JPS60178358A - Glucose detecting ink composition and tester formed by using the same - Google Patents

Abstract

PURPOSE:To enhance sensitivity and quantitative analytical capacity, by dissolving or dispersing a reagent composition, which consists of glycosidase, peroxidase, an oxidizable indicator, a binder and a stabilizer, in a non-aqueous solvent. CONSTITUTION:A compound having proper oxidation inhibiting activity such as tocopherols and a surfactant such as glycerol monoacetate are added to glycosidase, peroxidase, an oxidizable indicator such as o-toluidine and a binder such as a polyester resin to form a reagent composition which is, in turn, dissolved or dispersed in a non-aqueous solvent such as benzene to constitute a glucose detecting ink composition. By using this composition in a state applied to a support such as paper, the discoloration and coloration phenomenon of the oxidizable indicator by peroxide in the open air can be prevented.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field of the Invention The present invention relates to an ink composition that can be used to form a test body that can easily and quickly detect glucose in a solution, particularly a body fluid such as urine, and an ink composition that can be formed using the same. Regarding the tested specimen.

Technical background of the invention and its problems Quickly and easily knowing the amount of glucose in body fluids such as urine, blood, or lymph can be used for early detection of diabetes.
Essential for diagnosis and management.

Conventionally, the main methods for detecting glucose in body fluids, especially urine, are
Therefore, test specimens coated with glucose test reagents have been used. This test specimen had the advantage of being easy to use and to perform the determination in a short time.

By the way, in a test sample for detecting glucose in body fluids, glucose reacts with oxygen in the air due to the action of glucose oxidase, and is finally oxidized to gluconic acid and hydrogen peroxide. This hydrogen peroxide produces nascent oxygen through the action of peroxidase, which immediately reacts with an oxidizable indicator such as O-IJ to cause the indicator to develop a color.

A test sample for detecting glucose in body fluids using this principle is
A reagent composition consisting of a sugar oxidase, a peroxidase, and an oxidizable indicator is dissolved or dispersed in water or a water-alcoholic solvent, and a lip is impregnated with the resulting liquid, dried, and the mouth paper is Attach to glass film and apply I as appropriate.
It has been produced by cutting it into L size.

However, this method has the following problems.

(a) When an impregnation solution is prepared by dissolving or dispersing a reagent composition consisting of sugar oxidase, peroxidase, and an oxidizable indicator in water or a water-alcoholic solvent, the enzyme is unstable and deactivated; The impregnating liquid deteriorates rapidly. For this reason, it is necessary to impregnate the paper in multiple steps immediately after preparing the impregnating liquid. Furthermore, even if the paper is impregnated immediately, there is a problem that some of the enzymes are deactivated and the impregnating liquid is partially deteriorated.

(b) As mentioned above, since the impregnating liquid is unstable and the manufacturing process is complicated, it is difficult to maintain a constant quality of the test object obtained, and it is necessary to ensure the test accuracy and reliability of the test object. requires special care and skill, which reduces manufacturing efficiency and increases manufacturing costs.

For this reason, the development of inspection bodies that can simplify the manufacturing process and are suitable for mass production has been progressing, and Patent Publication No. 44-25953 V
C is a water-alcohol mixed solution containing enzymes in advance.
This is followed by an indicator, a pI buffer, a polymeric binder, and a water-absorbing carrier. A method has been proposed in which an ink composition having printing or coating properties is prepared by mixing C. There is a same C. However, in this method, the enzymes are partially dissolved in water and therefore unstable and rapidly deactivated, so it was necessary to print immediately after preparation. Furthermore, it was necessary to dry at a low temperature to prevent deactivation of the enzyme, and in order to obtain long-term storage stability, it was necessary to minimize the amount of residual moisture in the applied reagent layer.

Under these circumstances, this application was filed under Japanese Unexamined Patent Publication No. 58-20
No. 9995, enzymes are dispersed in a non-aqueous solvent that hardly dissolves the enzymes, and then an indicator,
We proposed a method of preparing an ink composition by dissolving or dispersing a buffer, a binder, and a water-absorbing carrier, and printing this ink composition on a support to produce a test object. According to this method, a specimen that exhibits excellent quantitative performance and sensitivity for glucose can be obtained, but this specimen gradually begins to take on a bluish tinge when exposed to the atmosphere for a long time. However, this test specimen indicates the presence of glucose by turning blue, so the discoloration that occurs when the test specimen is stored in the atmosphere can lead to incorrect judgments when testing body fluids. There was a strong desire for a solution to this problem.

As a result of the inventors' research to solve the above problems,
The blue color in the glucose detection test piece mentioned above is
This was discovered because the reagent composition, particularly the oxidizable indicator contained therein, is affected by peroxide substances present in trace amounts in the atmosphere. As a result of further research, it was found that this color reaction can be prevented by adding a stabilizer to the reagent composition. Has oxidizing activity
It has been found that certain surfactants as well as certain surfactants are particularly effective.

OBJECTS AND SUMMARY OF THE INVENTION The present invention seeks to overcome the drawbacks f:j')'i' associated with the prior art, as follows: 1. c. It has a purpose.

(a) To provide an ink composition capable of forming a glucose detector having excellent sensitivity and quantitative performance, and a glucose detector formed using the same.

To provide an ink composition for glucose extrusion which is stable even when stored in the atmosphere for a long period of time and does not cause discoloration, and to provide a glucose detector formed using the ink composition.

To provide a glucose extrudate that can be formed by a C1 coating method, particularly a printing method, and therefore to freeze the manufacturing process, and to provide an ink composition for glucose detection used in the formation thereof.

The ink composition for detecting glucose in body fluids according to the present invention includes:
A reagent composition consisting of a sugar oxidase, peroxidase, an oxidizable indicator, a binder, and a stabilizer is dissolved or dispersed in a nonaqueous solvent. Further, the glucose detector according to the present invention has a glucose detection area formed by coating a glucose detection ink composition having the above composition on a support.

First, the ink composition for glucose detection used in forming the test object according to the invention will be specifically explained below.

B) Principle Glucose in body fluids reacts with oxygen in the air through the action of glucose oxidases such as glucose oxidase IL, and is finally oxidized to gluconic acid and hydrogen peroxide. The generated hydrogen peroxide multiplies oxygen which is generated by the action of peroxidase, and this oxygen immediately reacts with an oxidizable indicator such as 0-tolidine, causing the indicator to develop a color. Depending on the degree of color development, the presence or absence of glucose in the body fluid and its amount are determined in the limbs 1j (-).

(Glucose oxidase) Glucose oxidase Id as a sugar oxidase - Purification model 1 Also in the form of a lyophilized product? - For example, if the enzyme is used with a titer of 100 units/m9, the enzyme should be used in an amount of 0.02 to 2%, preferably 0.02% based on the solid content of the ink composition. Preferably, it is present in an amount of 2 to 1.8% by weight.

c) Peroxidase Peroxidase is an enzyme that catalyzes the oxidation of four organic substances by hydrogen peroxide or organic peroxide, and is extracted from horseradish. For example, when a lyophilized product with a titer of 100 units/unit of activity is used, the enzyme content is preferably 0.002 to 0.02 to 0.2% based on the solid content of the ink composition. Preferably, it is present in an amount of 92%.

2) Oxidizable indicators Oxidizable indicators are indicators that develop color when oxidized by oxygen, and conventionally known compounds such as benzidines and N-alkylated benzidines can be widely used. Among these, 0-tolidine is particularly preferred, and this oxidizable indicator is 0-tolidine based on the solid content of the ink composition.
．． It is desirable to be present in an amount of 5 to 107% by weight, preferably 0.6 to 61% by weight.

e) Binder A binder is a substance that affects the components and pH of the body fluid to be tested, and also affects reagents, especially enzymes, and oxidizable indicators, but does not interfere with the color reaction. is required. Satisfying these requirements satisfies V(
(1) Polyester tree 1
1a, alkyd (Zaizuki, polyurethane resin, polystyrene resin, acrylic resin-epoxy resin, vinyl chloride resin, vinyl chloride copolymer resin, polyvinyl butyral resin, polyvinyl A/7 alcohol resin, polyvinylpyrrolidone resin, maleic anhydride type) Synthetic resins such as Kyodogoitai, (ii) Cellulose derivatives such as methylcellulose, ethylcellulose, hydroxyethylcellulose, and carboxymethylcellulose, (iii) Tenbun and Taa
Class 4I.

Natural polymers such as gelatin, casein, or sodium alginate are used. Moreover, two or more of these binders may be combined. This binder is preferably 0.1 to 20% by weight based on the solid content of the ink composition.
Preferably present in an amount of 0% by weight.

f) Stabilizer Stabilizer contributes to the stabilization of the reagent composition consisting of sugar oxidase, peroxidase, oxidizable indicator and binder. Among these, oxidizable indicators tend to change color due to the action of peroxides in the atmosphere, as mentioned above, but the main role of stabilizers is to prevent this. , a compound having a moderate antioxidant activity, a specific surfactant typified by glycerol esters, or a mixture thereof.

Substances that exhibit antioxidant effects include 2.6-di-butylmethoxyphenol, P-methoxyphenol, tonaphthol, pentamethylphenol, 2°2.5, 7.8
- Radical scavengers such as pentamethyl 6-hydroxychroman and tocopherol a, or reducing agents such as ascorbic acid are used, but these are effective at least in the reaction system for glucose detection (oxidizable indicator), which is the purpose of this test. It also has the property of inhibiting oxidation reactions and reducing sensitivity. Therefore, it is preferable to use an antioxidant substance that does not inhibit the reaction system and reduces the effects of peroxides yi in the atmosphere. Preferred antioxidant substances include radical scavengers, of which tocopherols (α-1β-1γ-1δ-) are particularly effective. The amount of tocopherol added is preferably 0.02 to 0.21% by weight based on the solid content of the ink composition. In this case, if it is less than 0.02%, the coloring phenomenon in the atmosphere can be effectively prevented, and if it exceeds 0.2% by weight, an adverse effect on the coloring reaction will begin to be observed and the sensitivity will decrease.

Furthermore, in addition to the above-mentioned substances that exhibit antioxidant effects, as another additive that prevents the coloring phenomenon of the reagent reaction layer in the atmosphere, certain specific additives such as glycerol esters are used, although the mechanism of action is unknown. There are surfactants. Examples of the glycerol esters include glycerol monoacetate, glycerol diacetate, glycerol monostearate, glycerol monostearate, glycerol monooleate, glycerol monolaurilenite, and nato(1) glycerol fatty acid ester. The addition +l″C is 0.5 based on the solid content of the ink composition.
A value of ~3% is desirable. If it is less than 0.5%, coloring phenomenon in the atmosphere cannot be effectively prevented.

Glycerol esters may be used in excess because they have almost no adverse effect on the color reaction.

g) Non-aqueous solvent Each of the above components is dissolved or dispersed in a non-aqueous solvent that does not substantially contain water. Such non-aqueous solvents include aromatic hydrocarbons such as (al benzene and toluene), aliphatic hydrocarbons such as (bl methyl ethyl ketone,
Esters such as Cl ethyl acetate or (d) n-
Alcohols such as butanol are used. Among alcohols, C1 to C2 lower alcohols are not preferred because they cause deactivation of the enzyme.

Conventionally, enzymes have been thought to be unstable and susceptible to deterioration in organic solvents, but the ink composition for glucose detection, which is made by dispersing freeze-dried enzymes in non-aqueous solvents as described above, is stable. Moreover, it was completely unexpected that it would not change easily. The reason for this is not necessarily clear, but water-soluble enzymes do not dissolve in non-aqueous solvents, but are only dispersed; therefore, enzymes in a dispersed state are
The active sites and structures of the proteins that make up this enzyme are not susceptible to displacement, and even if they are displaced near the interface with the solvent, it is difficult for the solvent to penetrate inside, so most enzymes lose their activity. It is presumed that this will not be lost.

As described above, it is preferable that the non-aqueous solvent contains substantially no water, and therefore it is preferable that the solvent is dehydrated before use.

h) Other components In addition to the above-mentioned components, a water-absorbing powder or a wetting agent may be added to the glucose detection ink composition depending on the case.

Addition of the water-absorbing powder has the function of increasing the water absorption of the reagent composition provided on the support, promoting contact between the analyte fluid and the reagent composition, and promoting the color reaction of the indicator.

Such water-absorbing powders, when in contact with water,
Those exhibiting extreme acidity or alkalinity are preferred, and those with high whiteness are preferred. in particular,
Kaolin, synthetic silica, glass, cellulose block,
Microcrystalline cellulose, ion exchange cellulose, ion exchange resins, calcium carbonate, magnesium carbonate, aluminum silicate, etc. can be used.

The water-absorbing powder has a content of 30 to 9 based on the solid content of the ink composition.
Preferably it is present in an amount of 0% by weight.

Wetting agents include nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, polyethylene glycols, etc., and these wetting agents are used to disperse each reagent. It can help promote the formation of a uniform reagent layer and improve water wettability. The wetting agent is preferably present in an amount of 0.5 to 5% by weight based on the solid content of the ink composition.

In addition, to make it easier to see the color tone of the indicator,
For example, a background dye such as oil yellow may be added.

Note that when detecting glucose in a body fluid by forming a glucose detection area with the glucose detection ink composition having the composition described above, reducing substances such as ascorbic acid, glutathione, or cysteine may be present in the body fluid. Even when these substances coexist, there is an advantage that there is hardly any adverse effect on the color reaction caused by these substances.

The ink composition for glucose detection as described above is applied onto a support to form a glucose detection area, thereby obtaining a test object according to the present invention. As the application technique, a printing method, a coating method (for example, roll coating, spray coating, dye coating, solid coating), etc. can be used. In the present invention, since it is preferable that the number of applicators of the ink composition is relatively large and the number of coated surfaces is one, the ink composition is applied onto the support by a silk screen printing method, an intaglio printing method, a gravure printing method, etc. It is preferable to provide one. Application h1 is Ingi+l1
Although it varies depending on the composition of Japanese oyster-1, generally the support 2-b is preferably a material that does not react with the reagent composition and inhibits the coloration of the reagent. Specifically, examples include paper, synthetic material, paper, nonwoven fabric, or synthetic material.
The first filter uses a 11ir film or a laminate of paper and @tM resin film. C The test body according to the present invention in which a glucose test area is provided on a support may be formed in the form of a stick-roll, tape, etc. A vine-like shape, for example, a tube-like shape, a test tube-like shape, a dish-like shape, a tray-like shape, or a dropper-like shape, is formed, and a glucose test area is provided on the support.
The test material according to the present invention may also be used as a test tree - Effects of the invention Since the glucose detector according to the present invention is formed using an ink composition for glucose detection containing a stabilizer for an oxidizable indicator, it can be used as follows. It has a great effect.

a) Stable and does not change color even if stored in the atmosphere for a long time 1. Elephants are never recognized.

b) It has excellent sensitivity and excellent quantitative performance.

C) Since the glucose detection area can be formed directly on the support by a coating method, particularly a printing method, it is advantageous for one-man production and the process can be shortened.

d) The ink composition for glucose detection is stable.

Easy to handle and easy to use.

The present invention will be explained below with reference to Examples.
The present invention is not limited to the examples of T et al.

Example 1 An ink composition for glucose detection having the following composition was prepared by pulverizing and dispersing it using a homomixer.

Glucose oxidase 0.50 parts by weight (Toyo Conjunctiva:
Qrodc II) Peroxidase 0.10 (Oriental Conjunctiva: Grode III) 0-Tolidine
2.0 1) Lα-tropherol 0.1 (chemical soap, Tween 20) fIlt crystalline cellulose 30 (Asahi Kasei, Avicel SF) rl-butanol 47 Solvent Yellow GO, 05 citric acid
3.2 Citrus Sodium 12.0 After sufficiently finely dispersing the above composition using a homomixer, it was printed on a white polystyrene sheet with a height of 250 μm to form a square shape of 5 mm on each side using a screen printing method. Printed. The screen version used was 100 meshes,
Total thickness of resist and screen gauze is 130μm
Met.

The obtained printed material was partially dried at 65° C. and then cut into a stick shape to produce a test piece for glucose detection.

When the obtained specimen was briefly immersed in urine with a known glucose concentration, it showed rapid and clear color development. This test sample has high sensitivity, and 20+++g/dl ~ 100
It had quantitative ability in the range of 0 vdl, and the color tone after immersion was extremely stable over time. Table 1 shows the results of measuring the color tone a leap second after immersion.

Table 1 This color tone display is based on 6 standard colors defined in JIS Z8721.

In addition, similar results can be obtained using a test solution to which ascorbic acid has been added up to 250 #V (ll).This makes the determination less susceptible to the presence of reducing substances in body fluids. I understand that.

Example 2 An ink composition for glucose detection having the following composition was prepared in the same manner as in Example 1.

Glucose oxidase 0.50 parts by weight (Toyo Conjunctiva: Grode II) Peroxidase 0.10 (Toyobo: Grode ill) 0-Tolidine
2,0 Stearic acid glycerol ester 15 (chemical soap Excel T-95) Polyoxyethylene sorbicun monoole 1.2 ate (chemical soap Tween 20) Microcrystalline cellulose 30 (Fishikasei Avicel 5F) n-butanol 47 Solvent Yellow6 0.05 Citric acid: 3.2 Sodium citrate 12.0 Using the above ink composition for glucose detection, a test body for glucose detection was produced in the same manner as in Example 1.

A test specimen for glucose detection was produced in the same manner as in Example 2 except that stearic acid-based glycerol ester was not added to the ink composition for glucose detection of Example 2.

Regarding the glucose concentration, the same coloring results as in Example 2 were obtained, but when the specimen was left in the air for several hours to several days, the coloring part of the test material obtained in Comparative Example 1 changed. Discoloration was observed. The results are shown in Table 2.

Table 2 Furthermore, the samples obtained in Example 1 or 2 were stable even when stored for a long period of time in a sealed bottle with a desiccant added, and no deterioration in a-potency was observed.7. It was c.

Applicant's agent Kiyoshi Inomata

Claims (1)

[Claims] 1. Sugar oxidase, peroxidase, oxidizable indicator,
An ink composition for glucose detection, characterized in that a reagent composition comprising a binder and a stabilizer is dissolved or dispersed in a non-aqueous solvent.2, the stabilizer is an antioxidant. The ink composition according to claim 1. 3. The ink composition according to claim 2, wherein the antioxidant is a tocopherol. 4. The ink composition according to claim 1, wherein the stabilizer is a glycerol ester and an alcohol. 5. Sugar oxidase, peroxidase, oxidizable indicator,
A glucose detection area comprising a glucose detection ink composition in which a reagent composition comprising a binder and a stabilizer is dissolved or dispersed in a non-aqueous solvent and coated on a support. Detected object. 6. The detector according to claim 5, wherein the stabilizer is an antioxidant. 7. The detection object according to claim 6, wherein the antioxidant is a tocopherol. 8. The detector according to claim 5, wherein the stabilizer is a glycerol ester.