JPS61151460A - Integral type multi-layered analysis element for analysis of calcium or magnesium - Google Patents

Abstract

PURPOSE:To obtain an integral type multi-layered analysis element for analysis of calcium or magnesium wherein the fixing property of reagents in a reaction layer is high and highly accurate measurement is eabled, by containing a specific basic substance in the reaction layer. CONSTITUTION:The title integral type multi-layered analytical element for the analysis of calcium or magnesium has a reaction bed containing a basic polymer having a repeated unit represented by formula (1) or (2) [wherein R<1> is a hydrogen atom or a methyl group, A is a group selected from a group consisting of structural formulae (2-1)-(2-5), X is an oxygen atom or -NH-, Q is a monovalent heterocyclic group represented by structural formula (3-1), R<2> and R<3> are a hydrogen atom or an 1-4C (alkyl) group, (n) is 0, 1, 2, 3 or 4, (m) is 0 or 1 and Z is an anion]. This basic polymer has affinity to a chelate agent or a color former and has action for holding said reagent in the reaction layer even at the time of detection. Therefore, highly accurate analysis is enabled.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to an integrated multilayer analytical element for the analysis of calcium or magnesium in liquid samples. More specifically, the present invention relates to an integrated multilayer analytical element suitable for measuring calcium or magnesium concentration in biological fluids simply and with high accuracy.

[Background of the Invention] Calcium and magnesium are both elements deeply involved in physiological phenomena in living organisms. Therefore, measuring their concentrations in biological fluids is extremely important in clinical testing.

A number of methods have been developed for analyzing calcium and magnesium in liquid samples, but in the field of clinical testing, doctors and other medical professionals involved in diagnosis have been demanding that they require only a small amount of sample and are easy to operate. There is a strong demand for an analysis method with sufficient measurement accuracy. In response to this, dry analysis methods that are easy to handle and operate are being used in place of the conventionally used wet analysis methods. As a dry analysis method, an analysis method using an integrated multilayer analysis element (hereinafter abbreviated as 1 analysis element), which is easier to operate and has sufficient accuracy, has been developed, and further improvements are being made. The analysis element is a sheet-like analysis tool in which a support, a functional layer such as a reaction layer containing a detection reagent, and a development layer are laminated.

When analyzing calcium or magnesium in a sample solution using an analytical element, there is a method that uses a reagent that forms a chelate with these ions and measures the color development of the chelate or the color change of the indicator caused by the formation of the chelate. Generally used.

The analytical elements using the above method include JP-A-54-2
Analytical elements that can be used for calcium analysis as described in Japanese Patent No. 9700 have already been proposed. however,
Several problems exist when using analytical elements to analyze calcium or magnesium in liquid samples.

First, the calcium or magnesium analyte and reagents such as chelating agents and coloring agents used in the analytical element are low molecular weight substances, so they settle after encountering moisture in the liquid sample. It has poor properties and causes diffusion of analytes and reagents into the developing layer.
There is a problem in that as the above-mentioned substances involved in measurement decrease, their abundance becomes unstable, resulting in a significant decrease in detection accuracy during measurement. Furthermore, the above reagents are generally known to have a detrimental effect on the layer formation of the analytical element, and it is necessary to prevent these reagents from diffusing into the developing layer.

The analytical element described in the above-mentioned Japanese Patent Application Laid-Open No. 54-29700 attempts to solve this problem by providing a radiation-transparent dye migration blocking layer between the developing layer and the reaction layer. . However, adding a new layer between the deployment layer and the reaction layer.

This is unavoidable and adversely affects the mobility of the sample to the reaction layer and the light transmittance during detection, which is undesirable from the viewpoint of handling operations and detection accuracy.

Second, commonly used chelating agents have low specificity for ions, particularly for calcium ions. In biological fluid analysis, calcium atoms and their ions and magnesium atoms and their ions are mutually important inhibitors of detection reactions. In order to compensate for the low specificity of chelating agents, methods such as using a masking agent for ions that act as inhibitors or adjusting the reaction conditions, especially the pH, to be most specific for each ion are available. be. For example, to improve the specificity of the chelating agent for calcium ions, magnesium! [Means such as adding 8-hydroxyquinoline or a derivative thereof as a masking agent and adding a basic substance to make the pH most specific to calcium ions (alkaline) are generally used.

However, it is generally difficult to add a basic substance to the reaction layer within the analytical element. That is, it has been pointed out that generally containing an alkali in a reaction layer formed from a polymer binder deteriorates the properties of the binder and also has an adverse effect on other layers.

[Summary of the Invention] The present inventor has attempted to solve the above-mentioned problems in an integrated multilayer analytical element for analyzing calcium or magnesium in a liquid sample, and has arrived at the present invention.

The first object of the present invention is to
An object of the present invention is to provide an integrated multilayer analytical element for calcium or magnesium analysis, which has high fixation properties for reagents and enables highly accurate measurement.

A second object of the present invention is to provide an integrated multilayer analytical element for calcium or magnesium analysis, which contains a basic substance in the reaction layer that does not adversely affect the layer structure.

The present invention provides an integrated multilayer analytical element comprising a liquid (water) impermeable, light-transparent support, a reaction layer in which at least one detection reagent is dispersed in a polymer binder, and a porous spreading layer. , the reaction layer has the following formula (1)
The present invention provides an integrated multilayer analytical element for calcium or magnesium analysis, which is characterized by containing a basic polymer having a repeating unit represented by (2).

-(CHt CI(t N H to (1) 10 spaces below CH2G-→--- co (2) [In formula (2), R1 is a hydrogen atom or a methyl group; A is the following formula ( 2-1) to formula (2-5)] -X-+CH2>]-Q (2-3) [Formula (
In the structural formulas from 2-1) to formula (2-5), X is an oxygen atom or -NH-; is a heterocyclic group; R2 and R3 are a hydrogen atom or an alkyl group having 1 to 4 carbon atoms is a group: n is 0.1,2
．． 3 or 4; m is 0 or 1; Z is an anion] [Effects of the Invention] The analytical element of the present invention is characterized in that the reaction layer contains a basic polymer. The above-mentioned basic polymer not only has a high fixing property by itself, but also has an affinity with chelating agents and coloring agents, and has the effect of retaining these reagents in the reaction layer even during detection. .

Therefore, in the analytical element of the present invention, the detection reagent does not flow out of the developing layer, and highly accurate analysis is possible. In addition, deterioration of the layer structure due to outflow of the detection reagent into the developing layer is less likely to occur.

Furthermore, since the basic polymer itself has little adverse effect on the layer structure, it can be added for the purpose of adjusting the pH to 11L. Therefore, the analytical element of the present invention has high specificity for metal atoms or ions to be analyzed, particularly for calcium, and exhibits excellent analytical precision.

[Detailed Description of the Invention] Specific examples of the liquid (water)-impermeable and light-transparent support (hereinafter referred to as support) constituting the integrated multilayer analytical element of the present invention include polyethylene terephthalate, bisphenol, etc. Made of a polymer such as A polycarbonate, polystyrene, cellulose ester (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) and has a thickness of about 501 Lm to about im.
, preferably in the range of about 80 Bm to about 3007 zm, transparent and substantially water-impermeable supports.

If necessary, an undercoat layer is provided on the surface of the support to strengthen the adhesion between the reaction layer provided on the support or other layers provided as necessary (e.g., water absorption layer) and the support. can do. Further, instead of the undercoat layer, the surface of the support may be subjected to a physical or chemical activation treatment to improve the adhesive strength.

A reactive layer is provided on the support (possibly via another layer such as a subbing layer). This layer is dispersed in a binder. The polymer binder is preferably a hydrophilic polymer that absorbs water and swells.

Hydrophilic polymers that can be used to manufacture the reaction layer include:
Generally, it is a natural or synthetic hydrophilic polymer having a swelling rate upon absorption of water in the range of about 150% to about 2000%, preferably about 250% to about 1500% at 30°C. An example of such a wood-loving polymer is JP-A-59-17186.
Gelatin (e.g., acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (e.g., phthalated gelatin, hydroxyacrylate grafted gelatin, etc.), agarose, pullulan, etc. disclosed in Publication No. 4, Japanese Patent Application No. 58-217428, etc. , pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and the like.

Preferably, the dry thickness of the reaction layer ranges from about IILm to about 100 lm, more preferably from about 3 Bm to about 30 pm. It is also preferred that the reaction layer is substantially transparent.

The analytical element of the present invention is characterized in that the reaction layer contains a basic polymer.The basic polymer used in the present invention is:
These are basic mordants and the like that are generally used in photographic materials. A specific example of the above basic polymer is the formula (
T) to a basic polymer having repeating units represented by formulas (VI).

1+CH2CH2N H to (I) 10H2-C-
(II) [In formula (■), R1 is a hydrogen atom or a methyl group; R2 and R3 are a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; X is an oxygen atom or -NH- : n is 0, 1.2.3 or 4] Among the repeating units represented by the above formula (n).

Amides (X is -NH-) are disclosed in JP-A-48-5
This is described in Japanese Patent No. 5723.

Specific examples of compounds that can be used as the repeating unit represented by the above formula (n) include N,N-(dimethylamino)ethyl acrylate, N,N-(dimethylamino)ethyl methacrylate, N,N-( diethylamino)ethyl acrylate.

Examples include N,N-('(dimethylamino)propyl]acrylamide, N,N-((diethylamino)propyl)acrylamide, and N,N-((dimethylamino)ethylcoacrylamide).

I [In formula (III), R1 is a hydrogen atom or a methyl group; X is an oxygen atom or -NH-; n is o, l, 2.3 or 4] 1i1 〒0 X(CH2) nQ [In formula (ff).

Hz is a hydrogen atom or a methyl group; X is an oxygen atom or -NH-; n is 0, l, 2.3 or 4; is a heterocyclic group; R2 is a hydrogen atom or the number of carbon atoms 1-4 alkyl group] 10H2-C-←-(V) ■ [In formula (V), R1 is a hydrogen atom or a methyl group: R2 is a hydrogen atom or a C1-4 alkyl group is an alkyl group] Specific examples of compounds that can be used as the repeating unit represented by the above formula (V) include l-acryloyl-
4-Methylbiperazine, l-methacryloyl-4-methylpiperazine, 1-acryloyl-4-ethylpiperazine, l-methacryloyl-4-ethylpiperazine, l-
Acryloyl-4-propylpiperazine.

Examples include 1-methacryloyl-4-propylpiperazine, 1-acryloyl-4-butylpiperazine, and -methacryloyl-4-butylpiperazine.

→OH, -C-To (Vl) View O [In formula (VI), R1 is a hydrogen atom or a methyl group; X is an oxygen atom or -NH-; Z is an anion: m is 0 or Among the repeating units represented by the above formula (VI), those in which amides (X is -NH-) are disclosed in JP-A-49-3263.
This is described in Publication No. 5.

The polymer containing repeating units represented by any of the above formulas (I) to (Vl) may be a homopolymer or a copolymer, and as a copolymer, , may be a copolymer consisting only of repeating units represented by any of the formulas (I) to (71) above, or may be a copolymer with other hexamers. When producing a copolymer with other monomers, it is preferable to contain at least 30 mol% of the above repeating units.Other monomers used in the copolymer include acrylamide, methacrylamide, and diacetone. Acrylamide, tert-butylacrylamide, N,N-dimethylacrylamide,

N-methylolacrylamide, acrylic acid (methacrylic acid) esters, styrene, vinyl acetate, vinyl alcohol, N-vinylpyronidone,
Examples include allylamine, glycidyl acrylate (methacrylate), and the like.

The basic polymer used in the present invention is the polymer synthesis method I.
J (edited by C, G. Oververger, translated by Hiroshi Minato, published by Tokyo Kagaku Dojin, 1968) or "Experimental Methods of R-Polymer Synthesis" (co-authored by Takayuki Otsu and Masayoshi Kinoshita, published by Tokyo Kagaku Dojin, 1972). It can be easily synthesized and manufactured.

Regarding the molecular weight range of the basic polymer, consider that if the molecular weight is too low, the polymer itself may diffuse, while if the molecular weight is too high, the compatibility with the wood-philic colloid will be poor. Determine depending on the type of coalescence. In general, the intrinsic viscosity measured at 30°C using 1% saline is 0.1 to 5.
It is preferable to use a basic polymer having a value of about 0, preferably about 0.3 to 2.5.

In addition, regarding the range of the content of the basic polymer in the reaction layer, if the content is too low, the effect of the present invention may be insufficient, while if it is too large, the coating solution forming the reaction layer may have poor wood affinity. It is determined by taking into consideration that the compatibility with the polymer will be poor. It generally ranges from about 1% to about 20%, preferably from about 2% to about 15%, by dry weight, based on the polymeric binder constituting the reaction layer.

The detection reagent contained in the reaction layer of the present invention forms a chelate with calcium ions or magnesium ions, and the chelate itself or the color forming agent develops a detectable color or changes color along with the formation of the chelate.

Specific examples of calcium or magnesium detection reagents include 2.2'-bibenzoxazoline, 1-(1-hydroxy-4-methyl-2-phenylazo)-2-naphthol-4-sulfonic acid, 2-(4- sulfophenylazo)-1,8-dihydroxy-3,6-naphthalenedisulfonic acid trisodium salt, 1-(1-hydroxy-4-methyl-2-phenylazo)-2-naphthol-4-sulfonic acid, 1- (2-Hydroxy-4-sulfo-1-naphthylazo)-2-naphthol-3,6-disulfonic acid trisodium salt.

? -Hydroxy-1-(2-hydroxy-4-sulfo-
1-naphthylazo)-3-naphthoic acid, cyclo-tris(7-(1-azo-8-hydroxynaphthalene-3,6
-disulfonic acid)] 6 sodium salt, purpuric acid 0 ammonium salt, 3.3'-bis[N,N-di(carboxymethyl)aminomethyl J fluorescein, 3.3'-bis[N,N-di( carboxymethyl)aminomethyl]phenolphthalein, 3,3'-bis[N
, N-di(carboxymethyl)aminemethyl]-〇-cresolphthalein, 3.3°-bis[N,N-di(carboxymethyl]aminomethyl]thymolphthalein, 3.3'-bis[N,
N-di(carboxymethyl)aminomethyl]-〇-cresolphthalein disodium salt 3.3'-bis[N,N-di(carboxymethyl)aminomethyl]-p-xylenolsulfonephthalein 4
Sodium salt, 3.3°-bis[N,N-di(carboxymethyl)aminoethyl]thymolphthalein sodium salt.

Examples include o-[2-[α-(2-hydroxy-5-sulfophenylazo)benzylidene]hydrazino]benzoic acid sodium salt, and 4-methylumbelliferone-8-methyleneiminodiacetic acid.

Further, as a specific example of the magnesium detection reagent, l-azo-2-hydroxy-3-(2,4-dimethylcarboxy7mido)naphthalene-1°-(2-hydroxybenzene-5-sulfonic acid) sodium salt etc.

When using the analytical element of the present invention for calcium analysis,
If necessary, a magnesium masking agent can be included in the reaction layer. Specific examples of magnesium masking agents include 8-hydroxyquinoline and its derivatives (e.g., 8-hydroxyquinoline and its derivatives).
hydroxyquinoline-5-sulfonic acid) and the like.

Furthermore, when the analytical element of the present invention is used for magnesium analysis, a calcium masking agent can be contained in the reaction layer, if necessary. Specific examples of calcium ion agents include glycol ether diamine-N, N, N',
Examples include No-tetraacetic acid.

Further, it is preferable that the reaction layer contains a surfactant, which is effective for controlling the permeation rate of a liquid sample. As a surfactant.

Although both ionic (anionic or cationic) surfactants and nonionic surfactants can be used, it is preferable to use nonionic surfactants. Examples include polyalkylene glycol derivatives of alkyl-substituted phenols such as p-nonylphenoxyglycerin, polyoxyethylene nonylphenyl ether, and polyethoxyethanol, and polyalkylene glycol esters of higher fatty acids.

The reaction layer is prepared by applying an aqueous dispersion of a basic polymer, a detection reagent, and a wood-philic polymer (containing reagents such as a surfactant as necessary) onto a support layer or an adhesive layer by a known method. It can be provided by coating and drying.

A light shielding layer can be provided on the reaction layer, if necessary. The light shielding layer is composed of fine particles or fine powder (hereinafter simply referred to as fine particles) having light shielding properties or both light shielding properties and light reflecting properties, dispersed and retained in a small amount of a wood-philic polymer binder having film-forming ability. A water-permeable or water-permeable layer. The light shielding layer measures the color of the aqueous liquid dotted onto the developing layer, which will be described later, when detectable changes (color change, color development, etc.) occurring in the reaction layer are reflected photometrically from the light-transmitting support side. In particular, when the sample is whole blood, it blocks the red color of hemoglobin, etc., and also functions as a light reflecting layer or a background layer.

Examples of fine particles that have both light-shielding and light-reflecting properties include titanium dioxide fine particles (rutile type, 7-natase type, or pulchite type with a particle diameter of about 0.1 gm to about 1.27 tm).
microcrystalline particles, etc.), barium sulfate microparticles, aluminum microparticles, or microflakes, and examples of light-shielding microparticles include carbon blur, silica, gas black, carbon microbeads, and the like. Among these, titanium dioxide fine particles and barium sulfate fine particles are particularly preferred.

Examples of hydrophilic polymer binders with film-forming ability include, in addition to the hydrophilic polymers that can be used for producing the reaction layer, weakly hydrophilic regenerated cellulose and cellulose acetate. Among them, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. Gelatin and gelatin derivatives can be used in combination with known hardening agents (crosslinking agents).

It is preferable that the light shielding layer contains a surfactant. As the surfactant contained in the light shielding layer, the same surfactant as that used in the reaction layer can be used.

The light-shielding layer is formed by applying an aqueous dispersion of light-shielding fine particles and a wood-loving polymer (containing reagents such as a surfactant as necessary) onto the reaction layer by a known method and drying it. can be provided. Further, instead of providing a light shielding layer, components of the light shielding layer may be contained in the porous development layer described later.

Note that an adhesive layer may be provided on the reaction layer, optionally via a layer such as a light shielding layer, in order to adhere and laminate a porous development layer, which will be described later.

11 when the adhesive layer is wet with water or contains water.
The porous spreading layer can be bonded when wet,
Examples of wood-philic polymers that can be used in the production of the adhesive layer, which preferably consist of hydrophilic polymers that allow the layers to be integrated, include hydrophilic polymers similar to those used in the production of the reaction layer. Examples include wood polymers. Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. The dry film thickness of the adhesive layer is generally about 0.5 lm to about 20 uLm, preferably about IB
m to about 10XLm.

In addition to the adhesive layer on the reaction layer or the light shielding layer,
The adhesive layer may be provided on a desired layer to improve the adhesion of other living areas.The adhesive layer is prepared by applying an aqueous solution containing a wood-philic polymer and a surfactant added as necessary to the reaction layer, etc. It can be provided by coating on top.

A porous spreading layer (hereinafter referred to as the spreading layer) constituting the integrated multilayer analytical element of the present invention is provided on these layers.The spreading layer of the present invention has a liquid sample measuring function. A spreading layer having a liquid sample metering function, which is preferable, means that the aqueous liquid sample that is spotted on its upper surface (the surface far from the support) is substantially free of the components contained therein. This layer has the function of spreading in the lateral direction and supplying a substantially constant capacity per unit area to the reaction layer without uneven distribution.

From the above points, materials constituting the matrix of the spreading layer of the present invention include paper, nonwoven fabric, woven fabric, knitted fabric, glass fiber! It is preferable to use filter paper, a membrane filter made of brushed polymer, or a three-dimensional lattice structure layer made of polymer microbeads.

The matrix of the developing layer is selected from these materials depending on the analysis conditions, etc., but when using a sample containing non-soluble substances (blood cells in the case of whole blood) that inhibit analysis, such as whole blood, it is necessary to It is preferable to use a woven or knitted fabric that has the ability to remove .

Examples of woven fabrics (woven fabrics) that can be used for the above-mentioned spreading layer include JP-A-55-164356 and JP-A-57-6.
No. 6,359 discloses a wide variety of woven fabrics. Among woven fabrics, plain woven fabrics woven with warp (warp) yarns and weft (weft) yarns are preferable, and among plain woven fabrics, thin fabrics, kanayama fabrics, broad fabrics, boblin fabrics, etc. are preferable. Examples of threads that can be used include threads made of the same material as the threads that make up the knitted fabric described later, and the forms of the threads include filament threads and spun threads (twisted threads).
Any of these can be used, and among these, spun yarn is preferred; the thickness of the yarn of the woven fabric is in the range equivalent to about 205 to about 1505, preferably about 405 to about 120S, expressed in terms of cotton spun yarn count, or Approximately 3 in silk denier
5D to about 300D, preferably about 45D to about 130D
0 equivalent range, the thickness of the woven fabric is from about 1100 JL to about 5007 zm, preferably from about 1207 tm to about 350 tm.
Bm ranges, the porosity of the woven fabric ranges from about 40% to about 90%, preferably from about 50% to about 85%.

In addition, there are a wide variety of knitted fabrics (knitted fabrics, that is, knitted cloth-like materials) that can be used for the above-mentioned development layer, and among them, warp (warp) knitted fabrics and horizontal (weft) knitted fabrics are available. Stockinette is preferred. Warp knitted fabrics include heavy atlas knitted fabrics, tricot knitted fabrics, double tricot knitted fabrics, Milanese knitted fabrics, Raschel knitted fabrics, etc.; as weft knitted fabrics, flat knitted fabrics, pearl knitted fabrics, etc. can be used. Fabric, rubber knitted fabric, double-sided knitted fabric, etc. can be used. Threads for knitting 1A fabrics include natural fiber threads such as cotton, silk, and wool, and regenerated cellulose such as viscose rayon and cupra.

Semi-synthetic organic polymers such as cellulose diacerate and cellulose triacetate, polyamides (various nylons), acetalized polyvinyl alcohol (vinylon, etc.)
Yarns made of fine fibers or single fibers of synthetic organic polymers such as polyacrylonitrile, polyethylene terephthalate, polyethylene, polypropylene, polyurethane, etc. Yarns made of mixed fibers of natural fibers and regenerated cellulose, semi-synthetic or synthetic organic polymer fibers can be given.

Thread types include filament yarn, spun yarn (twisted yarn)
Any of these can be used, and among these, spun yarn is preferred.The thickness of the yarn of the knitted fabric is from about 4O3 to about 15O5, preferably about 60S, expressed in terms of cotton spun yarn count.
to about 12O3, or from about 350 to about 1300, preferably from about 450 to about 900, expressed in silk thread denier.The gauge number during the knitting process of the knitted fabric is in the range of about 20 to about 50. The thickness of the knitted fabric is about 11001L to about 6001Lm, preferably about 1
50 gm to about 400 gm, the knitted fabric has a porosity of about 40% to about 90%, preferably about 50% to about 85%. Among warp knitted fabrics, tricot knit fabrics, raschel knit fabrics, and milanese knit fabrics are selected from the viewpoints of less expansion and contraction in the longitudinal direction, ease of operation in the lamination process of the knitted fabric layer, and the ability to keep the stitches intact during cutting. , double tricot knitted fabric is preferred.

The woven or knitted fabric used for the porous development layer is a woven or knitted fabric that has been subjected to degreasing treatment such as washing with water to substantially remove at least the oils and fats supplied or attached during yarn production, fabric production, or knitting. Furthermore, the woven or knitted fabric is subjected to at least one side of the fabric a physical activation treatment (preferably glow discharge treatment or corona discharge treatment, etc.) as disclosed in Japanese Patent Publication No. 57-66359, or No. 164356, JP-A-57-
The woven fabric or knitted fabric is made hydrophilic by carrying out a wood-loving treatment such as a hydrophilic polymer impregnation treatment disclosed in Japanese Patent Publication No. 66359, or by performing step 1 of these treatments sequentially, and the lower layer (the side closer to the support) is made hydrophilic. can strengthen the adhesive strength of

In order to adhere and laminate a spreading layer made of a woven or knitted fabric to a reaction layer or an adhesive layer, it can be prepared according to the method disclosed in JP-A-55-164356 and JP-A-57-66359. . That is, water (or water containing a small amount of surfactant) is supplied substantially uniformly to the reactive or adhesive layer while it is still wet after application, or to the dried layer to swell the layer, and then the fabric or adhesive layer is applied. The knitted fabric is adhered, laminated, and integrated onto the wet or wet layer substantially uniformly while applying light pressure.

In addition, when the spreading layer is made of a plush polymer or a membrane filter, Japanese Patent Publication No. 53-21677, etc., and when it is a three-dimensional lattice structure layer made of polymer microbeads, Japanese Patent Application Laid-Open No. 55-90859, etc. , when it is made of filter paper or non-woven fabric, Japanese Patent Application Laid-Open No. 57-1482
They can be provided according to the methods described in Japanese Patent No. 50 and the like.

When the wood-philic polymer/coating of the reaction layer or adhesive layer is gelatin or a gelatin derivative, the material constituting the porous spreading layer ( A method of bonding, laminating, and integrating fabrics (woven fabrics, knitted fabrics, etc.) can be adopted.

Further, the spreading layer in the present invention can contain reagents such as light-shielding fine particles and surfactants, if necessary.

As the surfactant contained in the spreading layer, the same surfactant as that used in the reaction layer can be used.

As described above, the integrated multilayer analytical element of the present invention consists of 81 layers of support layer, reaction layer, and development layer, and these support layer, reaction layer, and development layer are laminated in this order. Preferably.

Examples and comparative examples of the present invention are shown below.

The basic polymers used in the Examples and Comparative Examples were synthesized according to the following Synthesis Examples.

[Synthesis example of basic polymer] A solution for basic polymer synthesis having the following composition was placed in a 5JL reaction vessel equipped with a stirring device, and while blowing nitrogen gas, 1.0 g of potassium beloxonisulfate was added to 20 m of distilled water.
1 was added.

Basic polymer synthesis solution composition N,N-dimethylaminopropylacrylamide 150g Distilled water 600mJl Isopropyl alcohol 150mJl Sodium hydroxide aqueous solution (0.5N) IO, 6mJL Keep the temperature of the above solution at 35-50°C and stir for 3 hours. After the reaction was completed, 4 drops of acetonitrile were added, and the temperature was lowered to 15% while stirring.
It was lowered to ℃. The supernatant was removed, 300 ml of distilled water was added to redissolve the precipitate, and 4 drops of acetonitrile were added again to lower the temperature to -5°C to precipitate poly-N,N-dimethylacrylamide. When 200 mJL of distilled water was added and stirred, 756 g of a homogeneous liquid (solution) was obtained.

In order to calculate the amount of polymer produced, 2 g of the above solution was taken, dried with hot air at 100 ° C. for 3 hours, and the weight of the polymer was measured. The result was 362 mg, and the polymer content in the solution was:
It was 18.1%. Further, the dried polymer was dissolved in 1% saline and the solution viscosity was measured to determine the intrinsic viscosity, which was found to be 0.5 at 30°C. Furthermore, the pH of the solution produced
As a result of the measurement, a value of 10.5 was obtained.

[Example 1] A reaction layer (dry film thickness: 12 layer m) and a light shielding layer (dry film thickness: 4 m) were placed on a gelatin-subbed colorless transparent polyethylene terephthalate (PET) film (thickness 180 pm).
(m) was sequentially coated and dried. Furthermore, the surface coated with the above two layers was coated with p-nonylphenoxyglycerin 0.
Polyester and cotton blend fabric (100 count plain woven fabric; blend ratio, polyethylene terephthalate/cotton = 75/2
5) was pressed and dried to form a spreading layer to create an integrated multilayer analytical element for calcium measurement.6 The reaction layer and light shielding layer were prepared as shown below.

Reaction layer: Purified water 145m Vertical decalcified gelatin 20g Poly-N,N-dimethylaminopropylacrylamide (see synthesis example) (5% aqueous solution) 35g3.3'-bis[N,N-di(carboxymethyl)aminomethyl] -o- Cresolphthalein (2% aqueous solution) 45 g 8-hydroxyquinoline-5-sulfonic acid (5% aqueous solution) 72 g p-nonylphenoxyglycerin (5% aqueous solution) 4 g A mixed solution with the above composition was heated to dissolve gelatin, A coating solution adjusted to pH 10.5 with an aqueous sodium hydroxide solution was applied onto the PET film and dried.

Light shielding layer: Titanium oxide 6,2 g/m'Decalcified gelatin 1,2 g/rrlp-nonylphenoxyglycerin 50mg/rrf'Titanium oxide fine powder was dispersed in an aqueous P-nonylphenoxyglycerin solution, and then added to a decalcified gelatin solution. The coating solution prepared by dispersion was coated onto the reaction layer to the above coating amount and dried.

[Comparative Example] Comparison was carried out in the same manner as in Example 1 except that a reaction layer was provided using 35.6 g of triethanolamine (5% aqueous solution) instead of the poly N, N-dimethylaminopropylacrylamide of Example 1. I created an analysis element for

The following experiments were conducted on the analytical element of the present invention (Example 1) and the analytical element for comparison (Comparative Example).

Calcium concentration is 0.4.8.1O112.16mg
/ d l, the magnesium concentration is 2 mg/d! A control serum consisting of 7% by weight physiological saline containing human serum albumin L was prepared, and each of them was placed on the spreading layer of the analytical element of the present invention and the analytical element for comparison.
og was added vertically and incubated at 37° C. for 6 minutes, and then the reflected optical density was measured using 570 nm light. The measurement results are shown in Table 1.

Margin Table 1 below (mg/dll) Example 1 Comparative Example 0
0.40 0.48 4 0.52 0.57 8 0.65 0.73 10 0.72 0.6912
0.80 0.761 & 0.95
0.89 As is clear from the above results, a sufficient correlation was not observed between the comparative analytical element and the calcium concentration in the control serum. In contrast, one analytical element of the present invention was found to have a very good correlation with calcium concentration.

[Example 2] A reaction layer (dry film thickness: 14 Bm) and a light-shielding M layer (dry film thickness +
4pm) was sequentially applied and dried. Furthermore, the surface coated with the above two layers was coated with p-nonylphenoxyglycerin 0
．． A blended fabric of polyester and cotton (80 count plain woven fabric; blending ratio, polyethylene terephthalate/cotton = 75/2) that was wetted with a 2% aqueous solution and then treated to be hydrophilic by glow discharge.
5) was pressed and dried to form a spread layer, thereby creating an integrated multilayer analytical element for measuring magnesium. The methods for preparing the reaction layer and the light shielding layer are shown below.

Reaction M: Purified water 130mM decalcified gelatin 20g poly-N,N-dimethylamitube apyracryl 7mide (see synthesis side) (5% aqueous solution) 35g methylthymol blue (3% alkaline aqueous solution) 60g glycol-ether diamine- N,N,N',N'-tetraacetic acid (5% aqueous solution) 4gp-nonylphenoxyglycerin (5% aqueous solution) 4g A mixed solution of the above composition was heated to dissolve gelatin, and the solution was adjusted to pH 10.5 with an aqueous sodium hydroxide solution. A coating solution adjusted to the following was applied onto a PET film and dried.

xj Port 1 layer: Titanium oxide 6.2g/m''Decalcified gelatin 1.2g/m'p-nonylphenoxyglycerin 50mg/disperse fine titanium oxide powder in p-nonylphenoxyglycerin aqueous solution, and then dissolve it in decalcified gelatin aqueous solution. The coating solution prepared by dispersion was coated onto the reaction layer to the above coating amount and dried.

For the above analysis element of the present invention, magnesium concentration is 0.2.
．． At each concentration of 4.8 mg/di, a control serum consisting of human serum albumin Zti amount % physiological saline containing a calcium concentration of 10 mg/dl was prepared, and each of them was placed on the spreading layer of the above analytical element. After 10 drops and incubation at 37°C for 6 minutes,
Reflection optical density was measured using 600 nm light. Second result
Shown in the table.

Table 2 Mg concentration Reflective optical density (mg/d)
(600nm)0
0.62 2 0.67 4 0
．． 748 0.83 As is clear from the above results, it was observed that the analytical element of the present invention had a very good correlation with the magnesium concentration. In addition, the coloring of the developed layer to which human serum was dropped was not visible, and it was confirmed that the coloring agent did not migrate between the layers.

Patent Applicant Fuji Photo Film Co., Ltd. Agent Patent Attorney Yasushi Yanagawa Procedural Amendment March 20, 1985 Patent Application No. 278297 2, Title of Invention: Integrated multilayer analytical element for calcium or magnesium analysis 3. Relationship with the person making the amendment Patent applicant name (520) Fuji Photo Film Co., Ltd. 4 Agent address 8 Mitsuya Yotsuya Building, 2-14 Yotsuya, Shinjuku-ku, Tokyo
Floor 6: Number of inventions increased by amendment None 7: Subject of amendment: Column 8 of “Detailed Description of the Invention” in the description, Contents of amendment We will correct it as follows.

Ichichoichi (1) rN,N-(dimethylamino)ethyl acrylate 1 on page 17, line 7 to line 8 of the same page is corrected to r(dimethylamino)ethyl acrylate 1.

(2) Compensate rN,N-(dimethylamino)ethyl methacrylate J on page 17, line 9 to line 10 of the same page as r(dimethylamino)ethyl methacrylate.

(3) rN, N-( from page 17, line 11 to page 12, line 12)
Diethylamino)ethyl acrylate-N is corrected to r(diethylamino)ethyl acrylate 1.

(4) rN, N-( on page 17, line 13 to line 14 on the same page
(dimethylamino)propylcoacrylamide j rN
-[Correct with (dimethylamino)propylcoacrylamide 1.

(5) rN, N-[ on page 17, line 15 to line 16 on the same page
(diethylamino)propylcoacrylamide” rN
-[Correct with (diethylamino)propylcoacrylamide 1.

(6) rN, N-[ from page 17, line 17 to page 18, line 18
(Dimethylamide (7) Structural formula (VI) on page 20 of the specification
) is corrected as follows.

Before correction: I Correct with cresol sulfone phthalein 1.

(9) r Thymolphthalein J on page 25, line 4
Correct with thymolsulvonphthalein 1.

Correct with tase type 1.

(11) Page 33, line 3, cellulose diacerate 1”
Correct with r cellulose diacetate 1.

Claims (1)

[Claims] 1. An integrated multilayer analytical element formed by laminating a liquid-impermeable/light-transparent support, a reaction layer in which at least one detection reagent is dispersed in a polymer binder, and a porous development layer. , the reaction layer has the following formula (1) or (2)
An integrated multilayer analytical element for calcium or magnesium analysis, characterized by containing a basic polymer having repeating units represented by: -(CH_2CH_2NH)-(1) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(2) [In formula (2), R^1 is a hydrogen atom or a methyl group; A is the following formula (2-1 ) to formula (2-5)] ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (2-1) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (2- 2) -X-(CH_2)-_nQ(2-3) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(2-4) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(2-5) [Formula (2- In the structural formulas from 1) to formula (2-5), X is an oxygen atom or -NH-;
It is a monovalent heterocyclic group selected from the group represented by ▼ and ▲ where there are mathematical formulas, chemical formulas, tables, etc.; R^2 and R^3 are hydrogen atoms or carbon atoms 1 to 4
is an alkyl group; n is 0, 1, 2, 3 or 4; m is 0 or 1; Z is an anion] 2. The above basic polymer is a repeating group represented by the following formula: The analytical element according to claim 1, characterized in that it contains at least 30 mol% of the unit. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the above formula, R^1 is a hydrogen atom or a methyl group; R^2 and R^3 are a hydrogen atom or a carbon atom number of 1 to 4
X is an oxygen atom or -NH-; n is 0, 1, 2, 3 or 4] 3. The basic polymer is poly-N,N-(dimethylamino)ethyl Acrylate, Li-N,N-(dimethylamino)ethyl methacrylate, Poly-N,N-(diethylamino)ethyl acrylate, Poly-N,N-[(dimethylamino)propyl]acrylamide, Poly-N,N
The analytical element according to claim 2, which is a polyamine selected from the group consisting of -[(diethylamino)propyl]acrylamide and poly-N,N-[(dimethylamino)ethyl]acrylamide. 4. Claim 1, wherein the basic polymer is a polymer having an intrinsic viscosity in the range of 0.1 to 5.0 as measured at 30°C using 1% saline. Analysis elements described. 5. The analytical element according to claim 1, wherein the dry weight ratio of the basic polymer and the polymer binder in the reaction layer is in the range of 1:4 to 1:100. 6. The analytical element according to claim 1, which is used for calcium analysis, and wherein the reaction layer contains a magnesium masking agent.