JPH01231897A - Dry glucose-analyzing element - Google Patents

Abstract

PURPOSE:To provide a dry glucose-analyzing element capable of quantitatively analyzing glucose in a high accuracy up to a high concentration region of the glucose by adding specific components to a reagent layer and an enzyme- containing layer, respectively, disposed on a support and using a special material in a light-shielding layer. CONSTITUTION:A reagent layer, enzyme-containing layer, light-shielding layer and porous developing layer, all being water-permeable, are laminated in this order to a water and light-impermeable support. At least a coupler among a hydrogen peroxide-detecting coloring composition comprising a hydrogen donor and the couple is added to the reagent layer and gluxose oxidase, peroxidase and a mordant are added to the enzyme-containing layer. An oxygen- permeable and protein-impermeable material is used for the light-shielding layer. It is preferable that the light-shielding layer is a non-porous layer wherein titanium dioxide fine powder having a light-shielding property and a light- reflecting property are bonded with a hydrophilic polymer haivng a little film formability.

Description

DETAILED DESCRIPTION OF THE INVENTION Japanese Patent Application Laid-Open No. 60-82859 discloses a technique for a glucose quantitative analysis element having an oxidase layer. This dry analytical element for glucose determination is characterized by containing peroxidase in the reagent layer, but it has the disadvantage that the slope of the calibration curve decreases at high concentrations, resulting in a decrease in the accuracy of quantitative glucose analysis in the high concentration range. there were.

[Technical Problems to be Solved] The technical problems to be solved by the present invention are to provide a dry analytical element that allows the calibration curve to maintain the necessary slope up to high concentrations and enables quantitative analysis of glucose with high accuracy even in the high concentration range. It's about doing.

[Means for solving technical problems] The above-mentioned problems of the present invention were solved as follows.

A dry analytical element for glucose determination having a water-permeable layer on a water-impermeable, light-transparent support, wherein a reagent layer, an enzyme-containing layer, and a light-shielding layer, all of which are water-permeable, are provided on the support. layer and a porous development layer are laminated in this order, the reagent layer containing at least a coupler of a hydrogen peroxide detection color forming composition consisting of a hydrogen donor and a coupler, and the enzyme-containing layer containing glucose oxidase, peroxidase, and a mordant. A dry analytical element for quantifying glucose, wherein the light shielding layer is oxygen permeable and protein impermeable.

The hydrogen donor may be contained in the enzyme-containing layer, or may be contained in the reagent layer together with the coupler.

Hydrogen donors are, for example, 4-aminoantipyrine, 4-amino-2-methyl-3-phenyl to 1-(2,4,8,
-)lichlorophenyl)-3-pyrazolin-5-one.

Couplers are, for example, 1,7-hydroxynaphthalene, phenol, alpha-naphthol.

As the mordant, cationic polymers, ie, polymers with tertiary amine groups, polymers with quaternary ammonium groups, etc. are preferred. Its molecular weight usually ranges from 5,000 to 200,000.

For example, US Patent Co., Ltd., J-J! No. 2, ! J'
! .

'fi issue 30, same! , /4#, Ot1 issue, same 3.7! t
, l1It issue, Tokukai Sho! Vinylpyridinium cationic polymer disclosed in US Pat. No. 2-/3tt2, specification, etc.; US Pat. Otsu 2! , Otori ≠ issue, same 3. ? ! the law of nature.

No. 0, No. T, T, ISR, R3R, British Patent No. 1.

Core 7, ≠! Polymers capable of crosslinking with gelatin, etc. disclosed in US Pat. No. 3, etc.; the law of nature! ♂, Tatayo No. 2-, 72/.

rsx issue, 2.798', 01,3 issue, Tokukai Sho! Aqueous sol-type cationic polymers disclosed in Hiromu-//J-221, Ibid.≠-7≠jj2, Hiromu-/2t027, etc.; U.S. Patent No. 3. ♂r, o♂♂ issue and Tokukai Shoyo 513317
Water-insoluble cationic polymer disclosed in the specification of No. 2; U.S. Pat.
No. 33) Reactive mordants capable of co-synthesizing with the dyes disclosed in the specifications; furthermore, U.S. Pat.
rJ-1, same No. j, A! u, 44.1'j
No. J, 4!11.70, No. 3, !
! 7, No. 066, No. J, 27/, /≠7, No. J, J7/, /4#, JP-A-60-7/332,
same! No. 3-30321, same! 2-/! ! J-21, the same! 3-/ko! Examples include cationic polymers and polymers disclosed in Japanese Patent No. J3-10.

Other U.S. patents, Otsu 7J', j/-A, same! .

! #2, /! Also mentioned are the cationic polymers described in the specification.

Among these, those that are difficult to migrate from the hydrophilic colloid layer to other layers are preferred, such as those that crosslink with hydrophilic colloids such as gelatin, water-insoluble cationic polymers,
and aqueous sol (or latex dispersion) can be preferably used.

Particularly preferred cationic polymers are shown below.

(1) A group that has a primary ammonium group and can be covalently bonded to gelatin (e.g., aldehyde group, chloroalkyl group, chloroalkyl group, vinylsulfonyl group, hyridinium propionyl group, vinylcarbonyl group, alkylsulfoxy group) For example, (2) a copolymer consisting of a repeating unit of a monomer represented by the following general formula (■) and a repeating unit of another ethylenically unsaturated monomer, and a crosslinking agent. (e.g. bisalkanesulfonates, bisarenesulfonates).

Q; divalent group R3+ R4+ R5; Al, kyl group, aryl group,
At least two of R3-R5 may be bonded to form a petero ring.

X: Anion (the above alkyl groups and aryl groups include those that are substituted) (3) Polymer represented by the following general formula (■) - General formula (■) X: Approximately O, co! ~about 5 molt y; about 0 to about 0 molt 2; about 10 to about tatamolt 2; monomer B having at least two ethylenically unsaturated bonds: copolymerizable ethylenically unsaturated monomer Q; N'
D R1+ R2, R3 near redundant group, cyclic hydrocarbon, or at least two of R1-R2 may be combined to form a ring.

(These groups and rings may be substituted.) (4) Water-insoluble polymer having //J or more repeating units represented by the following general formula (IX) General formula (IX) R1+ R2T R3p sonzonn 7 /Lz −+
The total number of carbon atoms in R1-R3 is 12 or more. (The alkyl group may be substituted.) It will be done. 1! Protein impermeability refers to the ability of a layer to penetrate under analytical conditions, i.e., when water, the solvent of an aqueous liquid sample or blood sample, penetrates into this layer and wets or swells this layer. oxygen in the air (o2
) means that it is substantially permeable, while the protein is substantially impassable. In addition, the protein here refers to a protein in the usual sense with a molecular weight of about 5,000 or more,
Particularly, heme proteins represented by hemoglobin (molecular weight approximately 65,000), and complex proteins having hydrogen peroxide decomposition activity such as catalase (molecular weight approximately 250,000). The oxygen-permeable, protein-impermeable, light-shielding layer consists of fine titanium dioxide powder, which has light-shielding and light-reflecting properties, dispersed in a small amount of a wood-philic (or weakly hydrophilic) polymer binder that has the ability to form a film. It is a substantially non-porous layer. The light-shielding layer is used to measure color development or discoloration in the reagent layer from the light-transmissive support side by measuring the color of the blood sample spotted on the 5 development layer (described later), the red color caused by hemoglobin in the case of whole blood, etc. It shields and at the same time functions as a light reflective layer and a background layer.

The fine titanium dioxide powder used in the light shielding layer is aluminum oxide (alumina, AfL2o3) or hydrated oxide of aluminum (e.g. alumina hydrate AJI 203).
Surface treatment (mainly coating) using an aluminum compound containing trivalent aluminum and oxygen, such as R20, Au203, 3H20, etc., or a substance containing trivalent aluminum, another element (e.g., tetravalent silicon), and oxygen. titanium dioxide fine powder without alumina treatment, or titanium dioxide fine powder without surface treatment (in this specification, these are collectively referred to simply as titanium dioxide fine powder without alumina treatment),
Titanium dioxide fine powder is Anatase
, Rutile ffi, or Purkai)
(Brookite) IJ Any crystal type may be used.

The average particle size of the fine powder is approximately 0, which is commercially available.
．． 17Lm to about 1.0m, preferably about 0.151m
Approximately 0. ．． The range is 5JLm. Specific examples of fine titanium dioxide powder without alumina treatment include 1 fine titanium dioxide powder without surface treatment, fine titanium dioxide powder surface-treated with titanium hydroxide; -) 2nd/Surface 1/- Processing: 4 Sanitary/Acid/
Among these, titanium dioxide fine powder that has not been surface-treated is preferred.

Examples of lignophilic (or weakly hydrophilic) polymer binders that have the ability to form adhesion include gelatin (e.g., acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (e.g., phthalated gelatin, hydroxymethyl acrylate gelatin, etc.) etc.), polyvinyl alcohol, regenerated cellulose, cellulose acetate (e.g., cellulose diacetate), etc. Among these, gelatin, gelatin derivatives, etc. are preferable. Gelatin and gelatin derivatives should be used together with known hardening agents (crosslinking agents). Can be done. In addition,
When these polymers are used in the adhesive layer described below, the polymer binder for the light shielding layer can be selected from a wide range of wood-loving polymers, similar to those used for the indicator layer.

The ratio of fine titanium dioxide powder without alumina treatment to the polymer binder (when dry) in the light shielding layer is such that the light shielding layer is non-porous to the extent that oxygen permeability is maintained and at the same time protein impermeability is maintained. (This is smaller than the average pore size in which a developing or metering effect occurs in the porous spreading layer, and also includes microporosity of humidity that does not have a developing or metering effect.) can. Specifically, in terms of ff1ffl ratio, the polymer binder (dry weight) is about 0.6 to about 1.8, preferably about 0.8 to about 1.5, for 10 parts of fine titanium dioxide powder without alumina treatment. is within the range of

The dry thickness of the light speed 1i12 layer is about 3 pm to about 30 gm.

'IF is preferably in the range of about 5 pm to about 20 pm.

If necessary, an intermediate layer may be provided between the reagent layer and the light shielding layer, or between the reagent layer and the oxidase layer when an oxidase layer is provided, or between the oxidase layer and the light shielding layer. can. As the intermediate layer, a layer of a hydrophilic polymer having the same film-forming ability as that used for the reagent layer can be used. The thickness of the intermediate layer is approximately 0.24 m to approximately 10 m.
μ, preferably in the range of about 0.5 pm to about 7 JLm. If necessary, an adhesive layer can be provided between the light shielding layer and the porous expansion layer described below.

[Example 1 (1) Support and reagent layer An aqueous solution of the following composition was placed on a 180 μm thick colorless transparent polyethylene terephthalate (PET) smooth film coated with gelatin undercoat to a dry film thickness of about 15 μm by a conventional method. Apply it and let it dry.

(Reagent layer coating solution) Gelatin 33g 1.7-hydroxynaphthalene 1.03g Polyoxyethylene nonylphenyl ether 0.33g (2) Enzyme-containing layer Apply the following composition on the reagent layer so that the dry thickness is approximately 3 μm. An aqueous solution was applied and dried (enzyme-containing N).

(Enzyme-containing layer coating solution) Gelatin 15g Peroxidase 0.52g Glucose oxidase 0.22g Styrene/p-((1
-Methyl-1-piperazino)methyl)styrene/divinylbenzene copolymer 7.5 g Divinyl sulfone 0.40 g Polyoxyethylene nonylphenyl ether 0.33 g 4-Amino-
2-Methyl-3-phenyl-1-(2,4,6,-trichlorophenyl)-3-pyrazolin-5-one
A solution having the following composition was coated on the 5.2 g enzyme-containing layer to a dry thickness of approximately μ and dried to form an oxygen-permeable and protein-impermeable light-shielding layer.

(Light shielding layer coating liquid) Titanium dioxide 100g gelatin
10 g polyoxyethylene nonylphenyl ether 0.33 g A liquid having the following composition was applied onto the light shielding layer to a dry thickness of about 2 μm and dried to form an adhesive layer.

(Adhesive layer coating liquid) Gelatin 9.8 g Polyoxyethylene nonyl phenyl ether 0.33 g Next, distilled water was supplied to the entire surface of the adhesive layer at a rate of about 30 g/m'' to moisten it, and then a tricot made of polyester thread was applied. The knitted fabric was brought into close contact, passed through a laminating roll, and dried to produce an integrated multilayer analytical element for glucose determination [1].

[Example 2] In Example 1, 4-amino-2-methyl-3-phenyl-1-(2,4,6-)lichlorophenyl)-3-pyrazolin-5-one in the enzyme-containing layer was replaced with 4- Proceed in the same manner except for replacing aminoantipyrine (3,4 g>).
A glucose quantitative analysis element [2] was produced.

[Example 3] In Example 2, 4-aminoantipyrine in the enzyme-containing layer was removed, and 4-aminoantipyrine 2. A glucose quantitative analysis element [3] was prepared in exactly the same manner except that Og was added.

[Comparative Example 1] Based on the technique of a glucose quantitative analysis element having an oxidase layer disclosed in JP-A-60-82859, an analysis element was produced as follows.

Peroxidase was removed from the enzyme-containing layer of Example 1, and 0.23 g of peroxidase was added to the reagent layer coating solution. A glucose quantitative analysis element [4] was produced in the same manner as in Example 1 in other respects.

[Measurement example] Glucose is added to human whole blood to prepare samples with various glucose concentrations up to 1000 mg #f, and analysis element [1]
A calibration curve was created for [4].

The analytical elements [1] to [3] of the present invention have a calibration curve slope up to a high concentration, and the analytical elements [1] to [3] of the present invention have a slope of the analytical element. [4
] Quantitative performance at high concentrations was significantly improved.

(continued on next page) Table 1 Applicant: Fuji Photo Film Co., Ltd. Masashi Nakayama (voluntary) 1. Indication of the case Patent Application No. 57786 filed in 1988 2 Name of the invention Dry glucose analysis element 3 Person making the amendment Relationship to the JIF case Patent applicant Location 210 Nakanuma, Minamiashigara City, Kanagawa Prefecture Fuji Photo Film Co., Ltd. Tokyo Head Office Telephone (206) 2537 5. The statement in the claims section of the statement of contents of the amendment is amended as shown in the attached sheet.

The description in the detailed description of the invention section of the specification is amended as follows.

1) Line 1 of page 3 of the statement "Hydrogen donor and coupler" Correct it to ``a hydrogen donor and a coupler.''

Attachment Claims 1) A dry analytical element for glucose determination having a water-permeable layer on a water-impermeable, light-transparent support, wherein a reagent layer is provided on the support, both of which are water-permeable. , an enzyme-containing layer, a light-shielding layer, and a porous development layer are laminated in this order, the reagent layer containing at least a coupler of a hydrogen peroxide-detecting coloring composition consisting of and a blur, and the enzyme- and element-containing layer 1. A dry analytical element for quantifying glucose, comprising glucose oxidase, peroxidase, and a mordant, and wherein the light shielding layer is oxygen permeable and protein impermeable.

2) The analytical element according to claim 1, wherein the reagent layer contains a hydrogen donor and a coupler.

3) Claim 1) in which the enzyme-containing layer contains a hydrogen donor
analysis elements.

Claims (1)

[Scope of Claims] 1) A dry analysis element for glucose determination having a water-permeable layer on a water-impermeable, light-transparent support, wherein reagents, both of which are water-permeable, are provided on the support. layer, an enzyme-containing layer, a light-shielding layer, and a porous development layer are laminated in this order, the reagent layer containing at least a coupler of a hydrogen peroxide-detecting coloring composition consisting of a hydrogen donor and a coupler, and the enzyme-containing layer 1. A dry analytical element for quantifying glucose, comprising glucose oxidase, peroxidase, and a mordant, and wherein the light shielding layer is oxygen permeable and protein impermeable. 2) The analytical element according to claim 1, wherein the reagent layer contains a hydrogen donor and a coupler. 3) Claim 1) in which the enzyme-containing layer contains a hydrogen donor
analysis elements.