JP2000241433A - Integrated multi layered chemical analyzing element for quantitatively determining direct reacting bilirubin and quantitatively determining method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a highly sensitive integrated multi layered chemical analyzing element easy to operate and excellent in secular storage property for quantitatively determining direct reacting bilirubin in a liquid specimen, and a quantitatively determining method for the direct reacting bilirubin. SOLUTION: An integrated multi layered chemical analyzing element uses a system for determining quantitatively direct reacting bilirubin in a liquid specimen with diazo method. The integrated multi layered chemical analyzing element has a translucent support body 11 and a developing layer 31 made of a porous material with void ratio ranging 30 to 80% containing diazonium salt, 1,5-naphthalenedisulfone or its derivative, water-soluble zinc salt, and acid polymer as compositions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an integrated multi-layer chemical analysis element for direct bilirubin determination for determining direct bilirubin contained in a liquid sample such as plasma or serum.

[0002]

2. Description of the Related Art There are direct and indirect types of bilirubin in body fluids such as plasma and serum. Direct bilirubin is mainly a glucuronide of bilirubin in which one or two of the carboxyl groups of bilirubin is ester-bonded to glucuronic acid (conjugated type), and indirect bilirubin is free (unconjugated type) without such ester bond or the like. Is bilirubin. Total bilirubin is a general term for direct bilirubin and indirect bilirubin.

[0003] Blood bilirubin, which is measured as a clinical test at the time of disease, includes blood bilirubin concentration and its fractionation.
Urinary bilirubin and urobilin, urobilin in feces, etc. are the main. Among them, the dynamics of blood bilirubin are the most important. Diagnosis of diseases such as hepatitis, cirrhosis and biliary obstruction, and differentiation of obstructive jaundice It is important to.

[0004] The method of quantitative determination of bilirubin in blood by the wet method starts with a quantitative method utilizing a diazotization reaction by HIJMANS VAN DEN BERGH and the like, and is followed by KUL.
Many quantification methods are known, including the improved method by HANEK and the like, but all of these quantification methods are directed to total bilirubin and cannot directly quantify bilirubin. As a method for directly quantifying bilirubin,
A method using p-sulfobenzenediazonium-1,5-naphthalenedisulfonic acid salt is disclosed (JP-B-63-22262). According to this method, 1,5-naphthalenedisulfonic acid salt forms an ion pair with p-sulfobenzenediazonium salt having high specificity for bilirubin directly in the reaction system, and becomes the above-mentioned stable diazonium salt. However, in this method, it is practically difficult to directly separate and measure bilirubin from total bilirubin (particularly, it is remarkable for a sample containing no albumin or a sample containing a drug such as salicylic acid). Therefore, p-sulfobenzenediazonium salt has a problem that it cannot be stored for a long period of time because it is unstable.

Further, Japanese Patent Application Laid-Open No. Hei 5-276992 discloses an enzymatic method using bilirubin oxidase in the presence of a bilirubin measuring reagent. However, this method has problems such as insufficient fractionation measurement and stability of bilirubin oxidase, which have been pointed out above.

[0006] High performance liquid chromatography (HPLC)
Also known is a method for fractionation and quantification (Isra
elJournalofChemistryVol. 2
3, page 241-247, 1983). Although this method has a sufficient separation ability, it has problems such as the use of expensive and special equipment and the limitation of the number of samples that can be processed at one time.

The above-mentioned methods are all wet methods,
It is difficult to say that simplicity in use and quickness of analysis are sufficient.

On the other hand, JP-A-59-171864 and JP-A-61-71363 disclose a dry analytical element and a quantification method for measuring total bilirubin. These all use the diazo method,
The analytical element has, in this order, a light-transmissive support, a water-absorbing layer, and a porous reagent layer comprising a diazonium salt, a soluble substance (7- (2,3-dihydroxypropyl) theophylline), an arylsulfonic acid and an acidic polymer. By including a soluble substance, a sample containing bilirubin can be solubilized, and indirect bilirubin can be quantified. Acidic polymers have been used to improve the stability and shelf life of diazonium salts. Aryl sulfonic acids are also used to enhance the effect of this acidic polymer. However, the dry analytical elements described in these publications are:
The method is intended for total bilirubin, and has a problem that sufficient storage stability over time cannot be obtained and the measurement sensitivity and measurement accuracy are low with the above-mentioned coating amount.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a highly sensitive method for directly quantifying bilirubin in a liquid sample.
An object of the present invention is to provide an integrated multilayer chemical analysis element which is easy to handle and has excellent storage stability over time, and a method for directly determining bilirubin.

[0010]

According to the research of the present inventors,
An integrated multilayer chemical analysis element using a system for directly quantifying bilirubin in a liquid sample by a diazo method, comprising a light-transmitting support, a diazonium salt, 1,5-naphthalenedisulfone or a derivative thereof, a water-soluble zinc salt and The porosity containing the composition containing the acidic polymer is 30 to 80%.
It has been found that an integrated multilayer chemical analysis element characterized by having a spreading layer made of a porous material in the range described above can solve the above problem.

Preferred embodiments of the integrated multilayer chemical analysis element of the present invention are as follows. (1) An integrated multi-layer, wherein the spreading layer is provided on a light-transmitting support via an adhesive layer containing 1,5-naphthalenedisulfonic acid or a derivative thereof and a water-soluble zinc salt. Chemical analysis element. (2) An integrated multilayer chemical analysis element, wherein the water-soluble zinc salt is zinc sulfate. (3) An integrated multilayer chemical analysis element, wherein the acidic polymer is present in the spread layer at a coating amount of 3 to 10 g / m ² . (4) An integrated multilayer chemical analysis element, wherein the porous material is a knit.

The present invention is also characterized in that a liquid sample is spotted on the integrated multilayer chemical analysis element described above, and the color density in the analysis element is measured at a wavelength in the range of 550 to 670 nm. There is also a method for direct bilirubin determination in liquid samples.

[0013]

DETAILED DESCRIPTION OF THE INVENTION In the present invention, the concentration of direct bilirubin in a liquid sample is determined using a diazo method for colorimetrically determining the concentration of azobilirubin produced by the reaction of a diazonium salt with direct bilirubin in the sample. Quantify. The present invention provides a conventional integrated multilayer chemical analysis element, in which a water-soluble zinc salt is allowed to coexist in a developing layer of a porous material comprising a diazonium salt, an arylsulfonic acid and an acidic polymer, and has sensitivity,
It is characterized by improved accuracy and storage stability. In addition, the coating amount of the acidic polymer is increased,
The use of 5-naphthalenedisulfonic acid or a derivative thereof is also characterized in that higher storage stability was obtained.

FIG. 1 shows a schematic view of an integrated multilayer chemical analysis element of the present invention. The analysis element was a light-transmitting support (1
1) and a spreading layer (31) made of a porous material containing a composition containing a diazonium salt, 1,5-naphthalenedisulfonic acid or a derivative thereof, a water-soluble zinc salt and an acidic polymer. The spreading layer (31) is preferably provided on the light-transmitting support (11) via the adhesive layer (21).

The light-transmitting support is a water-impermeable light-transmitting support. Examples of the water-impermeable, light-permeable support which can be used in the present invention include JP-A-55-164356.
And those used for the support of the multilayer chemical analysis element described in Japanese Patent Application Laid-Open Publication No. H10-163, etc. can be used. The material of the support is a poorly or hydrophobic polymer such as polyethylene terephthalate, cellulose acetate, polycarbonate of bisphenol A, polystyrene, polymethyl methacrylate, or transparent glass. Preferably, it is polyethylene terephthalate. The support preferably has self-holding properties and satisfies the suitability for production. Specifically, in the case of a polymer support, its thickness is preferably in the range of 50 μm to 1 mm, and more preferably in the range of 80 to 400 μm. 100 μm to 2 mm for transparent glass
, And more preferably in the range of 150 μm to 1 mm.

The surface of the support may be subjected to a known physicochemical treatment (such as ultraviolet irradiation treatment, corona discharge treatment, or glow discharge treatment) if necessary, to further enhance the adhesive force with the reagent layer. Alternatively, a physicochemical treatment can be performed (or not), and a gelatin undercoat layer can be further provided to increase the adhesive force with the reagent layer.

The adhesive layer may be used if necessary.
It is made of a polymer having a function of bonding the support and the spreading layer and having a hydrophilic film forming ability. As the hydrophilic polymer, the hydrophilic polymers described in JP-A-55-164356, JP-A-57-208997 and the like can be used. It is preferable to use a neutral or acidic organic polymer substance. Specific examples include polyvinyl alcohol (PVA), gelatin, gelatin derivatives, pullulan, pullulan derivatives, agarose, polyvinylpyrrolidone, and polyacrylamide. It is particularly preferred to use PVA. The adhesive layer is made of 1,5 as described later.
-It is preferable to include naphthalenedisulfonic acid or a derivative thereof and a water-soluble zinc salt. The thickness of the adhesive layer is
It is preferably in the range of 1 to 500 μm,
More preferably, it is in the range of 0 μm.

The spreading layer is made of a porous material containing a composition containing a diazonium salt, 1,5-naphthalenedisulfonic acid or a derivative thereof, a water-soluble zinc salt and an acidic polymer. The thickness of the spreading layer is preferably in the range of 1 to 500 μm, and more preferably in the range of 5 to 300 μm.

The diazonium salt causes a coupling reaction with bilirubin directly in the developing layer. The integrated multilayer analytical chemistry of the present invention does not recognize indirect bilirubin. Examples of the diazonium salt include benzenesulfonic acid diazonium salt, dichlorobenzenediazonium salt, N-substituted sulfonamidobenzenediazonium salt, dichloronitrobenzenediazonium salt, substituted biphenylbisdiazonium salt, and alkoxycarbonyl group described in Japanese Patent Application No. 58-45547. It is preferable to use an aryldiazonium salt or the like containing an alkylaminosulfonyl group, an alkylaminocarbonyl group or the like, and it is particularly preferable to use a diazonium salt of benzenesulfonic acid. The diazonium benzenesulfonate is formed by the reaction between sulfanilic acid and sodium nitrite in the developing layer. The amount of the diazonium salt described above, which is generated in the spreading layer and directly reacts with bilirubin, is preferably in the range of 0.09 to 0.26 μmol.

A water-soluble zinc salt is added to stabilize the above diazonium salt. Arylsulfonate, tetrafluoroborate, hexafluorophosphate, etc. (JP-A-61-71363);
5-Naphthalenedisulfonic acid salt (JP-B-63-2226)
No. 2) is used for forming a counter ion with a diazonium salt to stabilize the diazonium salt, but these methods cannot be used in the present invention. In the present invention, a water-soluble zinc salt is used. It is presumed that this water-soluble zinc salt does not form a counter ion and contributes to stabilization, but contributes as a central metal ion having a diazonium ion as a ligand. A water-soluble zinc salt is one that dissociates into zinc ions in the analytical element.
Either can be used. The anion constituting the water-soluble zinc salt is preferably a sulfate ion, a chloride ion or a nitrate ion. As the water-soluble zinc salt, it is preferable to use zinc sulfate or zinc chloride, more preferably zinc sulfate, and particularly preferably zinc heptahydrate. The applied amount of the water-soluble zinc salt in the spread layer is about 1.6 to 3 g / m ^{2 in} a dry state after the application.
Is preferably within the range.

1,5-Naphthalenedisulfonic acid or a derivative thereof is the same arylsulfonic acid in that it is used to stabilize and stabilize azobilirubin formed in the spreading layer and make it difficult to diffuse and diffuse. Its role is different from that of sulfosalicylic acid (JP-A-61-71363), which is used to improve the stability and storage stability of diazonium salts. In addition, 1,5-naphthalenedisulfonic acid or a derivative thereof in the adhesive layer also contributes to stabilization of azobilirubin. The 1,5-naphthalenedisulfonic acid or a derivative thereof may be a 1,5-naphthalenedisulfonic acid salt or an ester thereof, but is preferably superior in hydrophobicity to sulfosalicylic acid and the like. The substitution position of the sulfone group of 1,5-naphthalenedisulfonic acid or a derivative thereof is not limited to this position. As 1,5-naphthalenedisulfonic acid or a derivative thereof, disodium 1,5-naphthalenedisulfonic acid is preferably used. The amount of disodium 1,5-naphthalenedisulfonic acid applied in the spread layer is preferably in the range of about 0.4 to 0.8 g / m ² in a dry state after application.

For the acidic polymer, see JP-A-59-1.
Nos. 71864 and 61-71363. selected from the group consisting of p-styrenesulfonic acid, acrylic acid, methacrylic acid, maleic acid, maleic anhydride, maleic acid monoamide, maleic acid monoester, N- (sulfoalkylacrylamide) and N- (sulfoalkylmethacrylamide) Or a copolymer of a monomer having one or more unsaturated bonds, or a copolymer of the above-described monomer and another monomer having an unsaturated bond. The acidic polymer has an effect of improving the storage stability and stability of the diazonium salt. The acidic polymer is preferably a polymer containing a sulfonic acid group (sulfoalkyl group or sulfophenyl group) in the repeating unit. This repeating unit is preferably contained in the polymer at least 10 mol% (more preferably, at least 30 mol%). Following general formula (I): (I) - a sulfonic acid group represented by _{- [C 2 H 3 (COOC} 3 H 6 OH)] 6 - [C 2 H 3 (CONHC 4 H 8 SO 3)] 4 The polymer having the repeating unit is particularly excellent in protecting the diazonium salt, and it is particularly preferable to use the polymer.

The amount of the acidic polymer to be applied is in the range of about 3 to 10 g / m ^{2 in} the developing layer after drying (10 to 10 g / m ² of the coating solution).
~ 15% by weight). When the amount of the polymer is about 3 g / m ² or less, it is estimated that a problem occurs in practicality.

The porous material has a porosity of 30 to 80%.
If it is in the range, it may be fibrous or non-fibrous. Examples of the fibrous porous material include a woven or knitted fabric, a nonwoven fabric, a filter paper, and an aggregate of short fibers. A typical example of the non-fibrous porous material is a membrane filter. A dispersion in which silicate or the like is dispersed in a binder, or an aggregate in which glass, resin, or the like is adhered to each other can also be used. It is preferable to use a fibrous porous material as the porous material. It is preferable to use a woven or knitted fabric as the fibrous porous material. Here, the woven or knitted fabric includes any woven or knitted fabric or the like.

As the woven fabric, a plain woven fabric woven with warp and weft yarns is preferred. As the yarn constituting the woven fabric, natural fibers such as cotton, kapok, flax, hemp, ramie, silk, wool,
Examples include chemical fibers such as nylon, tetron, rayon, cupra, acetate, vinylon, acryl, and polyethylene terephthalate. These may be used in combination of two or more. The thickness of the fiber depends on its type, but is preferably in the range of about 20 to 300 denier, and more preferably in the range of about 40 to 130 denier. As the form of the yarn, a filament yarn, a spun yarn or the like is preferable, and a spun yarn is more preferable. Preferably, the thickness of the fabric is in the range of about 100-500 μm, more preferably in the range of about 120-350 μm.

The knitted fabric is preferably a warp knit or a side knit. Examples of the fresh knitted fabric include a single atlas knitted fabric, a tricot knitted fabric, a double tricot knitted fabric, a Miranese knitted fabric, and a raschel knitted fabric. Examples of the weft-knitted fabric include plain knitted fabric, pearl knitted fabric, rubber knitted fabric, double-sided knitted fabric, and the like. The form of the yarn and the yarn constituting the knitted fabric is the same as that of the woven fabric. The thickness of the fiber depends on its type, but is preferably in the range of about 35 to 150 denier, more preferably in the range of about 45 to 120 denier. The number of gauges in the knitting process of the knitted fabric is preferably in a range of about 20 to 50. Preferably, the thickness of the knit is in the range of about 100-600 μm, more preferably in the range of about 150-400 μm. Porosity is 30-80%
It is preferable to use a tricot knitted fabric, a raschel knitted fabric, a Milanese knitted fabric, or a double tricot knitted fabric in the range described above.

The integrated multilayer chemical analysis element of the present invention is preferably manufactured by the following method. In order to form an adhesive layer on the roll-shaped light-transmitting support, an aqueous solution in which a hydrophilic polymer is dissolved is applied, dried, and wound up. After supplying water to the entire surface of the dried adhesive layer, a porous material is laminated by a known method. Thereafter, an aqueous solution containing the above-described reagent and the like is applied onto the porous material, and dried. As a coating method, various known coating methods can be used.

The concentration of direct bilirubin in the liquid sample can be determined by spotting the liquid sample on the integrated multilayer chemical analysis element of the present invention, performing incubation if necessary,
The color density is determined by photometry at a wavelength in the range of 70 nm.
The color development density is the color development density of azobilirubin having an absorption peak at about 570 nm, which is produced by directly reacting bilirubin with the diazonium salt in the analysis element. If a calibration curve is prepared in advance using a standard solution whose bilirubin content is directly known, the color development density can be converted into the bilirubin concentration in the sample. The sample that can be measured may be any sample as long as it directly contains bilirubin. It is preferably a biological liquid sample such as blood, plasma, serum, and urine. The concentration of direct bilirubin is 0-25 mg /
More preferably, it is performed on a sample in the range of dL. Photometry is performed from the side of the light-transmitting support (11) using a light ray (41) for measuring the color density (FIG. 1). The amount of spotted sample is preferably in the range of 1 to 10 μL.

The integrated multi-layer chemical analysis element of the present invention is held and fixed by a holding member (mount) according to the method described in Japanese Patent Application Laid-Open No. 57-63452. Holding member (mount)
Materials include polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyamide, polycarbonate, polyethylene terephthalate, ABS resin, polymethyl methacrylate (PMMA) resin, ceramics (eg, alumina, silica, etc.), metals (aluminum, stainless steel, titanium) Etc.).

[0030]

[Example 1] [Preparation of integrated multilayer chemical analysis element for direct bilirubin measurement] Application of each component in an aqueous solution having the following formulation on a transparent polyethylene terephthalate (PET) colorless transparent film having a thickness of 180 µm. The aqueous solution was applied and dried so that the amount became the following amount and the thickness after drying became 20 μm, thereby forming an adhesive layer.二 Disodium 1,5-naphthalenedisulfonic acid 2.01 g / m ² zinc sulfate Heptahydrate 1.00 g / m ² Purified polyvinyl alcohol (PVA) 24.6 g / m ² Next, water was supplied over the entire surface of the adhesive layer at a supply amount of about 30 g / m ² to wet the surface, and the P equivalent to 50 denier was applied.
Tricot knitted fabric with 36 gauge knitted ET spun yarn
(Thickness: about 350 μm) was pressed and dried. On this knitted fabric, an aqueous solution of the following formulation is applied almost uniformly so that the application amount of each component in the aqueous solution becomes the following amount, dried, and a developed layer is formed. The element was made. ────────────────────────────────── Sulfanilic acid 1.73 g / m ² Sodium nitrite 0.08 g / m ² 1,5-naphthalene disulfonic acid disodium 0.60 g / m ² of zinc sulfate heptahydrate 3.38 g / m ² acidic polymer 9.25g / m ² ──────────────酸性 The acidic polymer has a repeating unit represented by the following formula (I)
I): (II) - [ C 2 H 3 (COOC 3 H 6 OH)] 6 - [C 2 H 3 (CONHC 4 H 8 SO 3)] 4 - is a polymer represented by. The obtained integrated multilayer chemical analysis element is cut into a predetermined size (12 × 13 mm square),
A typical dry bilirubin measurement slide (1) of the present invention was set in a slide frame.

[Direct quantification of bilirubin]
Seven kinds of human serum solutions containing ditaurobilirubin having different concentrations from each other (a substance in which two carboxyl groups of free bilirubin are amide-bonded to taurine, which is a standard substance replacing direct bilirubin in a sample) were prepared. These were separately spotted on the slide (1) in an amount of 10 μL. Next, each was incubated at 37 ° C. for 5 minutes, and the color density of each was measured at 570 nm using Fuji Dry Chem 5000 (manufactured by Fuji Photo Film Co., Ltd.). A calibration curve showing the relationship between the concentration of zitaurobilirubin and the color density was prepared (FIG. 2).

FIG. 2 shows that the concentration of ditaurobilirubin was at least 20 mg / dL (for the concentration of direct bilirubin containing glucuronide-conjugated bilirubin as a main component, it was about 16 m / dL).
g / dL), good linearity can be obtained. Therefore, it is presumed that a sample having a direct bilirubin concentration of up to about 16 mg / dL can be quantified using the dry slide (1) for direct bilirubin measurement of the present invention.

[Confirmation of Repeat Reproducibility] On the slide (1), human serum solutions of three kinds of ditaurobilirubin having different concentrations (a: 0.2 mg / dL, b: 9.
8 mg / dL and c: 14.2 mg / dL), and incubated at 37 ° C. for 5 minutes.
The measurement was repeated 0 times. The measured coloring density was compared with the calibration curve shown in FIG. 2 to determine the concentration of ditaurobilirubin. Table 1 shows the results. The CV value represents a coefficient of variation, and is a value obtained by dividing a standard deviation by an average value.

[0034]

Table 1 Table 1-Concentration of ditaurobilirubin (mg / dL)試 料 Sample number abc ─────────── 10.2 10.1 13.8 2 0.3 9.9 14.1 3 0.2 9.7 14.0 40.2 9.7 14. 15 0.2 9.9 14.1 6 0.2 9.7 13.9 7 0.2 9.8 13.7 8 0.2 9.8 14.1 9 0.2 9.8 14. 2 10 0.2 9.5 14.2 11 0.2 9.9 14.5 12 0.2 9.8 14.4 13 0.2 9.7 14.3 14 0.2 9.9 14. 3 15 0.2 9.8 14.2 16 0.2 9.7 14.6 17 0.2 9.8 14.2 18 0.2 9.8 14.5 19 0.2 9.9 14.3 20 0.2 9.7 14.9} ─────────────────── Average value 0.21 9.80 14.22 ──────────────────── ─────────── standard deviation 0.022 0.123 0.278 ──────────────────────────── ─── CV value (%) 10.91 1.26 1.96 ─────────────────────────────────

From the above Table 1, it can be seen that the slide (1) of the present invention
Has good reproducibility even in the measurement of ditaurobilirubin at a concentration in the range of 0.2 to 14.2 mg / dL.

Comparative Example 1 A dry slide for direct bilirubin measurement was performed in the same manner as in Example 1 except that disodium 1,5-naphthalenedisulfonate was removed from the coating solution supplied to the developing layer. 2) was produced.

To each of the slide (2) obtained above and the slide (1) obtained in Example 1, 10 μL of a human serum solution having a concentration of 10 mg / mL of ditaurobilirubin was spotted. After incubation for 5 minutes, the respective color spectra were measured (FIG. 3). Curves 1 and 2 show the color development spectra when using slides (1) and (2), respectively.

[Evaluation of Analytical Slides (1) and (2)] From FIG. 3, it can be seen that the sensitivity of slide (1) is about 10% higher. This is presumably because the hydrophobicity of disodium 1,5-naphthalenedisulfonic acid prevented the diffusion and migration of azobilirubin and improved the analytical sensitivity.

Example 2 Example 1 was repeated except that the amount of the acidic polymer applied on the knitted fabric was changed to 4.63 and 3.08 g / m ² , respectively.
In the same manner as described above, dry slides (3) and (4) for direct bilirubin measurement were prepared.

On slides (1), (3) and (4),
Each human serum solution having a concentration of 10 mg / mL of ditaurobilirubin was spotted on each of 10 μL, and incubated at 37 ° C. for 5 minutes, and then the color development density was measured (FIG. 4).

[Evaluation of Analytical Slides (1), (3) and (4)] From FIG. 4, it was found that if the coating amount of the acidic polymer in the spread layer was in the range of 3 to 10 g / m ² , ditaurobilirubin was used. Although the sensitivity is high enough to measure the concentration of the acidic polymer, as shown in slide (1), the coating amount of the acidic polymer is about 9.2.
It is understood that it is desirable to be about 5 g / m ² .

Comparative Example 2 A dry slide for directly measuring bilirubin (5) was prepared in the same manner as in Example 1 except that zinc sulfate heptahydrate was removed from the coating solution applied on the knitted fabric. ) Was prepared.

[Evaluation of slides (1) and (5)] After incubating both slides (1) and (5) at 35 ° C. for 3 days, the concentration of ditaurobilirubin was 10 mg / min. 10 μL of each mL sample was spotted and incubated at 35 ° C. for 5 minutes to measure the color density of each sample. The same operation was performed for slides on which the above processing was not performed. The result is shown in FIG. Lines 3 and 4 show changes in color density with respect to the number of processing days when slides (1) and (5) were used, respectively.

FIG. 5 shows that the slide (1) has better stability over time than the slide (5).

[0045]

According to the present invention, a developing layer comprising a diazonium salt, an arylsulfonic acid and an acidic polymer is provided.
Further, by adding a water-soluble zinc salt and changing the arylsulfonic acid to hydrophobic disodium 1,5-naphthalenedisulfonic acid, the handling is simple and the measurement sensitivity, measurement accuracy and storage stability are excellent. An integrated multilayer chemical analysis element can be made. In particular, the measurement sensitivity can be further improved by setting the coating amount of the acidic polymer in the range of about 3 to 10 g / m ² . Further, direct bilirubin in a liquid sample can be quickly and sensitively quantified using the analysis element of the present invention.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a typical integrated multilayer chemical analysis element for direct bilirubin quantification of the present invention.

FIG. 2 is a graph of a calibration curve showing a relationship between a concentration of ditaurobilirubin and a coloring density.

FIG. 3 is a graph showing Comparative Example 1.

FIG. 4 is a graph showing Example 2.

FIG. 5 is a graph showing Comparative Example 2.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 11 Light-transmitting support body 21 Adhesive layer 31 Spreading layer 41 Light ray for color density measurement

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Kaoru Terashima 3-11-46 Izumi, Asaka-shi, Saitama F-Term in Fuji Photo Film Co., Ltd. (reference) 2G045 AA01 AA15 BA11 BB21 BB48 BB50 CA25 CA26 CB03 DA53 FA29 FB16 GC10 JA05

Claims (6)

[Claims] 1. An integrated multilayer chemical analysis element using a system for directly determining bilirubin in a liquid sample by a diazo method, comprising: a light-transmitting support; a diazonium salt;
An integrated multi-layer chemistry comprising a spreading layer of a porous material having a porosity in the range of 30 to 80% containing a composition containing naphthalenedisulfonic acid or a derivative thereof, a water-soluble zinc salt and an acidic polymer. Analysis element. 2. The developing layer is provided on a light-transmitting support via an adhesive layer containing 1,5-naphthalenedisulfonic acid or a derivative thereof and a water-soluble zinc salt. Item 1. The integrated multilayer chemical analysis element according to Item 1. 3. The integrated multilayer chemical analysis element according to claim 1, wherein the water-soluble zinc salt is zinc sulfate. 4. The integrated multilayer chemical analysis element according to claim 1, wherein the acidic polymer is present in the spread layer at a coating amount of 3 to 10 g / m ² . 5. The integrated multilayer chemical analysis element according to claim 1, wherein the porous material is a knit. 6. A liquid, wherein a liquid sample is spotted on the integrated multilayer chemical analysis element according to claim 1, and the color density in the analysis element is measured at a wavelength in the range of 550 to 670 nm. Method for direct bilirubin determination in samples.