JPS6014141A - Monolithically multilayered analyzing implement - Google Patents

Abstract

PURPOSE:To measure rapidly, simply, precisely and accurately a constituent concn. in a sample in spite of a very small quantity of the sample by using a monolithically multilayered analyzing implement in which a reagent layer and a detecting layer are laminated on one surface of photoreflective supporter. CONSTITUTION:In the monolithically multilayered analyzing implement 1, a reagent which is allowed to react with an objective component in a liquid sample and causes an optically detectable variation is dissolved in a binder, and resulting solution is painted on a film or a plate-shaped body to be a supporter 2 by painting apparatus and dried to form the part to be a reagent layer 3. Next, a material for composing the detecting layer 4 is laminated on an upper surface of the layer 3 by welding, etc. to unite the layers to one body. A supporter 2 supports the layers 3 and 4, prevents the further migration of the sample which has been allowed to migrate to the layer 3 through the layer 4 to another part from the layer 3, and acts as a reflector of the measuring light transmitted through the layer 4 in case of measuring an optical variation at the layer 4.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a multilayer integrated analytical device 6 for quantifying components in liquids, particularly body fluids.

A simple test method that has been well known in clinical testing is the so-called test strip method, and among these, urine test strips are widely used. Measuring urine components using urine test strips has become an indispensable screening test because it allows us to detect abnormalities in the body without any burden on the patient.

On the other hand, various test strips have recently been developed for measuring blood components, which require more accurate measurements. This is because test strips are called solid phase reagents in contrast to so-called liquid reagents, and are used to prepare reagents, prepare reaction vessels, etc. in clinical tests.
This is due to the fact that there is no need for preparation processes such as mixing samples and reagents, so there is growing demand in the field of blood biochemistry testing.

However, with conventional paper as a reagent carrier, measurement accuracy is insufficient due to the non-uniformity of the paper white body, and the reagents that can be used are limited due to lack of acid resistance and alkali resistance, which limits the measurement items. There was a drawback.

Therefore, a technique using a water-resistant polymeric substance as a holder for a reagent was developed (Japanese Patent Publication No. 33800/1989).

This involves mixing a reagent that reacts with the target component with a water-resistant polymeric substance and applying the mixture onto a plastic strip such as polyester to produce a water-resistant film. This film surface is more uniform and the measurement accuracy is better than the test paper, but because the surface is non-porous, the dropped sample hardly penetrates inside. Therefore, during measurement, it takes time and effort to remove excess samples, and automatic measurement using a machine is particularly difficult. Furthermore, because the amount of sample permeation is small, it is difficult to achieve high sensitivity, such as blood uric acid? It is not suitable for measuring IIt components.

Therefore, as a method to overcome these drawbacks, a multilayer analytical element as disclosed in Japanese Patent Publication No. 53-21677 was developed. In this method, a reagent is coated on a transparent support to form a reagent layer, and a spread layer made of a non-fibrous porous medium is further provided on top of the reagent layer. This spreading layer supplies a substantially constant amount of the sample per unit area to the reagent layer so that the components to be analyzed in the sample do not undergo chromatographic phenomena. The concentration of the target component in the sample can be determined by dropping the sample onto the spread layer and measuring the coloration generated in the reagent layer from the support side (the opposite side of the spread layer) after a predetermined period of time has elapsed. It is. Similar to this, JP-A-55--16
Multilayer analysis sheet seen in specification No. 4356, JP-A-5
There is a multilayer analytical element found in US Pat. No. 6-24576. The former uses a hydrophilized fabric for the spread layer as described above, and the latter uses a fibrous porous carrier, but the purpose is the same.

Although these multilayer analysis pieces having a spread layer have generally solved the drawbacks described above, they have created the following new problems.

First of all, the shape of the dropped sample spreads radially, which requires a relatively large area of the reagent layer, which not only causes economic loss of reagents and other materials, but also reduces the amount of components targeted for analysis in the sample. However, it is extremely expensive because it requires a spreading layer with a special structure so as not to cause the chroma I phenomenon. Second, since the sample is dropped from the top surface and photometry is performed from the bottom surface, the transparent support on the bottom surface should be free from scratches and blemishes that would cause optical noise, and a separate test chip should be used to prevent scratches and stains. Requires a protective holder. Furthermore, since the light that has passed through the reagent layer is not sufficiently reflected by the spread layer, there is a drawback that the measurement sensitivity is poor. Thirdly, since the reaction is carried out within the gel, the reactivity is poor and a relatively long reaction time is required.

In order to correct the second drawback mentioned above, a porous metal film with good light reflection efficiency was developed between the reagent layer and the spread layer (Japanese Patent Laid-Open No. 55
-26428) and metal masu (Japanese Unexamined Patent Publication No. 55-26429)
However, these measures make analytical elements increasingly complex, lowering productivity and increasing costs.

By the way, solid-phase reagents, including conventional ones that use paper as a reagent holder, those that use film as a holder, and those that have the various multilayer structures mentioned above, have the following two essential drawbacks. .

First, measurements using solid-phase reagents do not require a mixing step, and it is difficult to react with the sample and reagent in a completely mixed state, making it difficult to perform measurements with high reproducibility. Next, among the various components that are required to be measured in clinical tests, some are contained in relatively large amounts in the sample, while others are contained in extremely small amounts, but the ratio of the amount of the sample to the reagent is Since it is naturally limited, it can only be applied to the measurement of components within a limited concentration range.

The purpose of the present invention is to significantly improve such essential drawbacks, to provide a liquid sample analysis tool that can measure a small amount of sample easily, quickly, and precisely. The purpose is to provide an excellent multilayer analysis tool.

In order to achieve this objective, the present inventors have conducted intensive research and found that on one side of the light reflective support or support system,
A reagent layer containing a reagent that causes an optically detectable change by reacting with a target component in a liquid sample, and which becomes a solution or a sol by the solvent of the sample liquid; We have developed a multilayer integrated analysis tool that has a detection layer that has the function of uniformly diffusing optically detectable substances produced or reduced in the reagent layer as well as transferring them to the reagent layer.

The features of this analysis tool are: firstly, the reaction with the sample, which conventionally takes place in the reagent layer, and the retention of the reaction mixture are separated, and the latter takes place in the detection layer; and secondly, the reaction to the detection layer In order to ensure uniform and smooth diffusion of the resulting product, the reaction in the reagent layer is performed in a solution or sol state. According to the analysis tool of the present invention, the dropped sample reaches the reagent layer while being spread somewhat in the detection layer, and spreads laterally at the interface while melting each component of the reagent layer, quickly uniforming the entire reagent layer. melt or sol. In the reagent layer, a predetermined reaction proceeds smoothly, and a reaction mixture containing an optically detectable substance is easily diffused into the detection layer by capillary action. Here, the optically detectable substance is generated or decreased depending on the amount of the target component in the sample, so it can be detected in advance by irradiating the detection layer with light that is absorbed by the substance and measuring the amount of reflected light. The concentration of the target component can be determined from the established calibration curve.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The analysis tool of the present invention will be described in detail below based on embodiments shown in the drawings.

FIG. 1 shows an example of a multilayer integrated analysis tool (1) according to the present invention. In this case, a solution in which a reagent that reacts with the target component in a liquid sample and causes an optically detectable change is dissolved in a binder is coated onto a film or plate-like material that will become the support (2) using a well-known coating device. The part that will become the reagent layer (3) is formed by coating and drying, and then the material constituting the detection layer (4) is laminated and integrated by welding etc. on the upper surface of the part that will become the reagent layer (3). , with predetermined dimensions (e.g. 5 x 10m)
Cut into m square).

The analysis tool (11) may be fixed to the upper surface of the tip of a strip-shaped holder (5) with double-sided adhesive tape (6) as shown in Fig.
Multi-layer integrated analysis tool (1) that is convenient to use when fixed to
′ is obtained.

The support (2) in the present invention supports the reagent N (3) and the detection layer (4), and passes through the detection layer (4) to the reagent layer (3).
In addition to preventing the sample that has migrated from the reagent layer (3) from migrating to other parts, it also reflects the measurement light that has passed through the detection layer (4) when measuring optical changes in the detection layer (4). It also functions as a body. Note that since a part of the light irradiated to the detection layer (4) passes through the detection layer (4), the amount of change in light can be increased by reflecting this light on the support (2). Therefore, the support (2) reflects the measurement light to a certain extent, and the reagent N (3) and the detection layer (4)
It is preferable that a material that can support this property alone has this property, but a composite material may also have this property. Metals, plastics, glass, ceramics, etc. can be used as materials that have this property alone, but white plastic films and sheets are suitable from the viewpoint of processability and economy, and among them, polyester, polypropylene, polychloride, etc. A white vinyl film or sheet is preferred. As mentioned above, it is preferable to use a single material as a support, but a material that can support a reagent layer, such as a transparent polyester, polypropylene, or polystyrene film, may be coated with a white paint or a white paint to have a light-reflecting function. It is also possible to provide the support as a whole with the above-mentioned properties by not pasting film or paper together.

Furthermore, this light reflecting function can be achieved by using the holder (51) as shown in Fig. 2 or 3.
, the entire support system (7) including the support (2) and fixing means such as double-sided adhesive tape (6) may be provided.

That is, as long as the holder (5) or tape (6) has a light reflecting function, the support (2) may be made of transparent plastic film or sheet. As the material for the holder (5), in addition to the same material as the support (2), cardboard or the like may be used. However, if the support (2) itself is expanded to form a holder-like structure as shown in FIG. 4, the support (2) itself must have a light reflecting function. In the case of Fig. 4, the reagent layer (3) and the detection layer (
4) was directly laminated on a part of a sheet, etc., and cut into an appropriate size to obtain a multilayer integrated analysis tool (1).

Next, the reagent layer (3) consists of a reagent system that reacts with the target component to produce an optically detectable change, and a binder that holds the reagent system. Typical reagent systems include glucose oxidase and peroxidase for glucose measurement, for example.

Combination of oxidizable chromogen and p11 buffer; combination of diazobenzenesulfonic acid and organic acids for bilirubin measurement; alanine-α-ketogeltaric acid, pyruvate oxidase, and oxidizable chromogen for measurement of glutamic acid pyruvate transaminase. body.

Examples include a combination of pH buffers, a combination of pyruvate, reduced nicotinamide adenine dinucleotide (NADH), and pl+buffer for measuring lactate dehydrogenase, and a combination of urease and a pH indicator for measuring urea.

On the other hand, the binder is preferably one that holds the reagent and dissolves in or forms a sol with the liquid sample, such as hydrophilic polymers that are solid at room temperature, particularly sodium alginate, polyvinyl alcohol, and polyvinylpyrrolidone. Polysaccharides such as gelatin and agar are also useful, but they gel at room temperature, and even when a liquid sample is supplied, they do not dissolve and are included in the gel. It is preferable to carry out the reaction in a state where the temperature is raised to transform into a sol. However, in the case of the present invention, it is not an essential element that the reagent layer (3) is dissolved or solified by the liquid sample, and even if the binder is a hydrophobic polymeric substance or something that gels, it is not necessary to react and mix it with the detection layer (4). There is no change in the fact that the liquid diffuses, and it can be used even in these cases. However, dissolution or sol formation is advantageous in that the reaction can be easily carried out and the diffusion into the detection layer can be carried out quickly.

The detection layer (4) allows the dropped liquid sample to reach the reagent N (3) while slightly spreading it, spreads it laterally while dissolving each component of the reagent layer, and detects optical It is constructed of a material that receives a reaction mixture containing a detectable substance while uniformly diffusing it, and can be optically measured from the surface, that is, the side on which the sample is dropped. Examples of materials that absorb the analyte and diffuse the reaction mixture are membrane filter paper, cloth, nylon mesh, ceramic, metal mesh, and glass fiber filter paper, which meet this condition, but they are uniform and good. Chromatography filter paper, membrane filter, or cotton broadcloth is suitable because of its good diffusibility and processability.

Therefore, when using the analysis tool of the present invention, first, a certain amount (for example, 10~) of the sample is dropped onto the detection layer (4).
(A direction in Figure 1). Then, the sample reaches the reagent layer while expanding somewhat within the detection N (4), and spreads laterally at the interface while melting each component of the reagent layer.

Reagent N (3) reacts with the component to be analyzed in the form of a solution (or sol), and the reaction mixture containing optically detectable substances produced or reduced in the reagent layer is detected by capillary action. It is uniformly diffused into layer (4).

In this state, light that is absorbed by an optically detectable substance is
The target component is quantified by irradiating the detection M (4) from the direction and measuring the amount of reflected light.

The multilayer integrated analysis tool of the present invention has been described above, but
Examples are given below to further clarify the content of the present invention. Of course, this does not limit the invention.

Example 1. (Analysis tools for measuring uric acid) Uricase・・・・・・・・・・・・・・・・・・・・・
・・・・・・・・・・・・・・・・・・ 200mg peroxidase・・・・・・・・・・・・・・・・・・
・・・・・・・・・ 300mg 4-aminoantipyrine・
・・・・・・・・・・・・・・・・・・ 200mgN-
Ethyl-N(2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt・・・・・・・・・・・・・・・
...500mg polyvinylpyrrolidone...
・・・・・・・・・・・・・・・ 10g Triton X
−100−−−・・−−−−・−100 mgo, 1M phosphate buffer・・・・・・・・・・・・・・・・・・・・・
... 100 The reagent with the above formulation was uniformly coated on a white polyester film (manufactured by Toshi) to serve as a support to a coating thickness of 0.2 mm in a width of 501, and then dried at 45°C for 3 hours. to form a reagent layer. Next, wet the surface of the reagent layer with a membrane filter [Toyo Roshi side type: TM
-500) was crimped and then cut into 5 x 10 mm squares to obtain analysis tools. Next, this analysis tool was attached to the tip of a 5 x 70 mm polyethylene film holder using double-sided tape for convenient use. Five types of serum lOμ2 with known uric acid concentrations were added to this, and the light reflectance at 560 nm was measured 4 minutes after dropping [Measuring instrument: 2208 minutes light intensity with 1 integrating sphere for measuring reflectance manufactured by Hitachi, Ltd. Total]
However, the reflectance corresponding to the uric acid concentration was obtained as shown in Table 1. A standard curve showing the relationship between the uric acid concentration in the sample and the reflectance is shown in FIG.

Table-1 Example 2. (Analytical tool for measuring bilirubin) Sulfosalicylic acid ・・・・・・・・・・・・・・・・・・・・・
・・・ 6.0g sulfanilic acid ・・・・・・・・・
・・・・・・・・・・・・・・・ 1.0 g Sodium nitrite ・・・・・・・・・・・・・・・・・・・・・
...0.5 g carboxymethyl cellulose...
・・・・・・・・・ 1.5 g purified water ・・・・・・
・・・・・・・・・・・・・・・・・・ 100 m
A multilayer integrated analysis tool was prepared in the same manner as in Example 1 using the reagents with the above formulation. Two types of patient serum were dropped into this analysis tool, and the reflectance at a measurement wavelength of 565 nm was measured 10 times simultaneously after 3 minutes. As a result, good reproducibility was observed as shown in Table 2. .

Table-2 Furthermore, Eheri 7-? Roy method (J, Biol, Chem,
119,481゜1937), the concentration of bilirubin at both ends was investigated and A was 3.2 mg/d! , B is 9.1
mg/J.

Example 3. (Analysis tool for measuring lactate dehydrogenase) NAD
H (reduced nicotinamide adenine dinucleotide)... 60mg lithium pyruvate...
・・・・・・ 201I1g bridge 35 ・・・・・・
・・・・・・・・・・・・・・・・・・・・・・・・
...1.0g polyvinyl alcohol (#50
0)・・・・・・・・・15g0.1M phosphoric acid 1h
Solution (pH 7.5) --.=100ml A multilayer integrated analysis tool was produced in the same manner as in Example 1 using the reagents with the above formulation. Two types of control serums (Marchenzyme, manufactured by Hyland) were dropped into this analysis tool, and the reflectance at a measurement wavelength of 340 nm was measured after 1 minute. As a result, good reproducibility was obtained as shown in Table 3. Admitted. The displayed values of both control serums are 373 U// for A and 643 U// for B.
It was U/l.

Table 3 Example 4 (Analysis tool for glucose measurement) Glucose oxidase ・・・・・・・・・・・・・・・・・・
1ooa+g peroxidase・・・・・・・・・
・・・・・・・・・200mg 4-aminoantipyrine
・・・・・・・・・・・・・・・・・・10 axis g Sodium alginate (30 cps)・・・・・・・・・ 3
g3,5 dimethoxy-N-ethyl N-(1-hydroxy-3-sulfopropyl) aniline, sodium salt ・・・・・・・・・・・・・・・
・・・・・・300mg Tween 20 ・・・・・・・・・
・・・・・・・・・・・・・・・・・・・・・・・・
・100mgo, 1M citrate buffer (pl+ 5.5
)・・・・・・・・・1OOI Coat the above-mentioned reagent on a white polyester film [manufactured by Toshi@] to a thickness of 0.2 mm.
The reagent layer is coated uniformly to a width of 20 mm and then dried at 40° C. for 2 hours to form a reagent layer. Next, the surface of the reagent layer was lightly moistened with a 2 cm wide cotton cloth [Toyobo θm! ! The material was pressed onto the reagent layer and laminated. Further, the reagent portion is cut into 2 pieces along the coating direction, and then cut into 51 pieces perpendicular to the coating direction. In this way, the detection layer and reagent layer are 5 x 10 mm.
A multilayer integrated analysis tool for glucose measurement was fabricated.

10μ of various glucose aqueous solutions were dropped into this analysis tool, and the light reflectance after 3 minutes was measured in the same manner as in Example 1. A calibration curve showing the relationship between glucose concentration and light reflectance is shown in FIG.

10 μl of serum from 30 patients was dropped into this multilayer integrated analysis tool, and the reflectance was measured after 3 minutes, and the concentration was determined using the calibration curve shown in FIG. At the same time, measurements were also carried out using a glucose analyzer based on the C0D-oxygen electrode method [■ Kyoto Hako Kagaku Co., Ltd., trade name "Glucolizer"], and the relationship between the two was investigated. The results are shown in FIG. The correlation coefficient between the two is 0. Q92, the regression linear equation was y = (1,99x -0,6, and good consistency was observed. (The value measured by this analysis tool is y, and the value measured by the glucose analyzer is X.) Implementation Example 5. (Analytical tool for measuring glucose) 3 g of sodium alginate in the formulation of Example 4 was added to 15 g of gelatin.
A multilayer integrated analysis tool was produced by changing to g. During measurement, when the sample was incubated at 37°C immediately after dropping, the gelatin in the reagent layer was transferred from gel to sol and fluidized, and the blue substance generated in the reagent layer was diffused into the detection layer, increasing the glucose concentration in the sample. A blue color corresponding to the color was observed.

Then, as in Example 4, serum concentrations of various concentrations of glucose were measured 15 times using a calibration curve using a glucose standard solution prepared in advance. The coefficient of variation (CV%) was 1 to 1. It was 3%.

Although the preferred embodiments of the present invention have been described above, various changes can be made within the scope of the technical idea of the present invention.

First, any support may be used as long as it is light reflective and can maintain the strength of the analysis tool. Of course, the structure is not limited to a single layer, but may be a multilayer structure such as a transparent plastic film or sheet coated with paint or a colored plastic film not pasted together. Furthermore, in the case of an analysis tool (1)' using a holder (5), the holder (5) may be light reflective and a transparent plastic film or sheet may be used as the support (2). In short, if there is a boulder, it is sufficient that the entire support system including the boulder and fixing means such as adhesive tape has the above-mentioned properties.

Incidentally, the shape of this boulder (5) may be rectangular as shown in FIG. In this case, the analysis tool (1) may have the same shape as the holder (5).

Alternatively, as shown in Figure 4, the support (2) itself can be expanded to serve as a holder, and the reagent layer (3) and detection layer (4) can be directly attached to a part of a sheet, etc. that is thicker than the one in the previous example. Multilayer integrated analysis tools (11" may be made by laminating and cutting into appropriate sizes.Other analysis tools (1), (1)', (1)
Various shapes can be considered, such as a continuous tape shape, a slide shape with a frame, etc., depending on the device to be combined and the purpose.

On the other hand, the binder for reagent N(3) may be any binder as long as it can hold the reagent. The material used for the detection layer may be any material that is permeable to the sample and capable of retaining the reaction mixture containing the reagent layer and the optically detectable substance of the sample. Further, a wetting agent or a surfactant may be added to the reagent layer and the detection layer as desired.

As described in detail above, the multilayer integrated analysis tool of the present invention includes a support or a support system that is impermeable to liquid and has light reflective properties, a support that is located on one side of the support system, and a liquid sample. A reagent layer containing a reagent that reacts with the components therein to produce an optically detectable change, and a reagent layer located on the opposite side of the surface of the reagent layer that is in close contact with the support, so that the reagent can be optically detected. It is characterized by a stack of optical detection layers that uniformly diffuse substances.

Therefore, the analysis tool of the present invention does not require removal of excess sample, can uniformly transfer the sample to the reagent layer without the need for a spreading layer that requires complicated and delicate manufacturing conditions, and can be optically detected. Since the substance can be uniformly diffused into the detection layer, the concentration of the target component in the sample can be accurately determined by simply dropping the sample and waiting for the required time. Furthermore, by simply changing the thickness and material of the detection layer, the ratio between the amount of reagent and the amount of sample can be adjusted depending on the relative amount of the target component, even though it is a solid phase reagent.

In addition, since a hydrophilic polymer that dissolves or becomes a sol in the liquid sample is used as a binder in the reagent layer, the reaction with the component to be analyzed proceeds in the melt or sol state, which is different from conventional multilayer analysis elements. Because the reactivity is higher than when reacting in a gel state, the accuracy of analysis is improved and even trace components can be analyzed accurately.

As described above, according to the present invention, a small amount of sample can be used to quickly and
The concentration of components in a sample can be measured easily, precisely, and accurately.
Moreover, it makes it possible to manufacture a multilayer test tool with high productivity, which is excellent in processability and economical efficiency, and its practical value is extremely large.

[Brief explanation of the drawing]

FIG. 1 is an enlarged sectional view showing an example of a multilayer integrated analysis tool according to the present invention, FIGS. 2 and 3 are perspective views of the multilayer integrated analysis tool in which the analysis tool shown in FIG. 1 is attached to a holder,
Fig. 4 is a perspective view showing an example of an analytical tool in which a reagent layer and an optical detection layer are directly laminated on a holder-shaped support, Fig. 5 is a standard curve showing the relationship between uric acid concentration and reflectance, and Fig. 6 is a calibration curve showing the relationship between glucose concentration and reflectance, and FIG. 7 is a graph showing the correlation between the measured values by the analysis tool of the present invention and the measured values by the glucose analyzer based on the C0D-oxygen electrode method. . ■・１′・１″・・・・・・Multilayer integrated analysis tool 2・・・・
... Support 3 ... Reagent layer 4 ... Optical detection layer 5 ... Boulder 6 ... Double-sided adhesive tape 7 ... ...Support system A...Sample dropping direction and measurement direction 1st 3rd 4th figure (R'/ρ 5th uric acid 1st grade 60 (R0/.) (Secret procedure) Masaru Ichi: (Voluntary) September 21, 1981 1. Indication of the case 1988 Patent Application No. 122690 2. Name of the invention Multi-layer integrated analysis tool 3. Address: 57 Nishiakeda-cho, Higashikujo, Minami-ku, Kyoto Name: Kyoto Daiichi Science Co., Ltd. Representative Kozo Tamura, Department 4, Agent address: 2-3-1o Tenjinbashi, Kita-ku, Osaka Voluntary IIi
Correction 6, Statement of the subject of amendment 7, Detailed explanation of the invention section, page 23, line 2 of the description of the contents of the amendment, "Optically detectable substance as a reagent" has been added to the procedural amendment (own ship 1, Display of the case 1982 Patent Application No. 122690 2 Name of the invention Multi-layer integrated analysis tool 36 Person making the amendment Relationship to the case Patent applicant Address 57 Higashikujo Nishiakeda-cho, Minami-ku, Kyoto Name Kyoto Daiichi Co., Ltd. Scientific Representative: Kozo Tamura, Department 4, Agent Address: 6-6, 2-3-10 Tenjinbashi, Kita-ku, Osaka, ?ii Correct Subject Specification 7, Statement of Contents of Amendment, Page 10, Line 8 [Wanai. Correct -1 to "Wanai."

Claims (1)

[Claims] ] is used for spectrophotometric analysis of a liquid sample, wherein one surface of a light-reflective support or support system is coated with an optically reflective material that reacts with components in the liquid sample. A reagent layer consisting of one or more layers containing a reagent that causes a detectable change, and further comprising an optically detectable substance produced or reduced in the reagent layer on the side of the reagent layer opposite to the support or support system. A multilayer integrated analysis tool characterized by laminated detection layers for uniform diffusion. 2. The multilayer integrated analysis tool according to claim 1, wherein the binder of the reagent layer is a hydrophilic polymer that dissolves or becomes a sol in the solvent of the liquid sample.