate, polystyrene, to bisphenol
Transparent, i.e., wavelength-containing, polymers such as cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose tisetate bilobionate, etc.) having a thickness of from about 50 to about 1 mm, preferably from about 80 to about 300 um. At least some wavelengths within the range of about 200 nm to about 900 nm! A film-like (sheet-like) or flat support having a smooth surface that is transparent to the surrounding electromagnetic radiation can be used. Optical performance can be adjusted by dispersing fine particles of titanium dioxide, fine particles of barium sulfate, carbon black, etc. in the support, if necessary. If necessary, a known undercoat layer or adhesive layer may be provided on the surface of the support to strengthen the adhesion between the support and the water-absorbing layer provided on the support.

In the integrated multilayer analytical element of the present invention, these pores are designed to allow smooth passage of aqueous liquid from the upper side to the lower side between the fibrous porous sheet and the two layers of non-fibrous porous sheets. The adhesive sheets are substantially closely adhered and integrated (porous adhesion or microporous adhesion) to each other by an adhesive layer having a micro-porous structure (or a micro-porous structure). In this specification.

Uniform passage of an aqueous liquid means that an aqueous liquid that is dotted onto the upper surface of a fibrous porous sheet with continuous micro-pores is diffused by the metering action of the continuous micro-pores and reaches the lower surface of the layer. The passage of an aqueous liquid over the upper surface of an adjacent upper non-fibrous porous sheet while substantially maintaining its distribution, or the distribution of an aqueous liquid reaching the lower surface of an upper non-fibrous porous sheet. means that the aqueous liquid passes through the upper surface of the adjacent lower non-fibrous porous sheet while substantially maintaining the .

Adhesive layers with a micro-penetration structure include 2. For example, adhesive layers in the form of circles, squares, stars, irregular shapes, etc., arranged in an orderly manner similar to halftone dots in printing, parallel thin lines (wavy or In this case, those arranged in the form of crossed thin lines (wavy or straight).

microscopically dot-shaped like screenless dots (grain-like pattern),
Although the size and arrangement are the rung J1, an adhesive layer is used that is arranged so as to have a partial arrangement, such as one arranged so that uniformity can be perceived macroscopically.

The ratio of the total area occupied by adhesive dots and lines per unit area, that is, the adhesive area ratio, is the dot area ratio in printing (edited by the Japan Printing Institute, "Printing Engineering Handbook, published in May 1983, 2
According to the same definition as (pages 62-263), approximately 80% or less,
Preferably it is less than about 50%, most preferably in the range of about 5% to about 20%. The dot diameter or line thickness of the adhesive is desirably small within the range of about 501 to about II, as long as the adhesive strength is not impaired. The mutual spacing of the adhesive dots or lines can be determined experimentally in the range of about 30 um to about 3 mm, as long as no capillary gaps are created between the two opposing surfaces of the two porous sheets that are bonded together. 1
As an example, two membrane filters with a smooth surface of 150L and 1m in thickness can be used with a dot adhesive within the above numerical range. When aqueous 4α droplets are applied to one surface, the spread pattern and area of the liquid formed are the same as the surface on which the α droplets are applied. No substantial difference is seen on the opposite surface.

As an example, when bonding a smooth membrane filter with a thickness of 1 50 1 tm to a tricot knitted fabric with a thickness of 200 pm, apply adhesive only to the ridges of the threads on the surface of the latter and then paste the two together. When combined, the two can be bonded porously.

As the adhesive, liquid adhesives, non-Newtonian dry adhesives, hot melt adhesives, etc. can be used. Viscosity of liquid adhesive is approximately +000c
An adhesive with a ps or higher and less byssus is preferable because it is localized on the surface of the porous sheet and less penetrates into the interior of the porous sheet. Specific examples include polyvinyl alcohol aqueous solution, dextrin aqueous solution, and carboxymethyl cellulose aqueous solution. The liquid adhesive with non-Newtonian viscosity is preferably a liquid adhesive that is imparted with non-Newtonian viscosity by appropriately mixing solid particles or the like. Specific examples include starch paste, methylcellulose and starch paste, and vinyl acetate-butyl acrylate copolymer aqueous emulsion. The hot-melt adhesive is preferably an adhesive that is solid at a temperature of about 55°C or less. A specific example is ethylene-vinyl acetate copolymer.

There is an ethylene-isobutyl acrylate copolymer.

Hot-melt adhesives are thread-like or finely thread-like.

Granules, particulates, powders, etc. can be appropriately selected and used.

Printing is a preferred method for forming adhesive partial presence patterns, such as halftone dot patterns, parallel thin line (wavy or straight) patterns, intersecting thin line (wavy or straight) patterns, and grain patterns. There is a method.

Printing methods include forming an adhesive pattern directly on the surface of a porous sheet using an intaglio or gravure plate, or using an offset printing method to transfer the adhesive pattern onto a rubber roller, release paper, etc., and then print the adhesive pattern. There are methods of forming an adhesive pattern on the surface of a porous sheet directly or by an offset method using a screen printing method through a polyethylene terephthalate gauze screen or a metal screen. When using hot melt adhesives, these methods can be carried out at temperatures above the melting temperature of the adhesive.

Another method for forming a partial adhesive pattern is to use 7rAp!, a liquid adhesive that is given non-Newtonian viscosity by appropriately mixing solid particles, etc. on the surface of a temporary support. A screenless dot-shaped adhesive pattern in printing is formed on the surface of the membrane filter or made paper by coating and forming a membrane filter or made paper on it, and immediately peeling it off. There is a way.

Another method of forming a partial adhesive pattern is to apply a thin layer of adhesive on the surface of a temporary support and then apply a regular micro-textured surface such as a woven or knitted fabric. By stacking the porous sheets uniformly and immediately peeling them off, the adhesive is applied in dots only to the crests of the grain (the crests of the threads) of the woven or knitted fabric. There is a method of adhering it to another porous sheet with a smooth surface (membrane filter, etc.) to tightly integrate it (porous adhesion).

Another method of forming a partial adhesive pattern is to immerse the entire fibrous porous sheet into an adhesive solution, remove the sheet from the solution, squeeze the sheet to squeeze out the adhesive solution, and apply pressure to the surface of the fibers. There is a method of attaching the fibrous porous sheet to another porous sheet with a smooth surface (membrane filter, etc.) so that the adhesive is adhered to the surface of the sheet, and then bonding and integrating it (porous adhesion).

In this way, a porous sheet with a partial adhesive pattern formed on its surface is superimposed on another porous sheet (without an adhesive pattern formed on its surface) (when using a hot melt adhesive) By applying substantially uniform light pressure (at a temperature higher than the melting temperature of the adhesive) to bring opposing porous sheets into close contact with each other, the porous sheets form a mutually microporous structure (or microporous structure). are bonded and integrated in substantially close contact with each other by the adhesive layer (herein, 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 multilayer analysis element of the present invention, one or more layers of fibrous porous sheets or non-fibrous porous sheets can be further provided by porous bonding as needed. Examples of the various functional layers described in the aforementioned patent specifications include an indicator layer, a mordant layer or detection layer, and a semipermeable membrane layer.

Barrier layer, migration prevention layer, pretreatment layer or trap layer, light shielding layer or light repulsion layer (background layer)
, a water absorption layer, etc. can be appropriately selected and provided.

The integrated multilayer analytical element of the present invention is suitable for quantitative analysis of predetermined target components (analytes) in an aqueous liquid sample.
It is particularly useful for polymeric analytes such as various enzymes, analytes bound to polymers (proteins) such as bilirubin, and hydrophobic analytes such as cholesterol and triglycerides.

It is also useful for low-molecular analytes such as glucose, urea, uric acid, and creatinine in blood and urine. Furthermore, it is also possible to carry out quantitative analysis of antibodies or antigens by immunological methods by retaining antigens or antibodies in the porous layer of the multilayer analytical element. The integrated multilayer analytical element of the present invention is particularly useful for whole blood samples, but also for other aqueous liquid samples, such as plasma, serum, urine, and spinal fluid.

It can also be applied to foods, drinks, etc.

Example 10 of a specific example of an integrated multilayer analytical element of the present invention In the analytical element, from top to bottom: a fibrous porous sheet made of 1i material fabric (l>), a non-fibrous porous sheet made of a cellulose acetate membrane filter (2),
A non-fibrous porous sheet (3) made of a cellulose acetate membrane filter, a light-transparent water-impermeable support (5) whose hydrophilic polymer binder layer (water absorption layer) (4) made of polyvinyl alcohol is made of a polyethylene terephthalate transparent film. ), the same color reagent composition is glued onto the porous layer (2).
) and (3) are impregnated and retained in the porous sea) (1)
- (2) Between.

(2) - (3) are tightly integrated (porous adhesive) by forming finely penetrating voids using starch glue arranged in a halftone pattern. The fibrous porous sheet (1) mainly has a metering function, and the non-fibrous porous sheet (2)
is a porous reagent layer that impregnates and retains a coloring reagent composition, and also has a blood cell filtering effect and a light shielding effect.

The non-fibrous porous sheet (3) is a porous reagent layer that impregnates and retains a coloring reagent composition, and also serves as a coloring layer.

The water-absorbing N (4) has the function of absorbing water in the whole blood sample and promoting the transfer of the aqueous liquid component to the non-fibrous porous sheet (3).

In the analytical element of Example 2, which is another specific example of the integrated multilayer analytical element of the present invention, from the top, the following are: (1) a fibrous porous sheet made of a tricot knitted fabric; (1) a non-fibrous porous sheet made of a cellulose acetate membrane filter; sheet (2),
A non-fibrous porous sheet (3) consisting of a cellulose acetate membrane filter and a hydrophilic polymer binder layer (4) consisting of polyvinyl alcohol are placed on a light-transparent water-impermeable support (5) consisting of a polyethylene terephthalate transparent film. With an integrated multi-layer analysis element in the composition glued to the.

The same coloring reagent composition is impregnated into the porous layer (3) and the hydrophilic polymer binder layer (4).

Between porous sheets (1) and (2), porous sheet (2)
- (3) Porous adhesion is made with starch glue arranged in a halftone dot pattern. The fibrous porous sheet (1) mainly has a metering function, and the non-fibrous porous sheet (2) mainly has a blood cell filtering function and a light shielding function.

The non-fibrous porous sheet (3) is a porous reagent layer that impregnates and retains a coloring reagent composition, and also serves as a coloring layer.

The hydrophilic polymer binder layer (4) is a reagent layer that impregnates and retains a coloring reagent composition, and also serves as a coloring layer.

It also has the effect of roughly absorbing water in whole blood samples and promoting the transfer of aqueous liquid components to the non-fibrous porous sheet (3).

The multilayer analytical element of the present invention has a side of about 15 mm to about 30 mm.
Cut into small pieces such as squares or circles of approximately the same size,
Special Publication No. 57-28331, Utility Publication No. 56-142454
．． Japanese Patent Publication No. 57-63452. Utsukai Showa 58-32350.
It is preferable from various viewpoints such as manufacturing, packaging, transportation, storage, and measurement operations to use the slides as chemical analysis slides by placing them in the slide frame described in Japanese Patent Publication No. 58-501144. Depending on the purpose of use.

It can also be used in the form of a long tape and stored in a cassette or magazine, or small pieces can be attached or stored in a cart with an opening.

The multilayer analytical element of the present invention can analyze test components in liquid samples by the operations described in the aforementioned patent specifications.

That is, about 5ul to about 501J+, preferably 8μ
Droplets of aqueous liquid components such as whole blood, plasma, and serum in the range of 30 μm to 30 μm are deposited on the spreading layer and heated at a substantially constant temperature in the range of about 20° C. to about 40° C. for 1 to 10 minutes. The color, fluorescence,
The optical density of the reagent development layer is measured by reflection photometry using light at or near the turbidity or ultraviolet absorption maximum wavelength, and the analyte component in the liquid sample is measured using the principle of colorimetric measurement using a calibration curve prepared in advance. The content can be determined. By keeping the amount of aqueous liquid sample spotted, incubation time, and temperature constant, quantitative analysis of the test component can be performed with high precision. This measurement operation can be carried out with extremely easy operation and high precision using the chemical analysis apparatus described in JP-A-56-77746, JP-A-58-21566, JP-A-58-161867, and the like.

(Space below) Example 1 and Comparative Example 1 Integrated multilayer analytical element for quantifying total bilirubin Layer thickness after drying on gelatin-subbed colorless transparent polyethylene terephthalate (PET) film (support) with a thickness of 180 um A polyvinyl alcohol aqueous solution having an average degree of polymerization of 100 and a degree of saponification of about 88% was coated so that the thickness was 25 um, and dried to form a water-absorbing layer.

After wetting the surface of the water absorbing layer almost uniformly with water at about 25°C, the lower non-fibrous porous sheet was formed with a minimum pore diameter of 1.2.
Laminated with a cellulose acetate membrane filter measuring 1 m long, 140 umL thick, and having a porosity of about 80%.

After drying, the water absorption layer and membrane filter were closely integrated.

Separately, 100g of water '11 liquid and redaphyllin (Chemi)
calAbstracts Registry No.
479-18-5) 10g, sulfosalicylic acid 2g,
An aqueous solution of a coloring reagent composition for bilirubin analysis containing 0.2 g of 2.4-dichlorobenzenediazonium sulfosalicylate was prepared, and the aqueous solution was applied onto the lower membrane filter at a rate of 80 ml per 1 m2. After drying, the membrane filter was impregnated with the coloring reagent composition to form a first porous reagent layer.

The minimum pore size of the unidirectional coloring reagent composition aqueous solution is 3. Oum
A cellulose acetate membrane filter with a thickness of 140 um and a porosity of about 80% was immersed to fill the voids with an aqueous solution, and the membrane filter was taken out from the aqueous solution and dried as it was to impregnate and retain the coloring reagent composition.

Next, starch paste was applied to the surface of the lower membrane filter by screen printing through 100 mesh dots (dot area ratio of about 20%) at a solid content of about 3 g/m2.
Immediately upper non-fibrous porous sea! - Laminate the membrane filter impregnated with the coloring reagent composition described above, dry the starch paste, and bond the two membrane filters together with porous adhesive to form a second porous reagent layer. did.

Then, do the same thing and place it on the upper membrane filter.
Approximately 250mm thick made of PET spun yarn equivalent to S. ff
RL Tricot) The W fabric was porously bonded and integrated. In this way, an integrated multilayer analytical element for quantifying total bilirubin (Example 1) was prepared, in which three layers of porous sheets were closely adhered and integrated (porous adhesive) with an adhesive having a microporous structure. did.

On the other hand, Example 1 except that the upper membrane filter was omitted
An integrated multilayer analytical element for quantifying total bilirubin (Comparative Example IA; porous reagent layer IPj) in which two porous sheets were porously bonded together in the same manner as above (Comparative Example IA; porous reagent layer IPj) and the tricot knitted fabric layer were omitted,
An integrated multilayer analytical element for quantifying total bilirubin (Comparative Example 1B; porous A sex reagent F!IN) was prepared.

Separately, hematocrit value 45%, bilirubin content f10
．． 5mg/dl and assay (J endrassik-G
By rof method.

The whole blood was centrifuged to separate plasma and blood clots, and a portion of the plasma was added to an equal volume of control serum Omega Bilirubin (trade name) with a total bilirubin content of 21 mg/dl (tested value). Replace and mix with the blood clot again to give a hematocrit value of 45% and bilirubin content (m<test value).
Control whole blood samples of 4.1; 8.0; IEi, 2B/dl were prepared.

Next, in order to evaluate the above three types of elements, 20.1 of each of these whole blood samples and control whole blood samples was used in Example 1.
and on the plain weave fabric layer of each multilayer analysis element of Comparative Example 1, incubation was performed at 37°C for 6 minutes, and the center wavelength was 540.
The color development in the element was measured by reflection photometry from the PET support side using visible light of nm wavelength, and the results shown in Table 1 were obtained. In the multilayer analytical elements of Example 1 and Comparative Example 1, the whole blood sample developed well, but in the multilayer analytical element of Comparative Example 2, the whole blood sample remained in the raised droplet state at the time of spotting and did not develop. It was insufficient. The same measurement operation was carried out for 10 times on the multilayer analysis element of Example 1.
After repeated measurements, it became clear that there was very little variation in the measured reflected optical density values, and that the reproducibility of the measurements was good.

(Leaving space below) Table 1 From the results in Table 1, it is clear that the integrated multilayer analytical element for quantifying total bilirubin, which has two adjacent porous reagent layers impregnated with the same reagent composition of the present invention, has a low It is clear that the reflected optical density is small (less fog) in the content range, and the slope of the calibration curve is large, so the measurable range of bilirubin is wide and the measurement accuracy is good. This is because the analyte bilirubin sufficiently diffuses into the reagent layer, but the red blood cells are separated in the layer above the reagent layer.
This is presumed to be because reflected optical density measurement is not interfered with even without the light shielding layer.

On the other hand, the uppermost layer is a tricot knitted fabric layer, and the second layer is a membrane filter layer impregnated with a coloring reagent composition.
In Comparative Example IA, the reflected optical density value is large regardless of the bilirubin content, and the slope of the calibration curve is small, making it difficult to quantify total bilirubin for whole blood samples. It is clear that it cannot be put to practical use as an integrated multilayer analytical element. This is presumed to be because red blood cells are not completely separated by the tricot layer and reach the color reagent-impregnated membrane filter layer, interfering with reflection optical density measurement.

In addition, in Comparative Example IB of the multilayer analytical element, which has two membrane filter layers, and the lower membrane filter layer has one porous reagent layer that impregnates and retains a coloring reagent, the slope of the calibration curve is small, so the analysis accuracy is low. Unfortunately, it is clear from the behavior of the whole blood sample upon spotting that it cannot be put to practical use as an integrated multilayer analytical element for quantifying total bilirubin for whole blood samples.

Example 2 Integrated multilayer analytical element for albumin determination Polyvinyl having an average degree of polymerization too and a degree of saponification of about 88% was deposited on a gelatin-subbed colorless transparent PET support of 180 um thickness so that the layer thickness after drying was 25 IJm. An aqueous alcohol solution was applied and dried to form a polyvinyl alcohol layer.

Separately, water-methanol (weight ratio 1:3) mixed bacterial medium) Night 1
00g per libromcresol green 0.5g, Hayashi 4m
A solution containing 10.37 g of a coloring reagent composition for albumin analysis was prepared. This solution was applied at a rate of 50 ml/m 2 to the polyvinyl alcohol layer prepared in the above procedure, impregnated, and dried to form a first reagent layer.

Then, after wetting the surface of the first reagent layer almost uniformly with water at about 25°C, a lower non-fibrous porous sheet with a minimum pore diameter of 1
．． Two layers of cellulose acetate membrane filters with a thickness of 140 m and a porosity of 80% were laminated and dried to closely integrate the water absorption layer and the membrane filter.

Next, apply the same coloring reagent composition solution for albumin analysis as above to the lower membrane filter per 1112 13
A second reagent layer (porous reagent N) was formed by applying the coloring reagent composition at a ratio of 0 ml and drying to impregnate and retain the coloring reagent composition on a membrane filter.

Next, starch paste was applied to the surface of the lower membrane filter at a solid component ratio of 3811112 by screen printing through 100 mesh dots (w4 dot area ratio of about 20%), and immediately formed into an upper non-fibrous porous sheet. Minimum pore diameter 3
, 0 INI+, a cellulose acetate membrane filter with a thickness of 140 um and a porosity of 80% is laminated, and l! i The powder paste was dried and the two membrane filters were porously bonded and integrated.

Then, in the same way, apply 10O on the upper membrane filter.
Approximately 14014ITl thick made of PET spun yarn equivalent to S
The plain weave fabrics were porously bonded and integrated into one.

In this way, an integrated multilayer analytical element for quantifying albumin was prepared in which three layers of porous sheets were porously adhered.

Separately, hematocrit value 42%, albumin content ffi
A whole blood sample tested as 4.2 g/dl and the whole blood were centrifuged to separate plasma and blood clots, and a portion of the blood 5M was added to an equal volume of physiological saline or physiological saline containing 15% human albumin. Replaced and mixed with separated blood clot again, hematocrit value 42%, albumin content m<h constant value) 1.4
: 7.7; 10. An Og/old control whole blood sample was prepared.

Next, 20 ul of each of these whole blood samples and control whole blood samples were spotted on the plain weave fabric layer of the multilayer analytical element, incubated at 37°C for 6 minutes, and the center wavelength was 640 nm.
The color development in the element was measured by reflection photometry from the PET support side using visible light of m, and the results shown in Table 2 were obtained.

Table 2 From the results shown in Table 2, the integrated multilayer analytical element for albumin quantification in which the bromcresol green of the present invention is contained in two adjacent reagent layers has a large slope of the calibration curve and high accuracy in albumin quantification. That is clear. This is presumed to be because the analyte albumin in the whole blood sample quickly permeates into the porous reagent layer in a sufficient amount, so that the color reaction progresses sufficiently.

Example 3 Integrated multilayer analytical element for albumin quantification Integrated multilayer for albumin quantification in which three layers of porous sheets were porously adhered in the same manner as in Example 2 except that bromcresol purple was used instead of bromcresol green Analytical elements were prepared.

Regarding the obtained element, 20 ul of each of the whole blood sample and the control whole blood sample were spotted on the plain weave fabric layer of the multilayer analysis element in the same manner as in Example 2, and incubation was performed at 37°C for 6 minutes. The color development in the element was photometrically measured from the PET support side using visible light with a center wavelength of 600 nm, and almost the same results as in Example 2 were obtained.

From this result, it was found that the bromcresol purple of the present invention was used as the indicator dye for the adjacent 2J? ! It is clear that in the integrated multilayer analytical element for quantifying albumin contained in the reagent layer, the slope of the calibration curve is large, and the accuracy of quantifying albumin is high. This is presumed to be because the analyte albumin in the whole blood sample quickly permeates into the porous reagent layer in a sufficient amount, so that the color reaction progresses sufficiently.

Claims (7)

[Claims] (1) In an integrated multilayer analytical element having multiple layers on a light-transmitting and water-impermeable support, in order from the side farthest from the support, a fibrous porous sheet, an upper non-fibrous porous sheet, and a lower layer. It has a non-fibrous porous sheet and a hydrophilic polymer binder layer, and each of the three porous sheets has finely penetrated voids between its two adjacent surfaces so that the uniform passage of liquid is substantially unhindered. are substantially closely adhered and integrated by an adhesive partially arranged so as to constitute the support, and the coloring reagent composition is contained in two or more layers excluding the support. An integrated multi-layer analysis element characterized by: (2) The analytical element according to claim 1, wherein the partially arranged adhesive is an adhesive arranged in the form of dots, intersecting lines, parallel lines, or grains. (3) The coloring reagent composition comprises two layers of the hydrophilic polymer binder layer and the lower non-fibrous porous sheet, two layers of the hydrophilic polymer binder layer and the upper non-fibrous porous sheet, or two layers of the hydrophilic polymer binder layer and the upper non-fibrous porous sheet. An analytical element according to claim 1 contained in a non-fibrous porous sheet of the layer. (4) The analytical element according to claim 1, wherein the coloring reagent composition is contained in three layers: the hydrophilic polymer binder layer and the two layers of non-fibrous porous sheets. (5) The analytical element according to claim 1, wherein the coloring reagent composition is contained in three layers: the fibrous porous sheet and the two layers of non-fibrous porous sheets. (6) The analytical element according to claim 1, wherein the coloring reagent compositions contained in the two or more layers are substantially the same coloring reagent composition. (7) Claim 1, wherein the coloring reagent composition contained in the two or more layers includes two or more of the components, each of which is contained in a different layer. analysis elements.