JPS6394993A - Dry analytical element containing oxidized form coenzyme - Google Patents

Abstract

PURPOSE:To eliminate a bad influence of a reducing substance in a specimen and make accurate measurement possible, by adding an electron transmitting compound into a light shielding layer or a layer above said layer. CONSTITUTION:In a multilayered dry analytical element having layers containing a dehydrogenase, oxidized form nicotinamide coenzyme, electron transmitting compound and electron accepting dye compound on a support, a light shielding layer is provided on the upper side (opposite side to the support) of the layer containing the electron transmitting compound and an electron accepting dye- forming compound is further contained in the light shielding layer or in a layer above said layer. The electron accepting dye-forming compound is preferably a tetrazolium salt.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field of the Invention] The present invention relates to a dry analytical element containing an oxidized coenzyme.

[Background of the Invention] Reactions in which a dehydrogenase enzyme and a coenzyme are combined in a coupled reaction system are widely used in clinical chemical analysis, such as glycerin dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase.

A reaction system involving glutamate dehydrogenase, aldehyde dehydrogenase, α-glycerophosphate dehydrogenase, glucose-6-phosphate dehydrogenase, etc. is used to react with triglyceride, glycerin, cholesterol, lactic acid, glutamate, glycerin-3=phosphoric acid, glucose-6-phosphate, etc. Substrates, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH)
), amylase, creatine kinase (GK), and other enzymes, and is characterized by the ability to perform quantitative analysis by directly measuring the increase or decrease in reduced coenzymes.

However, the absorption maximum of commonly used NADH (cotinamide adenine dinucleotide) or NADPH (cotinamide adenine dinucleotide phosphate) is around 340 nm, so a photometer in the ultraviolet region is required for optical quantitative determination. This makes the equipment expensive. In addition, in the ultraviolet region, various compounds have absorption, so they are easily interfered with.

Instead of directly quantifying NADH (or NADPH) by ultraviolet absorption, an electron-accepting compound is reduced through an electron-transferring compound in the presence of NADH to form a compound (dye) that is detectable in the visible region. A reaction system has been proposed.

Tetrazolium salts are used as electron-accepting compounds, but they react with reducing substances coming from the sample, causing background coloration (sometimes called fog). To prevent this, blank values are measured and corrected, and ascorbic acid is decomposed using ascorbic acid oxidase to remove the effects, but these methods take time to measure and are expensive. There were some problems.

SUMMARY OF THE INVENTION The present invention provides a dry analytical element having a precipitation reagent system comprising an oxidized nicotinamide coenzyme, an electron transfer compound, and an electron-accepting dye-forming compound.

To reduce the influence of errors caused by color development of electron-accepting dye-forming compounds caused by reducing substances in the specimen by adding an electron-transfer compound within the light-shielding layer or in a layer above the light-shielding layer. It is in the dry analysis element that achieved this.

[Detailed Description of the Invention 1 The present invention can be applied to various known dry analysis elements. In particular, it can be applied to analytical elements that include a solid carrier that can be permeated by a dehydrogenase enzyme system, a reduced coenzyme detection system, and a test liquid.

The analytical element consists of a reagent layer containing an electron-transferring compound and a light-shielding layer (instead, an electron-accepting layer) containing a trace amount of an electron-accepting dye-forming compound above the reagent layer (a layer on the opposite side of the support). (a layer containing a dye-forming compound may be provided on the light shielding layer), a second reagent layer, a reflective layer, a porous development layer, a filtration layer, a detection layer, (Registra
It may be multiple layers, including a cation layer, a water absorption layer, a support, a subbing layer, and other layers known in the art.

When using a support, a practically preferable configuration is as follows: (1) The support has a color-forming reagent layer containing an electron-transfer compound and an electron-accepting dye-forming compound, and a light-shielding layer containing an electron-accepting dye. (2) A water-absorbing layer is added between the support and the color reagent layer in the layer structure of (1); (3) A color reagent layer and a light shielding layer are added to the layer structure of (2). (4) A filtration layer is inserted between the light shielding layer and the developing layer, or between the light shielding layer and the developing layer. (4) An electron-accepting dye layer is inserted between the light shielding layer and the developing layer in the layer configuration of (2). There are things, etc.

In the dry analytical element of the present invention, the detection reagent system contained in at least one reagent layer contains an oxidized nicotinamide coenzyme. Specifically, oxidized nicotinamide coenzyme refers to cotinamide/adenine dinucleotide oxidized type for NAD+ or cotinamide/adenine dinucleotide oxidized type for NADP+.
adenine dinucleotide phosphate oxidized form).

Which of NAD÷ and NADP+ to use is determined depending on the type of oxidoreductase involved in the detection reaction as an analysis target or as a detection reagent.

In the dry analytical element of the present invention, the detection reagent system contained in at least one reagent layer closer to the support than the light shielding layer contains an electron transfer compound.

In the present invention, the electron transfer compound refers to a compound that accepts and takes electrons from the reduced nicotinamide coenzyme (electron donor) produced by the reaction of the test substance, and reduces the electron-accepting dye-forming compound described below. means a compound having

Specific examples of the electron transporting compounds include N-methylphenazine methosulfates such as 5-methylzenazinium methylsulfate or 1-methoxy-5-methylzenazinium methylsulfate, and diaphorase (dihydro ribamide reductase, EC
1, 6, 4, 3,), etc. Among these electron transfer compounds, diaphorase is preferred.

The detection reagent system used in the dry analytical element of the present invention further contains an electron-accepting dye-forming compound. The electron-accepting dye-forming compound is a compound (dye) that is reduced by the electron-transferring compound and can be detected in a long wavelength region.
In the dry analytical element of the present invention, which is a substance that forms 0, it is preferable to use a tetrazolium salt as the electron-accepting dye-forming compound.

Specific examples of tetrazolium salts include 3.3'-(3,3'-dimethoxy-4゜4゛-biphenylene)-bis[2-(p-nitrophenyl)-2H-
Tetrazolium chloride] (NET).

3-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (
INT).

3-(4,5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT).

3.3'-(4,4'-biphenylene)-bis(2,5
-diphenyl) -2H-tetrazolium chloride; 3.3°-(3,3'-dimethoxy-4,4"-biphenylene)-bis(2,5-diphenyl)-2H-tetrazolium chloride; and 3.3'- (3,3'-dimethoxy-4,4°-biphenylene)-bis[2,5-bis(p-nitrophenyl)-2H-tetrazolium chloride] can be mentioned.

The electron-accepting dye-forming compound added within the light-shielding layer or in a layer above the light-shielding layer may be the same or different from the electron-accepting dye-forming compound added to the coloring reagent layer. Good, the amount to add is 5-170 mg/rn'
can be used, preferably 7-120 mg/m
'is.

For details of the reaction system containing the oxidized nicotinamide coenzyme, electron transfer compound, and electron-accepting dye-forming compound, see CLINICA C by A. L. Babson et al.
HIMICA AGTA, Vol, 12, 210-2
15 pages (1965).

R, J, Gay etc. CLINICAL GHEM
ISTRY Vol., 14 (8), pp. 740-753 (
196B) ; and 0LINICAL CHEMISTRY by R, D, Capps II, etc., Vo
l, 12(7), pp. 406-413 (1966);
It is described in the literature such as

The present invention can be used for the purpose of measuring the concentration of a substance to be measured in a liquid sample by containing an oxidoreductase in at least one layer.The dry analytical element of the present invention reacts with an oxidoreductase (dehydrogenase). It can be used to measure all substances in which the oxidized nicotinamide coenzyme acts as an electron acceptor.
When measuring glycerin dehydrogenase and breast disfigurement, each measurement becomes possible by adding lactate dehydrogenase.

The detection reagent system used in the dry analytical element of the present invention can also be used to measure various types of oxidoreductase activities in liquid samples. That is, by using the dry analytical element of the present invention, it is possible to measure all oxidoreductases (dehydrogenases) in which oxidized nicotinamide coenzyme serves as an electron acceptor.

When the dry analytical element of the present invention is used to measure the activity of an oxidoreductase as described above, a substrate for the oxidation reaction (dehydrogenation reaction) catalyzed by the oxidoreductase is further added to the detection reagent system. For example, when using the dry analytical element of the present invention for measuring lactate dehydrogenase activity,
The detection reagent system further includes lactic acid. Glucose-
In the case of 6-phosphate dehydrogenase, glucose-6-phosphate is added as substrate.

The light-shielding layer is preferably a water-permeable layer in which light-reflecting fine particles are dispersed using a wood-philic polymer having film-forming ability as a binder. detectable changes (color change,
When performing reflection photometry from the light-transmissive support side for color development, etc., the color of the aqueous liquid dotted onto the developing layer, especially the red color of hemoglobin when the sample is whole blood, is blocked and the light is reflected. It can also be used as a layer or background layer.

As examples of the light-reflecting fine particles, titanium dioxide fine particles and barium sulfate fine particles are preferable.

In the present invention, the above-mentioned light-shielding fine particles may also be contained in the spreading layer, if necessary.

The hydrophilic polymer used in the water absorption layer, reagent layer, filtration layer, and reflection layer that can be used in the present invention generally has a swelling rate of about 1.5 to 20 at 30°C, preferably about 2.5 to 2.5. 15
Examples of natural or synthetic hydrophilic polymers in the range of
Examples include gelatin (eg, alkali-treated gelatin, ugly-treated gelatin, etc.), gelatin derivatives (eg, phthalated gelatin, etc.), agarose, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and the like. Furthermore, a surfactant (cationic, amphoteric, or nonionic surfactant) can be included if necessary.

An adhesive layer may be provided on the water absorption layer, light shielding layer, filtration layer, reagent layer, etc. for adhering and laminating the spreading layer. When the adhesive layer is moistened with water, , or a wood-philic polymer that can adhere to the spreading layer when swollen with water. Examples of hydrophilic polymers that can be used in the adhesive layer include those used in the water-absorbing layer. Examples include hydrophilic polymers similar to . Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. The dry film thickness of the adhesive layer generally ranges from about 0.5 JLm to about 20 JLm, preferably from about 1 pm to about 10 JLm. In addition to the detection layer, the adhesive layer may be provided on other desired layers to improve adhesive strength.The adhesive layer contains a wood-philic polymer and a surfactant added as necessary. The aqueous solution can be provided by a known method, such as by applying the aqueous solution onto the reagent layer or the like.

An adhesive layer for adhering and laminating the spreading layer may be provided on the layers such as the water absorption layer, light shielding layer, filtration layer, and reagent layer. The adhesive layer is preferably made of a wood-philic polymer that is capable of adhering the spreading layer when wetted with water or swollen with water. Examples of wood-philic polymers that can be used in the adhesive layer include hydrophilic polymers similar to those used in the water-absorbing layer. Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred. The dry film thickness of the adhesive layer is generally approximately 0.5JL.
m to about 20 pm, preferably from about 1 gm to about 10 gm. Note that the adhesive layer may be provided not only on the detection layer but also on a desired layer in order to improve the adhesion between other layers.
The adhesive layer can be provided by a known method such as applying an aqueous solution containing a hydrophilic polymer and a surfactant added if necessary onto the reagent layer.

A spread layer is a layer in which a liquid sample is dotted onto its surface and spread out at a rate of approximately constant amount per unit area in the lateral (horizontal) direction without substantially unevenly distributing the components contained therein. It has an effect.

Materials constituting the matrix of the spreading layer include filter paper,
Membrane filter 1 or polymer formed from nonwoven fabric, woven fabric (e.g., plain weave such as broadcloth, boblin, etc.), woven fabric (e.g., tricot knit, double tricot knit, Milanese knit, etc.), glass fibrosis paper, plush polymer It is preferable to use a three-dimensional lattice structure made of microbeads, etc. Among these, it is particularly preferable to use a fibrous layer typified by woven fabrics and knitted fabrics in terms of retention of reagents. .

The woven fabric or knitted fabric that can be used in the dry analysis element of the present invention is a woven fabric or knitted fabric that has been subjected to degreasing treatment such as washing with water to substantially remove at least oils and fats supplied or attached during yarn production, fabric production, or knitting. Fabric is preferred.

When the above-mentioned woven or knitted fabric is used as a spreading layer of an integrated multilayer analytical element, the woven or knitted fabric is further subjected to physical activation treatment (preferably glow discharge treatment or corona discharge treatment, etc.) on at least one side of the fabric, or wood-filtering treatment such as hydrophilic polymer impregnation treatment disclosed in JP-A-55-164356, JP-A-57-66359, etc., or these treatments. By performing the steps sequentially, the woven or knitted fabric can be made hydrophilic and its adhesive strength with the lower layer (the side closer to the support) can be strengthened.

In order to adhere and laminate the spread layer of an integrated multilayer analysis element made of woven or knitted fabric on the above-mentioned water absorbing layer or adhesive layer, Japanese Patent Application Laid-open Nos. 55-164356 and 57-66 are disclosed.
It can be created according to the method disclosed in each publication such as No. 359. That is, water (or water containing a small amount of surfactant) is substantially uniformly supplied to the water-absorbing layer or the adhesive layer while it is still wet after being applied, or after drying, to swell the layer.
The woven or knitted fabric is then bonded, laminated, and integrated onto the wet or swollen layer substantially uniformly and under gentle pressure.

In addition, when the spreading layer is made of butech polymer or membrane filter, Japanese Patent Publication No. 53-21677, etc., and when it is a three-dimensional lattice structure made of polymer microbeads, Japanese Patent Application Laid-Open No. 55-90859 is disclosed. etc., when it is made of filter paper or non-woven fabric, Japanese Patent Application Laid-Open No. 57-14825
They can be provided according to the methods described in Publication No. 0 and the like.

The spreading layer can be impregnated with a hydrophilic polymer or a surfactant to control the spreading.

Examples of wood-philic polymers include cellulose derivatives, polyvinylpyrrolidone, and polyvinyl alcohol.

Examples include polyacrylamide.

In addition, surfactants include nonionic, amphoteric, cationic,
It can be appropriately selected from anionic surfactants depending on the analysis item. Furthermore, the developing layer may contain a reaction reagent or an enzyme for a detection substance (lipophilic high molecular weight substance) that is difficult to pass through the coating layer.

When the dry analysis element of the present invention is made into an integrated multilayer analysis element, it is cut into small pieces such as squares of about 15 mm to about 30 mm or circular pieces of approximately the same size.
No. 3452, Japanese Unexamined Patent Publication No. 156079/1983, Japanese Unexamined Patent Publication No. 1983
Manufacturing, packaging,
It is preferable from all viewpoints such as transportation, storage, and measurement operations.

When using the dry analysis element of the present invention, an aqueous liquid material of about 5 to about 30 degrees, preferably about 8 to about 15 degrees, is dotted onto the spreading layer, and if necessary, about 30 degrees of aqueous liquid is applied. 20℃
Incubation is carried out at a substantially constant temperature ranging from about 45°C to about 45°C. Thereafter, detectable changes such as color changes in the dry analytical element from one side (from the light-transmissive support side in the case of integrated multilayer analytical elements) are measured by reflectance photometry in the liquid sample using the principle of colorimetry. Analyze the component to be measured.

[Example] A reagent coloring layer was coated on a 180 pm thick polyethylene terephthalate (PET) film with a gelatin undercoat so that the dry film thickness was 13 μm using the following formulation.

Alkali-treated gelatin 9.4g3.3'-(
3,3'-dimethoxy-4,4' (biphenylene)-bis[2-(p-nitrophenyl-2H-tetrazolium chloride)] 0.04g ATP O, 79gNAD
O, 3g Dotite Nitrotetrazolium Blue 0.2g Sodium Citrate 3g Diaphorase 2800U Glycerol Kinase 880U Glycerol Triphosphate Dehydrogenase 5400U Note Nido-Tite Nitrotetrazolium Blue's chemical name is 3.3'-
(3,3'-dimethoxy-4,4'-biphenylene)bis[2-(p-nitrophenyl)-5-phenyl-2H
-tetrazolium] dichloride.

Note that the reagent layer contains alkali-treated gelatin with a concentration of 17.5
It was applied in an amount of g/1rrr' (support surface ff1).

Next, apply a light shielding layer with the following recipe to a thickness of 7gm.
Dry.

Alkali-treated gelatin 7g Rutile titanium dioxide 35g 3.3'-(3,3°-dimethoxy-4,4" (biphenylene)-bis[2-(p-nitrophenyl-2H-tetrazolium chloride)] 0.1g Water 50g
Note that the light shielding layer contains alkali-treated gelatin with a content of 5.0%.
It was applied in an amount of g/lrn' (support surface area).

Next, water was supplied as dampening water at a rate of 30 g/m' onto the coating layer to wet it, and then a tricot knitted fabric (average thickness 250 Bm) made of polyethylene terephthalate spun yarn (50 denier) made hydrophilic by glow discharge. ) was pressure-bonded and laminated to provide a development layer. Add 1r of the following prescription solution on top of this.
200 ml per rI'' was applied and dried to form a multilayer analytical element for neutral fat detection.

water 50
0mM polyvinylpyrrolidone (K2O) 20
g Polyoxyethylene nonylphenyl ether (n=40) 5 g2-
Amino-2-methyl-1,3-propanediol 9g Riboprotein lipase 11400U [Comparative example] A comparative sample was prepared using the same method as in the example except that the electron-accepting dye-forming compound was removed from the light-shielding layer. . The completed analysis element was cut into square chips of 1.5 cm x t and scm, and placed in a plastic mount disclosed in JP-A-57-63452 to complete a chemical analysis slide for quantifying neutral fat.

[Evaluation of analysis slides] Each slide shows the neutral fat concentration value after heparin blood collection and centrifugation, 51.130.196.322°421.523
One LOg of human plasma at mg/di (value determined by solution analysis using the glycerol triphosphate oxidase method) was applied, and after incubation at 37°C for 6 minutes, color was developed from the PET support side using measurement light with a center wavelength of 640 nm. The reflected optical density of the sample was measured and a calibration curve was created.

The calibration curve obtained for each individual analysis slide is shown in Table 1.

Table 1 Example Comparative Example 51 0.420 0.418130
0.551 0.531196 0.672
0.681322 0.860 0.87
1421 0.996 0.990523
1.112 1.081 Next, the influence of bilirubin was investigated by developing color in the same manner as the calibration curve using human samples with different bilirubin concentrations.

The results obtained are shown in Table 2.

As is clear from the results in Table 2, the analysis slides of the Examples are less affected by bilirubin than the analysis slides of the Comparative Examples.

Table 2 Neutral fat concentration Bilirubin concentration Example Comparative example 54
2.0 54 54132 1
2.5 131 150210 3.2
211 211122 1.0 12
0 121 Next, ascorbic acid was added to the human sample using O15.
, 15, and 20 mg/dl, respectively, were used to examine the effects on each analytical slide.

The results are shown in Table 3.

Table 3 0 5 15 20IIg/d sentence example 75
78 80 83 Comparative example 74 82 9
1104 It is clear that analytical slides using analytical elements configured according to the invention exhibit excellent performance.

Claims (1)

[Scope of Claims] 1. A multilayer dry analytical element having a layer containing a dehydrogenase, an oxidized nicotinamide coenzyme, an electron transfer compound, and an electron-accepting dye-forming compound on a support, comprising: It has a light-shielding layer above the layer containing the electron-transfer compound (on the opposite side from the layer containing the electron-transfer compound and the support), and further has an electron-accepting layer on the light-shielding layer or a layer above the light-shielding layer. An analytical element characterized in that it contains a dye-forming compound. 2. The analytical element according to claim 1, wherein the electron transfer compound is diaphorase. 3. Oxidized nicotinamide coenzyme is NAD or NA
The analysis element according to claim 1, characterized in that it is a DP. 4. The analytical element according to claim 1, wherein the electron-accepting dye-forming compound contained in the light-shielding layer or a layer above the light-shielding layer is a tetrazolium salt.