JPS59230160A - Bilirubin analyzing element - Google Patents

Abstract

PURPOSE:To obtain a bilirubin analyzing element which has high sensitivity and accuracy and is simple to operate by providing a reagent layer contg. a diazonium salt and a polymer acid material on a base and a developing layer thereon and incorporating nonionic surface active agent into the reagent layer or the developing layer. CONSTITUTION:A reagent layer contg. an acid material for med by (co)polymerization of monomers having COOH, SO3H and phosphono group, for example, a polyacrylic acid, styrene sulfonic acid/styrene copolymer and a diazonium salt is provided on a transparent base and a porous developing layer is provided on the reagent layer to make a bilirubin analyzing element. A good difference in coloring density is exhibited with respect to a difference in the concn. of bilirubin by incorporating the polymer acid layer into said layer and the reaction of the bilirubin and the diazonium salt can be further accelerated by incorporating a nonionic surface active agent into either of the reagent layer or the developing layer or into both layers. The sensitivity of quantitative analysis and the accuracy in measurement are thus improved and the operation is made simple.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an analytical element, particularly an analytical element for analyzing a predetermined specific component in a fluid, and more particularly to an analytical element for analyzing a predetermined specific component in a biological fluid sample. This invention relates to a dry analytical element for analyzing bilirubin.

[Prior art]

Many methods have been developed to analyze analyte components in fluid samples, but they can be broadly divided into two types: reaction systems in which the reaction takes place in a solution, and reaction systems in which the reaction takes place in a homogeneous carrier. be able to. Analytical reactions in solution systems (hereinafter abbreviated as wet chemistry) are widely known, from analytical methods called manual methods that do not use any machinery at all, to automatic analytical instruments. In particular, progress has been remarkable in the field of clinical chemistry, and in recent years various automatic quantitative analysis instruments for clinical tests have been introduced into clinical laboratories of hospitals.

However, the above-mentioned methods have various drawbacks because the reaction is basically carried out in the form of an aqueous solution. That is,
The analysis process requires a large amount of water, especially purified pure water or distilled water, which inevitably leads to increased energy consumption, and automatic analyzers for various types are themselves extremely expensive. It is not a stupid system that requires a great deal of skill to operate, and a large amount of time and effort.
The waste liquid inevitably has the drawback of causing environmental pollution.

On the other hand, analytical methods using solid-phase analytical reactions (hereinafter abbreviated as dry chemistry) are also widely used, but these are provided in the form of a sheet of paper or the like impregnated with reagents.

The above-mentioned paper is made by impregnating a reagent solution into a water-absorbing fibrous carrier such as a paper as described in, for example, U.S. Patent No. 5,050,575 or U.S. Pat. No. 3,06 L525. It is made by drying it. These are generally referred to as analytical test papers or simply test strips, and a fluid sample is dropped onto the test strip or the test strip is immersed in the fluid sample, and the change in color or concentration of the test strip is determined visually. or a reflection densitometer to determine the concentration level of a particular component in a fluid sample.

Although these test pieces are useful because they are easy to handle and provide immediate results, their composition limits them to semi-quantitative or qualitative analysis.

On the other hand, multilayer analytical elements are known that use dry chemistry that is easier to operate than the conventional analytical methods described above, and that also have high quantitative performance. For example, Tokuko Sho 5
No. 5-21677, JP-A-55-164556, JP-A-55-164556
The multilayer analytical element described above is described in the specifications of Japanese Patent No. 7-125847, No. 57-197466, and No. 58-90167.

According to each of the above specifications, all the reagents used in the analytical reaction are contained in one analytical element, and a certain volume of serum or whole blood is dropped onto the above element, and after being kept at a constant temperature for a certain period of time, It is possible to measure the reflection density from the support side and determine the substance concentration from the reflection density.

The above method has a much higher analytical accuracy than the conventional test strip type method, and can be used without adjusting reagents in advance, and has performance equivalent to or better than wet chemistry.

Several methods for analyzing bilirubin using such analytical elements have been disclosed.

For example, in JP-A No. 55-89796, bilirubin is quantified by mordanting sibilirubin using a mordant composition, sinking the absorption wavelength, increasing the molecular extinction coefficient by 50 or more, and conducting spectroscopic measurements. It describes how to do this. Furthermore, in Japanese Patent Application Laid-open No. 53-89797, a complex of a dye and albumin is placed on a single layer of a hydrophilic binder in advance, taking advantage of the property of bilirubin to strongly bind to albumin, etc., and a sample solution is prepared. A method for quantifying bilirubin is described based on the principle that the dye molecules forming the complex are replaced by the free bilirubin contained therein, and the released dye is measured spectroscopically. Furthermore, JP-A-57-37262 describes a method for extracting bilirubin using a hydrophobic bilirubin extraction composition containing a hydrophobic amine and quantifying sibilirubin by spectrometry. .

However, it cannot be said that the analytical elements described in each of these specifications are necessarily satisfactory in terms of quantitative sensitivity.

[Purpose of the invention]

An object of the present invention is to provide an analytical element for measuring bilirubin that has excellent quantitative sensitivity and measurement accuracy and is easy to operate.

[Structure of the invention]

As a result of extensive studies, the present inventors were able to overcome the above drawbacks by using an analytical element having the following configuration.

That is, to summarize the present invention, the present invention relates to a bilirubin analysis element, and the analysis element includes at least a reagent layer and a spreading layer above the reagent layer on a support.
The present invention is characterized in that the reagent layer contains a diazonium salt and a polymeric acid substance, and the reagent layer and/or the spreading layer further contains a nonionic surfactant.

Examples of polymeric acid substances used in the analytical element of the present invention include polymers containing at least 5% by weight or more of copolymerizable ethylenically unsaturated acid monomer units and having an average molecular weight of +, 0.
000,000 to 1,000,000, preferably 2,000 to 500,000.

In the present invention, preferred copolymerizable ethylenically unsaturated acid monomers are those having at least one carboxyl group, sulfo group or phosphono group. Among those having a carboxyl group or a sulfo group, those having one or two carboxyl or sulfo groups are preferred, and monomers represented by the following general formula (1) are particularly preferred.

General Formula (1) In the formula, R1 represents a carboxyl group, a sulfo group, or a group having a carboxyl group or a sulfo group, R1 represents a hydrogen atom or an alkyl group, and R3 represents a hydrogen atom or an alkoxycarbonyl group.

As the group having a carboxyl group or a sulfo group in R1, a sulfophenyl group or a sulfoalkyloxycarbonyl group (this alkyl group has 1 to 4 carbon atoms) is preferable.
Preferred is a hesulfoalkylcarbamoyl group (this alkyl group preferably has 1 to 5 carbon atoms).

The alkyl group in R2 may have a substituent, and preferred substituents include an alkoxycarbonyl group and a carboxyl group. Preferred alkyl groups include a methyl group, an alkoxycarbonylmethyl group containing an alkoxy group having 1 to 4 carbon atoms, and a carboxymethyl group.

The alkoxycarbonyl group represented by R3 preferably has 2 to 5 carbon atoms.

Examples of the monomer represented by the general formula (J) include acrylic acid, methacrylic acid, itaconic acid, and itaconic acid monoalkyl esters (preferably alkyl esters having 1 to 4 carbon atoms, such as monomethyl itaconate and monobutyl itaconate). ], maleic acid monoesters (preferably alkyl esters having 1 to 4 carbon atoms, such as monomethyl maleate, monobutyl maleate, styrene sulfonic acid, acryloyloxyalkanesulfonic acid (
For example, acryloyloxypropanesulfonic acid, acryloyloxyethanesulfonic acid, methacryloyloxyalkanesulfonic acid (for example, methacryloyloxypropanesulfonic acid, methacryloyloxybutanesulfonic acid), acrylamidealkanesulfonic acid (for example, 2
-acrylamido-2-methylethanesulfonic acid, 2-
Acrylamido-2-methylpropanesulfonic acid, 2-
Acrylamido-2-methylbutanesulfonic acid), methacrylamide alkanesulfonic acid (for example, 2-methacrylamido-2-methylethanesulfonic acid).

Among the above-mentioned copolymerizable ethylenically unsaturated acid monomers, 1 is preferable among monomers having a phosphono group.
In particular, it has the following general formula (I
I).

General formula (It) [wherein, R4 represents a hydrogen atom or a methyl group, R5 represents an aliphatic hydrocarbon group having 2 to 10 carbon atoms or -(R, -0smR7-, and R7 represents a carbon atom] represents an aliphatic hydrocarbon group of numbers 2 to 6, R6 represents a hydrogen atom, an alkali metal, or a -NH4 group, and m is an integer of 1 to 5.] An aliphatic hydrocarbon group represented by R or R7 An alkylene group is preferred, and this alkylene group may be branched or may have a halogen atom such as a chlorine atom, a lower alkoxy group such as an ethoxy group, or an aryl group such as a phenyl group as a substituent.

Examples of the monomer represented by general formula (n) include, for example,
2-acryloyloxyethyl phosphate, 1-methyl-2-acryloyloxyethyl phosphate, 2-
7Cryloyloxyethoxyethyl phosphate, 4-
Acryloyloxybutyl phosphate, 2-methacryloyloxyethyl phosphate, 1-methyl-2-methacryloyloxyethyl phosphate, 1-chloromethyl-2-methacryloyloxyethyl phosphate,
2-methacryloyloxyethoxyethyl phosphate 7ate and 4-methacryloyloxybutyl phosphate).

The ethylenically unsaturated acid monomer constituting the polymeric acid substance may be used alone or in copolymerization of two or more types, and further copolymerized with other copolymerizable ethylenically unsaturated monomers. is also possible.

Other preferable copolymerizable ethylenically unsaturated monomers constituting the polymeric acid substance used in the present invention include ethylenically unsaturated nitriles, styrenes, ethylenically unsaturated acid esters, and ethylene Examples include polyunsaturated acid amides, conjugated dienes, vinyl heterocyclic compounds, and crosslinkable monomers.

As the ethylenically unsaturated nitriles, the following general formula CI
Those indicated by [ ] can be cited as preferred.

General formula (ffl) [In the formula, Ra and R9 are a hydrogen atom or a hydrogen atom, Rlo is a hydrogen atom, a hydrogen atom, an alkyl group, an alkoxy group, an acyloxy group, an aryl group, a cyano group, or a carnocomoyl group. represents a group. ] Preferred as R8 and R9 are a hydrogen atom and a fluorine atom, with a hydrogen atom being particularly preferred. Examples of the nitrogen atom that can be used as RIO include a fluorine atom, a chlorine atom, and a bromine atom. The alkyl group represented by R1 (i) is preferably a lower alkyl group having 1 to 5 carbon atoms, such as a methyl group, trifluoromethyl group, ethyl group, 1so-propyl group, n-propyl group, n-pentyl group. can be mentioned.RIO
The alkoxy group represented by has 1 to 1 carbon atoms.
3 is preferable, and for example, a methoxy group can be mentioned. The acyloxy group represented by Rse is preferably an alkylcarbonyloxy group having 2 to 4 carbon atoms, such as an acetoxy group. The aryl group represented by RIG includes a phenyl group (this phenyl group has a substituent such as a cyano group or a chlorine atom), a rogene atom, a lower alkyl group such as a methyl group, or a lower alkoxy group such as a methoxy group. ) are preferred, such as phenyl group, methoxyphenyl group, methylphenyl group, chlorophenyl group,
Mention may be made of cyanophenyl groups. Preferred among RIO are hydrogen atom/% rogen atom or alkyl group, with hydrogen atom being particularly preferred.

Specific monomers represented by general formula (1) include:
The following can be mentioned:

Acrylonitrile, methacrylonitrile, α-chloroacrylonitrile, α-bromoacrylonitrile, α-fluoro7-IJO nide IJ/l/, α-chloro-β-difluoro-acrylonitrile, σ-trifluoromethylacrylonitrile, α-ethyl acrylonitrile, α−
Ingropylacrylonitrile, α-n-propylacrylonitrile, α-n-pentylacrylonitrile, α
-Methquin acrylonitrile, α-acetoxyacrylonitrile, α-phenylacrylonitrile, α-cyanphenylacrylonitrile, α-chlorophenylacrylonitrile, α-methylphenylacrylonitrile, α
-methoxyphenylacrylonitrile and vinylidene cyanide.

Preferred examples of styrenes include those represented by the general formula [1v] below.

General formula [■■ C In the formula, R11 represents a hydrogen atom, a halogen atom, or an alkyl group, and Fin represents a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, a cycloalkyl group, an alkoxycarbonyl group, an acyloxyalkyl group, n is 1
It is an integer of ~5. ] The alkyl group represented by RH preferably has 1 or 2 carbon atoms.

The alkyl group represented by R12 has 1 carbon atom
〜6, the alkoxy group has 1 to 6 carbon atoms.
4, the cycloalkyl group has 5 or 6 carbon atoms, the alkoxycarbonyl group has 1 or 2 carbon atoms, and the acyloxyalkyl group has 1 or 2 carbon atoms. Number 2 or 3 is the alkyl group of the acyloxyalkyl group,
Those having 1 or 2 carbon atoms are preferred. n is preferably 1 or 2.

Specific examples of the styrenes represented by the general formula (IV) include the following.

Styrene, p-methylstyrene, α-methylstyrene,
p-chlorostyrene, p-chloromethylstyrene, m-
Chloromethylstyrene, m-methylstyrene, m-ethylstyrene, P-ethylstyrene, 2,5-dimethylstyrene, 3゜4-dimethylstyrene, 3,5-diethylstyrene, m-1so-propylstyrene, p-1
80-propylstyrene, p-butylstyrene, m-t
-butylstyrene, p-hexylstyrene, p-cyclohexylstyrene, m-methoxystyrene, 4-methoxy-3-methylstyrene, p-ethylstyrene, 3,4
-dimethoxystyrene, m-chlorostyrene, 5.4-
Dichlorostyrene, 3,5-dichlorostyrene, m-bromostyrene, p-bromostyrene, 5,5-dipromostyrene, 4-fluoro-3-trifluoromethylstyrene, 2-bromo-4-trifluoromethylstyrene,
p-methoxycarbonylstyrene, p-a7toxymethylstyrene/.

As the ethylenically unsaturated acid esters, ethylenically unsaturated carboxylic esters are preferable, and those represented by the following general formula [V] are particularly preferable.

General formula (V) [wherein, RIs represents a hydrogen atom or a methyl group, R
14 represents an alkyl group or a phenyl group. The alkyl group preferably has 1 to 8 carbon atoms, particularly 1 to 4 carbon atoms.

Examples of those represented by the general formula (V) include the following.

Methyl acrylate, ethyl acrylate, acrylic acid-n
-butyl, n-propyl acrylate, 1 acrylate
Ni-O-butyl, 5ec-butyl acrylate, 2-hydroxyhectyl acrylate, 2-hydroxypropyl acrylate, 2-ethylhexyl acrylate, hexyl acrylate, 2-phenoxyethyl acrylate, acrylic acid -2-chloroethyl, dimethylaminoethyl acrylate, benzyl acrylate, cyclohexyl acrylate, phenyl acrylate, 2-hydroxypropyl acrylate, tetrahydronerufuryl acrylate, 2,6-cyhydroxyglobyl acrylate, Methyl methacrylate, ethyl methacrylate, n-butyl methacrylate, propyl methacrylate, 2-human xyethyl methacrylate, 2-hydroxypropyl methacrylate, is-methacrylate.

-butyl, acetoxyethyl methacrylate, dimethylaminoethyl methacrylate, chlorobenzyl methacrylate, octyl methacrylate, furfuryl methacrylate, and phenyl methacrylate.

In the present invention, preferred as the single copolymerizable conjugated diene is a monomer represented by the following general formula (Vl).

General formula [■1] [In the formula, RIIS to Rgo are each a hydrogen atom,] 10
Gen atom, alkyl group, aryl group, cyano group or -C
! OOR, represents a group (ll represents an alkyl group). ] The halogen atom represented by R□ to R2° is preferably a chlorine atom or a bromine atom.

The alkyl group represented by R11 to R11 is preferably a lower alkyl group, particularly preferably a lower alkyl group having 1 to 4 carbon atoms. Among these, methyl group is particularly preferred. Also, R1! i'' The alkyl group represented by R21 may have a substituent, but is preferably unsubstituted.

The preferred aryl group represented by RlB''R2O is a phenyl group, and the phenyl group may have a substituent. Preferred substituents include a halogen atom (preferably a chlorine atom, a bromine atom and a farkyl group (preferably a bromine atom). has 1 to 5 carbon atoms, with methyl being particularly preferred).

Above, Fjts, +y R1 (various atoms and groups are listed as 1, but the most preferred among them is hydrogen atom).
It is a rogene atom or an alkyl group.

Furthermore, the total number of carbon atoms in the monomer represented by the general formula (Vl) is preferably 4 to 12, more preferably 4 to 9, particularly preferably 4 to 6.
belongs to. Preferably, at least two hydrogen atoms from Rss to Rgo are present.

Examples of the monomer represented by the general formula [V1] include the following.

1,3-butadiene, alkyl (preferably lower alkyl having 1 to 4 carbon atoms)
1,3-butadiene, 2-n-propyl-1,3-butadiene, 2-n-butyl-1,5-butadiene, 2,3
-dimethyl-1゜3-butadiene, 2-methyl-1,5
-pentadiene, 4-methyl-1,5-pentadiene, aryl (preferably phenyl) substituted 1,5-butadiene (e.g. 1-phenyl-1,5-butadiene, 2
-Phenyl-1,5-butadiene, 1-(p-chlorophenyl)-1,3-butadiene, 1-phenyl-2-carbomethoxy-1,5-butadiene, 2-p-)dyl-
1,3-butadief, etc., halogen (preferably chlorine,
bromine]-substituted 1,3-butadiene (e.g., 1-chloro-
1,3-butadiene, 2-lytetra-1,3-butadiene, 1-promo 1,3-butadiene, 2-promo 1.
3-butadiene, 1,1-dichloro-1,3-butadiene, 2,5-dichloro-1,5-butadiene, 2.5-
dibromo-1,5-butadiene, 1.1.2-trichloro-1,3-butadiene, +, 1.2.3-tetrachloro-1,3-butadiene, etc.), cyano-substituted 1,3-butadiene (e.g. 1-Sia/-1,5-butadiene, 2
-cyano-1,3-butadiene, etc.].

Examples of ethylenically unsaturated acid amides include acrylamide and methacrylamide. Examples of acrylamides include acrylamide, diacetone acrylamide, methylol acrylamide, and methyl acrylamide.

Examples of meta-crylamides include meta-crylamide and benzyl meta-V-crylamide.

Examples of the vinyl heterocyclic compound include N-vinylpyrrolidone, N-vinylimidazole, and vinylpyridines (eg, 4-vinylpyridine, 2-vinylpyridine, etc.).

Examples of the crosslinkable monomer include divinylbenzene, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, and pentaerythritol trimetaphorylate.

The ethylenically unsaturated acid monomer or the polymer composed of the ethylenically unsaturated acid monomer and other ethylenically unsaturated monomers constituting the polymeric acid substance used in the present invention is composed of the various monomers mentioned above. Preferably, the ethylenically unsaturated acid monomer is contained in a proportion of about 5 to 100% by weight, and the other ethylenically unsaturated monomers are contained in a proportion of O to about 95% by weight. more preferably about 8% to 100% by weight of ethylenically unsaturated acid monomers and 0% to about 92% by weight of other ethylenically unsaturated acid monomers. .

Next, specific examples of polymeric acid substances used in the present invention are shown.
The present invention is not limited to these in any way.

Exemplary polymer acids +11 Polyacrylic acid (2) Polymethacrylic acid (3) Itaconic acid-styrene copolymer (wt% 5
0:50) (4) Acrylic acid-butyl acrylate copolymer (weight% 75:25) (5) Acrylic acid-vinylidene chloride copolymer (weight% 80:20) (6) Ethylene-monobutyl maleate copolymer Polymer (
Weight % so: 5o) (7) Styrene-motuptil maleate copolymer (
Weight% 50:50) (8) Monobutyl maleate-methyl ethyl keto y
Copolymer (wt% 50:50) (9) Monobutyl maleate-isobutylene copolymer (wt% so:so) aQ methacrylic acid-divinylbenzene (wt% 98
: 2) (IEI Acrylic acid-styrene-divinylbenzene (wt% 90:8:2) (6) Polystyrene sulfonic acid (2) Polyacrylamide-2-methylethanesulfonic acid θ◆ Polymethacryloyloxypropanesulfonic acid tetrastyrene sulfone Acid-styrene copolymer (wt%
80:20) aQ Styrenesulfonic acid-divinylbenzene copolymer (weight% 90:10) 0 - Acrylamide-2-methylethanesulfonic acid-
Butyl acrylate-styrene copolymer (weight% +5:
80:5) (To) Methacryloyloxypropanesulfonic acid-2
,3-dimethyl-1,5-butadiene-acrylonitrile copolymer (wt% Bnia 5:17) α-styrene sulfonic acid-vinylidene chloride (wt%
75:25) to) methacryloyloxypropanesulfonic acid-methyl methacrylate (wt% 50:50) Qυ Poly 2-acryloyloxyethyl phosphate@Poly 2-methacryloyloxyethyl phosphate 1212-acryloyloxyethyl phosphate-methyl acrylate copolymer Combined (weight% 80:20) G! 4) 4-methacryloyloxybutyl phosphate-styrene-divinylbenzene copolymer (wt%
50:45:5) (c) 2-methacryloyloxyethoxyethyl phosphate-ethyl acrylate-acrylonitrile copolymer (wt% 15 near Q: 15) (1) 2-acrylamido-2-methylpropanesulfonic acid to acrylic acid Butyl copolymer (weight% 50:
50) @2-acrylamide-2-methylpropanesulfonic acid-acrylamide copolymer weight% 75:25J Commercially available strong acid and weak acid cation exchange resins can also be used. For example, Amber Light 15, Amber Light CG-50, Amber 2 Itoe Re-50 (
The product name is Rohm and Haas M). These resins can be used as they are or after being crushed.

The above polymeric acid substances can be easily synthesized by appropriately using conventional radical polymerization, such as solution polymerization, emulsion polymerization, suspension polymerization, etc. It is also possible to introduce or convert the acid moiety by polymer reaction.

The polymeric acid material used in the present invention need only be present in an amount to provide an appropriate pH for the coupling reaction between dorirubin and diazonium salt in the fluid sample; It is sufficient if it is present in an amount of .1 to about 4.

Examples of diazonium salts used in the present invention include p-nitrobenzenediazonium! , 2,5-dichlorobenzenediazonium salt, etc. Among them, p-nitrobenzenediazoela salt is preferable because it has a high ability to identify bilirubin.

Moreover, as the salt, tetrafluorobalate and hexafluorosinate are suitable.

The diazonium salt can be used in widely selected amounts, but in the range from 0.5 to 200 mmol/m2, preferably from 2 to 50 mmol/m2.

The nonionic surfactant used in the present invention is used to promote the reaction between bilirubin and diazonium salt,
Examples include alkylphenoxypolyethoxyethanol (also known as alkylphenyl monoether of polyethylene glycol). In particular, inoctylphenoxypolyethoxyethanol having 5 to 40 ethylene oxide units in the molecule is preferred, and this type is commercially available, for example, from Rohm and Haas as Triton X-100 or Triton X-405. Nonionic surfactants can be used in widely selected amounts, but in the range from 5 mW to 50 f/mj, preferably from 0.52 to 2097 m''.

The spreading layer according to the present invention has the function of (1) uniformly distributing a fixed volume of a fluid sample per unit area to the reagent layer; Moreover, in addition,
The performance described in Specification No. 7, that is, (2) the ability to remove substances or factors that inhibit analytical reactions in fluid samples;
and/or (3) it is preferable that it has the function of performing a background effect of reflecting measurement light transmitted through the support when performing spectrophotometric analysis. Therefore, the expansion layer according to the present invention is a layer having only the function (1) above;
In addition to (11), it can be either a layer having the functions (2) and/or (3), or (1
), it is also possible to separate multiple functions as appropriate and use a separate layer for each function. Furthermore (1), (2)
Of the functions (3) and (3), a layer having two functions and a layer having the remaining one function may be used in combination. For example, a spread layer of a non-fibrous porous medium called a plush polymer consisting of titanium dioxide and cellulose diacetate is described in the above-mentioned Japanese Patent Publication No. 55-21677; Developed layer of hydrophilized fabric, JP-A-57-9465
No. 8, No. 57-125847, No. 57-197466
Examples include the fiber structure spreading layer described in each specification such as JP-A No. 11g58-90167 and the particle combination structure spreading layer described in JP-A No. 11g58-90167. In particular, the fiber structure spreading layer and particle combination structure spreading layer are particularly useful as materials capable of rapidly transporting blood cell portions.

The film thickness of the spreading layer in the analytical element of the present invention should be determined by its porosity, but is preferably about 100 ml.
~about 500 μm1, more preferably about 150-550 μm
It is. Also, the porosity is preferably about 20 to about 85 inches.

The reagent layer according to the present invention refers to a layer that causes a quantitative reaction with bilirubin, which is a sample component to be analyzed, or contains reagents that are involved in the reaction, and includes hydrophilic colloids or reagents that are involved in the reaction. A layer using a polymer substance soluble in an organic solvent as a medium is suitable.

As the reagents to be contained in the reagent layer, the above-mentioned diazonium salts and polymeric acid substances are mentioned as essential ones. Furthermore, the above-mentioned nonionic surfactant and, if necessary, other additives (eg, hardener, preservative, etc.) can also be added to the above-mentioned reagent layer.

The above-mentioned reagent layer is different from the conventional method in which a carrier such as paper is impregnated with reagents, and has the advantage that it is possible to uniformly contain reagents and that the content of reagents can be freely controlled. have. The hydrophilic colloid substance used in the reagent layer according to the present invention is preferably a natural or synthetic polymeric substance, and more preferably 7
Gelatin derivatives such as talized gelatin, polysaccharides such as agarose and glulan, cellulose derivatives such as carboxymethylcellulose and hydroxyethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polymethacrylamide, or combinations of these with other vinyl monomers. Examples include copolymers. Among these, phthalated gelatin, polyacrylamide, polyvinylpyrrolidone, polyvinyl alcohol and the like are particularly advantageous.

These hydrophilic colloid materials preferably have a swelling degree of about 150 to about 500 μm, and the film thickness can be selected as desired, and is at least about 5 μm.
It is necessary that the number is greater than or equal to m.

Examples of polymers soluble in organic solvents include polystyrenes, polyacrylic esters, polymethacrylic esters, alkyl celluloses such as methyl cellulose and ethyl cellulose, polyvinyl alcohol, and polyvinyl carbonate.

Furthermore, it is also possible to use the polymeric acid substance used in the present invention as it is as a binder.

A reagent can be contained in the reagent layer by a dispersion method such as a direct dispersion method, dissolution, etc. depending on the characteristics of the reagent used.

The support according to the present invention may be of any conventionally known type, and suitable ones include those that are liquid-impermeable and light-transparent, such as cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene. A variety of polymeric materials are suitable for this purpose. Furthermore, in addition to the above-mentioned polymer materials, inorganic materials such as glass can be used as well. Although the thickness of the support according to the present invention is arbitrary, it is preferably about 50 to about 250 μm. Further, one side surface of the observation side of the support according to the present invention can be arbitrarily processed depending on the purpose. Furthermore, it is possible to improve the adhesion between the reagent layer and the support by using a light-transmitting undercoat layer on the support surface on which the reagent layer is laminated, depending on the case.

The analytical element of the present invention may include, for example, the reflective layer described in US Pat. No. 5,992,158, the undercoat layer,
The radiation blocking layer described in U.S. Pat. No. 4,042j35, the barrier layer described in U.S. Pat. No. 4,066,405, the scavenger layer described in JP-A-55-90859, etc. They can be combined to form any configuration suitable for the purpose of the present invention.

The various layers of these analytical elements are sequentially laminated on the support according to the present invention by appropriately selecting and using slide hopper coating methods, extrusion coating methods, dip coating methods, etc., which are conventionally known in the photographic industry, according to the desired configuration. By doing so, a layer of arbitrary thickness can be applied.

The analytical element of the present invention configured as described above supplies a fluid sample from the developing layer side, then observes the analytical reaction of the reagent layer from the transparent support side or the opposite side, and measures the reflection density.
The amount of unknown bilirubin can be determined by applying the obtained measured values to a calibration curve prepared in advance.

The amount of fluid sample applied to the analytical element of the present invention can be determined arbitrarily, but is preferably about 5 μt to about 50 μt.
t, more preferably from 5 μt to 20 μt.

It is preferred to apply a typical 10 μt fluid sample.

The analytical element of the present invention can be used for the analysis of whole blood, serum, and plasma without any disadvantage. Furthermore, other body fluids such as urine and feces can be used without any inconvenience. If whole blood is used, a radiation blocking layer or other reflective layer as described above may be provided, if necessary, to prevent interference of the radiation for detection by blood cells.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 One or two reagent layers having the composition shown in Table 1 were provided on a transparent subgrooved polyethylene terephthalate support with a film thickness of 180 μm, and then a spreading layer having the composition shown below was provided on each reagent layer. Analytical elements 1 to 7 of the present invention and comparative analytical elements 1 and 2 shown in -H were created.

Spreading layer (8-1) Powder 1 paper (c) 9 j,
O97m” (Toyo Ichishi Co., Ltd. 600 mesh or more) Styrene-glycidyl methacrylate copolymer 1!
LO/fn” (weight ratio 9:1) Triton X −+ 00
9. 097m" Table-■ For the analytical elements 1 to 7 of the present invention and comparative analytical elements 1 and 2, 10μ of bilirubin control (manufactured by Dito, USA) of bilirubin standard solutions of different concentrations prepared by After dropping it onto the developing layer and keeping it warm at 37°C for 10 minutes, the reflection density at 540 nm was measured from the support side.
Results between tables were obtained.

Table - ■ As is clear from the results in Table -
The analytical element using nitrobenzene diazonium salt showed a better coloring density difference with respect to the difference in bilirubin concentration than Comparative Analytical Elements 1 and 2 prepared using a comparative low-molecular-weight acid substance, and showed good discrimination and shading. It can be seen that the quantitative sensitivity is high.

Example 2 A reagent layer having the composition shown in Table IV was provided on a transparent unfinished polyethylene terephthalate support having a film thickness of 180 μm, and then a spreading layer having the composition shown below was provided on each reagent layer, and the book shown in Table II was prepared. Invention analytical element 8.9 and comparative analytical element 3.4 were created.

Table-1■ Development layer (S-2) Titanium dioxide 30.0t/m Cellulose diacetate 179/m Bone tatami Tokko Showa 5
Coating according to the method described in No. 5-21677.

Table - ■ Reflection density was measured for the analytical element 8.9 of the present invention and the comparative analytical element 5.4 in exactly the same manner as in Example 1,
The results shown in Table Vl were obtained.

Table-■1 As is clear from the results of Table-Vl above, analytical element 8.9 prepared using the nonionic surfactant according to the present invention
shows a good coloring density difference with respect to the difference in bilirubin concentration compared to Comparative Analytical Element 3 prepared without using a comparative surfactant and Comparative Analytical Element 4 prepared using a cationic surfactant. It can be seen that the reaction between bilirubin and diazonium salt is significantly promoted.

〔Effect of the invention〕

As described above in detail, the analytical element of the present invention has remarkable effects in that it is an analytical element for measuring bilirubin that has excellent quantitative sensitivity and measurement accuracy, and is easy to operate.

Patent applicant Hiroshi Nakamoto, agent of Konishiroku Photo Industry Co., Ltd.

Claims (1)

[Claims] 1. In an analytical element having at least a reagent layer and a developing layer above it on a support, the reagent layer contains a diazonium salt and a polymeric acid substance, and the reagent layer and/or the developing layer contains a non-containing material. A bilirubin analysis element characterized by containing an ionic surfactant.