JPH03131757A - Dry type analyzing element for analyzing whole blood sample - Google Patents

Abstract

PURPOSE:To facilitate processing at the time of production (molding) and to allow rapid measurement as well as to enhance the reproducibility of an analyte measured value by laminating a non-woven fabric having a prescribed average fiber diameter and density and a detecting function layer. CONSTITUTION:The non-woven fabric having 0.2 to 10mum average fiber size and 0.01 to 0.2g/cm<2> density and the detecting function layer are laminated or a porous layer as a development layer is held in place therebetween to form the dry analyzing element for the whole blood sample. There are no particular limitations on the fiber blank material of the non-woven fabric and spinning method. Filter paper, non-woven fabric, woven fabric, knitted fabric, etc., are usable for the porous layer and the woven fabric and knitted fabric are more preferable. A reagent layer, light shielding layer and non-woven fabric are laminated on a base to form the analyzing element. The reagent layer is the layer contg. the compsn. which can generate the coloration, discoloration or the change in absorption wavelength optically detectable in the presence of the component to be detected. This layer is formed by incorporating the reagent compsn. into a water permeable layer or pours layer consisting of a hydrophilic polymer binder.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a dry integrated multilayer analytical element used for quantifying blood components using whole blood as a sample. This is related to body type multi-layered analysis elements.

(Technical background) Various components in body fluids that are important in clinical medicine, such as glucose, bilirubin, BUN (urea nitrogen), uric acid, cholesterol, various enzymes (flare tincture (11 (2)), GOT (glutamate oxaloacetate transaminase) , GPT (glutamate pyruvate transaminase) 4, etc., there are two methods: wet method and dry chemical analysis.Dry chemical analysis uses analysis elements such as test pieces, analysis slides, and analysis tapes containing dry analytical reagent systems. This is an analytical method that uses liquid reagents, and is superior to wet methods that use solution reagents in terms of ease of operation and rapid analysis.

By further advancing this dry method, a dry integrated multilayer analytical element has been developed as an analytical means that can quickly perform high-precision chemical analysis on a minute amount of liquid sample. For example, Japanese Patent Publication No. 53-21677 (US3.992.158
), JP 55-164356 (US 4,292,2
72) Japanese Patent Publication No. 6O-222769 (EP 01623Q
There are analysis elements described in 2A).

An integrated multilayer analytical element (also referred to as a multilayer analytical element) is composed of, for example, a transparent support, a reagent layer, a light-reflecting layer (light-shielding layer), and a developing layer. The reagent layer coated on the transparent support (for example, a polymer sheet) contains a reagent that reacts with the test component contained in the liquid sample and develops or changes color to an optical density depending on the amount of the component. The spreading layer spreads the deposited liquid sample uniformly (without substantially unevenly distributing the components contained therein (at an almost constant rate per unit area in the plane direction). measurement
g) Action is also called spreading or spreading action.

When a liquid sample, for example, a small amount (e.g., approximately 10μ℃) of blood is spotted on the surface of the developing layer of such a dry analytical element, the blood developed in the developing layer passes through the light-reflecting layer. It reaches the reagent layer, where it reacts with the reagent and develops or changes color.

After incubating the multilayer analytical element at a certain temperature for a certain period of time, the amount of color development or color change is measured reflectively from the transparent support side, and quantitative analysis is performed based on a calibration curve. The light reflecting layer has the role of eliminating the influence of the color of the liquid sample in the developing layer and forming a white background during optical density measurement.

However, even in such dry chemical analysis methods, serum or plasma obtained by removing red blood cells from whole blood is often used as a sample, as in the wet method. The operation of separating red blood cells from blood requires a lot of effort, time, and equipment. Therefore, it is particularly desired that the dry method, which is characterized by speed and convenience, be able to directly analyze whole blood as a sample.

(Prior art) As a dry multilayer analysis element for whole blood samples,
3-21677 (US 3,992.1581, JP-A-60=111960 (US 4,637,978) and JP-A-60-27980, etc., a filtration layer for separating blood cells and high molecular weight components in blood is provided. Something has been proposed.

These are porous polymer membranes such as cellulose acetate membranes (
A membrane filter) or a porous layer having continuous voids formed of microparticles is used as a filtration layer to trap and remove blood cell components at or near the surface of the filtration layer. Therefore, in order to sufficiently remove blood cells, it is necessary to use a material with a sufficiently small average pore diameter. However, when the average pore size is made small, it takes time for plasma to pass through the filter layer, which significantly impairs the speed of measurement. In addition, if the pore size is small, the background due to hemoglobin pigment will increase due to hemolysis, and the leakage of blood cell white components will adversely affect the color reaction in the detection layer, resulting in variations in measured values.
There was also the inconvenience of poor reproducibility.

On the other hand, glass fiber filter paper is also known to have a high blood cell/plasma separation function that separates blood cells and plasma from whole blood, and analytical elements using this glass fiber filter paper were also developed in Japanese Patent Application Laid-Open No. 57-53.
661 (EP 0045476A), JP-A-6L-96
466 (EP 0159727A), etc.

These methods are excellent in that they require less bloodletting, but because they use highly brittle glass fibers, they require careful processing during the production of analytical elements, resulting in poor manufacturing suitability. Further, since the glass fiber filter paper (or glass fiber layer) is simply placed in close contact with the detection reagent layer or the development layer, its adhesive strength is low and it is easily peeled off. When peeling occurs, the amount of plasma transferred to the detection reagent layer becomes uneven, which causes a problem of poor reproducibility of measured values.

(Problem to be solved by the invention) The present invention was made in view of the above circumstances, and
An object of the present invention is to provide a dry analytical element for whole blood sample analysis for quantitative analysis that is easy to process during molding, can be measured quickly, and has high reproducibility of analyte measurement values.

(Means for Solving the Problems) The above purpose is to obtain fibers with an average fiber diameter of 0.2 to 10 μm and a density of 0.2 μm.
This is achieved by a dry analytical element for analyzing whole blood samples, which is formed by laminating a nonwoven fabric with a weight of 0.01 to 0.2 g/cm and a detection function layer.

The above object also includes a nonwoven fabric having an average fiber diameter of 0.2 to 10 μm and a density of 0.01 to 0.2 g/am3, and a porous layer.
This can also be achieved by a dry analytical element for whole blood sample analysis in which a detection function layer is laminated in this order.

(Function) The present invention was made based on the findings of the present inventors who discovered that a certain type of fine fiber nonwoven fabric specifically separates blood cells and plasma without hemolysis. It is constructed using plasma separation effect.

Although the separation mechanism is not clear, the nonwoven fabric does not trap blood cells only on its surface, but as it permeates through its thickness, first large blood components and later small blood components gradually become entangled in the fiber structure. This is thought to be due to the so-called volumetric filtration effect, which removes blood cells over the entire length in the thickness direction.

Although the porous layer is intended to function as a spreading layer, the fine fiber nonwoven fabric itself can also exhibit a certain degree of spreading effect, so when a nonwoven fabric alone is sufficient, the nonwoven fabric can have both blood cell separation and spreading effects. If the porous layer is used, the porous layer can be omitted.

(Detailed explanation of means for solving the problem) The basic composition of the dry analysis element of the present invention is a lamination of a fine fiber nonwoven fabric and a detection function layer, or a porous layer as a spread layer between the two. The smallest unit is what is sandwiched. However, the analytical elements of the present invention may be formed on a transparent support. In addition, the detection function layer includes at least one or more reagent layer, and in order to facilitate the detection of the dye produced as a result of the color reaction, it may be transferred to another detection layer for detection. explain.

The fiber material of the jade heather nonwoven fabric is not particularly limited. This is because the blood cell/plasma separation function of nonwoven fabric is not due to the chemical properties of the fibers.
Rather, it is thought that this is due to the fabric structure of non-woven fabric. Therefore, the present invention can be achieved by selecting fiber diameter and density that allow sufficient separation of blood cells and plasma. - For boat fishing, the average fiber diameter is 0.2 to 10 μm and the density is 0. It is desirable that the content be in the range of -0.2 g/cm3.

The thickness (average diameter) of the fibers of the cloth used is approximately 0.2 μm
to about 10 μm, preferably from about 0.5 μm to about 5 μm. The density of the cloth is about 0.01g/Cm3 to about 0.2
g/cm3, preferably from about 0.02 g/cm3 to about 0.02 g/cm3.
1g/cm” range, cloth thickness is about 100μm to about 20μm
00gm, preferably about 150LLm to about 11000L
In the range of L, the porosity of the fabric ranges from about 75% to about 99.5%, preferably from about 85% to about 98%.

Nonwoven fabrics can be manufactured using dry methods such as the spunlace method, spunbond method, melt blow method, knee pan method, and binder method, as well as wet methods that have been conventionally used. As a method for producing a nonwoven fabric, a method can be employed in which a nonwoven fabric is produced by spinning fine fibers and then using a spunbond method, or a method in which a nonwoven fabric is produced directly from the raw material by a melt blowing method.

The material of the fiber used in the spunbond method may be any material as long as it becomes fibrous. The spinning method is direct spinning (
Direct spinning of fine fibers), splitting (dividing a multi-component composite spun product to obtain fine fibers), and extraction (extracting and removing self-spinning components from a multi-component composite spun product using a solvent). to obtain fine fibers).

The material of the nonwoven fabric in the case of melt blowing may be any polymer that can be melt-spun, such as polyethylene terephthalate (generally referred to as polyester), polybutylene terephthalate, polyethylene, polypropylene, and nylon.

The porous layer is a nonwoven fabric that uniformly spreads blood plasma separated from blood cells, and supplies the plasma to the detection function layer below. It is a layer separate from the nonwoven fabric, and may be formed of either a fibrous porous layer or a non-fibrous porous layer.

In the case of forming a fibrous porous layer, filter paper, nonwoven fabric, woven fabric (for example, hand-woven fabric), knitted fabric (for example, tricot knitted fabric), etc. can be used. Among these, woven fabrics and knitted fabrics are preferred. Woven fabrics, knitted fabrics, etc. are disclosed in Japanese Patent Application Publication No. 57-66359+US 4,783.315)
Apply glow discharge treatment as described in . be able to. Or Tokuko Sho 59-11709, Tokuko Sho 5
Woven fabrics, knitted fabrics, etc. treated with alkali etching (alkali weight loss) as described in No. 9-24233 can also be used.

In the case of a non-fibrous porous layer, Japanese Patent Publication No. 53-216
77 (US 3,992.158), US Pat.
Preferred are plush polymer layers (membrane filters) of cellulose esters, such as cellulose acetate, cellulose acetate butyrate, and cellulose nitrate, as described in US Pat. No. 21,341. In addition, polyamides such as 6-nylon and 6.6-nylon; microporous membranes such as polyethylene and polypropylene; JP-A-62-27006
A microporous membrane made of polysulfone as described in 1 can also be used. In addition, polymer microparticles, glass microparticles, diatomaceous earth, etc. described in Japanese Patent Publication No. 53-21677, Japanese Patent Application Laid-open No. 55-90859 (US 4,258.0011, etc.) are bonded with hydrophilic or non-water-absorbing polymers in a point adhesive manner. Porous layers with continuous voids can also be used.

When the fine fiber nonwoven fabric exhibits a spreading action, the multilayer analytical element of the present invention may be constructed by omitting this porous layer. However, when a fine fiber nonwoven fabric and a porous layer are laminated by partial adhesion (porous adhesion), which will be described later, these two layers are harmoniously integrated to have a blood cell separation effect, a plasma expansion (liquid measurement) effect, and a plasma distribution function. It is a preferred embodiment of the present invention to have the function of supplying to the lower detection function layer.

It is desirable that this porous layer be impregnated with or contain a hydrophilic polymer or surfactant as described below to make its spreading action more uniform.

■Hydrophilic polymer JP-A-6O-222770 (EP 0162301Al
Hydrophilic cellulose derivatives described in JP-A-63-21533 (EP O254202A1: methylcellulose, ethylcellulose: polyvinyl alcohol; polyvinylpyrrolidone; polyacrylic acid, etc.; JP-A-62-182652 (DE 3717913A1)
Hydrophilic polymers such as acrylamide, homopolymers or copolymers of N-substituted or N,N-disubstituted acrylamide, etc.
, JP 55-164356 (US 4,292.27
Nonionic surfactants such as nonylphenoxypolyethoxyethanol described in JP-A No. 60-222770, nonionic surfactants with an HLB value of 10 or more as described in JP-A-60-21533 (EP 0254202Al), Alcohol ester ethylene oxide adducts; polyethylene glycol fatty acid esters; higher alcohol ethylene oxide adducts; alkylphenol ethylene oxide adducts; nonionic surfactants such as higher fatty acid alkanolamides: JP-A-62-6167 (Chemical Abst)
racts 107゜171961w), JP-A-63-
These wood-philic polymers and surfactants, such as the fluorine-containing surfactants described in EP 0278496A1 and the like, can also be used in combination.

In addition, if the porous layer is omitted, or if the nonwoven fabric also has a spreading effect even when used together with the porous layer, these wood-loving polymers and surfactants may be incorporated into the fine fiber nonwoven fabric. is desirable.

The detection function layer receives plasma supplied from the porous layer (or directly from the nonwoven fabric layer) and has the function of detecting the test component. It consists of at least one reagent layer, but it is also possible to allow only a color development (or color change) reaction to occur in the reagent layer, and the resulting dye to be transferred to a detection layer, in which the amount of dye is detected. .

Achievement I The reagent layer is a layer containing a composition (reagent composition) that causes optically detected color development or color change, or a change in absorption wavelength in the ultraviolet region, in the presence of a test component, and is a hydrophilic layer. In some cases, a reagent composition is contained in a water-permeable layer or a porous layer made of a transparent polymer binder.

■When the reagent layer is composed of a water-permeable layer made of a hydrophilic polymer binder, examples of the hydrophilic polymer binder include gelatin and its derivatives (e.g., phthalated gelatin); cellulose derivatives (e.g., hydroxyethyl cellulose); agarose. :
Polyacrylamide, bonometacrylamide, copolymers of acrylamide or methacrylamide and various vinyl monomers; polyvinyl alcohol: polyvinylpyrrolidone, etc. can be used. All or some of the components of the reagent composition (
In this case, the remaining components are contained in other layers) substantially uniformly.

The reagent layer using a wood-philic polymer as a binder is
21677 (US 3,992.158), Japanese Patent Publication No. 1983
-164356 (US4, 292.272), JP 54-101398 (US 4,132.528), JP 61-292063 ((:chemical Abs
tracts 106210567y), by applying an aqueous solution or aqueous dispersion containing a reagent composition and a tree-philic polymer onto a support or other layer such as a detection layer and drying it. Can be done. The dry thickness of the reagent layer using wood-philic polymer as a binder is approximately 3 μm.
to about 50 μm, preferably from about 5 μm to about 30 μm, with coverage ranging from about 3 g/m 2 to about 50 g/m 2 , preferably from about 5 g/m" to about 30 g/m".

■When the reagent layer is composed of a porous layer containing a reagent composition (
Porous reagent layer), in this case, the porous reagent layer may be a porous layer similar to the above-mentioned porous layer, or a porous layer as described in JP-A-59-120957 (EP 01
A porous structure layer containing solid fine particles and a polymeric binder as described in [144Q3A] and the like can be used.

The reagent composition is contained in the porous reagent layer by impregnation or coating with a solution (or dispersion) of the reagent composition. The porous reagent layer containing the reagent composition can be used in conjunction with other water-permeable layers, such as a second reagent layer, a water-absorbing layer,
Laminated on top of the detection layer or adhesive layer. Note that this lamination is
JP 55-164356 (US 4,292.272
), a porous reagent layer can be adhered by lightly pressing onto a lower layer that has been wetted almost uniformly with water.

Alternatively, after adhering a porous layer on the water absorption layer, detection layer, or adhesive layer by the method described in JP-A-55-164356, the solution or dispersion of the reagent composition is applied to the porous reagent layer. It may be applied to

A known method can be used to impregnate or apply a reagent composition solution to the porous reagent layer. For example, dip coating,
Doctor coating, hopper coating, curtain coating, etc. can be selected and used as appropriate.

(2) Reagent Composition The reagent composition may be any composition as long as it can produce an optically detectable substance (for example, a pigment) in the presence of the test component. Even if it reacts directly with the test component to develop or change color, it may be necessary to use a reagent composition containing the test component and other reagents [e.g.
(upper right column of page) (EP 0226465A, page 5 line 48 ~
A composition (indicator) that produces an optically detectable substance (pigment or dye) as a result of the reaction with an intermediate produced by the reaction with a reagent composition (such as the reagent composition described on page 6, line 40)
But that's fine. For example, i) compositions that produce dyes by oxidation of leuco dyes (e.g., triarylimidazole leuco dyes described in U.S. Pat. No. 4,089,747, etc.; (diarylimidazole leuco dyes); ii) diazonium salts; 1ii) compositions containing compounds that, when oxidized, produce dyes by coupling with other compounds (e.g. 4-aminoantipyrines and phenols or naphthols);
; iv) A compound consisting of a compound capable of producing a pigment in the presence of a reduced coenzyme and an electron transfer agent can be used.

Furthermore, in the case of an analytical element for measuring enzyme activity, a self-developing substrate that liberates a colored substance such as p-nitrophenol can be included in the reagent layer or porous layer.

The reagent composition may contain an activator, a buffer, a hardening agent, a surfactant, etc., if necessary. Examples of buffers that can be contained in the reagent layer of the analytical element of the present invention include carbonates, borates, phosphates, and
Examples include Good's buffer described in TryJ, Vol. 5, pp. 467-477 (1966). These buffers are used in "Basic Experimental Methods for Proteins and Enzymes"
(Takeshi Horio - Baka, Nankodo, 1981), rBio
ChemistryJ Vol. 5, pp. 467-477 (19
You can make a selection by referring to literature such as 1966).

If the coloring reaction proceeds in multiple stages, the reagent layer may be divided into two or more parts, and each layer may contain a reagent composition corresponding to each reaction stage.

Group 1 1 The detection layer is a layer in which dyes generated in the presence of the test component in the reagent layer diffuse to reach this layer and are optically detected through the transparent support, and must be composed of a wood-philic polymer. Can be done.

If necessary, the detection layer may include a mordant, such as a cationic polymer for anionic dyes, to facilitate the detection of colored dyes.

A water absorption layer may be provided below the detection layer.

The water absorption layer generates a kind of diffusion negative pressure by absorbing water, which promotes the movement, diffusion, and penetration of plasma into the porous layer and reagent layer, and the movement of dye into the detection layer. A layer in which the dye produced in a layer does not substantially diffuse into the layer. This water-absorbing layer can be made of a wood-philic polymer that easily swells with water.

Further, a light shielding layer may be provided above the detection layer and at least below the nonwoven fabric or porous layer. This light shielding layer shields the red color of hemoglobin of red blood cells in blood when measuring detectable changes (color change, color development, etc.) occurring in the detection layer, reagent layer, etc. from the transparent support side. A layer that functions as a light reflective layer or background layer, containing titanium dioxide,
It is a layer made of a hydrophilic binder containing light-reflecting fine particles such as barium sulfate, or a porous layer containing light-reflecting fine particles.

Subsection 1 A preferred material for the support is polyethylene terephthalate. Cellulose esters such as cellulose triacetate, polystyrene, polycarbonate of bisphenol A, etc. can also be used. A support that is light-transparent (transparent) and water-impermeable is generally used, but water-permeable, water-permeable, or light-impermeable (opaque) supports can also be used. The support is usually provided with an undercoat layer or subjected to a hydrophilic treatment in order to firmly adhere the hydrophilic layer laminated thereon.

However, if the fine fiber nonwoven fabric layer has a physical strength that can maintain the structure of the element, the support may be omitted.

Complete responsibility The analytical elements of the invention can have various configurations.

For example, Japanese Patent Publication No. 49-53888 (US 3,992,
158 + Japanese Patent Publication No. 55-164356 (US 4,292
, 272), JP-A-60-222769 (EP Ro1
62302Al, JP 62-138756, JP 62-138757, JP 62-138758 (
The layer structure of a multilayer analysis element described in EP 0226465A) etc. can be referred to. Note that in one aspect of the present invention,
As described above, the porous layer adjacent to the fine fiber nonwoven fabric may be omitted.

Practically, for example, (1) a reagent layer containing a hydrophilic polymer binder and a layer of fine fiber nonwoven fabric are placed on the support in this order; (2) a reagent layer containing a hydrophilic polymer binder and a porous layer are placed on the support. (3) On the support, place the water absorption layer, the reagent layer containing hydrophilic polymer binding, the porous layer, and the layer of the fine fiber nonwoven fabric in this order. (4) On the support. (5) Place a detection layer, a reagent layer containing a hydrophilic polymer binder, a porous layer, and a fine fiber nonwoven fabric layer in this order on the support. It is useful to have (6) a detection layer, a reagent-containing porous layer, and a fine fiber nonwoven fabric layer on a support in this order (the support may include an undercoat layer).

. Next, the partial contact (or porous adhesion) between the fine fiber nonwoven fabric and the porous layer or the detection function layer, which is a feature of the multilayer analytical element of the present invention, will be explained.

Partial adhesion is described in Japanese Patent Application Laid-Open No. 61-4959 (EP 0166).
365A) JP-A-62-138756-8 (EP 02
A mode of adhesion between two adjacent porous layers or between an adjacent porous layer and a non-porous layer described in 26465A) etc. )
"The adhesive is substantially tightly bonded and integrated with the adhesive disposed on the two adjacent surfaces, and is configured such that the uniform passage of liquid between the two adjacent surfaces is substantially unobstructed." In the multilayer analysis element of the present invention, the interface between the adjacent fine fiber nonwoven fabric and the porous layer, or the interface between the adjacent fine fiber nonwoven fabric and the detection function layer is partially bonded.

- For boat fishing, adhesive is placed partially on the fine fiber nonwoven fabric, and then the fine fiber nonwoven fabric is bonded to the porous layer or the detection function layer while applying light pressure uniformly. Conversely, it is also possible to place an adhesive partially on the porous layer or the detection function layer, and then attach the fine fiber nonwoven fabric to the porous layer or the detection function layer while uniformly applying light pressure. Furthermore, an adhesive is placed partially on the fine fiber nonwoven fabric to form a porous layer, and the fine fiber nonwoven fabric is laminated uniformly and lightly under pressure to a porous sheet-like material to form a porous layer. After placing adhesive partially on the porous sheet material, and then applying fine fiber nonwoven fabric uniformly and applying light pressure to the porous sheet material, the porous sheet material is uniformly applied to the detection function layer. It can also be attached to.

Methods for partially disposing an adhesive on a fine fiber nonwoven fabric, a porous layer, or a detection function layer are described in JP-A-61-4959, JP-A-62-138756, JP-A-64-23160 (
Various methods described in DE 3721236A) etc. can be used. Among these methods, the printing method is preferred. Among the printing methods, a method of transferring and adhering an adhesive to a porous layer or a detection functional layer using a printing plate (preferably a Clavier printing plate or an intaglio) roller and a method of pasting two adjacent layers together include, for example, Edited by the Printing Society of Japan, “Printing Engineering Handbook J” (Gihodo Publishing (Kiku, 1983), 83)
It can be carried out using the known apparatus and method described on pages 8 to 853.

Adhesives that can be used include various adhesives described in JP-A-62-138756, as well as those described in the above-mentioned "Printing Engineering Handbook,"
! Known adhesives such as those described on pages 1839 to 853 can be used. As the adhesive, a water solvent type adhesive, an organic solvent type adhesive, or a thermal adhesive (hot melt type or heat sensitive) adhesive can be used.

Examples of water-based adhesives include water-based glues such as starch glue; aqueous solutions such as dextrin, carboxymethyl cellulose, and polyvinyl alcohol; and vinyl acetate-butyl acrylate copolymer emulsions. As the organic solvent type adhesive, one whose solvent evaporates slowly is suitable. Thermal adhesion (
Hot melt or heat sensitive) adhesives are particularly useful.

As the heat-adhesive hot-melt adhesive, the hot-melt adhesive described in "Industrial Materials" Vol. 26 (No. 11), pages 4-5, etc. can be used. Examples include ethylene copolymers such as ethylene-vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, and ethylene-acrylic acid copolymer; polyolefins such as low molecular weight polyethylene and active polypropylene; and nylon. Thermoplastic rubbers such as polyester copolymers and styrene block copolymers such as SBS; styrene-butadiene rubber, butyl rubber, urethane rubber; rosin, petroleum resins, terpene resins; and synthetic waxes.

(Left below) (Example 1 and Comparative Example 1) Analytical element A was prepared by laminating a reagent layer, a light shielding layer, and a nonwoven fabric in this order on a lucose-type layer support.

First, an aqueous solution of a reagent composition was coated on the surface of a colorless and transparent polyethylene terephthalate (PET) sheet (support) having a thickness of 180 μm so as to have the following coating amount and dried to form a reagent layer.

Wiping agent 1m” Deionized gelatin 26g Glucose oxidase 2000U Peroxidase 3300U4- [4-(
dimethylamino)phenyl]-5-phenethylimidazole (leuco dye) acetate 600mg 1,7-hydroxynaphthalene 660m
g60mg nonylphenoxy polyethoxyethanol 0
(Contains oxyethylene units) 270 mg An aqueous dispersion of the following composition was applied onto this reagent layer to form a light-shielding layer (dry thickness: about 7 μm) with the following coverage.

The surface of this light shielding layer is uniformly swollen with water, and then the surface of the light shielding layer is uniformly swollen with water, Polyester melt-blown nonwoven fabric (average fiber diameter 0.8 μm, density 0.13 g
/cm3. The film was laminated with a thickness of about 40 OLLm while applying light pressure, and was dried to form a tight and integrated structure.

On top of the nonwoven fabric, apply an ethanol solution of 20% by weight polyvinylpyrrolidone (average molecular weight 360,000) to a dry coating amount of 4g.
/m'' and dried to obtain a -type multilayer analytical element (Element A).

As a comparative example, a cellulose acetate membrane filter with a nominal pore diameter (average effective pore diameter or minimum pore diameter) of 1.2 gm, a thickness of about 140 μm, and a porosity of about 80% was used instead of the polyester nonwoven fabric, and the other aspects were the same as Element A. An integrated multilayer analysis element (Element B) was created.

Elements A and B include normal human whole blood (hemacrit value 42%, plasma glucose concentration 122 mg/mf;!,
) 20μβ was spotted. The element was incubated at 37° C. for 6 minutes, and the reflected absorbance within the element was immediately measured from the support side using visible light with center wavelengths of 540 nm and 640 nm.

This measurement was repeated 10 times using 10 elements each, and the average value and standard deviation (SD) were determined. The results are shown in Table 1.

The reflected absorbance at 540 nm, which corresponds to the absorption of surface red blood cell hemoglobin, was not observed at all in Element A using a nonwoven fabric, whereas Element B, a comparative example, showed a large value of o, 46. This indicates that in Element B, blood cell separation was insufficient or hemolysis occurred, whereas in Element A using a nonwoven fabric, red blood cells were completely removed and hemolysis hardly occurred.

In addition, the 640 nm reflection absorbance corresponding to the glucose value in plasma was as high as 1.118 for element A, and the variation was small (SD = 0.004). - For blade element B, 0
．． It was small at 762, and the variation was large (SD・0
．． 036). This is considered to be because in element B, hemolysis occurred within the analytical element, inhibiting the pigment production reaction.

Thus, element A of the present invention exhibits excellent blood cell separation properties and non-hemolytic properties. Furthermore, the measurement sensitivity itself was excellent, and the reproducibility was also excellent.

(Example 2 and Comparative Example 2) Analytical element C for quantifying total bilirubin was prepared by laminating a first water absorbing layer, a second water absorbing layer, a reagent layer, and a nonwoven fabric in this order on an elementary support.

First, a first water-absorbing layer (dry thickness approximately 15 μm) and a second water-absorbing layer (dry thickness About 10 μm) was coated from an aqueous solution in this order and dried.

Amount of 1st layer 1 m2 Polyvinyl alcohol (degree of saponification 88%, viscosity of 4% aqueous solution at 20°C 5 cps) 4 g Nonylphenoxy polyglycidol (contains average 1O glycidol units) 350 mg
2nd layer 1 m2 Polyvinyl alcohol (degree of saponification 99%, viscosity of 4% aqueous solution at 20°C 5 cps) 9.4 g Nonylphenoxy polyglycidol (contains average 1O glycidol units) 300 mg
A broad woven fabric made of 100-count double-thread cotton yarn, in which the second water-absorbing layer is uniformly moistened with water, and then treated to make it hydrophilic with an aqueous solution of nonylphenoxy polyglycidol (containing an average of 10 glycidol units) and dried. were uniformly laminated with light pressure and dried to form a porous reagent layer matrix.

Next, the first reagent composition coating solution was applied onto the textile fabric in the following coating amount, and the fabric was impregnated and dried.

Further, a second reagent composition coating solution was applied onto the textile fabric in the following coating amount, impregnated, and dried to form a light shielding layer on the upper edge of the textile fabric (porous reagent layer).

First reagent Amount of acid 1 m”?-(2,3-dihydroxypropyl)theophylline [
CA Registry No. 479-18-5]
25g sulfosalicylic acid
5g Hydroxypropyl methacrylate-N-(
α-sulfomethyl-α-methylethyl) acrylamide (6:41 copolymer 2.5g
2.4-Dichlorobenzenediazonium sulfosanotylate 320mg2:
Amount of acid 1m” Titanium dioxide fine particles 11g7-
(2,3-dihydroxypropyl)theophylline 0 g Nonylphenoxypolyglycidol (contains an average of 10 glycidol units) Ig On top of this woven fabric (porous reagent layer), fibers produced by spunbond method have an average fiber diameter of 3.7 μm and a density of 0 .05g
/cm”, a nylon non-woven fabric with a thickness of about 500 LLm was placed closely on the integrated multilayer analysis element for quantifying total bilirubin (
Element C) was obtained.

As a comparative example, a nylon nonwoven fabric with a nominal pore diameter of 1.
An integrated multilayer analysis element (Element D) was prepared in the same manner as Element C except that a cellulose acetate membrane filter having a filter width of 2 μm, a thickness of about 140 μm, and a porosity of about 80% was used.

Elements C and D include normal human whole blood (hematocrit value 42%, plasma median bilirubin concentration 0.98 mg/dβ
) 20μβ was spotted. The element was incubated at 37° C. for 6 minutes, and the reflected absorbance within the element was immediately measured from the support side using visible light with center wavelengths of 600 nm and 540 nm.

This measurement was repeated 10 times using 10 elements each, and the average value and standard deviation (SD) were determined.

The reflection absorbance at 600 nm, which corresponds to the amount of surface pigment, was as high as 0.617 (and the variation was small (S
For D=0.0021°-blade element angle, it was as small as 0.357, and the variation was large (SD 0.030). This is thought to be due to the fact that hemolysis occurs in the element and the pigment production reaction is inhibited.

The reflected absorbance at 540 nm is the sum of the reflected absorbance of both the produced pigment and the red blood cells. Element C had a large value of 1.018, compared to 0.888. As described above, although the dye formation i (absorbance at 600 nm) was lower than that of element C, the absorbance at 540 nm showed a high value. This indicates that blood cell separation is insufficient with elemental separation. On the other hand, Element C of the present invention exhibits excellent blood cell separation properties and non-hemolytic properties, and also has excellent measurement sensitivity and reproducibility.

(Example 3 and Comparative Example 3) A first reagent layer (coloring reagent layer), a second reagent layer (
Analytical element A was prepared by sequentially laminating a porous reagent layer) and a nonwoven fabric.

First, a coloring reagent layer coating solution was applied to the surface of a colorless transparent PET sheet (support) with a thickness of 180 μm in the following coating amount, and dried to form a coloring reagent layer (dry thickness of approximately 20 μm).
μm).

Color test amount of 1m2 [leuco dye oily organic solvent solution] 2~(4-hydroxy-3,5-dimethoxyphenyl)
-4-[4-(dimethylamino)phenyl 1-5-phenethylimidazole (leuco dye), acetate 15 mg Same as above leuco dye, hydrochloride 60 mgN,
N-diethyl laurylamide 7.2g [gelatin aqueous solution] Deionized gelatin 22g bis[(
220 mg of the above gelatin aqueous solution (gelatin content: 15.8% by weight) was added with the above leuco dye oily organic solvent solution while stirring it in an emulsifier, and about 3
Stirring was continued for 0 minutes to prepare a dispersion emulsion (coloring reagent composition aqueous dispersion), which was applied to a support and dried to form a coloring reagent layer6.Then, the surface of the coloring reagent layer was uniformly coated with water. Keep it moist with
On top of that, the nominal pore size (effective pore size or average value of the minimum pore size)
Cellulose acetate membrane filters of 1.21 tm, thickness of about 1404 tm, and porosity of about 80% were laminated while applying light pressure uniformly and dried to closely integrate the coloring reagent layer and the membrane filter.

Next, an aqueous dispersion of a cholesterol measuring reagent composition having the following composition was applied onto the membrane filter in the following coating amount, impregnated, and dried to form a second reagent layer (porous reagent layer).

Cholesterol measurement reagent Acid coverage: 1 m” Recholesterol esterase 7.7 kU Cholesterol oxidase 3.6 kU Lipoprotein lipase 5.3 kU Peroxidase 83 kU Potassium ferrocyanide 490 mg Condensate of nonylphenoxy polyethoxyethanol and formaldehyde
400mg Next, on the membrane filter of this laminate, average fiber diameter 2.6μm, density 0.03g/Cm
”, a polypropylene melt-blown nonwoven fabric with a thickness of 670 μm was subjected to glow discharge treatment (electrical energy: 1.
6 kw/m'', oxygen concentration 0.1 Torr, for 30 seconds) were partially adhered by gravure printing to prepare an integrated multilayer analytical element (element E) for quantifying total cholesterol.

For partial adhesion, hot melt adhesive Nittite H635 is used.
2-2A (manufactured by Nitta Gelatin ■: Styrene-book diene copolymer; softening point 76°C, viscosity at 140°C 4100c
PS ) was melted at 135°C and a gravure plate with the pattern shown in Figure 1 (a pattern with perfectly circular dots arranged at each vertex of a square; each dot had a depth of 50 μm and a diameter of 0.4 m) was prepared.
m, shallow U-shaped cross section, vertical and horizontal dot center spacing (pitch)
0.8 mm 2 ) onto a membrane filter, and the membrane filter of element 3 was immediately crimped and laminated onto a nonwoven fabric.

As a comparative example, an integrated multilayer analytical element (element F )It was created.

Elements E and F include normal human whole blood (hematocrit value 48%, plasma cholesterol concentration 151 mg/d).
12) 20u 12 was spotted. element is 37°C
Incubate for 6 minutes at
The reflected absorbance within the element was measured using visible light.

This measurement was repeated 10 times using 10 elements each, and the average value and standard deviation (SDI) were determined.

The reflected absorbance at 540 nm, which is absorption by superficial red blood cells, was as large as 0.49 in Element F, indicating that blood cell separation was insufficient. On the other hand, the reflected absorbance of Element E using a nonwoven fabric was at the Pack-Graland level, indicating that red blood cells were almost completely removed and hemolysis hardly occurred.

In addition, the 640 nm reflection absorbance corresponding to the total cholesterol value was as high as 1.00 for element E, and the variation was small (SD=0.007). - for blade element F, 0.3
The score was small at 5, and the variation was large (SD・0.02
81.

This is considered to be because element F was hemolyzed within the analytical element and the pigment production reaction was inhibited.

As described above, Element E of the present invention exhibits excellent blood cell separation properties and non-hemolytic properties, and also has excellent measurement sensitivity and reproducibility.

(Effects of the Invention) As described above, the integrated multilayer analysis element of the present invention is designed to remove and separate blood cells using a fine fiber nonwoven fabric.
Shows excellent blood cell separation and non-hemolytic properties. Therefore, the measured values do not vary and have excellent reproducibility. Furthermore, since a non-woven fabric with low brittleness is used, the analytical elements are easy to manufacture and process.

[Brief explanation of the drawing]

Figure 1 shows the pattern (square) of the gravure printing plate used to place the adhesive using the gravure printing method when partially bonding (porous bonding) fine fiber nonwoven fabrics in the integrated multilayer analytical elements of Examples 1 to 3. A pattern with perfectly circular dots arranged at each vertex; each dot has a depth of 50 μm and a diameter of 0.4 mm.
, shallow U-shaped cross section, vertical and horizontal dot center spacing (pitch)
0.8 mm).

Claims (2)

[Claims] (1) Average fiber diameter 0.2-10 μm, density 0.01-0
．． A dry analytical element for analyzing whole blood samples, which is made of a laminated layer of 2g/cm^3 nonwoven fabric and a detection function layer. (2) Average fiber diameter 0.2-10 μm, density 0.01-0
．． A dry analytical element for whole blood sample analysis, comprising a nonwoven fabric of 2 g/cm^3, a porous layer, and a detection function layer laminated in this order.