JPS63119694A - Dry analysis element containing oxidized coenzyme and production thereof - Google Patents

Abstract

PURPOSE:To improve the preservability of an analytic element containing an oxidized nicotinamide coenzyme, an electron transfer compound and a compound forming electron-accepting dye, without separating each element, by adding an alkali component as a buffering agent. CONSTITUTION:A hydrophilic polymer binder layer containing an oxidized nicotinamide coenzyme and a compound forming electron-accepting dye is formed on a light-transmitting water-impermeable substrate. The compound forming electron-accepting dye is tetrazolium salt. A layer containing an alkaline pH-buffering agent or an alkali agent is formed by coating a layer above the hydrophilic polymer binder layer with an alkali agent or an alkaline pH- buffering agent dissolved or dispersed in an organic solvent which does not swell the hydrophilic polymer binder layer. The alkali agent is e.g. ethanolamine, trimethylamine, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a dry multilayer analytical element containing an oxidized coenzyme and a method for producing the same.

[Prior Art] Reactions in which dehydrogenase enzymes and coenzymes are combined in a coupled reaction system are widely used in clinical chemical analysis. Examples include glycerin dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, glutamate dehydrogenase, aldehyde dehydrogenase, A reaction system involving α-glycerophosphate dehydrogenase, glucose-6-phosphate dehydrogenase, etc. is used to react with substrates such as triglyceride, glycerin, cholesterol, lactic acid, glutamate, glycerol-3-phosphate, glucose-6-phosphate, and aspartate amino acid. It is used to quantify enzymes such as transferase (AST), alanine amine transferase (ALT), lactate dehydrogenase (LDH), amylase, and glutinin kinase (CK), and is quantified by direct measurement of the increase or decrease in reduced coenzyme. It has the ability to be analyzed. 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 quantification. 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 via an electron-transferring compound in the presence of NADH, resulting in a flexible compound (dye) that can be detected in the visible region. A reaction system has been proposed to form

However, if the oxidized coenzyme, electron transfer compound, and electron acceptor compound are allowed to coexist, the sensitivity of the reaction deteriorates significantly over time. In particular, the Special Public Interest Publication Act 53-216
This problem is encountered when incorporating the above reaction system into a dry integrated multilayer analytical element such as those described in Japanese Patent Application Laid-open No. 77, Japanese Patent Application Laid-open No. 55-164356, and Japanese Patent Application Publication No. 60-222769.

JP-A-49-11395 proposes disposing the electron transporting compound among the three components in different layers. Furthermore, Japanese Patent Application Laid-Open No. 59-44658 discloses a method in which a hydrophobic substance is dispersed in a hydrophilic dispersion medium so that an electron transfer agent and a color-forming substance cannot substantially react with each other.

JP-A No. 59-88096 discloses that among the above three components, an electron-transporting compound and a color-forming precursor (an electron-accepting compound, which is the same as the pigment-forming substance) are substantially reacted with each other. In order to prevent
As in the publication, it is proposed that one and both particles be placed in the same layer as separate particles. However, in this way, there are two other requirements for electron transporting compounds;
Upon separation from k, the progress of the detection reaction is determined by the separated reagent (
In particular, it is controlled by the diffusion rate of electron-transferring compounds).
This leads to a decrease in reaction rate and therefore detection sensitivity.

The above NADH (or NADPH) detection composition is applied to a dry integrated multilayer analytical element (for example, Japanese Patent Publication No. 53-21677, Japanese Patent Application Laid-Open No. 55-164356, Japanese Patent Application Laid-open No. 60-2
It has been found that when applied to the reagent layer of the multilayer analytical element for automatic chemical analysis described in Japanese Patent No. 22769, the dye formation rate of the composition decreases within several weeks when stored at room temperature.

When the cause of this was investigated, it was found that the main cause was not the deterioration of the electron-accepting compound as mentioned in JP-A-57-132061, but the main cause was the deterioration of NAD (or NADP). It turns out that there is. JP-A-59-8239 as a method for preventing deterioration of coenzymes in buffer solutions
A method of adding an azide and an azide together as disclosed in Japanese Patent Application No. 8 is known. However, it is not effective unless an azide having explosive properties and J properties is used, which is undesirable from the viewpoint of safety and pollution.

In order to prevent coenzyme NAD (or NADP) from deteriorating during storage, it is known that the pH value of the reagent layer containing the coenzyme is preferably near neutral, including during coating. Also,
Diaphorase is known to be inactivated at pH below about 6.0. On the other hand, it is also known that alkaline pH (I'm) is preferable in reactions involving dehydrogenases.

No dry analytical element or integrated multilayer analytical element that simultaneously satisfies these contradictory pH conditions has been proposed.

[Summary of the Invention] An object of the present invention is to provide a dry analytical element having a detection reagent system containing an oxidized nicotinamide coenzyme, an electron-transfer compound, and an electron-accepting dye-forming compound. The object of the present invention is to improve the storage stability of oxidized nicotinamide coenzyme while maintaining a sufficient reaction rate without separating it from its components.

The present invention has an oxidized nicotinamide coenzyme, an electron-transfer compound, and an electron-accepting dye-forming compound as a detection reagent system; In a dry multilayer analytical element that contains the forming compound in the same layer, the electron transfer compound is acid-etched nicotinamide coenzyme, and the electron accepting dye forming compound is kept near neutrality during storage and placed in a separate layer. It is an integrated dry analysis element that has improved storage stability by arranging an alkaline component as a buffer.

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

The element may consist of a single layer or may include a reagent layer, a reflective layer, a porous spreading layer, a light shielding layer, a filtration layer, a detection layer (regi
stration 1a7er), water absorption layer, support,
Such analytical elements, which may be multi-layered including a subbing layer and other layers known in the art, are described in Japanese Patent Publication No. 53-2167.
Publication No. 7, JP-A-51-40191, JP-A-55
There are disclosures in Japanese Patent Application Laid-open No. 164356 and Japanese Patent Application Laid-Open No. 60-222769.

When using a support, what is the practically preferred configuration?

(1) A support with a spreading layer that also serves as a reagent layer. (2) A water absorbing layer on a support and a spreading layer that also serves as a reagent layer on the support. (3) A reagent layer and a spreading layer on a support. (4) Items that have a water absorption layer, reagent layer, and developing layer on the support (5) Items that have a reagent layer, reflective layer or pass layer, and development layer in this order on the support ( 6) A support having a water absorption layer, a reagent layer, a filtration layer, and a development layer in this order.

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, the oxidized nicotinamide coenzyme means an oxidized form of cotinamide adenine dinucleotide (NAD+) or an oxidized form of cotinamide adenine dinucleotide phosphate (NADP÷).

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

Specific examples of the electron-transferring compounds include N-methylphenazine methosulfates such as 5-methylzenadinium methylsulfate or l-methoxy-5-methylzenazinium methylsulfate, and diaphorase (dihydrocarbon). ribamide reductase, E
C1,6,4,3,), etc. can be mentioned.

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 3.3'-(3,3'-dimethoxy-4°4'-biphenylene)-bis[2-(P-nitrophenyl-2H-tetrazolium chlorite] (=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 may be mentioned. can.

For details of the reaction system containing the oxidized nicotinamide coenzyme, electron transfer compound, and electron-accepting dye-forming compound, please refer to the Clinica Chimica by A. L. Babgon et al.
Actor [0LINICA (HIMIGA AC A)
Volume 12 (1965) pp. 210-215; Clinical Chemistry by R, J, Ga7 et al. [
GLINICAL CHEMISTRYI Vol.
, 14, No. 8, 1968°740-7
Page 53: and.

R, [1, Clinical Chemistry by Capps et al., Vol. 12. No. 7, 1966,
406-413; and others.

In the present invention, the concentration in a liquid sample can be determined by including an oxidoreductase of the substance to be measured in at least one layer.
It can be used for measuring purposes. The dry analysis element of Kidori J is capable of measuring all substances that react with oxidoreductase (dehydrogenase) and for which oxidized nicotinamide coenzyme becomes an electron acceptor. For example, when measuring glycerin, glycerin dehydrogenase can be measured, and when measuring lactic acid, lactate dehydrogenase can be added.

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, the dry analytical element of the present invention is capable of measuring all oxidoreductases (dehydrogenase 2k) 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 oxidoreductase as described in Section L, the detection reagent system further contains a component of the oxidation reaction (dehydrogenation reaction) catalyzed by the oxidoreductase. For example, when the dry analytical element of the present invention is used for measuring lactate dehydrogenase activity, lactic acid is further included in the detection reagent system. In the case of glucose-6-phosphate dehydrogenase, glucose-6-phosphate is added as substrate.

Light transmittance Φ that can be used in the dry analytical element of the present invention
Examples of water-impermeable supports include polymers such as polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) with a thickness of about 50
gm to about 1mm, preferably about 80#Lm to about 30
Examples include a film in the range of 0 gm or a transparent support in the form of a sheet.

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

The light shielding layer is preferably a water-permeable layer in which light-reflecting fine particles are dispersed using a hydrophilic polymer having a film-forming layer as a binder. When measuring detectable changes (color change, color development, etc.) that occur in the blood from the light-transmitting support side, the color of the aqueous liquid dotted onto the developing layer, especially if the sample is whole blood, is measured. In some cases, it shields the red color of hemoglobin and also serves as a light reflective 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.

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.
The adhesion layer is preferably made of a hydrophilic polymer capable of adhering the spreading layer when wet with wood or swollen with water; a hydrophilic polymer that can be used for the adhesion layer. Examples include hydrophilic polymers similar to those used in water absorption layers. Among these, gelatin, gelatin derivatives, polyacrylamide, etc. are preferred, and the dry film thickness of the adhesive layer is generally about 0.5 m.
to about 20 lm, preferably from about 1 lm to about 10 lm. In addition to the detection layer, the adhesive layer may be provided on a desired layer in order to improve the adhesion between other layers. It can be provided by a known method such as by coating an aqueous solution containing the reagent layer on the reagent layer or the like.

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.

+1 for the material that makes up the matrix of the expansion layer!
Membrane filters made of paper, nonwoven fabric, woven fabric (e.g., broadcloth, boblin, etc.), knitted fabric (e.g., tricot knit, double tricot knit, Milanese knit, etc.), glass semi-wave paper, plush polymer. It is preferable to use a three-dimensional lattice structure made of 1 or polymer microbeads. Among these, it is particularly preferable to use a fibrous layer typified by woven fabrics and knitted fabrics from the viewpoint of retention of reagents.

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 hydrophilic treatment such as hydrophilic polymer impregnation treatment disclosed in JP-A-55-164356, JP-A-57-66359, etc., or these treatment steps. By sequentially carrying out these steps, the woven or knitted fabric can be made into a parent material, and the adhesive force with the lower layer (the side closer to the support) can be strengthened.

In order to adhere and laminate the development layer of an integrated multilayer analysis element made of woven or knitted fabric on the above-mentioned water absorbing layer or adhesive layer, please refer to JP-A-55-164356! =) and Japanese Unexamined Patent Publication No. 1983
It can be created according to the method disclosed in each publication such as No.-66359. That is, water (or wood containing a small amount of surfactant) is substantially uniformly supplied to the water-absorbent or adhesive layer while it is still wet after application, or to the dry layer to swell the layer, and then the fabric or adhesive layer is applied. The knitted fabric is 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 a plush polymer or a 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, 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.

In addition to the spreading layer, the reagent layer, water absorbing layer 1 reflective layer, adhesive layer, etc. can also contain a surfactant.

Examples include nonionic surfactants. Specific examples of nonionic surfactants include p-octylphenoxypolyethoxyethanol, p-nonylphenoxypolyethoxyethanol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, p-
Examples include nonylphenoxy polyglycidol and octyl glucoside. By containing a nonionic surfactant in the spreading layer, the spreading action (metering action) of the aqueous liquid sample becomes better. By including a nonionic surfactant in the reagent layer or the water absorption layer, the wood in the aqueous liquid sample can be absorbed substantially uniformly into the reagent layer or the water absorption layer during analysis operations, and the liquid may not come into contact with the developing layer. becomes quickly and substantially uniform.

If the analyte is difficult to pass through the uniform coating layer of the polymer bisoder (e.g. lipophilic analyte, polymer analyte), the porous development layer may contain some of the detection reagent components, especially degrading enzymes. It is preferable to let

Examples of the alkaline buffer or alkali agent contained in the developing layer include 2-amino-2methyl-1°3-propanediol, 2-amino-2-ethyl-1,3-propanediol, jetanolamine, and ethanolamine. , 2-
Amino-2-methyl-sama-propanediol, tris(
Examples include hydroxymethyl)aminomethane, trimethylamine, and the like. Other buffers that can be used include [
“Chemical Handbook Basic Edition” edited by the Chemical Society (Tokyo, Maruzen, 1
Published in 1966) Pages 1312-1320 R, M, G,
Dawsonet a1 edition rData for Bia
chemical research J 2nd edition (Ox
ford at the C1arendon Pre
ss, published in 1969) pp. 476-508,
r BiochemistryJ 5. From page 467 (
1966), rAnalytical Bioche
There is a pH buffer system described in, for example, 1st Try J, p. 4300-310 (1980). The porous spreading layer preferably contains a hydrophilic polymer and/or a surfactant for the purpose of controlling the aqueous liquid sample from spreading too much in the porous spreading layer.

Examples of hydrophilic polymers that can be used include polyvinylpyrrolidone, polyvinyl alcohol, polyacrylamide, polyacrylic acid, and hydrophilic cellulose derivatives. Hydrophilic cellulose derivatives are cellulose ethers in which part or all of the hydroxyl groups are etherified with a lower alkyl group having 1 to 3 carbon atoms or a hydroxy-substituted lower alkyl group having 1 to 4 carbon atoms. Examples of cellulose ethers include water-soluble methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, and hydroxybutylmethylcellulose. Among these, preferred hydrophilic polymers are polyvinylpyrrolidone, polyvinyl alcohol,
and water-soluble cellulose ethers are preferred, and two or more types of hydrophilic polymers can be used in combination. The content of the hydrophilic polymer in the porous spread layer is 1
from about 0.5 g to about 15 g per rrr', preferably about 0.5 g to about 15 g per rrr'.
The surfactant in a range of 7 g to about 10 g can be appropriately selected from nonionic, cationic, anionic, or amphoteric surfactants. Among these, nonionic surfactants are generally preferred. Nonionic surfactants include polyhydric alcohol ester ethylene oxide adducts (condensates), polyethylene glycol 7-esters, polyethylene glycol diesters, high quaternary alcohol ethylene oxide adducts (condensates), alkylphenol ethylene oxide adducts (lii compounds), and higher There are fatty acids, etc. Two or more of these nonionic surfactants can be used in combination. When a nonionic surfactant is used in combination with a hydrophilic cellulose derivative, one having an HLB value of lo or higher is preferred.

The content of the nonionic surfactant in the porous spreading layer is about 0.1 to about 3 g per m', preferably about 0.2 to about 3 g per m'.
It is in the range of about 2g.

A multilayer analytical element can have layers other than these. A water absorption layer can be provided between the support and the reagent layer. The water-absorbing layer is a layer whose main component is a hydrophilic polymer that absorbs water and swells, and is a layer that can absorb water from an aqueous liquid sample that has reached or penetrated the interface of the water-absorbing layer, and when using a whole blood sample. has the effect of promoting the penetration of crystals, which are aqueous liquid components, into the reagent layer. The hydrophilic polymer used in the water absorption layer may be selected from those used in the reagent layer described in ln7. Generally, it is preferable to use gelatin or a gelatin derivative, polyacrylamide, or polyvinyl alcohol, and among these, gelatin ( Deionized gelatin) is most preferred.

The dry thickness of the water absorption layer is about 3 pm to about Zo.

JLm, preferably in the range of about 5 tile m to about 30 lm, and coverage in the range of about 3 g/rrf to about to Og/m', preferably about 5 g/rrf' to about 30 g/rrf. The water absorption layer can contain a pH buffering agent, a known basic polymer, etc., which will be described later, to adjust the pH during use (when performing analysis operations).

A light-shielding layer (or light-reflecting layer) can also be provided between the developing layer and the reagent layer. The light shielding layer is made of a hydrophilic polymer binder similar to that used in the water absorption layer or a cross-linked hydrophilic polymer binder in which light-reflecting fine particles such as titanium dioxide or barium sulfate are dispersed. This is a layer that reflects light, thereby blocking the color of an aqueous liquid sample such as blood in the developing layer and providing a stable background. It is generally preferable to use gelatin or a gelatin derivative, polyacrylamide, polyvinyl alcohol, etc. for this light shielding layer, and among these, gelatin (deionized gelatin) is the most preferable hydrophilic polymer used for the light shielding layer. can be appropriately crosslinked and cured using a known crosslinking agent (curing agent) to form a reflective layer. The dry thickness of the light shielding layer ranges from about 51 Lm to about 50 gm, preferably about 7 m to about 30 gm, and the coverage ranges from about 5 g/rn' to about 50 g/rrl', preferably about 7 g. /rrI' ~ approx. 3
It is in the range of 0g/lrf.

On the light shielding layer, a known adhesive layer made of a hydrophilic polymer similar to that used in water absorbing layers such as gelatin can be provided for the purpose of firmly adhering and integrating the spreading layer. The dry thickness of the adhesive layer ranges from about 0.5 gm to about 5 pm.

The multilayer analytical element of the present invention can be prepared by known methods as described in the aforementioned patent specifications.

In the method of the present invention, an alkaline pH buffer (or alkaline agent) and a hydrophilic polymer used together if necessary are used in a manner that does not substantially dissolve the coloring reagent composition, and a hydrophilic polymer in the coloring reagent layer. The feature is that the binder is dissolved or dispersed in an organic solvent that does not substantially swell the binder and then added to the porous development layer.

Any organic solvent may be used as long as it satisfies these conditions, but in general, polar solvents with a boiling point of 100°C or lower are preferred, such as aliphatic alcohols (e.g., methanol, ethanol, propatool, butanol, impropyl, etc.). Alcohols), dialkyl keto/(eg, acetone), dialkyl ethers (eg, dimethyl ether), aliphatic cyclic ethers (eg, tetrahydrofuran, dioxane), and the like are suitable. Among these, aliphatic alcohols are preferred, and alcohols with low toxicity to the human body, such as ethanol, propatool, butanol, and isopropyl alcohol, are particularly preferred in consideration of the working environment. The concentration of the solution or dispersion is preferably as high as possible within a range that allows easy inclusion and uniform penetration into the porous spreading layer. and, depending on the type of hydrophilic polymer used in combination if necessary, for example, about 0.2% to about 10%, preferably about 0.2% to about 10%.
A range of 3% to about 7% is suitable. The solution may be prepared by a conventional method, and wood may be mixed with an organic solvent to the extent that migration of the coloring reagent composition to the developing layer does not substantially occur.

When a surfactant is contained in the spreading layer, a mixed solution of the pH buffer (with a hydrophilic polymer if necessary) and the surfactant may be used, or they may be contained separately in separate solutions. .

It is preferable to add the pH buffer to the porous spreading layer after laminating the spreading layer. If the spreading layer is a structure such as a microfilter, knitted fabric, or woven fabric, it should be added first and then laminated. However, in that case, there is a problem that it is difficult to control the content. Therefore, it is advantageous in terms of analytical accuracy and manufacturing cost to incorporate the pH buffer into the developing layer after providing the developing layer on the reagent layer (directly or via an adhesive layer or the like). The content may be applied, for example, by uniformly coating or spraying onto the porous spreading layer by a known method.

Drying may be performed by air drying or reduced pressure drying.

When the dry analytical element of the present invention is made into an integrated multilayer analytical element, it is cut into small pieces such as squares of approximately 15 mm to approximately 30 mm in diameter or circular pieces of approximately the same size.
-63452, JP-A No. 54-156079, JP-A-56-142454, JP-A-58-32350, and JP-A-58-501144, etc., and use it as an analysis slide by placing it in the slide frame disclosed in each publication. It is preferable from all viewpoints such as manufacturing, packaging, transportation, storage and measurement operations.

In the dry analysis element of the present invention, an aqueous liquid sample of about 5 to about 30 degrees, preferably about 8 to about 15 degrees, is applied to the developing layer, and if necessary, from about 20°C. Incubation is performed at a substantially constant temperature in the range of about 45°C. Then, detectable changes such as color changes and color development within the dry analytical element are measured by reflection photometry from one side (from the light-transmitting support side in the case of integrated multilayer analytical elements), and the liquid sample is measured using the principle of colorimetry. Analyze the component to be measured inside.

[9. Effect of light] In the dry analytical element of the present invention, the detection reagent layer containing the oxidized nicotinamide coenzyme, the electron-transferring compound, and the electron-accepting dye-forming compound and the alkaline component used as the buffer are separated into layers. By arranging the oxidized nicotinamide coenzyme, the storage stability of the oxidized nicotinamide coenzyme is significantly improved, and the formation of fog during storage is significantly prevented, thereby improving the reproducibility and detection accuracy of one analysis result.

Examples and comparative examples of the present invention are shown below.

[Example 1] Colorless clear 180 pm thick coated with gelatin
A coloring reagent layer for detecting neutral fat was coated on a polyethylene terephthalate (PET) film using an aqueous solution so as to have the following coating amount, and was dried.

Alkali-processed gelatin 17 g7m? Nonylphenoxypolyethoxyethanol (contains 15% oxyethylene) 75 mg/rn
'ATP 1.5g/magnesium gosulfate 1.2g/rn'NAD
280mg/rrrI3.3゛, -(3
,3'-dimethoxy-4,4"-biphenylene)-bis[2-(p-nitrophenyl)-2H-tetrazolium 1 dichloride 550 g/sodium citrate 3.7 g/m diaphorase (EC1,6,4, 3) 6000U
/ Goglycerol Kinase (EC2,7,1,30) 1800 U
/goglycerol-3-phosphate dehydrogenase 1o000tJ/go(EC1,1
, 99, 5) IN sodium hydroxide aqueous solution was added dropwise to this solution to pH 6,
Adjusted to 8. A light shielding layer was applied onto the coloring reagent layer using an aqueous dispersion in the following coating amount and dried.

Alkali-treated gelatin 5,0 g/rr? Rutile titanium dioxide 28 g/m'nonylphenoxypolyethoxyethanol (contains 15 oxyethylene on average) 100 mg/rrI'
Next, an adhesive layer was applied onto the light shielding layer using an aqueous solution in the following coating amount and dried.

Alkali-treated gelatin 2.7 g/m'' Nonylphenoxypolyethoxyethanol (containing an average of 15 oxyethylene) 270 g/m' Next, water was supplied onto the adhesive layer at a rate of 30 g/rn' as a dampener. A knitted fabric (average thickness 250 JLm) made of polyethylene terephthalate spun yarn (thickness 50 denier) that was moistened and then made hydrophilic by glow discharge treatment.
A spread layer was provided by pressure laminating.

On top of this, an alkaline agent-containing ethanol dispersion having the composition shown below was applied at a rate of 200 mM per lrn' and dried to complete an integrated multilayer analytical element for neutral fat quantitative analysis.

Ethanol 500 m 1 Polyvinylpyrrolidone (average molecular weight 360,000) 20 g/rrl'
Nonylphenoxypolyethoxyethanol (containing 40 oxyethylene on average) 5g/d2-amino-1-methyl-i,3-propanediol 9g/m'Next, the same amount of wood was used in place of ethanol in the alkali substance-containing dispersion. A comparative analysis element (1) was prepared in the same manner as in Example 1, except that the aqueous solution containing the alkaline agent was used.

In addition, 2-amino-
A comparative analysis element (2) was prepared in the same manner as in Example 1 except that 2-methyl 1,3-propanediol was not added.

The completed element was cut into square chips of 1.5 mm x 1.5 mm and placed in a plastic mount disclosed in Japanese Patent Application Laid-open No. 57-63452 for chemical analysis slides for triglyceride quantification and chemical analysis slides for triglyceride quantification for comparison. Analysis slide (
1) and (2) have been completed.

After leaving the three types of slides in a dry state at -25°C and 45°C for one week, 1041 human sera with different neutral fat concentrations were added.
After spotting and incubating at 37°C for 6 minutes,
Reflection optical density was measured from the PET support side using 1) 1 constant light of 540 nm.

The neutral fat concentration of the serum was separately determined by the glycerol-3-phosphate oxidase method, a calibration curve was prepared, and the result was compared with a comparative example.

For neutral fat concentration of 0, a 7% human albumin solution was spotted and the optical density was measured.

Table 1 Neutral fat in serum Example 1 Comparative example 1 Comparative example 2 Concentration (m
g/di) 0 0.17810.1800.29510.503
0.32610.51565 0.30510.30
70.41110.6030.43810.52038
1 0.83510.8500.962/1.030
0.99110.940525 1.045/1.0
G11.102/1.0811.11210.932(
-25°C/45°C) The integrated multilayer analytical element for neutral fat determination according to the present invention
Even after one week of forced aging at 5°C, the increase in background coloring optical density is negligible.
and a wide range of triglyceride content (approximately 3.3
(up to 2 times).

There was no decrease in color optical density and a calibration curve with a large slope was obtained, indicating that the quantitative analysis accuracy was high.

Claims (1)

[Claims] 1. A dry multilayer analytical element containing a dehydrogenase, an oxidized nicotinamide coenzyme, an electron transfer compound, and an electron-accepting dye-forming compound as a detection reagent system, comprising: an alkaline pH buffer or an alkaline pH buffer; An analytical element characterized in that an alkaline agent is contained in a layer different from a layer containing both the coenzyme and the dye-forming compound. 2. Claim 1, wherein the alkaline agent or alkaline pH buffer is capable of maintaining the pH value within the element within the range of 7.5 to 10.0 during analysis operations. Analysis elements described in. 3. The analytical element according to claim 1 or 2, wherein the electron-accepting dye-forming compound is a tetrazolium salt. 4. In producing a dry multilayer analytical element containing a dehydrogenase, an oxidized nicotinamide coenzyme, an electron-transfer compound, and an electron-accepting dye-forming compound as a detection reagent system, the coenzyme and the dye-forming compound are used. A hydrophilic polymer binder layer containing a hydrophilic compound is provided on a light-transparent water-impermeable support, and then an alkaline agent or an alkaline agent is dissolved or dispersed in an organic solvent that does not substantially swell the hydrophilic polymer binder layer. By applying a pH buffering agent to the layer above the hydrophilic polymer binder layer, the alkaline pH can be adjusted.
A method for producing a dry multilayer analytical element, which comprises providing a layer containing a buffer or an alkaline agent. 5. The hydrophilic polymer binder layer containing the coenzyme and the dye-forming compound is formed using a coating method using a coating liquid having a pH within the range of 6.0 to 7.0. A method for manufacturing a dry multilayer analytical element according to claim 4.