JPH04106469A - Testing device - Google Patents

Abstract

PURPOSE:To achieve higher stability against change with time by catching a radical which will cause a coloring during a preservation to add an agent for trapping deactivating free radical into a reagent layer. CONSTITUTION:A testing device 1 is provided with a reagent layer 3 on one side of a support 2. For example, the support 2 is shaped in a plate with a thickness of 20-500mum and constructed of material inactive to a sample. Component material of the support 2 employs various resins such as polyethyleneterephtharate or glass or the like. The reagent layer 3 has a reagent supported on a carrier made of a porous material. The carrier is preferably those made on non-fibrous or fibrous porous material. The composition of the reagent is determined properly by a specified component to be detected in the sample. Moreover, a radical scavenger of free radical trapping agent is added into the reagent layer 3. This enables the catching and deactivating of a substance affecting the coloring of a leuco pigment such as radical thereby achieving longer stability.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a test device for detecting a specific component in a specimen by a color reaction.

<Prior art> When detecting a specific component in a specimen, such as glucose or cholesterol in blood, or glucose or hemoglobin in urine, a reagent carrying a reagent that reacts with the specific component to develop a color is used. A test device with layers is used.

The color intensity (color density) of this test device depends on the amount of the specific component in the sample, so it is possible to quantify the specific component in the sample by measuring the color intensity of the test device. can.

For example, when measuring the amount of sugar in a sample, the coloring principle of the reagent layer is as shown in the formula below.
It is decomposed into gluconic acid and hydrogen peroxide by an enzyme called D), and this hydrogen peroxide is decomposed into water and active oxygen by an enzyme called peroxidase (POD), and this active oxygen reacts with a chromogen (color former). It becomes a pigment.

GOD glucose +0 Cholesterol ester in the sample is decomposed into cholesterol and free fatty acids by cholesterol esterase (COE), and this cholesterol and cholesterol in the sample are decomposed into Δ4-cholestenone and hydrogen peroxide by cholesterol oxidase (COD), and this peroxide Hydrogen is decomposed into water and active oxygen by peroxidase (POD), and this active oxygen reacts with a chromogen (coloring agent) to become a pigment.

CUE - Cholesterol ester - - → Cholesterol 10-L + Free fatty acid OD Cholesterol - Luna 02 - - - Δ 4-Cholestenone + H2O2OD H20, - [0 Co + HzO [0] Decachromogen - Pigment like this A problem with test devices is that the reagent layer develops color during the manufacture of the test device or during storage in an unused state.

In particular, those using leuco dyes as color formers have high sensitivity, so their stability during separation is poor, and the phenomenon of color development during storage is noticeable.

<Problems to be Solved by the Invention> The present invention has been made in view of the above-mentioned drawbacks of the prior art, and its purpose is to provide a test device with excellent stability over time.

Means for Solving the Problems> Such objects are achieved by the following inventions (1) to (4).

(1) A test device having a reagent layer supporting a reagent containing a leuco dye as a coloring agent, wherein the reagent layer contains a free radical scavenger.

(2) The test device according to (1) above, wherein the leuco dye is benzidine or a benzidine-based compound.

(3) The test device according to (1) or (2) above, wherein the free radical scavenger is at least one selected from the group consisting of dithioerythritol, dithiothreitol, glutathione, and 2-mercaptoethanol.

(4) The amount of the free radical scavenger added is determined in the reagent layer III.
(1) to (3) above, which is 1 to 1000 mg per +2
The test device described in any of the above.

<Function> The present inventors studied the cause of color development of the reagent layer during the manufacture and storage of the test device, and found the following.

For example, 0-tolidine, which is an example of a leuco dye, reacts with active oxygen to form oxidized o-tolidine as shown in the following formula and develops a color.

(0-tolidine) (oxidized o-tolidine) In this case, a cation radical of 0-tolidine is generated during the reaction, which produces color (green: λmax 630
nm).

In the reagent layer, for example, high molecular weight polymers and surfactants used as binders and thickening agents coexist, but these agents contain unavoidable substances such as oxidizing agents, reaction initiators, and polymerization initiators. Impurities remain, and these impurities react with other coexisting substances to generate radicals, which oxidize 0-tolidine and cause color development.

Such color development may be promoted by environmental conditions such as temperature, humidity, and light.

Therefore, the present invention aims to improve stability over time by adding a free radical scavenger to the reagent layer that captures and deactivates radicals that cause color development during storage.

<Example> Hereinafter, the test device of the present invention will be described in detail based on a preferred example shown in the accompanying drawings.

FIG. 1 is a cross-sectional front view showing an example of the configuration of the test device of the present invention. As shown in the figure, the test device 1 has a reagent layer 3 disposed on one side of a support 2.

The support 2 has a plate shape with a thickness of, for example, about 20 to 500 μs, and is made of a material that is inert to the specimen.

Specific constituent materials of the support 2 include polyethylene terephthalate, cellulose ester (cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), polycarbonate of bisphenol A, polymethyl methacrylate, polyvinyl chloride, polypropylene, polystyrene, Examples include various resins such as polyvinyl alcohol, glass, and the like. Further, the support 2 may be a stack of sheets made of two or more of the above materials.

The reagent layer 3 is composed of a carrier supporting a reagent to be described later.

The carrier is preferably made of a non-fibrous or fibrous porous material.

Typical examples of non-fibrous porous materials include filter paper and membrane filters, as well as dispersions of porous materials such as diatomaceous earth and microcrystalline materials dispersed in a binder, and microspherical beads of glass or resin that are bonded to each other. Examples include porous aggregates bonded at points.

Furthermore, examples of the fibrous porous material include woven or knitted fabrics, nonwoven fabrics, short fiber aggregates, and the like.

Examples of the fiber material include natural fibers such as cotton, silk, and wool, and resins such as glass and nylon.

The thickness of the carrier, that is, the thickness of the reagent layer 3 is not particularly limited, but is about 0.1 to 2 mm, particularly 0.2 to 1III.
It is preferable to set it to about 1.

The composition of the reagent is appropriately determined depending on the specific component to be detected (quantified) in the specimen. For example, when detecting glucose in a sample, the enzyme glucose oxidase (
COD) and peroxidase (poD), and a color former (chromogen) are the main components of the reagent. In addition, when measuring the total cholesterol amount in a sample, the enzymes cholesterol esterase (COE), cholesterol oxidase (COD), and peroxidase,
The color former is the main component of the reagent.

In addition, other enzymes may be used instead of the above enzymes, such as uricase and peroxidase, riboprotein lipase and glycerol oxidase and peroxidase, phospholipase D, choline oxidase and peroxidase.

Leuco dyes are used as coloring agents, such as o-
Tolidine, m-tolidine, benzidine, 3,3° 5
．． Benzidine or benzidine compounds such as 5°-tetramethylbenzidine, 0-methylbenzidine, 0-dianisidine, 2.7-diamitsufluorene, 4.4°-diaminodiphenyl, 0-phenylenediamine, m-phenylenediamine, p -phenylenediamine, 2.3-)
lylenediamine, 2.4-) lylenediamine, 2.5-
Various substituted phenylene diamines such as lylene diamine and 2,6-lylene diamine, phenols such as amorphous gallic acid, gallic acid, phloroglucinol, hydroquinone, and leucoindophenol, guaiacol, pyridine derivatives, substituted azines, Examples include leucomalachite green.

Such leuco dyes are easily affected by radicals and easily develop color during storage, so they are effective in applying the present invention.

Among the leuco dyes, the benzidine or benzidine-based compound is particularly easy to develop color during storage, and is therefore preferable for application of the present invention.

A free radical scavenger is added to such a reagent layer 3.

As a result, substances such as radicals that affect the color development of the leuco dye are captured and deactivated, and stability over time is improved.

Examples of the free radical scavenger include dithioerythritol, dithiothreitol, glutathione, 2-mercaptoethanol, etc., and various substances having similar functions, among which dithioerythritol, dithiothreitol and Glutathione is particularly effective and preferred.

In addition to the function of capturing radicals, these free radical trapping agents have the function of stabilizing the function of enzymes coexisting in the reagent layer 3. In other words, enzymes contain various metal ions, and when these are oxidized, the enzyme becomes inactive, but the free radical scavenger prevents the oxidation of the metal ions in the enzyme and maintains the enzyme activity. Appropriate color reaction can be obtained during measurement.

Examples of enzymes that have the effect of maintaining activity are, for example,
Peroxidase, glucose oxidase, cholesterol esterase, cholesterol oxidase, glycerol oxidase, choline oxygase, urease, creatine amidinohydrolase, biroglutamyl peptigase, protocatechuate 3゜4-dioxygenase, proline dehydrogenase, leucine dehydrogenase, glutathione Examples include peroxidase, β-glucosidase, α-glucosidase, and the like.

The amount of the free radical scavenger added should be such that it can sufficiently capture the radicals in the reagent layer 3 and not inhibit the color reaction during measurement (specifically, 1 m2 of the reagent layer). ~1
000 mg, especially 10 to 100 mg.

Moreover, a pH adjuster, a surfactant (wetting agent), a light-reflecting substance, a stabilizer, a sensitizer, a viscosity agent, etc. may be added to the reagent layer 3 as necessary.

As a pH adjuster, phosphate buffer, citrate buffer,
Examples include borate buffer, Tris buffer, Gud buffer, and the like.

The surfactant (wetting agent) is used to uniformly wet (penetrate) the sample liquid when the reagent layer 3 and the sample come into contact, and includes polyvinylpyrrolidone, sodium cholate, sodium lauryl sulfate, and sodium dodecyl sulfate. , alkyl sulfates such as sodium tetradecyl sulfate, alkyl benzene sulfonates such as sodium dodecylbenzenesulfonate, sodium dioctyl sulfosuccinate, sodium dibutyl sulfosuccinate, sodium di-2-ethylhexyl sulfosuccinate (Aerosol-OT)
) and other dialkyl sulfosuccinates.

The light-reflecting substance is used to eliminate the influence of red blood cells on color density measurement when the sample is whole blood, and includes fine particles such as titanium oxide, barium sulfate, aluminum, and various ceramics.

The stabilizer is for improving color stability after coloring, and includes a copolymer of methyl vinyl ether and maleic anhydride, a half ethyl ester thereof, and the like.

The sensitizer enhances the activity of the coloring reaction of the coloring agent by peroxidase, and includes quinoline and its derivatives, such as quinine, cinchonine, 6-medoxyquinoline, quinaldine, 8-amino-6-medoxyquinoline. , 2-quinolitool, isoquinoline, benzo(f)quinoline, 3-aminoquinoline, and the like.

The presence of such a sensitizer usually accelerates the oxidation reaction and increases the color intensity of the oxidized color former, thereby increasing the sensitivity of the test device.

The thickening agent is for preventing the drug from flowing out from the carrier of the reagent layer 3, and includes polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, acrylate,
Examples include polymers such as polyacrylamide, poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), carboxymethyl cellulose, gelatin, and gum arabic.

The above reagent (including additives) may be supported on a carrier by impregnating the carrier with a liquid containing the reagent (e.g., immersing the carrier in an immersion liquid), and then drying it.

When there are two or more types of liquids, impregnation and drying are repeated.

Note that such a reagent layer 3 can be formed by applying, for example, double-sided adhesive tape (not shown), adhesive, or other holding device (for example, the holding member described in Japanese Patent Application No. 63-334198) to the surface of the support 2. Fixed by

Further, the reagent layer 3 may be obtained by applying a liquid containing a reagent or the like to the surface of the support 2 and drying it.

In this case, the reagent layer 3 contains (disperses) a predetermined reagent in a hydrophilic binder such as gelatin, polyvinyl alcohol, polyvinylpyrrolidone, agarose, sodium polyvinylbenzenesulfonate, polyurethane, or polyvinylpropionate. ) consists of a diluted composition.

In addition to the various additives described above, a hardening agent (crosslinking agent) may be added to such a coating type reagent layer 3.

As a hardening agent, it crosslinks gelatin etc.
For example, N,N'-hexamethylene-1,6-bis(
1-aziridinecarboxamide) (HDU), trimethylolbroban tri-β-aziridinylpropionate (TAZM), tetramethicolmethane-tri-β
-Aziridine derivatives such as aziridinyl propionate (TAZO).

The thickness (dry film thickness) of such a coating type reagent layer is not particularly limited, but it is preferably about 1 to 100 p, particularly about 5 to 30 p.

In the illustrated example, there is only one reagent layer 3, but a plurality of reagent layers each containing reagent components of different compositions may be laminated. In particular, a layer in which the light-reflecting substance is dispersed (light-reflecting layer) may be separately provided on the surface of the reagent layer 3.

Although the test device of the present invention has been described above with reference to the configuration example shown in the drawings, the present invention is not limited thereto. In particular, the layer structure of the test device can be arbitrary, and may include a developing layer, a light reflecting layer, an absorbing layer (sample amount adjustment layer), a protective layer, an oxidizing agent-containing layer, and other layers provided for various purposes. There may be layers.

The test device of the present invention is used for testing specimens (e.g. whole blood,
In addition to detecting glucose and cholesterol in body fluids such as plasma, serum, urine, feces, saliva, lymph, and cerebrospinal fluid, it also detects BUN, creatinine, calcium, oxalic acid, triglycerides (neutral fats), free fatty acids, Ketone bodies, bilirubin, urobilinogen, protein, nitrite, ascorbic acid, hemoglobin, myoglobin, white blood cells, G
It can also be applied to the detection of OT%GPT1ALP, γ-GTP, etc., and can also be applied to other fields such as analysis of food and environmental samples.

<Example> Examples of the present invention will be described below.

(Example 1) Solutions 1 and 2 having the composition shown below were prepared, and the thickness was about 0.
A 32 mm knitted fabric (T-1000 manufactured by Seiren ■) was first impregnated with Solution 1 and dried (40°C, 60 minutes), and then impregnated with Solution 2 and dried (40°C, 5 minutes).

Thereafter, this was cut into a size of 9 mm x 13 mm and adhered to a polystyrene sheet support with a thickness of 0.3 mm using double-sided adhesive tape to obtain a test device for measuring the total amount of cholesterol in blood.

The amount of free radical scavenger supported in this test device was 30 mg per reagent layer 1+a''.

1 indigo polyethylene glycol 4000 60mg cholesterol esterase 2000u cholesterol oxidase 500u peroxidase 11000uI Linshun buffer (p
i(6,0) 1.5mj distilled water
4.5mj dithioerythritol (free radical scavenger) IIIg soluble i
3.3',5.5-tetramethylbenzidine 70 g (color former) Aerosol-OT (surfactant)
100mg acetone 7
mC (Example 2) Dithiothreitol: 1 mg of free radical scavenger in solution 1
The procedure was the same as in Example 1 except that .

(Example 3) The procedure was the same as in Example 1 except that the free radical scavenger in Solution 1 was replaced with 4 mg of glutathione.

(Example 4) The procedure was the same as in Example 1 except that the free radical scavenger in Solution 1 was replaced with 2-mercaptoethanol (1 mg).

(Example 5) The procedure was the same as in Example 1 except that the coloring agent in Solution 2 was replaced with 0 mg of 0-tridinenia.

(Comparative Example) The same procedure as Example 1 was carried out except that Solution 1 did not contain a free radical scavenger.

Each test device of Examples 1 to 5 of the present invention and the comparative example was stored for a long time in a bright room at a temperature of 22° C. and a humidity of 65% RH.

When the coloring status of the reagent layer was examined with the naked eye, all of the test devices of Examples 1 to 5 showed that no coloring was observed even after 5 hours had passed, and the storage stability was good. However, in the test device of the comparative example, green color development was observed after 5 hours had passed.

Further, for the test devices of Example 1 and Comparative Example, the surface of the reagent layer was irradiated with white light from two directions at an angle of 45°, and the spectrum of the reflected light (direction perpendicular to the surface of the reagent layer) was measured.

The results are shown in the graph of FIG.

The device used for the measurement was MCPD-2 manufactured by Otsuka Electronics.
It was type 00.

As shown in the figure, color development due to radicals (maximum wavelength 670 nm) was observed in the comparative example, but in Example 1, the radicals were captured and deactivated, so no color was developed at all.

In addition, for each test device of Examples 1 to 5, the cholesterol concentration was 50.100.200.300.400.
When cholesterol concentration was measured using serum samples adjusted to 500 mg/dj and 500 mg/dj, there was almost no difference in the measured values with either test device.
I was able to perform normal measurements.

<Effects of the Invention> As described above, the test device of the present invention has excellent stability over time and does not develop color even when stored for a long time.

In addition, when detecting and measuring a specific substance in a specimen, it is possible to perform proper detection and measurement without adversely affecting the color reaction.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional front view showing an example of the configuration of the test device of the present invention. FIG. 2 is a graph showing the reflection spectrum of the reagent layer. Explanation of symbols 1...Test device 2...Support 3...Reagent layer

Claims (4)

[Claims] (1) A test device having a reagent layer supporting a reagent containing a leuco dye as a coloring agent, characterized in that the reagent layer contains a free radical scavenger. (2) The test device according to claim 1, wherein the leuco dye is benzidine or a benzidine-based compound. (3) The test device according to claim 1 or 2, wherein the free radical scavenger is at least one selected from the group consisting of dithioerythritol, dithiothreitol, glutathione, and 2-mercaptoethanol. (4) The amount of the free radical scavenger added is 1 m^ of the reagent layer.
The test device according to any one of claims 1 to 3, wherein the amount is 1 to 1000 mg/2.