JPH0653075B2 - Glucose detection ink composition and test body formed using the same - Google Patents

Description

Description: TECHNICAL FIELD OF THE INVENTION The present invention relates to an ink composition suitable for forming a test body capable of simply and quickly detecting glucose in a body fluid such as a solution, particularly urine, and an ink composition formed using the same. Regarding the inspected body.

TECHNICAL BACKGROUND OF THE INVENTION AND PROBLEMS THEREOF To quickly and easily know the amount of glucose in body fluid such as urine, blood or lymph is indispensable for early detection, diagnosis and management of diabetes.

In order to detect glucose in body fluids, especially urine, a test body coated with a glucose test reagent has been mainly used in the past. This inspection body has the advantages that it is easy to operate when used and the determination can be made in a short time.

By the way, in the test body for detecting glucose in body fluid, glucose reacts with oxygen in the air to be finally oxidized to gluconic acid and hydrogen peroxide by the action of glucose oxidase. This hydrogen peroxide produces nascent oxygen by the action of peroxidase, and this oxygen immediately reacts with an oxidizable indicator such as O-tolidine to cause the indicator to develop color.

The test body for glucose detection 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-alcohol solvent, the obtained liquid is impregnated with a paper roll, and then the paper is dried. It has been produced by adhering it to a plastic film and cutting it into an appropriate size.

However, this method has the following problems.

(a) When a reagent composition comprising a sugar oxidase, a peroxidase, and an oxidizable indicator is dissolved or dispersed in water or a water-alcohol solvent to prepare an impregnating liquid, oxygen is unstable and easily deactivated, and The impregnating liquid changes in quality rapidly. For this reason, it is necessary to carry out impregnation of the paper in multiple steps immediately after the preparation of the impregnating liquid. Further, even if the paper was impregnated immediately, there was a problem that a part of the enzyme was inactivated and the impregnating solution was partially denatured.

(b) As described above, since the impregnating liquid is unstable and the manufacturing process is complicated, it is difficult to keep the quality of the obtained inspection body constant, and in order to ensure the test accuracy and reliability of the inspection body. Requires special attention and skill, and the manufacturing efficiency is likely to be lowered, resulting in an increase in manufacturing cost.

For this reason, the development of an inspection body that can simplify the manufacturing process and is suitable for mass production is promoted, and Japanese Patent Publication No.
An enzyme is preliminarily dissolved in an alcohol mixed solution, and an indicator, a pH buffer, a polymer binder, a water-absorbing carrier and the like are mixed to prepare an ink composition having printing or coating suitability. A method of producing an inspection body by printing (including coating) the composition on a support and then drying the composition has been proposed. However, with this method,
The enzymes were partly dissolved in water, and therefore unstable and rapidly deactivated, so it was necessary to print immediately after preparation. In addition, it was necessary to dry at a low temperature in order to prevent inactivation of the enzyme, and it was necessary to reduce the residual water content in the applied reagent layer as much as possible in order to obtain long-term storage stability.

Under such circumstances, the applicant of the present invention has filed Japanese Patent Application Laid-Open No. 58-209995.
In the publication, the enzymes are dispersed in a non-aqueous solvent that hardly dissolves the enzymes, and then an indicator, a buffer,
A method has been proposed in which a binder and a water-absorbent carrier are dissolved or dispersed to prepare an ink composition, and the ink composition is printed on a support to produce a test body. According to this method, an inspection body which has an excellent quantitative performance for glucose and an excellent sensitivity can be obtained, but this inspection body gradually becomes bluish when exposed to the atmosphere for a long time. However, in this test body, the presence of glucose is shown by coloring it in blue.Therefore, the banding to blue due to the storage of the test piece in the atmosphere may cause an incorrect judgment when examining the body fluid. There has been a strong demand for a solution to this problem, which could cause the issue.

The present inventors have conducted research to solve the above problems,
The banding color to blue in the glucose detection test piece described above is
This has been found out because the oxidizable indicator contained in the reagent composition, in particular, is affected by the action of a peroxidative substance present in a trace amount in the atmosphere. As a result of further research, it was found that this color reaction is prevented by adding a stabilizer to the reagent composition, and as a stabilizer for an oxidizable indicator, it has an appropriate anti-oxidation effect. It has been found that compounds having oxidative activity as well as certain surfactants are particularly good.

Object of the Invention and Outline thereof The present invention is intended to solve the drawbacks associated with the prior art, and has the following objects.

(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.

(b) To provide a glucose detection ink composition which is stable even after being stored in the air for a long time and in which a discoloration phenomenon is not observed, and a glucose detection body formed using the same.

(c) To provide a glucose detector which can be formed by a coating method, particularly a printing method, and therefore can simplify the manufacturing process, and to provide a glucose detecting ink composition used in the formation thereof.

The ink composition for detecting glucose in body fluid according to the present invention,
A reagent composition comprising a sugar oxidase, a peroxidase, an oxidizable indicator, a binder and a stabilizer is formed by being dissolved or dispersed in a non-aqueous solvent. The glucose detector according to the present invention has a glucose detection region formed by applying the glucose detection ink composition having the above composition onto a support.

Detailed Description of the Invention First, the glucose detection ink composition used for forming the test body according to the present invention will be specifically described below. B) Principle Glucose in body fluid reacts with enzymes in the air by the action of glucose oxidase such as glucose oxidase, and is finally oxidized to gluconic acid and hydrogen peroxide. The produced hydrogen peroxide produces nascent oxygen by the action of peroxidase, and this oxygen immediately reacts with an oxidizable indicator such as O-tolidine to cause the indicator to develop color. The presence or absence of glucose in the body fluid and its amount are semi-quantitatively determined by the degree of color development. (B) Glucose oxidase Glucose oxidase as a sugar oxidase is used in the state of a purified lyophilized product. This enzyme is preferably present in an amount of 0.02 to 2% by weight, preferably 0.2 to 1.8% by weight, based on the solid content of the ink composition, when the enzyme having a titer of 100 unit / mg is used. . C) Peroxidase Peroxidase is an enzyme that catalyzes the oxidation of various organic substances by hydrogen peroxide or organic peroxide, and is mainly extracted from horseradish. This enzyme is preferably present in an amount of 0.002 to 1% by weight based on the solid content of the ink composition when a freeze-dried product having an activity of 100 unit / mg is used.
It is desirable to be present in an amount of 0.02-0.2% by weight. D) Oxidizable indicator The oxidizable indicator is an indicator that is oxidized by oxygen to develop a color, and conventionally known compounds such as benzidines and N-alkylated benzidines can be widely used. Of these, O-tolidine is particularly preferable. This oxidizable indicator is 0.5 relative to the solid content of the ink composition.
It is desirable to be present in an amount of -10 wt%, preferably 0.6-6 wt%. E) Binder The binder should not affect the components in the sample liquid and pH, and should not affect the reagents, especially the enzyme and the oxidizable indicator, and should not interfere with the color reaction. Required. Binders confirmed to meet such requirements include (i) polyester resin, alkyd resin, polyurethane resin, polystyrene resin, acrylic resin, epoxy resin, vinyl chloride resin, vinyl chloride copolymer resin, polyvinyl butyral. Resins, polyvinyl alcohol resins, polyvinylpyrrolidone resins, synthetic resins such as maleic anhydride copolymers, (ii) methyl cellulose, ethyl cellulose, hydroxyethyl cellulose,
Cellulose derivatives such as carboxymethyl cellulose,
iii Starch, polysaccharides, gelatin, casein, natural polymers such as sodium alginate, etc. are used. Further, two or more kinds of these binders may be combined. The binder is preferably present in an amount of 0.1 to 20% by weight, preferably 0.5 to 10% by weight, based on the solids content of the ink composition. F) Stabilizer The stabilizer is a donation for the stabilization of a reagent composition comprising a sugar oxidase, a peroxidase, an oxidizable indicator and a binder. Of these, the oxidizable indicator tends to discolor due to the action of peroxide substances in the atmosphere as described above, but the main role of the stabilizer is to prevent this, and as a stabilizer, A compound having a proper antioxidant activity, a specific surfactant represented by glycerose esters, or a mixture thereof is used.

Substances exhibiting antioxidative activity include 2,6-di-t-butylmethoxyphenol, P-methoxyphenol, tonaphthol, pentamethylphenol, 2,2,5,7,8-pentamethyl6-hydroxychroman, tocophenols. A radical scavenger such as or a reducing agent such as ascorbic acid may be used, but at least these inhibit the reaction system of glucose detection (oxidation reaction of oxidizable indicator), which is the purpose of this test, and lower the sensitivity. It also has the property of causing Therefore, it is preferable to use an antioxidant substance that does not hinder the reaction system and reduces the action of the peroxide substance in the atmosphere. A radical scavenger is mentioned as a preferable antioxidant, and among them, tocophenols (α-, β-, γ-, δ-) are particularly effective. The amount of tocophenol added is preferably 0.02 to 0.2% 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 cannot be effectively prevented,
On the other hand, if it exceeds 0.2% by weight, an adverse effect on the color reaction starts to be recognized and the sensitivity is lowered.

In addition to the above-mentioned substances exhibiting an antioxidant action, as another additive for preventing the coloring phenomenon of the reagent reaction layer in the atmosphere, its action mechanism is unknown, but a specific additive typified by glycerol esters is used. There is a surfactant. Examples of the glycerol esters include glycerol monoacetate, glycerol diacetate, glycerol monostearate, glycerol monopaltimylate, glycerol monooleate, glycerol monolaurate,
And glycerol fatty acid esters such as. The amount added is 0.5 to 3% by weight based on the solid content of the ink composition.
Is desirable. If it is less than 0.5%, the coloring phenomenon in the atmosphere cannot be effectively prevented. Glycerol esters may be used in excess since 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 (a) benzene, aromatic hydrocarbons such as toluene,
(b) Aliphatic hydrocarbons such as methyl ethyl ketone, (c) esters such as ethyl acetate, or (d) alcohols such as n-butanol are used. Of the alcohols, C ₁ -C ₂ lower alcohols are not preferred because they lead to inactivation of the enzyme.

Conventionally, it has been considered that the enzyme is unstable and easily deteriorated in an organic solvent, but the glucose detection ink composition obtained by dispersing the freeze-dried enzyme in the non-aqueous solvent as described above is stable. And it is quite unexpected that it is hard to change. The reason for this is not clear, but the water-soluble enzyme is not dissolved in the non-aqueous solvent and is only dispersed, and thus the enzyme in the dispersed state is
The active site and its structure of the proteins that make up this enzyme are not susceptible to displacement, and even if displacement occurs near the interface with the solvent, it is difficult for the solvent to penetrate even into the interior, so most enzymes are active. It is estimated that they will not lose.

As described above, the non-aqueous solvent preferably contains substantially no water, and therefore, the solvent is preferably dehydrated before use. H) Other components In some cases, in addition to the above components, a water-absorbing powder or a wetting agent can be added to the glucose detection ink composition.

The addition of the water-absorbing powder has a function of enhancing the water absorption of the reagent composition provided on the support, promoting the contact between the analyte liquid and the reagent composition, and promoting the color reaction of the indicator.

Such a water-absorbent powder, when contacted with water,
Those showing extreme acidity or alkalinity are not preferable, and those having high whiteness are preferable. In particular,
Kaolin, synthetic silica, glass, cellulose block,
Microcrystalline cellulose, ion exchange cellulose, ion exchange resin, calcium carbonate, magnesium carbonate, aluminum silicate and the like are used.

The water absorbing powder is preferably present in an amount of 30 to 90% by weight based on the solid content of the ink composition.

As the wetting agent, nonionic surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, polyethylene glycols, etc. are used, and this wetting agent helps disperse each reagent and uniformly. It is possible to promote the formation of a simple reagent layer and improve the wettability with water. 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 order to make the color tone of the indicator easier to see,
For example, a background dye such as oil yellow may be added.

Incidentally, when glucose is detected in the body fluid by forming a glucose detection region with the glucose detection ink composition having the composition as described above, ascorbic acid in the sample fluid, reducing substances such as glutathione or cysteine coexist. Even if it is present, there is an advantage that these substances have almost no adverse effect on the color reaction.

The glucose detection ink composition as described above is applied onto a support to form a glucose detection region, and the test body according to the present invention is obtained. As a coating technique, a printing method, a coating method (for example, roll coating, spray coating, deep coating, solid coating) or the like can be used. In the present invention, since the coating amount of the ink composition is relatively large and the coating amount is preferably constant, the ink composition is applied to the support by a silk screen printing method, an intaglio printing method, a gravure printing method, or the like. It is preferably provided above. The coating amount varies depending on the type of ink composition, but is generally preferably ² to 150 g / m ² (when dried).

The support is preferably one that does not react with the reagent composition and does not inhibit the coloration of the reagent. Specifically, for example, paper, synthetic paper, non-woven fabric or synthetic resin film or paper and synthetic resin film is used. A laminated body or the like is used.

The test body according to the present invention in which the glucose test area is provided on such a support may be formed into a shape such as a stick shape, a roll shape, or a tape shape. Alternatively, according to the present invention, the support itself is formed in a form capable of collecting a sample liquid, for example, a cup shape, a test tube shape, a dish shape, a tray shape, a dropper shape, and a glucose test area is provided on the support body. It may be an inspection body.

EFFECTS OF THE INVENTION Since the glucose detector according to the present invention is formed by using the glucose detection ink composition containing the stabilizer of the oxidizable indicator, it has the following effects.

a) It is stable even when stored in the air for a long time, and no discoloration phenomenon is observed.

b) It has excellent sensitivity and quantitative performance.

c) Since the glucose detection region can be formed directly on the support by a coating method, particularly a printing method, it is advantageous for mass production and the process can be shortened.

d) The glucose detection ink composition is stable and easy to handle.

The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1 An ink composition for glucose detection having the following composition was prepared by homogenizing and dispersing with a homomixer.

Glucose oxidase 0.50 parts by weight (Toyobo: GrodeII) Peroxidase 0.10 (Toyobo: GrodeIII) O-Tolidine 2.0 Isobutylene / maleic anhydride copolymer tanol ester compound 2.5 (Kuraray Isoprene Chemical, Isoban # 10) DLα-Trof Errol 0.1 Polyoxyethylene sorbitan monooleate 1.2 (Kao soap, Tween 20) Microcrystalline cellulose 30 (Asahi Kasei, Avicel SF) n-Butanol 47 Solvent Yellow 6 0.05 Citric acid 3.2 Sodium citrate 12.0 Sufficiently mix with a homomixer After being finely dispersed, a white polystyrene sheet having a thickness of 250 μm was printed by a screen printing method so as to form a quadrangle having a side of 5 mm. The screen plate used was 100 mesh, and the total thickness of the resist and screen mesh was 130 μm.

The obtained printed matter was dried at 65 ° C. for 30 minutes and then cut into a stick shape to manufacture a glucose detection test body. When the obtained test body was quickly immersed in urine having a known glucose concentration, it showed a rapid and clear coloration. This test specimen had a high sensitivity and had a quantitative ability in the range of 20 mg / dl to 1000 mg / dl, and the color tone after immersion was extremely stable over time. Display 1 shows the result of measuring the color tone 30 seconds after the immersion.

The display of this color tone is based on the standard color tone specified in JIS Z8721.

Similar results could be obtained by using a test solution containing ascorbic acid up to 250 mg / dl. From this, it can be seen that the judgment is not easily affected even if the reducing substance is present in the body fluid.

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 (Toyobo: GrodeII) Peroxidase 0.10 (Toyobo: GrodeIII) O-tolidine 2.0 Butanol esterified product of isobutylene / maleic anhydride copolymer 2.5 compound (isoban # 10 manufactured by Kuraray isobutylene chemical) Stearic acid-based glyce Loose ester 1.5 (Kao soap Exel T-95) Polyoxyethylene sorbitan monooleate 1.2ate (Kao soap, Tween 20) Microcrystalline cellulose 30 (Avicel SF manufactured by Asahi Kasei) n-butanol 47 Solvent Yellow 6 0.05 Citric acid 3.2 Sodium citrate 12.0 Using the above-mentioned glucose detection ink composition, a glucose detection test body was produced in the same manner as in Example 1.

Comparative Example 1 (Effect of Addition of Glycerol Esters) A glucose detection test sample was produced in the same manner as in Example 2 except that the stearic acid-based glycerol ester was not added to the glucose detection ink composition of Example 2.
Coloring results similar to those of Example 2 were obtained with respect to the glucose concentration, but the test material obtained in Comparative Example 1 showed a colored portion when the test body was left in the air for several hours to several days. Discoloration was observed. The results are shown in Table 2.

Further, the test bodies obtained in Example 1 or 2 were stable even when stored in a bottle containing a desiccant for a long period of time in a sealed state, and no deterioration in performance was observed.

Claims (6)

[Claims] 1. Discoloration of an indicator due to (a) glucose oxidase, (b) peroxidase, (c) an oxidizable indicator, (d) a binder, and (e) (a) an oxidizing substance in the atmosphere. In addition, either an antioxidant consisting of a radical scavenger that does not hinder the reaction system between the test target substance and the indicator at the time of test and (b) a surfactant that prevents coloring of the reagent reaction layer in the atmosphere. An ink composition for glucose detection, wherein a reagent composition comprising a stabilizer containing one or both of them is dissolved or dispersed in a non-aqueous solvent. 2. The ink composition according to claim 1, wherein the antioxidant is tocopherol. 3. The ink composition according to claim 1, wherein the surfactant is a glycerol ester. 4. Discoloration of an indicator due to (a) glucose oxidase, (b) peroxidase, (c) an oxidizable indicator, (d) a binder, and (e) (a) an oxidizing substance in the atmosphere. In addition, either an antioxidant consisting of a radical scavenger that does not hinder the reaction system between the test target substance and the indicator at the time of test and (b) a surfactant that prevents coloring of the reagent reaction layer in the atmosphere. A reagent composition comprising a stabilizer containing one or both of them, and having a glucose detection region formed by coating a glucose detection ink composition dissolved or dispersed in a non-aqueous solvent on a support. Characteristic, glucose test body. 5. The glucose test product according to claim 4, wherein the antioxidant is tocopherol. 6. The glucose test body according to claim 4, wherein the surfactant is a glycerol ester.