JPH0229986B2 - TASOKAGAKUBUNSEKIZAIRYO - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a multilayer analytical material for the purpose of quantitative analysis of components in aqueous liquids, particularly biological fluids. A wide variety of methods have been proposed for analyzing components in water, foodstuffs, and biological fluids. The method of producing a colored substance in proportion to the amount of the analyte in a sample solution and measuring its color density is also a well-known photometric principle, and is not limited to solution analysis methods. Used in dry analysis method. An example of this is a dry analysis sheet with a principle and shape similar to so-called PH test paper, in which paper or an absorbent carrier is impregnated with a reagent that changes color upon contact with a test substance. Multilayer chemical analysis materials that can be analyzed by dry processing are also known. For example, JP-A-49-33800, JP-A-49-53888, JP-A-50-137192, JP-A-Sho.
No. 51-40191, JP-A-52-3488, JP-A-53-
In the examples described in specifications such as No. 89796 and JP-A No. 53-131089, a single or multiple reagent layer is provided on a support, and a non-fibrous porous layer is further provided on the support. It has a laminated structure. When an aqueous liquid sample is deposited on the developing layer, the aqueous liquid sample permeates into the reagent layer while maintaining a uniform amount per unit area and causes a color reaction. By measuring the change in color density after a certain period of time, the concentration of the test substance in the aqueous liquid sample can be determined. An object of the present invention is to provide a novel analytical material that utilizes a reaction system that produces products whose diffusivity in a hydrophilic binder is significantly different depending on the action of the analyte. Biological fluids such as blood, urine,
Various hydrolytic enzymes contained in intestinal fluid, saliva, cerebrospinal fluid, pancreatic juice, etc. are typical examples of such test substances. Although a wide variety of hydrolases are known, this method is particularly useful for analyzing enzymes that use high-molecular-weight compounds as substrates and produce diffusible low-molecular-weight compounds through hydrolysis reactions. . Quantitative analysis of these hydrolytic enzymes
It is important as a clinical test item, and there is a high demand for the development of a simple and accurate analysis method. Specific examples of these hydrolases include amylase, lipase, protease, 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 abundance and concentration in a sample solution is calculated by a method of measuring its properties, that is, enzyme activity. A conventionally known method for measuring amylase activity involves adding a sample solution containing amylase to a solution containing a certain amount of a substrate for amylase, such as starch.
After reacting under certain conditions for a certain period of time, the enzyme activity is calculated from the ratio of the amount of the remaining unreacted substrate or low molecular weight reaction product generated by hydrolysis to the high molecular weight substrate that was added in advance. It is customary to do so. In order to measure the quantitative ratio between the high molecular weight substrate added in advance and the low molecular weight compound produced as a result of the enzymatic reaction, it is necessary to separate the two by some method. Usually, after stopping the reaction, a precipitant that has the effect of selectively precipitating a high molecular weight substrate from the solution is added.
A method is used in which unreacted high molecular weight substrate is removed as a precipitate in addition to the reaction solution. If it can be separated into a solution containing only unreacted substrate or a solution containing only reaction products, it is relatively easy to quantify either one by a method that leads to a color reaction. If carried out correctly, this method provides highly accurate and reliable results, but in reality, the separation of unreacted substrate and reaction product and the color reaction after separation are complicated. Due to problems such as being time-consuming, there was a desire to develop a simpler analytical method. The so-called chromogenic starch method allows photometric analysis to be performed immediately by binding a dye to the substrate in advance and separating it using a precipitant after the reaction is complete.
The method (method) has become widely practiced because the process is simpler than the basic operations described above. Furthermore, as a method for simplifying the separation operation, a method (blue starch method) in which a substrate to which a dye has been bound in advance is dispersed as fine particles to further facilitate the reaction and separation of unreacted substances after the reaction is completed is also known. . The method described in JP-A No. 53-131089 dry-processes these reaction systems and at the same time provides a material for measuring amylase activity using extremely simple operations. In this method, a reagent layer comprising a non-diffusible substrate for amylase, such as starch, to which a detectable chromophore, such as a dye, has been bound;
A laminated analysis material is used that includes a registration layer for receiving the test substance, that is, a reaction product that has become diffusible due to the action of amylase. In the disclosed technology, a non-diffusible substrate to which a detectable moiety has been bonded in advance is hydrolyzed by the hydrolytic action of an enzyme to produce a diffusible low molecular weight compound, which diffuses through the layer and finally The method utilizes the principle that since the dye is received and fixed in the registration layer, the amount of the reaction product can be determined by photometrically quantifying the color density corresponding to the amount of dye received in the registration layer. This method takes advantage of the inherent characteristics of the reaction system, in which a high molecular weight substrate is inherently non-diffusible and further produces a diffusible product through an enzymatic reaction, in a normal solution reaction. This method avoided the unavoidable complicated separation operation, and made it possible to provide a level of simplicity unimaginable with conventional methods. However, in the disclosed technology, since a detectable chemical group such as a dye is bonded to a non-diffusible substrate in advance, there is no new method for photometrically distinguishing between an unreacted substrate and a reaction product. It required a lot of ingenuity. In the disclosed technology, several methods for distinguishing between unreacted substrates and reaction products are exemplified. One method is to provide a light shielding layer (radiation blocking layer) containing fine titanium oxide powder between a reagent layer containing a non-diffusible unreacted substrate and a registration layer that receives reaction products. After completion of the reaction, only the reaction products can be measured by reflection photometry. Furthermore, a method is disclosed in which a reagent layer and a registration layer are prepared in advance in a form that can be easily separated, and after the reaction operation is completed, the two are separated, and the optical density of only the registration layer is measured. The specification of JP-A-51-40191 contains the above-mentioned JP-A-53-40191.
This technology relates to a multilayer analytical material having a layer configuration similar to the multilayer analytical material disclosed in the specification of No. 131089, but as specified in the specification, the reactive reagent layer is composed of a test substance (test in the original text). The essential condition is that it consists of an interactive substance that provides a diffusible and detectable chemical species in its presence, and that it includes a detection layer that detects this chemical species. In other words, the basis of this technology is the process in which a reaction in the reaction reagent layer converts a chemical species that cannot be detected by the means normally detected in the detection layer into a diffusible and detectable chemical species through the action of the analyte. It is in a place that includes. On the other hand, in the technique disclosed in JP-A No. 53-131089, the reaction reagent layer contains a detectable chemical species in advance, and the action of the test substance is due to the fact that the chemical species is diffusible. It uses the reaction that becomes However, JP-A-51-40191 and JP-A-53
The function and material of the detection layer (or registration layer) disclosed in 131089 are exactly the same, and its function is to compensate for the diffusible and detectable chemical species generated in the reaction reagent layer. The purpose is to facilitate or accept the detection by optical measurement, etc., and a so-called mordant polymer layer is mentioned as the only specific material. The role of these mordant polymers is to enhance the efficiency of diffusible detectable chemical species formed in the reaction reagent layer, make them non-diffusive, and in some cases increase the absorbance or shift the absorption wavelength due to mordant action. Shift (usually less than 10nm)
The object of this invention is to make quantitative determination by optical measurement easy and reliable. In any case, the detection layer or registration layer in the above two disclosed techniques is a layer for the mere reception of diffusible chemical species, and does not include any coloring reaction. Therefore, in order to clearly distinguish between detectable chemical species remaining or generated in the reaction reagent layer and chemical species captured or accepted in the detection layer or registration layer, and to obtain quantitative measurement results, it is necessary to It was essential to provide a light-scattering radiation blocking layer between the two layers for the purpose of color shielding. In the material of the present invention, the diffusible compound generated in the substrate layer by the action of the analyte is substantially colorless, and the diffusible compound forms a pigment by reaction with the chromogen compound, so the analyte The distinction in optical measurement of substrate and product before and after being acted upon by a substance becomes very clear, and the analysis disclosed in JP-A No. 51-40191 or JP-A No. 53-131089 A major feature of the material is that there is no need for a special layer to distinguish between the substrate layer and the product, a radiation blocking layer, or a method for peeling off the substrate layer and detection layer prior to photometry. are doing. Also, for uniform application of mordant polymers, acetone,
It is necessary to use a polar organic solvent such as alcohol as a coating solvent. Organic solvent-based coating differs from aqueous solution coating of gelatin, polyacrylamide, etc. in terms of process, so special consideration is required in terms of manufacturing equipment. In addition, since simultaneous multilayer coating, which is advantageous in the production process, is not possible, the production of the material of the present invention is entirely done by coating an aqueous solution or aqueous dispersion, which is extremely disadvantageous in terms of production efficiency. It is advantageous in comparison. The present invention provides a multilayer chemical analysis material in which at least two reagent layers are laminated on a light-transparent water-impermeable support, in which the two reagent layers are arranged in order from the side farthest from the support (a) substantially A substantially colorless diffusible compound that is essentially colorless and has a dye-forming reactive group that can react with a chromogen compound to be described later to form a dye, and that has the dye-forming reactive group by the action of a test substance. a substrate layer comprising a non-diffusible substrate capable of producing
and (b) a coloring reaction layer comprising a chromogen compound capable of reacting with the dye and the forming reactive group to form a dye. The structure and materials of the multilayer chemical analysis material of the present invention will be specifically explained below. Examples of layer configurations in specific embodiments of the multilayer chemical analysis material of the present invention are shown in FIGS. 1 to 4. The multilayer chemical analysis material of FIG. 1 is arranged on a light-transmissive water-impermeable support 10, in order from the side farthest from the support, a substantially colorless pigment capable of reacting with a chromogen compound to form a pigment. A non-containing compound that has a reactive group that forms the dye under the action of the test substance and that is substantially colorless and that can form a diffusible compound that can diffuse through a hydrophilic binder in the presence of water. A substrate layer 50 comprising a diffusible substrate in a first hydrophilic binder and a chromogenic compound capable of reacting with said dye-forming reactive group to form a dye in a second hydrophilic binder. The color reaction layer 20 is laminated. The first and second hydrophilic binders may be the same or different from each other, and may be in any combination; An appropriate hydrophilic binder can be selected depending on the dye used or the combination thereof. The multilayer chemical analysis material shown in FIG. 2 has a structure in which a diffusion prevention layer 30 is provided between a color reaction layer 20 and a substrate layer 50. The function of the diffusion prevention layer will be explained in detail later. FIG. 3 is a conceptual diagram of the layer structure to show the principle of the material of the present invention, in which a coloring reaction layer 20, a diffusion prevention layer 30, a substrate layer 50 are formed on a light-transparent water-impermeable support 10.
It has a structure in which the development layers 70 are laminated in this order. When a drop of an aqueous sample solution containing a test substance (eg, amylase) is applied to the development layer, it is spread almost uniformly in the development layer and penetrates into the substrate layer 50 . In the substrate layer, a diffusible compound is generated from the non-diffusible substrate by the action of the test substance, and the diffusion prevention layer 3 is formed from the substrate layer.
0 and diffuses into the coloring reaction layer 20. Since a dye-forming reactive group is bonded to the diffusible compound as a reaction product, when it reaches the coloring reaction layer 20, the dye-forming reactive group and the chromogen compound react to form a dye. When the test substance is a hydrolase, the enzyme has a high molecular weight, so it cannot pass through the diffusion prevention layer and remains in the substrate layer together with the non-diffusible substrate. As a result, a pigment is formed in proportion to the enzyme activity, so the enzyme activity can be determined by measuring the amount of pigment produced. Optical measurement using transmission or reflection in the absorption wavelength region of the dye is suitable for measuring the amount of the dye, but depending on the purpose and required precision, visual determination is also possible. In order to make the explanation easier to understand, the multilayer chemical analysis material with the configuration shown in Figure 1 will be used as an example below, and additional functions provided in the multilayer chemical analysis materials shown in Figures 2 to 4 will be added as necessary. The present invention will be described in detail, including a description of the layers. Substrate layer 50 is colorless because the non-diffusible substrate is substantially colorless. The color reaction layer can also be made colorless by selecting a colorless compound as the chromogen compound. Both layers are transparent since their layer thickness is at most about 50 μm, and therefore the multilayer chemical analysis material shown in FIG. 1 is transparent. When a sample solution (aqueous solution, i.e., a solution or dispersion containing water as a solvent or dispersion medium) containing an analyte is deposited onto a multilayer chemical analysis material, non-oxidation occurs in the substrate layer due to the action of the analyte. Diffusible substrates undergo chemical reactions, typically hydrolysis, to produce colorless diffusible compounds. A colorless diffusible compound is present in the hydrophilic binder matrix of the substrate layer 50 and in the color reaction layer 20.
Diffuses through the hydrophilic binder matrix in the presence of water derived mainly from the solvent contained in the sample solution or water as a dispersion medium, and reaches the chromogen compound present in the color reaction layer. , react to form a pigment. After reacting for a certain period of time under certain conditions, the amount or activity value of the test substance contained in the sample solution can be determined by photometrically quantifying the amount of the dye produced in the color reaction layer. As mentioned above, the material of the present invention is used for measuring hydrolase activity in aqueous liquid samples, particularly biological fluids such as blood, urine, saliva, cerebrospinal fluid, intestinal fluid, and pancreatic juice. To measure the enzymatic activity of hydrolytic enzymes such as amylase in blood, conventional methods involve separating plasma or serum from collected whole blood by a method such as centrifugation, and using this as a sample solution. It's here. The reason for this is that interference during photometry due to hemoglobin, which exists in large amounts in whole blood, could not be eliminated. By incorporating a light-shielding layer as one of the functional layers, the material of the present invention enables measurement of enzyme activity using whole blood directly as a sample solution, which was impossible with conventional analysis methods. It makes it possible. Since the material of the present invention is an analysis material in which a color reaction proceeds only after the sample solution is attached, photometry after the reaction may be performed from the bottom of the analysis material, and may be either transmission photometry or reaction photometry. In the multilayer chemical analysis material of the present invention, a light-shielding layer with high light scattering properties is provided between the color reaction layer and the substrate layer to block interference caused by red blood cells and hemoglobin on the upper surface of the substrate layer, and the material is passed through the transparent support. Direct measurement using whole blood as a sample solution is also possible by reflective photometry of the color density in the color reaction layer. An example of the layer structure of materials in such a case is shown in FIG. Since the light-shielding layer itself also functions to prevent the diffusion of diffusible interference substances and/or chromogenic compounds, there is no need to provide a special diffusion-preventing layer.
In addition, hemoglobin, bilirubin, and chyle, which are often found in serum, are detected in serum analysis.
However, the diffusion prevention layer used in the material of the present invention completely blocks the diffusion of these interfering substances to the lower layer, so if a light shielding layer is provided, hemolysis and high Analysis of serum samples containing bilirubin and chyle can also be performed without error. The non-diffusible substrate used in the material of the present invention has physical properties such as molecular size, shape, and degree of dissociation that change significantly due to the action of the analyte, which is the object of analysis.
As a result, it refers to a substance whose diffusivity in the hydrophilic binder layer of a multilayer chemical analysis material increases significantly.
Specific examples of non-diffusible substrates include substrates for various enzymes contained in biological fluids. Among these, the multilayer chemical analysis material of the present invention can be particularly effectively applied to the following:
Specific examples of hydrolytic enzymes include protease, amylase, lipase, and pectinase. When these hydrolases are used as test substances, the non-diffusible substrate used in the present invention is a substantially colorless pigment-forming reactive group on the substrate of each hydrolase, i.e., protein, amylose, glyceride, or pectin. A combination of these is used. The substrate layer is composed of a layered structure of a non-diffusible substrate. When the material of the present invention is formed by coating, the substrate is dissolved or dispersed in a binder solution consisting of one or more types of polymers, and then the solution is coated and dried. However, since the substrate layer of the material 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 a pretreatment such as impregnating a paper, cloth, porous plastic membrane, etc. with a substrate, it is also possible to overlay it on a support containing a color reaction layer by means such as lamination. The first hydrophilic binder polymer that can be used in the substrate includes various hydrophilic binders. Among these, those that can be used in the material of the present invention include natural hydrophilic polymers such as gelatin, agarose, sodium alginate, carboxymethylcellulose, and methylcellulose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, sodium polyacrylate, and polyhydroxy Examples include hydrophilic synthetic polymers such as ethyl methacrylate, copolymers containing acrylic acid, and copolymers containing maleic acid. An appropriate binder is selected from among these, taking into account usage conditions, characteristics of the test substance, coating characteristics, etc. For example, protein binders such as gelatin are completely inappropriate as binders for protease analysis materials. Furthermore, when the molecular weight of the test substance is large, diffusion of the test substance into the substrate layer becomes a problem, and it is necessary to use a binder with a high swelling ratio. Among the hydrophilic binder polymers, particularly suitable as binders for the substrate layer of the materials of the invention are agarose, polyacrylamide, sodium polyacrylate, copolymers containing acrylic acid. The substrate layer contains a non-diffusible substrate, a hydrophilic binder, a surfactant, a pH adjusting reagent, and a fine powder to improve various properties such as coating performance, diffusibility of diffusive compounds, reactivity, and storage stability. , antioxidants, and various other organic or inorganic additives can be added. The thickness of the substrate layer is not particularly limited, but in order to provide it as a coating layer, it is suitably in the range of about 1 μm to 50 μm, particularly preferably in the range of 2 μm to 30 μm. However, when a method other than coating is used, such as lamination, the film thickness can vary widely from several tens of micrometers to several hundred micrometers. The color-forming reaction layer of the material of the present invention is a layer containing a chromogen compound that reacts with a dye-forming reactive group in a diffusible compound (reaction product) to form a dye and a second hydrophilic binder. Various combinations of the non-diffusible substrate in the substrate layer and the chromogen compound in the color reaction layer are possible, as will be detailed later, but a specific combination that best matches the performance of the target analytical material is recommended. need to be selected. As mentioned above, the diffusion of the chromogen compound in the color reaction layer into the substrate layer in the process of producing the material of the present invention results in color development, that is, "fogging" that is not caused by the action of the test substance. Because it reduces analytical performance,
This must be definitely prevented. For this purpose, it is necessary to take various measures to make the chromogen compound diffusion resistant. There are two methods of making couplers diffusion-resistant, which are well known in the field of photochemistry: bonding a long-chain diffusion-resistant group to the coupler, or dissolving the coupler in a hydrophobic oil and placing it in a hydrophilic binder. A dispersion method can be applied. That is, it has a layer in which a chromogen compound to which a long-chain diffusion-resistant group is bonded is dispersed in a hydrophilic binder, and a layer in which oil particles containing dissolved chromogen compounds are dispersed in a hydrophilic binder. It can be used as a color-reactive layer. Furthermore, a method of fixing a chromogen compound to a solid surface by means such as adsorption to make it resistant to diffusion can also be applied. The second hydrophilic binder used in the coloring reaction layer is also a hydrophilic polymer similar to the binder for the substrate layer, and one having approximately the same properties as the binder for the substrate layer is suitable. The first and second hydrophilic binders may be the same or different. Of course, various additives may be used. The layer thickness of the color-reactive layer can vary widely and ranges from about 1 μm to about 50 μm, preferably from about 2 μm to about 30 μm. Combinations of dye-forming reactive groups and chromogenic compounds that can be used to create the materials of the present invention and methods for synthesizing non-diffusible substrates are detailed below. As used herein, the term chromogenic compound refers to any compound capable of forming a dye by reaction with a dye-forming reactive group. Techniques regarding diffusion-resistant couplers and oil-protecting couplers, which are well known in the technical field of photographic science, are also effective means for the dye-forming reaction used in the material of the present invention. The basic principle is to reduce the coupler's diffusivity in water by attaching a bulky diffusion-resistant group or increasing its hydrophobicity while maintaining the coupler's reactivity. A concrete example is ``The Theory of
the Photographic Process” 4th edition (New
York.The Macmillan Company.Published in 1977)
Described on pages 335-372. For coupling reactions using diazonium compounds, see "Light Sensitive Systems" by J. Kosar.
(New York, John Wiley and Sons, Inc.,
Published in 1965) on pages 194-258. A dye-forming reactive group refers to a single atom or a group of atoms that can be bonded by a chemical reaction with a chromogenic compound (resulting in the formation of a dye). The chemical reaction for bonding the dye-forming reactive group and the chromogen compound may be any reaction, and the bond may be any bond such as a covalent bond, an ionic bond, or a coordinate bond. In short, the dye produced by combining the dye-forming reactive group and the chromogen compound through a chemical reaction exists stably to the extent that it meets the purpose of photometry, preferably substantially permanently after being produced. Any combination and chemical reaction of a dye-forming reactive group and a chromogen compound can be used in the present invention as long as a dye having a bond such as that present in the dye is produced. In these combinations, the dye-forming reactive group is a substantially colorless group (substantially has no absorption in the visible light region), but the chromogenic compound may be colorless, and may interfere or cause interference when photometrically measuring the dye. It is acceptable to have a color that does not prevent damage (has absorption in the visible light range that does not interfere with or prevent damage in the visible light range of the dye). As the combination of a dye-forming reactive group and a chromogenic compound, a combination of many compounds known as a group of compounds having the ability to carry out a coupling reaction to form a dye can be used in the material of the present invention.
For example, in the above-mentioned bibliography edited by James, 335
- Combinations of conventional silver halide photographic color developer couplers and oxidizing agents described on page 361, and various coupling reagents using diazonium salts as diazo components as described on pages 194-258 of the literature by Kosar. as a coupling component (coupler), and a combination of a nitrogen-containing heterocyclic quaternary ammonium salt, which is often used in photographic sensitizing dyes, and dialkylaminobenzaldehyde or a similar compound (a styryl dye is formed). A combination of these can be preferably used in the material of the present invention because the dye-forming reaction is fast. Further, combinations of various leuco dyes used in pressure-sensitive copying papers, heat-sensitive recording materials, etc. and various phenol compounds as color development promoters can also be used in the materials of the present invention. As is clear from the principle, there is no reason to uniquely limit each element of the color-forming reaction combination to the dye-forming reactive group and the chromogen compound that reacts with this reactive group to form a dye. Regarding each element, a pigment-forming reactive group is inevitably selected depending on the inhibition of enzyme reaction, the stability of synthetically difficult compounds, etc., but since it diffuses together with a diffusible compound (substrate with a low molecular weight), a pigment-forming reactive group is It is preferable that it is diffusible. The dye-forming reactive group selected above is bonded to the non-diffusible substrate using the "reactive dye" technology widely used in the dye industry. In the dye industry, in addition to physically adsorbing dye compounds on various natural fibers, chemical bonds are intentionally formed to obtain a more favorable dyeing state. The dye used at this time is a reactive dye, and K.Venkataraman
This reactive dye is described in detail in The Chemistry of Synthetic Dyes, ed., Volume 1 (New York, Academic Press, 1972).
In particular, the "linking group" that connects dye molecules to molecules of natural polymers such as polysaccharide cellulose and protein fibers such as wool and silk has been described in detail, and these techniques can be used in the non-diffusible substrate used in the present invention. It can be used for manufacturing. That is, this linking group can be used to link a dye-forming reactive group and a non-diffusible substrate to obtain a compound useful in the present invention. Since the dye-forming reactive group is reduced in molecular weight by an enzymatic reaction and undergoes diffusion migration in a hydrophilic binder to perform a coloring reaction, it preferably has a soluble group such as a sulfone group, a carboxyl group, or a hydroxyl group. Even if this soluble group is introduced into a part of the linking group, favorable results can be obtained. The dye-forming reactive group that is bonded to the non-diffusible substrate through the linking group is usually a variety of couplers used in photographic systems, such as pyrazolin-5-one, naphthol, phenol, acylacetanilide, etc., and these need to be bonded to the linking group. Therefore, it is necessary to have an amino group, a hydroxyl group, etc.
Two-equivalent couplers having a leaving group in the coupling position, which are well known in the photographic industry, can also be used. When the chromogen compound is a diazonium salt, an arylamine or the like can be used in addition to the above-mentioned couplers as a coupling component. Diazotized benzene, naphthalene, anthraquinone derivatives and N,N-disubstituted-p-phenylenediamine derivatives, heterocyclic quaternary ammonium salts, aryl as chromogen compounds that react with dye-forming reactive groups to form dyes. Examples include aldehyde derivatives. These compounds are preferably synthesized using anti-diffusion techniques, which are well known in the photographic industry. For example, a long-chain fatty acid derivative such as a stearoyl group, a long-chain alcohol, or an amine chain may be bonded as a long-chain diffusion-resistant group, or an aromatic compound such as a 2,4-di-t-butylphenoxy group may be introduced. It can be made diffusible. Also, like pigments, it can be made insolubilized to make it resistant to diffusion. Furthermore, the chromogen compound can be made resistant by compound or physical adsorption onto porous surface active compounds such as silica gel or alumina. Furthermore, the chromogen compound can be made diffusion resistant by being chemically or physically adsorbed onto a polymer compound or by being encapsulated in a polymer matrix. Specific examples of dye-forming reactive groups include the following groups. (5-pyrazolone means 2-pyrazolin-5-one.) 4,6-bis(8-hydroxy-3,6-disulfo-1-naphthylamino)-s-triazine-2
-yl group 4-anilino-6-(8-hydroxy-3,6-
disulfo-1-naphthylamino)-s-triazin-2-yl group 4-chloro-6-(8-hydroxy-3,6-disulfo-1-naphthylamino)-s-triazin-2-yl group 4-chloro -6-(1-p-sulfophenyl-5
-pyrazolone-3-ylamino)-s-triazin-2-yl group 4,5-dichloro-6-(8-hydroxy-3-
Sulfo-1-naphthylamino)-2-pyrimidyl group 4-chloro-6-[3-(1-m-sulfophenyl-5-pyrazolon-3-ylamino)anilino]-s-triazin-2-yl group 4-m -Sulfoanilino-6-(2-hydroxy-5-chloroanilino)-s-triazine-2-
yl group 4-(8-hydroxy-3,6-disulfo-1-
naphthylamino)-6-(1-phenyl-5-pyrazolone-3-ylamino)-s-triazine-
2-yl group 4-[β-(N-ethyl-4-trichloromethylanilino)ethoxy]-6-(8-hydroxy-
3,6-disulfo-1-naphthylamino)-s-
Triazin-2-yl group 4-p-sulfoanilino-6-(3-p-sulfophenyl-4,4-dichloro-5-pyrazolone-
Alkali metal cations (e.g. Li, Na,
A group in which a sulfonate group (-SO ₃ ) to which K ) is bound (1,3,3-trimethyl-6-p-formylphenyl-6-azoniaoctyl)oxy group and a chlorine anion as a counterion . Specific examples of chromogen compounds include the following compounds. 2-tetradecyloxy-5-sulfonatobenzenediazonium 5-(N-octadecylsulfamoyl)-2-
Methoxybenzenediazonium tetrafluoroborate 5-(N-octylsulfamoyl)-2-methoxybenzenediazonium 1-naphthalenesulfonate 2-dodecyloxy-4-nitrobenzenediazonium tetrafluoroborate 5-tetradecyloxycarbonyl-2-methoxybenzenediazonium Tetrafluoroborate 9,10-anthraquinone-1-diazonium 2-
Naphthalenesulfonate 2-methyl-3-tetradecylbenzothiazolium Perchlorate 5-dodecyloxycarbonyl-2,3,3-trimethyl-1-(γ-sulfonatopropyl)indolenium 1-(2,4-di- t-amylphenoxy)-2
-Oxo-6-(4-amino-2-methylphenyl)-3,6-diazaoctane. The light-transmitting and water-impermeable support of the multilayer chemical analysis material of the present invention includes polyethylene terephthalate, cellulose ester (cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), polycarbonate,
A known transparent support such as a plastic film such as polymethyl methacrylate or a glass plate having a thickness of about 50 μm to about 2 mm can be used. If the support is hydrophobic and the adhesion with the hydrophilic binder of the reagent layer is insufficient, treatment to make the surface of the support hydrophilic (e.g., ultraviolet irradiation, electron beam irradiation, flame treatment, hydrolysis with alkali, etc.) ) or provide a subbing layer on the surface of the support made of a substance that has an appropriate adhesive strength to both the support and the hydrophilic binder of the reagent layer, or provide the surface of the support with a subbing layer that does not significantly reduce the light transmittance. Known auxiliary treatments such as forming fine irregularities within a range (brushing, electrolytic etching, etc.) can be applied. In addition to the support and the reagent layer, the material of the present invention can be used by laminating other functional layers for the purpose of preventing diffusion, uniformly spreading the sample, shielding light, adhesion, etc., if necessary. The function of the diffusion prevention layer is to completely prevent the diffusion of the non-diffusible substrate to other layers before being affected by the test substance, 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 chromogen compound in the color reaction layer from diffusing into the substrate layer. The materials used for the anti-diffusion layer are hydrophilic polymers similar to those used for the substrate layer. Particularly suitable polymers among these are gelatin, polyvinyl alcohol, which have properties suitable for revealing diffusivity differences between non-diffusible substrates and products (diffusible compounds). are doing. Various additives can be added to the diffusion prevention layer as well as those mentioned regarding the substrate layer. As the aqueous liquid sample spreading layer (hereinafter simply referred to as the spreading layer) of the multilayer chemical analysis material, non-fibrous isotropic porous materials described in the aforementioned patent specifications and literature can be used, as well as hydrophilic It is possible to use fabrics that have been subjected to chemical treatment. Non-fibrous isotropic porous materials include brush polymers (commonly known as membrane filters), materials in which a microporous material such as diatomaceous earth or a microcrystalline material (e.g. microcrystalline cellulose) is uniformly dispersed in a binder, glass or Porous material made of microspherical beads of synthetic polymeric material held in point contact with each other by a binder, TiO ₂ or BaSO ₄
There are also brush polymers in which fine powders such as, etc. are uniformly dispersed. As a textile that has been made hydrophilic,
The fabric is thoroughly washed with water, degreased and dried, and the fabric is impregnated with a small amount of surfactant, wetting agent, hydrophilic polymer, or hydrophilic polymer with dispersed TiO ₂ or BaSO ₄ fine powder after washing with water and degreasing. be. For details on the technology and fabrics using hydrophilic-treated fabrics as a spreader, please refer to JP-A-55-
164356) is specifically described in detail in the specification, and can be applied to the present invention according to the description. The thickness of the spread layer ranges from about 50 μm to about 500 μm, preferably about 50 μm for non-fibrous isotropic porous materials.
The thickness ranges from 80 μm to about 300 μm, and in the case of a hydrophilized fabric, the thickness of the fabric after hydrophilic treatment and air drying is about 80 μm to about 1 mm, preferably about 100 μm to about 400 μm. . When the spreading layer is a non-fibrous isotropic porous material, a non-fibrous etc. is placed on the substrate layer as described in the specifications of JP-A-49-53888, JP-A-50-137192, etc. The spread layer can be provided by applying and drying a solution or dispersion capable of forming a diagonal porous layer, or by gluing a thin layer of non-fibrous isotropic porous material. When the spreading layer is made of a hydrophilized fabric, the spreading layer can be provided by bonding the hydrophilized fabric onto the substrate layer. Non-fibrous isotropic porous materials or hydrophilized fabrics can be bonded to a substrate layer by utilizing the properties of the hydrophilic binder polymer in the substrate layer when the substrate layer is semi-dry or when the substrate layer is dry. A method in which the surface of the substrate layer is wetted with water or water containing a surfactant, and a non-fibrous isotropic porous material or a hydrophilized fabric is brought into close contact with the surface of the substrate layer, and an appropriate pressure is applied as necessary. can be adopted. Another method for adhering a non-fiber isotropic porous material or a hydrophilic-treated fabric to a substrate layer is to use an adhesive that can permeate an aqueous liquid sample, and to use an adhesive layer that can permeate an aqueous liquid sample, which will be described later. A method can be adopted in which the substrate layer is provided on the substrate layer. The multilayer analysis material of the present invention can be provided with a diffusion prevention layer, if necessary. Further, in the multilayer chemical analysis material of the present invention, a color-shielding layer or a light-reflecting layer can be provided between the substrate layer and the color reaction layer. Further, an aqueous liquid sample-permeable adhesive layer may be provided between the developing layer and the substrate layer, color shielding layer, or light reflecting layer for the purpose of firmly adhering the developing layer.
The color shielding layer, the light reflecting layer, and the adhesive layer are described in detail in the above-mentioned patent specification, and can be provided in the present invention according to the description. The color-shielding layer or light-reflecting layer is made by dispersing white fine powder such as TiO ₂ fine powder or BaSO ₄ fine powder in a hydrophilic binder polymer and has a thickness of about 1 μm to about 50 μm, preferably about 2 μm to about 20 μm. A layer having a thickness of about 2 μm to about 50 μm, preferably about 2 μm to about 20 μm, consisting of a white or light-colored metallic luster fine powder such as aluminum dispersed in a hydrophilic binder polymer; Made of light-colored metal with a thickness of about 5 nm to approx.
A thin porous metal layer permeable to an aqueous liquid sample of 100 nm, preferably from about 5 nm to about 50 nm, can be provided. The adhesive layer is made of a polymer of the same type as the aqueous 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 0.5 μm to about 10 μm, preferably about 0.7 μm. Layers ranging from μm to about 5 μm can be provided. To adhere the spread to an adhesive layer made of a hydrophilic polymer, an aqueous solution of the hydrophilic polymer is applied onto the substrate layer, color-blocking layer or light-reflecting layer, and the surface of the adhesive layer is then coated during or after drying. is moistened with water or water containing a surfactant, a non-fibrous isotropic porous material or a hydrophilized fabric is brought into contact with the surface, and an appropriate pressure is applied to uniformly contact the adhesive layer. Can be layered. Furthermore, a multilayer chemical analysis material in which the spreading layer is firmly adhered can also be obtained by applying a solution or dispersion capable of forming a non-fibrous isotropic porous layer onto the adhesive layer. In order to explain the material of the present invention more specifically and in detail, a multilayer chemical analysis for measuring amylase activity will be described in detail below as a specific example. A multilayer chemical analysis material for amylase analysis, which is an embodiment of the material of the present invention, consists of a non-diffusible substrate layer in which a dye-forming reactive group is bonded to a compound containing an amylose bond, which is a substrate for amylase, such as starch, and a hydrophilic binder. A substrate layer consisting of a substrate layer, a diffusion prevention layer, and a coloring reagent layer consisting of a hydrophilic binder and a chromogen compound that reacts with a dye-forming reactive group bonded to the substrate to form a dye, are layered on a transparent plastic film. It has a painted composition. Starch molecules are naturally very large and non-diffusible and do not diffuse into the coloring layer prior to reaction, so the dye-forming reactive groups and chromogenic compounds are kept separate. The multilayer chemical analysis material is virtually colorless. When an aqueous sample solution containing amylase, which is a test substance, is attached to the substrate layer, the 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 cut and reduced to lower molecules, the diffusivity in the layer increases, and the oligosaccharides (diffusible compounds) to which the pigment-forming reactive groups are bound undergo a coloring reaction. It diffuses into the layer, where it undergoes a coupling reaction with a chromogen compound, forming a pigment. 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 with attached pigment-forming reactive groups diffused into the color-forming reaction layer. Therefore, the activity of amylase can be determined by photometrically measuring the amount of dye formed within a certain period of time. As is clear from the above explanation, in the material of the present invention, the pigment formation reaction rate is measured, and from this and a calibration curve prepared in advance using a known amount of amylase, the amylase in the test solution is determined. Measure activity. In this method, the test solution 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. A dye substrate is used in which a dye is previously bound to starch, similar to blue starch, which was used in the well-known blue starch method or the dry analysis method known from JP-A No. 53-131089. It becomes unnecessary to completely separate the unreacted substrate and the reacted substrate during photometry, which is necessary in the method. For example, JP-A-53-
In the invention disclosed in No. 131089, the optical properties before and after the reaction are completely or almost the same, so it is not only necessary to separate the layers using diffusivity, but also to separate the layers using non-diffusivity. A light shielding layer is essential between the substrate layer containing the dye substrate and the detection layer containing the polymer mordant to completely optically separate the two. Further, in this method, reflection photometry from the detection layer side is an essential condition. On the other hand, with the material of the present invention, it is possible to measure using transmitted light without using a light shielding layer, and it is understood that this method is more suitable for quantitative measurement. Specific embodiments based on the material principles of the invention have already been described in detail. Since the material of the present invention utilizes a pigment-forming reaction,
Prior to the reaction with the test substance, the non-diffusible substrate containing the dye-forming reactive group and the chromogen compound must be kept completely separated. What is important is complete separation of the non-diffusible substrate and the chromogen compound, and one way to achieve this is to ensure that the substrate itself is non-diffusible and at the same time the chromogen compound itself is also non-diffusible. It is. Various known methods and principles can be used to keep the chromogen compound non-diffusible. A very effective means for making the chromogen compound difficult to diffuse is to dissolve the chromogen compound in a hydrophobic oil in advance, or to disperse it in a hydrophilic binder while being localized in particles. Details of the manufacturing method and implementation technology for such particles can be found in JP-A-56-
It is described in the specification of No. 8549. It goes without saying that techniques known in the field of photographic science can also be used. The hydrophobic solvent used as a solvent for the chromogen compound must have a high boiling point, be able to exist in a stable dispersion state in a hydrophilic binder, be hydrophobic and not only contain the chromogen compound. It also has high solubility or hydrophilicity for the produced pigment. Examples of such oils include liquid plasticizers such as phthalate esters (e.g., dibutyl phthalate, dicyclohexyl phthalate, didodecyl phthalate, dioctyl phthalate), phosphate esters (e.g., triethyl phosphate, tributyl phosphate, triphthalate), enyl phosphate),
Adipic acid esters (e.g. diisodecyl adipate, dioctyl adipate), long chain fatty acid amides (e.g. N,N-diethyldodecanamide, N,N
-dimethyloctanamide, N,N-diethyloctanamide). Organic solvents with low or high boiling points (e.g. toluene, benzyl alcohol, ethyl acetate, butyl acetate, acetone, methyl ethyl ketone, alkylnaphthalene, acetonitrile, methylene chloride), animal oils, vegetable oils, minerals to improve solubility and stability. Oil etc. can also be used alone or in a mixed state. The particle size of the hydrophobic oil particles containing the chromogen compound or the particle size of the hydrophobic oil particles containing the chromogen compound is approximately
0.1μm to about 30μm, preferably about 0.1μm to about 10μm
is within the range of Fine particles of hydrophobic oil containing a chromogen compound or fine particles containing a chromogen compound are dispersed in the coloring reaction layer. A color reaction layer formed by dispersing fine particles of hydrophobic oil containing a hydrophobic chromogen compound in a hydrophilic binder is one preferred embodiment of the color reaction layer in the multilayer chemical analysis material of the present invention. In particular, when the test substance is amylase and the non-diffusible substrate is starch with a pigment-forming reactive group, the diffusible compound produced in the substrate layer by the action of amylase is an oligosaccharide with a pigment-forming reactive group, and a small amount of Due to the hydrophilic nature of saccharides, they easily diffuse through the hydrophilic binder and reach the color reaction layer. The oligosaccharide having a pigment-forming reactive group that has reached the color-forming reaction layer undergoes a coupling reaction between the pigment-forming reactive group and the chromogen compound of the hydrophobic oil particles to form a pigment. The concentration of oligosaccharides with forming reactive groups decreases rapidly around hydrophobic oil particles containing chromogenic compounds,
Eventually, the concentration of oligosaccharides with pigment-forming reactive groups in the color-forming reaction layer decreases, and when looking at the concentration of oligosaccharides with pigment-forming reactive groups, a concentration gradient exists from the substrate layer side to the color-forming reaction layer. It turns out. Therefore, the diffusion of oligosaccharides having dye-forming reactive groups from the substrate layer to the color-forming reaction layer is constantly promoted. Therefore, unless the dye formation reaction rate in the color reaction layer is extremely slow, the dye formation reaction is fast, and as a result, there is a remarkable effect that the time required for quantifying amylase activity using the multilayer chemical analysis material of the present invention is short. There is. In addition, by selecting a combination of a dye-forming reactive group and a chromogen compound so that the dye formed in the color-forming reaction layer is a dye that is difficult to dissolve in water, a hydrophilic binder, or a hydrophobic oil, Since the dye formed near the surface of the hydrophobic oil particle is precipitated around the hydrophobic oil particle, the dye concentration within the hydrophobic oil particle does not increase, and the dye formed near the surface of the hydrophobic oil particle does not increase. The chemical reaction that forms the pigment is accelerated, and as a result, the effect of increasing the rate of pigment formation is achieved. On the other hand, by selecting a combination of a pigment-forming reactive group and a chromogen compound so that the pigment is easily soluble in hydrophobic oil, the formed pigment is concentrated inside the fine particles of hydrophobic oil. The optical density of the dye in the color reaction layer increases, and the amount of change in optical density value per unit activity value increases.
The effect is achieved that the precision of quantification is increased. Synthesis Example 1 (1) Synthesis of dye-forming reactive group (reactive coupler) Method of JTThurtston et al. ("J.Amer.Chem.
Soc., 73 (7), 2981-2983 (1951)), cyanuric chloride and 1-amino-8-hydroxynaphthalene-3,6-disulfonic acid (H acid) monosodium salt (hereinafter referred to as H acid Na salt) The reactive coupler 2-[8-hydroxy-3,6-
Synthesis of bis(sodium sulfonato)-1-naphthylamino]-4,6-dichloro-s-triazine 36.9 g of cyanuric chloride was dissolved in 150 ml of hot acetone, then poured into 500 ml of ice water and finely divided into a suspension. did. Then 140g H acid Na salt, 16g
A solution of sodium hydroxide in 300 ml of water was added dropwise to the above cyanuric chloride suspension at 0 to 4°C. Then, 250 ml of 2N aqueous sodium hydroxide solution was added dropwise. The reaction solution was gradually warmed to room temperature and stirred for 1 hour. This reaction solution was poured into acetone (4) to cause crystallization, and the precipitate was collected. The obtained compound exhibited ultraviolet absorption different from that of H acid, and high performance liquid chromatography confirmed that the content of the obtained compound (reactive coupler) was 90% or more. (2) Synthesis of a non-diffusible substrate (coupled starch) having a pigment-forming reactive group Dissolve 20g of sodium hydroxide in water 2,
Furthermore, when 46 g of cornstarch was added and stirred, it became a transparent paste. To this was added 12 g of the reactive coupler obtained above, and the mixture was stirred at room temperature for 8 hours. After neutralizing the reaction solution with dilute hydrochloric acid, 1.5% acetone was added to precipitate starch. The supernatant was decanted and then dissolved in two volumes of distilled water. After dissolution, 5 g of sodium chloride was added and reprecipitated with 1.5 g of acetone. This operation was repeated three times to remove raw materials and low molecular weight compounds. The precipitate obtained was coupler-formed starch, and this precipitate was freeze-dried. The coupler-formed starch obtained here was easily dissolved when it was swollen with water and then made into fine particles using a disperser. It was also uniformly dissolved in a 1% aqueous sodium hydroxide solution, and its absorption spectrum matched that of the reactive coupler. When the same reaction procedure was carried out by changing the reaction charge ratio of the reactive coupler, the reactive coupler was introduced at different ratios, that is, the ratio of the number of reactive coupler molecules to the number of glucose units increased from 1/18 to 1/18. Coupled starches with various ratios in the range of 1/50 were obtained. In order to assay the enzymatic activity of the obtained coupler starch, the color density was measured photometrically using a method similar to that used for Dyamyl-L, a commercially available dyed starch for quantifying amylase. Couplerized starch 460 mg, monopotassium phosphate 140
mg, and 176 mg of dipotassium phosphate were dissolved in 25 ml of distilled water to prepare a couplered starch aqueous solution. Commercially available Fast Red B Salt is used as a coloring liquid.
C-37125 (diazonium salt content approximately 20%) 300
mg was dissolved in 5 g of distilled water and used. Saliva is used as amylase solution for assay at 0.9%.
diluted with an aqueous solution containing 7% sodium chloride and 7% albumin to give 200 Somogyi units/dl.
A sample that was certified as Procedure: Add 1 part of coupler-formed starch solution to two test tubes (A, B).
ml of each sample was weighed out, and 1 ml of Diamil-L dyed substrate solution was weighed out into two other test tubes (C and D). Each test tube was pre-incubated at 37°C for 3 minutes, test tubes A and C were each filled with 100μ of amylase solution for assay, and test tubes B and D were each filled with 100μ of amylase solution for assay.
100μ of an aqueous solution containing 0.9% sodium chloride and 7% albumin was added. Each test tube was then incubated at 37°C for 10 minutes and treated with Dyamyl-L precipitant.
The enzyme reaction of amylase was stopped by adding 4 ml of Precipitant. Next, 100 µm of the diazonium salt coloring solution was added to test tubes A and B to develop color. All test tubes were centrifuged at 3000 rpm for 10 minutes, and the supernatant was exposed to light with a wavelength of 530 nm for A and B, and wavelengths of C and D for 530 nm.
Optical density values were determined by photometry using 540 nm light.

[Table] When similar assay experiments were carried out with couplered starches having different binding ratios to the reactive coupler glucose units, the optical densities of the supernatants corresponding to test tube A varied over a wide range of values from 0.23 to 1.9. It was done. Through this verification experiment, it was concluded that amylase acts normally on this couplered starch. Example 1 Colorless transparent poly(ethylene terephthalate (PET) film (thickness
180 μm) by coating in the following layer order. Color reaction layer A solution 2-methoxy-5-tetradecyloxycarbonylbenzenediazonium tetrafluoroborate 4.0g N,N-dimethylcaprylic acid amide 5.0g N,N-diethyllauric acid amide 1.0g acetonitrile 8.0g dichloromethane 8.0g B Liquid polyacrylamide (average degree of polymerization 18,000)
(5% aqueous solution) 140g Gelatin (10% aqueous solution) 30g p-nonylphenoxy polyglycidol (25%
aqueous solution) 2g bis(vinylsulfonylmethyl)ether (1
% acetonitrile solution) 2g The uniformly dissolved solution A was added to solution B, sufficiently dispersed with a homogenizer, coated with a small coating machine, and dried to form a colored reaction layer. The thickness of the colored reaction layer after drying was 8 μm. Diffusion prevention layer Water 50ml TiO ₂ fine powder 80g p-nonyl phenoxy polyglycidol (25%
Aqueous solution) 0.5g The above mixture was completely ground with a ball mill type grinder, then 300g of 10% gelatin aqueous solution was added to this, mixed lightly, and then applied and dried on the reaction layer to form a diffusion prevention layer. I let it happen. The thickness of the diffusion prevention layer after drying was 8 μm. Substrate layer Coupled starch obtained in Synthesis Example 1 10g Dipotassium phosphate 2.6g Monopotassium phosphate 2.1g Water 105g Polyacrylamide (5% aqueous solution) 80g p-nonylphenoxy polyglycidol (25%
aqueous solution) 2g The above mixture was sufficiently dispersed with a homogenizer,
The solution was dissolved, and the undispersed particles were passed through a nylon mesh, and then applied onto the diffusion prevention layer and dried to form a substrate layer. The thickness of the substrate layer after drying was 10 μm. Developing Layer A polyester/cotton blend fabric (blending ratio polyester/cotton = 75/25) was immersed in an aqueous solution having the following composition to perform hydrophilic treatment. Polyacrylamide (average degree of polymerization 18,000)
(0.8% aqueous solution) 150g p-nonylphenoxy polyglycidol (25%
Aqueous solution) 1g P-nonylphenoxyglycerin was added to the coated surface of the PET film with the substrate already obtained.
After moistening with a 0.2% aqueous solution, this hydrophilized blended fabric was pressed and dried to form a developing layer, completing a multilayer chemical analysis film for measuring amylase activity. This multilayer chemical analysis film showed a color optical density proportional to amylase activity of 30 to 2000 Somogi units/dl in human serum and human whole blood, and it was revealed that amylase activity could be quantified. Example 2 A color reaction layer was provided on a PET film in the same manner as in Example 1. However, the thickness of the color reaction layer after drying was 15 μm. Diffusion prevention layer Gelatin (10% aqueous solution) 30g Polyacrylamide (5% aqueous solution) 70g Nonylphenoxypolyglycidol (25% aqueous solution) 500mg The above mixture was placed on the coloring reaction layer so that the thickness after drying was
It was applied to a thickness of 4 μm and dried. Substrate layer Coupled starch was produced from starch (TAPON) consisting of almost 100% amylopectin using the same method as in Synthesis Example 1, and the substrate layer had a thickness of 10 μm after drying using the same method as in Example 1. A multilayer chemical analysis film was obtained by coating in this manner. Separately, the centrifuged saliva supernatant was mixed with 7% bovine serum albumin as an amylase sample solution with different concentrations.
diluted with an aqueous solution containing 0.9% sodium chloride,
Amylase 56, 120, 550, 1100, 3500 respectively
A standard solution containing Somogi units/dl was prepared. The multilayer chemical analysis film was placed on a hot plate kept at 37°C, and 10μ of the amylase sample solution was gently dropped onto the substrate layer. After 5 minutes, the mixture was brought into contact with paper impregnated with 0.1% zinc chloride to stop the reaction, and the color optical density was measured photometrically. The color optical density (OD) proportional to the amount of amylase activity was obtained as follows. Somogi units/dl 56 120 550 1100 3500 OD 0.35 0.45 0.53 1.04 1.27 When this relationship between amylase content and OD was plotted on a graph, it became clear that amylase content could be quantified using this multilayer chemical analysis film. Ta. Example 3 In the same manner as in Example 1, a color reaction layer, a diffusion prevention layer, and a substrate layer were formed on a PET film by sequential coating. Next, a membrane filter, Fuji Micro Filter FM-500, was pressed as a developing layer in the same manner as in Example 1 to prepare a multilayer chemical analysis film for amylase analysis. Various amylase concentration standard solutions were prepared by adding saliva to a commercially available enzyme standard serum diluted appropriately in the same manner as in Example 2. The amylase activity of each standard solution was assayed using a commercially available amylase activity measuring reagent, Pharmacia Diagnostics' blue starch method, and the results were 36, 61, 136, 236, 530,
It was 856, 1533, 2964 Somogi units/dl. Apply 10μ of each standard solution to the developing layer and then
Incubation was carried out for 10 minutes at 37°C. Next, when the color optical density was measured, a value almost proportional to amylase activity was obtained. Somogi units/dl 36 61 136 236 OD 0.30 0.35 0.43 0.53 Somogi units/dl 530 856 1533 2964 OD 0.68 0.87 1.06 1.25 These results revealed that amylase activity could be quantified using this multilayer chemical analysis film. Example 4 A multilayer chemical analysis film was prepared in the same manner as in Example 1, except that spinner coating was performed with the following composition to form a color reaction layer. 2-Methoxy-5-tetradecyloxycarbonylbenzenediazonium tetrafluoroborate 100mg Diacetyl cellulose 100mg Methylene chloride 5g A 50-fold dilution of saliva was attached to this multilayer analysis film, and the measurement was carried out after incubation at 40°C for 30 minutes. Similar to Example 1, the relationship between color optical density and amylase activity was obtained. Example 5 4-[p-(sodium sulfonato)anilino]-6-[3-p-(sodium sulfonato)phenyl-4,4-dibromo-2-pyrazolin-5-one-3- as couplerized starch [ylamino]-s-triazin-2-yl group-bonded cornstarch was used as the substrate layer, and 1-(2,4-di-t-amylphenoxy)- was used as the chromogen compound.
A multilayer analytical film for quantifying amylase activity was prepared in the same manner as in Example 2 using 2-oxo-6-(4-amino-2-methylphenyl)-3,6-diazaoctane in the coloring reaction layer. It was revealed that this multilayer analysis film developed a magenta color approximately in proportion to amylase activity, and that amylase activity could be quantified.

[Brief explanation of drawings]

1 to 4 are conceptual cross-sectional views of the multilayer chemical analysis material of the present invention. FIG. 1 shows a configuration in which a coloring reaction layer 20 containing a chromogen compound and a substrate layer 50 containing a non-diffusible substrate having a dye-forming group are provided in this order on a light-transmitting and water-impermeable support 10. FIG. 2 is a cross-sectional conceptual diagram of a multilayer chemical analysis material. FIG. 2 is a conceptual cross-sectional view of a multilayer chemical analysis material in which a diffusion prevention layer 30 is provided between a color reaction layer 20 and a substrate layer 50. FIG. 3 is a cross-sectional conceptual diagram of a multilayer chemical analysis material configured as shown in FIG. 2, in which a spreading layer 70 for the purpose of uniformly spreading an aqueous sample solution is further provided on the substrate layer of the multilayer chemical analysis material shown in FIG. It is. FIG. 4 is a conceptual cross-sectional view of a multilayer chemical analysis material having a structure in which a light shielding layer 40 is provided in place of the diffusion prevention layer 30 of the multilayer chemical analysis material having the structure shown in FIG. In FIGS. 1 to 4, the numbers represent the following: 10...Light-transparent water-impermeable support, 20
...Coloring reaction layer, 30...Diffusion prevention layer, 40...Light shielding layer, 50... Substrate layer, 70... Development layer.

Claims (1)

[Claims] 1. A multilayer chemical analysis material in which at least two reagent layers are laminated on a light-transparent water-impermeable support, in which the two reagent layers contain (a) a substantially colorless chromogen compound as described below; It has a dye-forming reactive group capable of reacting to form a dye, and comprises a non-diffusible substrate capable of producing a substantially colorless diffusible compound having said dye-forming reactive group by the action of a test substance. A multilayer chemical analysis material comprising: a substrate layer; and (b) a color-forming reaction layer comprising a chromogen compound capable of reacting with the dye-forming reactive group to form a dye.