JPS62297A - Multi-layer analytical element - Google Patents

Abstract

PURPOSE:In measuring activity of a hydrolase in a liquid specimen, to obtain an analytical element having high precision and improved shelf stability, by laminating both a substrate layer containing a pH color changing agent and a coloring layer containing a pH buffering agent to a light transmitting and water impermeable support. CONSTITUTION:A coloring layer and a substrate layer are laminated to a light transmitting and water impermeable support consisting of a polymer such as polyethylene terephthalate, cellulose ester, etc. The coloring layer is a layer wherein an undiffusing high polymer substance which can be set to pH different by >=1.5 based on pH unit from the substrate layer is dispersed into a polymer binder and gelatin agarose, etc., is used as the polymer. In the coloring layer, a substrate in the substrate layer is hydrolyzed by the action of ANARAITE in a specimen, the decomposition product is permeated into the coloring layer and change in color is produced by pH of the layer. The substrate layer contains a material obtained by bonding a colorless pH color changing agent to color by pH change to a nondiffusing substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to an integrated multilayer analytical element for measuring the activity of hydrolytic enzymes in liquid samples. More specifically, the present invention relates to hydrolytic enzymes such as proteases contained in biological fluids such as blood, urine, saliva, pancreatic juice, intestinal fluid, and cerebrospinal fluid.
The present invention relates to an integrated multilayer analytical element suitable for easily and highly accurately measuring the activities of amylase, lipase, pectinase, etc.

[Background of the Invention] A wide variety of methods have been proposed for analyzing components in biological fluids. Among them, the method of proceeding in a sample solution to produce a colored substance proportional to the amount of the analyte to be analyzed and measuring the color concentration is a well-known analytical principle. It is used not only as a solution analysis method (wet method) but also as a dry analysis method. An example of this is a dry analysis sheet having a shape similar to a so-called pH test paper, in which an absorbent material such as a filter paper is impregnated with a reagent that changes color upon contact with an analyte.

Further, sheet-like or film-like analysis elements having a single-layer or multilayer structure are also known, which enable highly accurate quantitative analysis of analytes while utilizing dry operation.

Among these, various types of multilayer analysis elements are known.
For example, Tokko Shotar 33100, Tokko Sho≠tarr3r
rr issue, Tokukai Sho-jo-is7iy co-issue, Tokukai Sho! l−≠
0/ri No. 7, Tokukai Sho! 2-3≠rr, Tokukai Shoyo 3-1
Patent publications such as JP-A No. 7-3 and JP-A No. 1999/101-1 disclose multilayer analytical elements having a structure in which a non-fibrous porous spreading layer is laminated on a support.

In these multilayer analysis elements, when an aqueous liquid sample containing an analyte is spotted on the development layer, the aqueous liquid sample enters the reagent layer while maintaining a uniform amount per unit area, causing a color reaction. .

By measuring the color density after a certain period of time, the concentration of the analyte in the aqueous liquid sample can be determined.

Typical examples of analytes for analysis using the above multilayer analysis element include biological fluids such as blood, urine, saliva, pancreatic juice,
Various hydrolytic enzymes contained in intestinal fluid, cerebrospinal fluid, etc. can be mentioned.

Various kinds of the above-mentioned hydrolytic enzymes are known, but the multilayer analysis element uses an enzyme that uses a high molecular weight compound as a substrate and produces a diffusible low molecular weight compound through a hydrolysis reaction. Particularly useful for analysis. Quantitative analysis of these hydrolytic enzymes is important as a clinical test item, and there is a strong demand for the development of simple and highly accurate analysis methods. Examples of these hydrolytic enzymes include proteases,
Examples include amylase, lipase, (eg, trypsin, chymotrypsin, pepsin, etc.), pectinase, and various kinases.

For example, measuring the amount of amylase present in blood is clinically extremely important as a means of testing pancreatic function. Amylase is an enzyme that has the property of hydrolyzing amylose binding moieties such as starch to produce low molecular weight polysaccharides, oligosaccharides, or monosaccharides. Generally, the relative value of the amount present and the concentration in the sample solution is calculated by a method of measuring the property, that is, the enzyme activity.

Conventionally known measurement of amylase activity involves adding a sample solution containing a substrate for amylase, such as a substrate such as starch,
After reacting under certain conditions for a certain period of time, the enzyme activity is determined from the ratio of the remaining unreacted substrate or low molecular weight reaction product produced by hydrolysis 1c, c to the high molecular weight substrate added in advance. Usually, it depends on the method of calculation.

The above-mentioned Japanese Unexamined Patent Publication No. 3-I3IO describes a dry multilayer analytical material (analytical element) that allows amylase activity to be measured with extremely simple operations. This multilayer analytical element consists of a reagent layer containing a non-diffusive substrate for amylase, e.g., starch to which a detectable chromophore, such as a dye, has been attached, and a reagent layer that has become diffusible through the hydrolytic action of the analyte, i.e., amylase. This is a laminated multilayer analysis element that includes a resist region layer for receiving reaction products. In the method of analyzing amylase activity using this multilayer analytical element, first, a detectable chromophore (dye, etc.)
The non-diffusible substrate to which is bound is hydrolyzed by the action of the enzyme in the sample solution, thereby producing a diffusible low molecular weight compound having a chromophore. This low molecular weight compound diffuses through the layer and is finally accepted and fixed in the resist region layer, thus increasing the tvc corresponding color density of the diffusible low molecular weight compound with chromophore received in the resist 74732 layer. The amount of the reaction product can be measured by photometric quantification.

The above-mentioned Japanese Patent Application Laid-open No. Shoj/-uO/ri No. 7 is
J-/J-/Or No. 9 discloses an analytical element having a layer structure similar to the analytical element disclosed. In the former method, a chemical species that cannot normally be detected by the detection layer is prepared in the reaction reagent layer, and this is converted into a diffusible and detectable chemical species in the reaction reagent layer by the action of an analyte. The difference is that the latter includes a process of detecting this with a detection layer, whereas the latter includes a process in which a detectable chemical species is contained in the reaction reagent layer in advance.

In these VC technologies, the purpose of the detection layer or the registration layer is simply to receive the diffusible character species, and no color reaction is expected within the layer. Therefore, in order to clearly distinguish between the detectable chemical species remaining or generated in the reaction reagent layer and the chemical species captured or accepted in the detection layer or resist region layer, and to obtain quantitative measurement results. It was essential to provide a light-scattering radiation-blocking layer between the two layers for the purpose of color shielding.

Japanese Unexamined Patent Application Publication No. 7-07-07 describes a multilayer analytical element that improves the above-mentioned drawbacks. In this multilayer analytical element, the diffusible compound produced by the reaction of the substrate and analyte contained in the substrate layer prepared therein is substantially colorless, and this diffusible compound is
It reacts with the chromogen previously contained in the analytical element, and a dye is formed for the first time. This chromogen is usually introduced in advance into the multilayer analytical element in a state contained in a coloring layer that is laminated on a substrate layer. Therefore, it is characterized in that it is extremely easy to optically distinguish between the substrate and the product before and after being affected by the analyte. However, in the above disclosed technology, when a diazonium compound is used as a chromogen compound that forms a dye by reaction with a diffusible compound, the stability of the diazonium compound is poor, and it also causes decomposition reactions and various undesirable side reactions. It has been found that this tends to reduce the quantitative nature of the analysis.

[Summary of the invention]

The present inventor sought to solve the above problems in an integrated multilayer analytical element for analyzing hydrolytic enzymes in biological samples,
We have arrived at the present invention.

An object of the present invention is to provide an integrated multilayer analytical element for nine-hydrolytic enzyme analysis that has high stability over time and excellent quantitative analysis.

The present invention provides a multilayer analytical element in which a coloring layer and a substrate layer are laminated on a light-transmissive/horizontal-transmissive support.
a) a substrate layer comprising a non-diffusible substrate capable of producing a diffusible compound having a color-changing agent; and b) a pH relative to the substrate layer.
In units/! Non-diffusive p
) An integrated multilayer analytical element for hydrolytic enzyme analysis is provided, which is characterized by comprising a coloring layer containing an I buffer.

〔Effect of the invention〕

Since the multilayer analysis element of the present invention is a dry method, the measurement operation is extremely simple, and even a person with little measurement experience can perform measurements with extremely high accuracy.

Since the multilayer analytical element of the present invention does not contain a diazonium salt, it is excellent in storage over time and can provide measurement results with good reproducibility over a long period of time.

[Detailed description of the invention]

The multilayer analytical element of the present invention has a basic structure in which a coloring layer and a substrate layer are sequentially laminated on a light-transmitting/horizontal-transmitting support.

Specific examples of light-transparent/horizontal-transparent supports (hereinafter referred to as supports) include polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, and cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate). Transparent and substantially water-impermeable supports having a thickness in the range of about 10 μm to about 300 μm, preferably about 10 μm to about 300 μm, are made of polymers such as Nate et al.

If necessary, an undercoat layer may be provided on the surface of the support to strengthen the adhesion between the coloring layer provided on the support and the support. Further, instead of the undercoat layer, the surface of the support may be subjected to a physical or chemical activation treatment to improve the adhesive strength.

A coloring layer is provided on the support (depending on the case, via another layer such as the lower a layer).

The coloring layer constituting the integrated multilayer analytical element of the present invention has a pH unit of 1.0% compared to the substrate layer. This layer is made by dispersing in a polymer binder a non-diffusible polymeric substance that can be set to a pH different by J or more.

In the coloring layer, the substrate in the substrate layer is hydrolyzed by the action of the analyte in the sample, and the decomposition products penetrate into the coloring layer, causing a significant change in color tone depending on the pH of the layer.

The polymer used in the coloring layer is preferably a hydrophilic polymer that absorbs water and swells. Examples of hydrophilic polymers include gelate/(acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (phthalated gelatin, hydroxyacrylate grafted gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone. etc. can be mentioned.

The dry film thickness of the coloring layer is in the range of about 100 μm, preferably in the range of about 10 μm to 30 μm. Moreover, it is preferable that the coloring layer is substantially transparent.

The analytical element of the present invention is characterized in that the coloring layer contains a non-diffusible pH buffering substance. The pH to be set is a substance that binds to the substrate and changes color depending on the pH change (hereinafter referred to as p).
(referred to as a color change agent) due to the pH dependence of the color change.

In the first group, the pH is set above μ, and in the second group, the pH is set below μ. By using a non-diffusive substance as the substance that sets the pH, the pH of the adjacent layer can be adjusted.
The coloring layer can be set to a desired pH without affecting the pH of the coloring layer.

Basic polymers can be mentioned as the first group of substances that set the pH above 100 ml. Specific examples of the basic polymer are basic polymers having repeating units represented by formulas (1) to (Vl) described below.

+CH2CH2NHner (1) In summer: H2-C-→-(If) [Formula (nu: where R1 is a hydrogen atom or a methyl group: R2 and R3 are a hydrogen atom or an alkyl group with a number of carbon atoms/~≠ group; X is an oxygen atom or -NH-; n is 0./, λ, 3- or Hiro] the above formula (II)
Amides (X is -N
H-) is described in Japanese Patent Application Laid-Open No. 1-63713.

Specific examples of compounds that can be used as the repeating unit represented by the above formula (n) include (dimethylamino)ethyl coacrylate;

[(dimethylamino)ethyl]methacrylate, [(diethylamino)ethyl coacrylate, N-[(dimethylamino)propylcoacrylamide N-[(diethylamino)propylcoacrylamide, and N-[(dimethylamino)ethyl] Core acrylamide and the like can be mentioned.

-4-CF(2-C-+-(III) [In formula (1), R is a hydrogen atom or a methyl group; X is an oxygen atom or -NH-; n is 0./, co, [In formula ([1), R1 is a hydrogen atom or a methyl group; XVi, an oxygen atom or -NH-; nf 'io, /, 2.3 or ≠] Q is a heterocyclic group; R2 is an atom of water 5 or an alkyl group with a number of carbon atoms ~μ] 1←CH2-C→-(V) [ In formula (V), R1 is a hydrogen atom or a methyl group; R2 is a hydrogen atom or an alkyl group having l-≠ carbon atoms] Specific examples that can be used as the repeating unit represented by the above formula (V) Examples of compounds include l-acryloyl-≠-methylbirazine, l-methacryloyl-≠-methylbiperazine, l-acryloyl-≠-ethylpiperazine, l-methacryloyl-≠-ethylpiperazine, l-methacryloyl-≠-ethylpiperazine,
Examples include -acryloyl-≠-propylpiperazine, l-methacryloyl-≠-propylpiperazine, l-acryloyl-≠-butylpiperazine, and /-methacryloyl-≠-butylpiperazine.

Ichiha CHz C-)-(Vl)! [In formula (M), R1 is a hydrogen atom or a methyl group; X is an oxygen atom or -NH-; Z is an anion; m is 0 or /] Represented by the above formula (Vl) Among the repeating units, those in which amides (X is -Nu-t-) are disclosed in Japanese Patent Application Laid-Open No.
This is described in Publication No. 2tJj.

The polymer containing the repeating unit represented by any one of formulas (1) to (■) above may be a homopolymer or a copolymer. The copolymer may be a copolymer consisting of repeating units represented by any of the above formulas (I) to (Vl), or a copolymer with other monomers. You can. When producing a copolymer with other heptamers, it is preferred to contain at least 30 mol of the above repeating unit. Other monomers used in the copolymer include acrylamide, methacrylamide, diacetone acrylamide, tert-butylacrylamide, N,N-dimethylacrylamide, N-methylolacrylamide, acrylic (methacrylic) esters, stele/, Vinyl acetate, vinyl alcohol, N-vinylpyrrolid/,
Examples include acrylamine, glycidyl acrylate (methacrylate), and the like.

The basic polymer used in the present invention is prepared according to “Polymer Synthesis Method 1j
(Oveargargar (C,G.

Oververger), translated by Hiroshi Minato, Tokyo Kagaku Doujin, published in 2010) or ``Experimental Methods of Polymer Synthesis'' (
It can be easily synthesized by referring to Otsu Tsuyoshi, co-authored by Kinoshita Masayoshi, Tokyo Kagaku Doujin, published in 1972).

Regarding the molecular weight range of the basic polymer, we take into consideration that if the molecular weight is too low, the polymer itself may diffuse, while if the molecular weight is too high, it will have poor compatibility with hydrophilic colloids. Determine depending on the type. In general, the intrinsic viscosity measured at 300C using 7% saline is 0. /-j
, about 0, preferably 0.3 to 2. ,! It is preferable to use a basic polymer that has a certain value.

In addition, regarding the range of the content of the basic polymer in the coloring layer, if the content is too small, the effect of the present invention may be insufficient, while if the content is too large, the hydrophilic polymer may It is determined by taking into consideration that the compatibility with the polymer will be poor. Generally, the dry weight range is from about λ to ≠0, preferably from about j to 30% based on the polymer binder constituting the coloring layer.

Acidic polymers are examples of substances that set the pH of the first group to below j. Examples of the acidic polymer include high molecular weight carboxylic acids, high molecular weight sulfonic acids, and carboxylic acid sulfonic acid copolymers.

Examples of the polymeric carboxylic acid include polyacrylic acid, polymethacrylic acid, acrylic acid copolymers, methacrylic acid copolymers, and maleic acid copolymers. Examples of the polymeric sulfonic acid include polystyrene sulfonic acid, ho+)ethylene sulfonic acid, styrene sulfonic acid copolymer, and vinyl sulfonic acid copolymer.

The content of carboxylic dispersion units and sulfonic acid units is approximately! ~10
0 mol, preferably in the range of 30 to 100 mol. The molecular weight is preferably in the range of about 30,000 to 1,000,000.

Specific examples of acidic polymers include polyacrylic acid, polymethacrylic acid, methyl vinyl ether maleic acid copolymer (/:/) methacrylic acid-acrylic acid copolymer (/:/) polystyrene sulfonic acid polyethylene sulfonic acid styrene-maleic acid Copolymer (/:/)2-acrylamide-λ-methylpropanesulfonic acid polymer and copolymers containing the same can be mentioned.

The substrate layer laminated on top of the coloring layer is substantially colorless and p
) Dispersed in a hydrophilic binder is a non-diffusible pH color change agent that has a pH color change agent that changes color upon a change in I and is capable of producing a diffusible compound that can diffuse in the presence of water by the action of an analyte. It is a layer.

The non-diffusible substrate used in the element of the present invention has physical properties such as molecular size, shape, degree of dissociation, etc. that change significantly depending on the preparation of the analyte that is the object of analysis. Specific examples of non-diffusible substrates, which are substances whose diffusivity in a hydrophilic binder layer increases significantly, include substrates for various enzymes contained in biological fluids. Among these, the multilayer analysis element of the present invention can be particularly effectively applied to hydrolytic enzymes, specific examples of which include protease, amylase, lipase, and pectinase. When these hydrolases are used as analytes, the non-diffusible substrate used in the present invention is a substrate of the respective hydrolase, i.e., protein, amylose, glyceride, or pectin, bound to a substantially colorless pH color-changing agent. The one that was made is used.

PH discoloration It is preferable that the material is substantially colorless at the pH of the substrate layer and becomes colored at the pH of the coloring layer, but even if it is colored at the pH of the substrate layer, it can be clearly identified by the pH of the coloring layer. A material that changes color tone can also be used.

A pH indicator is well known as a color-changing agent that changes color tone depending on a change in pH.

The first group of color-changing drugs are alkaline and color-changing, and include, for example, phenolphthalein, thymol phthalein, O-cresol phthalein, thymol blue, neutral red, phenol red, cresol red brome, phenol blue, and bromine. Examples include thymol blue p-xylenol blue, metacresol purple, and bromcresol purple.

The second group includes leuco dyes that change color when acidic, such as triphenylmethane dyes, spiropyran dyes, xanthine dyes, and fluoran dyes.The leuco dyes used in pressure-sensitive paper are especially popular. Specific examples include l-methylamino-t-diethylaminofluorane, λ-ethylamino-6-ethylaminofluorane, λ-dimethylamino-2-methylaminofluorane, and λ-dimethylamino-monoethylaminofluorane. It will be done.

Among the color changing agents listed in the first group, the following can also be used in the second group.

Thymol blue, p-xylenol blue, meta-cresol (-blue) The substrate containing the pH color-changing drug contained in the substrate layer diffuses into the coloring layer without being affected by the analyte, which is due to the action of the analyte. This refers to the occurrence of unreliable coloration, ie, "fogging", which degrades analytical performance, and must be completely prevented.

The pH color changing agent selected according to the purpose is bound to the non-diffusible substrate using the "reactive dye" technology widely used in the dye industry.

In the dye industry, more preferable dye-like Bk is obtained by physically adsorbing dyed objects on various natural fibers or by forming chemical bonds. The dye used at this time is a reactive dye, edited by K. Venkatara.
(ed.) [The Chemistry of Synthetic Dye Chemistry]
try of Synthetic Dyes)J
Volume ■, New York, Academic Press, (Aca
This reactive dye is described in detail in the demic Press) published in 1972). In particular, the "linking group" that connects dye molecules with molecules of various types of cellulose, which are natural polymers, and wool and silk, which are protein fibers, is described in detail, and the "linking group" that binds dye molecules to molecules of various types of cellulose, which are natural polymers, and protein fibers, such as wool and silk, is described in detail. For this purpose, the technique of Koran et al. can be used. That is,
This linking group can be used to link the H color change agent and the non-diffusible substrate to yield compounds useful in the present invention. P
If the amount of the H color change agent bound to the substrate is small, the amount of the pH color change agent produced by the hydrolysis action will be small and the sensitivity of detection will be impaired. The greater the amount of binding, the higher the sensitivity of detection, but if the amount is too large, the properties as a substrate will be impaired. When attaching a pH color-changing drug to starch, the pHi changes at a rate of 7 units per ~100 units of glucose in starch.
The coloring agents are bound together, and the coloring agents are combined.
The range is one per unit.

When forming an edge on the substrate layer, vc is the substrate layer /fn
Alternatively, a solution obtained by dissolving or dispersing two or more polymers in a binder solution is applied and dried. However, since the substrate of the present invention has a high molecular weight itself, it can also be formed by methods such as coating or impregnation without using a binder. For example, after pretreatment such as impregnating filter paper, nonwoven fabric, woven fabric, knitted fabric, glass fiber filter paper, membrane filter, etc. with a substrate, it is also possible to laminate the colored layer by VC such as lamination. .

There are various types of hydrophilic binder polymers that can be used in this substrate layer. Among these, those that can be used in the present invention include natural hydrophilic polymers such as Nachiuchi/, agarose, sodium alginate, calfoxymethylcellulose, and methylcellulose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and sodium polyacrylate. , polyhydroxyethyl methacrylate, a copolymer containing acrylic acid,
Examples include hydrophilic synthetic polymers such as copolymers containing maleic acid.

An appropriate binder is selected from among these, taking into consideration the characteristics of the analyte, coating characteristics, etc.

For example, gelatin is not suitable as a binder for protease analysis materials, and agarose is mainly used. Furthermore, when the analyte has a large molecular weight (for example, amylase), it is necessary to use a binder with a high swelling ratio, such as polyacrylamide, in order to effectively diffuse the analyte into the substrate layer. Among the hydrophilic binder polymers, particularly suitable as binders for the substrate layer of the invention are agarose, polyacrylamide, sodium polyacrylate, and copolymers containing acrylic acid.

In addition to non-diffusible pH color changing agents and hydrophilic binders, the substrate layer contains surfactants, pH adjusting reagents, antioxidants, and other organic substances for the purpose of improving various properties such as storage stability.
Various additives made of inorganic substances can be studied.

The thickness of the substrate layer should be 2~jOμ in order to provide it as a coating layer.
Appropriately, the thickness is approximately 30 μm, preferably in the range of 10 μm to 30 μm. When a method other than coating, such as lamination, is used, the film thickness can vary widely in the range of several tens to hundreds of micrometers.

The multilayer analysis element of the present invention can further prevent diffusion, if necessary.
It can be formed by laminating other functional layers for the purpose of uniform spread of the sample, light shielding, adhesion, etc.

The function of the diffusion prevention layer is to completely prevent the diffusion of other layers of the non-diffusible substrate before being affected by the analyte, and also to prevent the diffusion of reaction products (diffusible compounds) that have become diffusible. It's in the absence of it. Of course, it also has the function of preventing the polymer p)T buffer substance in the coloring layer from being mixed into the substrate layer. The materials used for the anti-diffusion layer are hydrophilic polymers similar to those used for the substrate layer. Among these, particularly suitable polymers are gelatin, agarose, and polyvinyl alcohol, which are used to define diffusivity differences between non-diffusible substrates and products (diffusible compounds). It has suitable characteristics. Various additives can be added to the diffusion prevention layer as well, similar to those mentioned regarding the substrate layer.

As can be understood from its function, the anti-diffusion layer is provided between the substrate layer and the coloring layer, and typically has a θ,! It has a thickness of ~μm.

The multilayer analytical element of the present invention can be provided with a spreading layer on the substrate layer.

As the spreading layer of the present invention, it is preferable to use filter paper, nonwoven fabric, woven fabric, knitted fabric, glass fiber filter paper, a membrane filter made of plush polymer, or a porous layer containing continuous voids made of polymer microbeads or the like.

Examples of woven fabrics (woven fabrics) that can be used for the spreading layer include JP-A-Sho zs-it≠3-6 and JP-A-37-4T.
J! A wide variety of woven fabrics are disclosed in each publication of No. 2.

Among the woven fabrics, plain woven fabrics are preferred, and silk fabrics, all-through fabrics, broadcloth fabrics, boblin fabrics, etc. are preferred. Thread thickness is 2oS, 1soS, preferably 4cO8-/20
The range is equivalent to S. The thickness of the woven fabric is ioo~zoo
μm, preferably in the range of 720 to 319 μm.

The knitted fabric is preferably knitted, and heavy atlas knitted fabric, tricot knitted fabric, double tricot knitted fabric, Raschel knitted fabric, etc. can be used. Thickness of yarn of knitted fabric is l10S~/J-(1)S, preferably tO
The range is equivalent to 8 to /20S.

The thickness of the knitted fabric is 100-400 μm, preferably /
The range is jO~≠00 μm.

Raw yarns for making woven fabrics and knitted fabrics include natural fiber yarns such as cotton, silk, and wool, viscose rayo/, regenerated cellulose such as Kyupra, semi-synthetic organic polymers such as cellulose diacetate and cellulose triacetate, and polyamide ( (nylons), acetalized polyvinyl alcohol (vinylon, etc.), polyacrylonitrile, polyethylene/terephthalate, polyethylene, polypropylene, polyurethane, etc. Threads made of fine fibers or single fibers of synthetic organic polymers, natural fibers and regenerated cellulose, Mention may be made of threads consisting of synthetic or mixed fibers with synthetic organic polymer fibers.

The woven or knitted fabric used for the porous development layer may be subjected to a physical activation treatment (preferably glow discharge treatment, corona discharge treatment, etc.) as disclosed in Japanese Patent Application Laid-Open No. 1987-1999, or at least one side of the fabric may be subjected to Or Tokukai Sho! j-/64
c3j No. 6, Tokukai Sho! The woven fabric or knitted fabric is made hydrophilic by hydrophilic treatment such as impregnation with a hydrophilic polymer disclosed in Publication No. 7-6t3j, etc., or by sequentially performing these treatment steps, and the lower side (the side closer to the support) is made hydrophilic. The adhesion between the layers can be strengthened.

To bond and laminate a spreading layer made of woven or knitted fabric to a substrate layer or adhesive layer, please refer to JP-A-Sho! It can be produced according to the methods disclosed in J-/A Reference Nos. 3 and 4 and Japanese Patent Application Laid-Open No. 17-46319. That is, water (or water containing a small amount of surfactant) is supplied substantially uniformly to the coater of the substrate layer or the adhesive layer while the coater is still wet or after drying to swell the layer, and then the fabric or adhesive layer is coated. The knitted fabric is bonded and laminated onto the wet or swollen layer substantially uniformly and under gentle pressure to integrate it.

If the developing layer is made of plush polymer or a mesh/bra/filter, it is special! Publication J-27677, etc.
In the case of a three-dimensional lattice structure layer consisting of polymer microbeads, JP-A-Sho! In cases where it is made of filter paper or non-woven fabric, such as in the J-ta orz publication, JP-A-57-/μt2!
They can be provided according to the methods described in Publication No. 0 and the like.

The multilayer analytical material of the present invention can be provided with a color-shielding layer or a light-reflecting layer between the substrate layer and the coloring layer.

Furthermore, a liquid sample permeable adhesive layer can be provided between the developing layer and the color shielding layer or the light reflecting layer for the purpose of firmly adhering the developing layer.

As the color-shielding layer or the light-reflecting layer, a layer having a thickness in the range of about λ to 20 μm and made by dispersing white fine powder such as TiO2 fine powder or B a S 04 fine powder in a hydrophilic binder polymer can be provided. .

The adhesive layer is made of a polymer of the same type as the liquid sample-permeable hydrophilic polymer used as a binder in the substrate layer, color shielding layer, light reflection layer, etc., and has a thickness of about 01! ~
Layers in the range of jμm can be provided. A hydrophilic polymer aqueous solution is applied onto the substrate layer, color shielding layer, or light reflecting layer, and the surface of the adhesive layer is moistened with water or water containing a surfactant during or after drying. ,
A porous material can be brought into contact with the surface and uniformly laminated onto the adhesive layer by applying appropriate pressure. Furthermore, by applying a solution or dispersion capable of forming a porous layer onto the adhesive layer, the multilayer analytical element to which the developing layer is firmly adhered can be spread.

In order to explain the elements of the present invention more specifically and in detail, a multilayer analysis element for measuring amylase activity will be described in detail below as a specific example. The multilayer analytical element for amylase analysis, which is one embodiment of the multilayer analytical element according to the present invention, has a hydrophilic and non-diffusible substrate in which a pH color changing drug is bound to a compound containing an amylose bond, which is a substrate for amylase. A substrate layer consisting of a binder and a pH color-changing drug bound to the substrate.
It has a structure in which a coloring layer made of a polymeric pH buffering agent that changes color tone depending on the change is coated on a completely transparent plastic film.

Because starch molecules are naturally very large and non-diffusible, they do not diffuse into the color layer prior to reaction, so the pH color change agent and polymeric pH buffer are kept separate. The multilayer analytical element is substantially colorless. When an aqueous sample solution containing amylase as an analyte adheres to the substrate layer, amylase diffuses and permeates into the substrate layer together with the aqueous medium.

Then, as the hydrolytic action of amylase progresses and the starch molecules are cleaved and reduced to lower molecules, the diffusivity in the layer increases, and oligosaccharides (diffusible compounds) to which p[-1 color-changing drugs are bound appear. It diffuses into the color layer, changes color tone due to the pH gap between the two layers, and develops a color. The activity of amylase is proportional to the rate of starch hydrolysis, while the rate of pigment formation is proportional to the amount of oligosaccharides bound to pF (f color) diffused into the color layer. Amylase activity can be determined by photometrically measuring the amount of nine pigments.

As described above, in the multilayer analytical element of the present invention, the rate of pigment formation reaction is measured, and the amylase activity in the test liquid is measured from this and a calibration curve prepared in advance using a known amount of amylase. In this method, the test liquid is almost colorless before application, and the optical density value obtained by photometry after the reaction is directly proportional to the amount of dye formed as a result of the enzymatic reaction.

Examples of the present invention are shown below.

[Synthesis method of basic polymer]

A solution for basic polymer synthesis having the following composition was placed in a JL reaction vessel equipped with a stirrer, and while blowing nitrogen gas,
Potassium Beroxonisulfate/01 was dissolved in 20 g of distilled water and added.

Solution composition for basic polymer synthesis N-[(dimethylamino)propylcoacrylamide/10F distilled water
100 Pisopropyl Alcohol /! ON Sodium hydroxide aqueous solution (O, tN) io, A Temperature of the above solution fJ! The temperature was maintained at -30'C and stirring was continued for 3 hours.

After the reaction was completed, 1 liter of acetonitrile was added, and the temperature was lowered to -j0C while stirring.

The supernatant was removed, 300 ml of distilled water was added, the precipitate was redissolved, and acetonitrile μl'r was added again to lower the temperature to -1'c to precipitate poly-N-[(dimethylamino)propylcoacrylamide gold. When distilled water λ00ytlf was added and stirred, a homogeneous liquid (solution) 7j4f was obtained.

In order to calculate the total amount of polymer produced, the above solution 21 was taken and dried with hot air at IOO 0C for 3 hours, and the weight of the polymer was measured to be 362 ~, and the polymer content in the solution was lIr , / became. In addition, the dried polymer'/
1 was dissolved in saline solution and the solution viscosity was determined to be 30°.
It was 0.2 in C.

Furthermore, when the pH of the generated solution was measured, it was found to be 10, z
The value of was obtained.

[Synthesis method of neutral red modified starch]j 0
Add cyanuric chloride to a 0rnl reaction vessel. jf, distilled water j
Add atA' and 710 ml of acetate and cool to -1o0c. Next, neutral red (3-amino-7
- dimethylamine-cometherphenazine hydrochloride)! ,
! Add a solution consisting of f, 2 ml of distilled water and sodium hydroxide, i, or, stir at -100C for 3 c minutes, and continue stirring at room temperature for λ hour. Next is acetone 2! Oat
Add, filter, and dry. A reaction product of Hirot. was obtained.

Next is soluble starch hiro? , dissolve sodium hydroxide≠1 in distilled water, add the above reaction product 2,
Stir at room temperature for io hours.

After the reaction was completed, it was reprecipitated with acetone, the supernatant liquid was removed, it was redissolved in distilled water, the pH was adjusted to 7, and it was freeze-dried. Yield is Hiro, 32° Neutral Red introduction rate into starch is J30nm
As a result of measuring the absorbance at , it was found that 7 neutral red molecules were introduced into 20 units of glucose.

[Method for synthesizing λ-methylamino-6-dinithylaminofluorane modified starch] Cyanuric chloride≠, oy in a 500 ml reaction vessel.

Add j ml of distilled water and acetone λO, -lo 0
Cool to c and stir. Next, -monomethylamino-6
-diethylaminofluorane t,oy, distilled water 20ml
, add a solution consisting of sodium hydroxide/, Of, -1
Stir at 00C for 30 minutes and at room temperature for 2 hours. Next, acetate 72jOmlf is added, and the product is filtered and dried. yield t
, λ2゜ Furthermore, in order to react with starch, soluble starch? , add sodium hydroxide to 1 liter of distilled water.
Add 52ff of reactive fluorane, and add i at room temperature.
Stir for o hours. Finally, excess acetone was added for precipitation and the product was dried. Yield J, Jt.

AO of the starch derivative obtained to measure the reaction rate
The absorption at Onm was measured and it was confirmed that 9 molecules of fluorane were introduced per 3 λ units of starch.

(Example 1) (1) Preparation of multilayer analysis film for measuring amylase activity A coloring layer (dry film thickness lλμm), a diffusion prevention layer ( Dry film thickness λ
μm) and substrate layer (dry film thickness/μrrL) were formed by sequential coating and drying. Further, the surface coated with the above two layers was wetted with an aqueous solution of p-nonylphenoxyglycerin O and flathead, and then a blended fabric of polyester and cotton (plain woven fabric with an IOO count; blending ratio: polyethylene terephthalate/polyethylene terephthalate/ Crimp cotton = 7j/21)
It was dried to form the entire development layer, and an integrated multilayer analytical element for measuring amylase was prepared.

The method for preparing the coloring layer and substrate layer is shown below.

Coloring layer purified water λ4 cOml deionized gelatin 2j 1 Poly-N-[(dimethylamino) propylcoacrylamide (see the synthesis method of basic polymers) (10 Aqueous solution) 3o yp-nonylphenoxyglycerin (J Aqueous solution) it Dissolve gelatin by heating and add io, ovr to pHt sodium hydroxide solution.
-Adjust the coloring layer coating solution.

Diffusion prevention layer purified water jo dTI
O2 fine powder toyp-nonylphenoxyglycerin (co!chi aqueous solution) o, ztThe above mixture was completely ground in a ball mill type grinder, and then gelatin 1
0chi aqueous solution 100? was added to this and uniformly dispersed using a homogenizer.

Substrate layer neutral red modified starch (see synthesis method) ≠ tHEPES (
N-2-hydroxyethyl-hiheracin-N'-ethanesulfonic acid /,
! ? Purified water ≠2 d polyacrylamide (j aqueous solution) 32 tp-nonylphenoxyglycerin (! aqueous solution) 4'? Dissolve the above mixture completely and adjust the pH of the coating solution to 7.0 with sodium hydroxide. Adjusted to /.

(2) Measurement of amylase activity Human pancreatic amylase was diluted stepwise with t-7% human albumin/physiological saline to prepare an amylase standard solution having the following amylase activity measured by the blue starch method.

A Tan λ Somogyi U/I%I B 3ri/ (: 101!4c Apply 10μl of amylase standard solution to the film immediately after coating and the film that has been incubated for 4!O"c for 7 days. The total reflected optical density was measured using a Macbeth densitometer using a Macbeth densitometer with light of u!Onm, and the following results were obtained.

Reflection optical density value From the above results, the film for measuring amylase activity of the present invention has color development performance that corresponds to the activity of amylase.
It was also found that it exhibits excellent stability even when stored at high temperatures.

(Example 2) m Preparation of a multilayer analysis film for measuring amylase activity An integrated multilayer analysis film for measuring amylase activity was carried out in the same manner as in Example 1, except that the coloring layer and substrate layer in Example 1 were changed to the following formulations. All films were made.

Coloring layer purified water /71 rrtl polyvinyl alcohol 20f polyacrylic acid (23% solution) 20f p-nonylphenoxy7glycerin (jti aqueous solution) pHt of coating solution
ri2. It was ri.

Substrate layer λ-methylamino-2-diethylaminofluorane-modified starch/ (see synthesis method) ≠ t HEPES /, zy purified water ≠ 24 polyacrylamide (j ti aqueous solution) 32 t p-nonylphenoxyglycerin (j ti aqueous solution) ≠ 1 coating Adjust the pH of the solution to 7. with sodium hydroxide. Adjusted to /.

(2) Measurement of amylase activity The following results were obtained in the same manner as in Example 1. Note that the reflection optical measurement was performed using jllOnm.

Reflection optical density value From the above results, the film for measuring amylase activity of the present invention has color development performance that corresponds to the activity of amylase.
It was also found that the gold exhibits excellent stability even when stored at high temperatures.

Patent Applicant: Fuji Photo Film Co., Ltd. Procedural Amendment (Showa to January 1, 1999 - Day 1, Case Indication: Showa to Year, Patent Application No. 1 JI773)
No. 2, Title of the invention Multi-layer analysis element 3, Relationship with the case of the person making the amendment Patent applicant 4, Date of amendment order February 2, 1930 (shipment date) 5, Subject of amendment Description 6, Amendment We will submit an engraving of the detailed statement of contents (with no changes to the contents).

Claims (5)

[Claims] (1) In a multilayer analytical element in which a coloring layer and a substrate layer are laminated in this order on a light-transmitting/horizontal-transmitting support, a) pH discoloration that changes in color due to pH change due to the action of the test substance; a substrate layer comprising a non-diffusible substrate capable of producing a drug-bearing diffusible compound; and b) a pH that differs by 1.5 or more pH units compared to the substrate layer.
1. A multilayer analysis element characterized in that it is a coloring layer comprising a non-diffusible pH buffer that can be set to . (2) The multilayer analytical element according to claim 1, wherein the pH color changing agent is a pH indicator that changes color on a highly alkaline side, and the non-diffusible pH buffer is a basic polymer. (3) Claim 1, wherein the pH color changing agent is a pH indicator or a fluoran leuco dye that changes color on the acidic side, and the non-diffusible pH buffer is a polymeric carboxylic acid or a polymeric sulfonic acid. Multi-layer analysis elements described. (4) The multilayer analytical element according to any one of claims 1 to 3, wherein the analyte is one selected from protease, amylase, lipase, and pectinase. (5) The multilayer analytical element according to any one of claims 1 to 3, wherein the non-diffusible substrate is starch and the analyte is amylase.