JPH0328199B2 - - Google Patents

Description

[Detailed description of the invention] [Industrial application field]

TECHNICAL FIELD The present invention relates to a test piece for testing body fluid components, which is used when testing pathological components contained in body fluids such as blood, lymph, and urine.

[Conventional technology]

2. Description of the Related Art Examining pathological components contained in body fluids such as blood, lymph, and urine is essential in disease diagnosis and health management. As methods for testing the pathological components, there are two methods: one using tablets for evaluation and the other using test pieces, which are easy to operate during testing, and
At present, methods for testing pathological components using test pieces are becoming mainstream because the time required for determination is short. Conventional test strips for testing the above-mentioned pathological components utilize a change in color due to a chemical reaction between a target component in body fluids and a reagent, but in recent years, Testing methods that use enzymes, which are physiologically active substances, are often used because they do not require the use of strong acids or alkalis, and can test for pathological components in body fluids under relatively mild conditions. It is becoming more and more common. For example, in Japanese Patent Publication No. 1878-1978, a copolymer of a liquid enzyme, a substance having a peroxidation reaction, a buffer, orthotolidine dihydrochloride, polyvinylpyrrolidone, etc. is added to a hygroscopic material. A glucose detection test strip is described that is obtained by impregnating a liquid mixture consisting of a compound and a surfactant. In the conventional test piece, the color tone of the test reagent layer portion changes depending on the content of the target substance to be tested.
After impregnating a hygroscopic material such as filter paper in multiple stages with a solution in which enzymes, indicators, etc. are dissolved in a mixed solution of water and alcohol, the resulting impregnated paper is cut into appropriate sizes. , which is manufactured by adhering it to a test substrate.

[Problem to be solved by the invention]

By the way, in the test piece using the conventional enzyme, water/water is not added during the process of obtaining the test piece.
Since the enzyme dissolved in the alcohol-based mixed solution is unstable and deteriorates rapidly over time, it is necessary to impregnate the hygroscopic material in multiple stages immediately after preparing the impregnation solution. The manufacturing process is complicated, and there are drawbacks such as the need for special skill in order to obtain reliable test pieces with good test accuracy. In contrast, the test piece for body fluid component testing of the present invention uses an ink composition when forming the test area layer, so it is extremely easy to produce reliable test pieces with good test accuracy and consistent quality. To provide a test piece for testing body fluid components that can be obtained in the following manner.

[Means to solve the problem]

The test piece for body fluid component testing of the present invention has a test piece base and a test area layer formed on the surface of the test piece base, and the test area layer contains an enzyme, an indicator, and a binder. It is formed by printing or coating an ink composition in which water-absorbing fine particles are dispersed and kneaded in an organic solvent, and then dried. The components used to form the test area layer of the test piece for testing body fluid components of the present invention having the above-mentioned configuration will be described below. Enzymes Pathological components contained in body fluids, such as glucose, cholesterol, urea, neutral fats, phospholipids, ammonia, lactic acid, pyruvic acid, sialic acid, GPT, GOT, coryphonesterase, leucine aminopeptidase , amylase, LCAT,
Used are those that react with clenatiphos kinase and the like to cause a color change in the indicator, or those that cause a substance produced by the reaction to react with another enzyme and cause a color change in the indicator. Indicator An indicator is used in which the aforementioned enzyme acts together with a pathological component and its color changes. The enzyme and indicator are appropriately selected and used from among the many combinations known for detecting pathological components. For example, for glucose detection, enzyme...
Glucose oxidase peroxidase indicator... A benzidine-based indicator is used; glucose oxidase oxidizes glucose, and the hydrogen peroxide produced at the same time causes the benzidine-based indicator to change color in the presence of peroxidase, thereby detecting glucose. . Further, for uric acid detection, uric acid is detected by allowing uricase to act and causing the generated hydrogen peroxide to develop color in a reaction system similarly equipped with a chromogen. Furthermore, for cholesterol detection, cholesterol is detected by causing cholesterol oxidase to act and coloring the generated hydrogen peroxide in a reaction system in which a chromogen is arranged. Binder The binder is used to pattern and fix enzymes and indicators on the surface of the test strip substrate, and to prevent the enzymes and indicators fixed on the surface of the test strip substrate from eluting into the test liquid. It is of course necessary to use a substance that does not affect the pathological components, enzymes, indicators, etc. contained in the body fluids mentioned above, and does not interfere with body fluid tests. It is. Regarding the binder, it is desirable to check whether it satisfies the above-mentioned conditions each time it is used, but in the experiments of the present inventors, for example, polyacrylic acid ester poly, phenyl acetal, Synthetic polymers such as polymethacrylate, polyacrylamide, polyurethane, polyvinyl chloride, polystyrene, maleic acid copolymers, polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as methylcellulose, ethylcellulose, propylcellulose, starch ether, etc. Semi-synthetic polymers such as, starch, casein, natural polymers such as polysaccharides, etc. can be used. Among them, when using a maleic acid copolymer such as isobutylene-maleic anhydride copolymer or styrene-maleic acid copolymer as a binder,
This is advantageous because the binder has good shape retention performance and good liquid permeability. Water-absorbing fine particles Water-absorbing fine particles increase the water absorption and water retention of the test area layer formed on the surface of the test strip substrate, and absorb enzymes, indicators, and pathological components that are the target components of the test object. For example, inorganic or organic fine powders such as silica clay, calcium carbonate, aluminum silicate, glass, and cellulose are used. In the test strip for body fluid component testing of the present invention, the ink composition used to form the test area layer is an organic solvent containing an enzyme, an indicator, a binder, and water-absorbing fine particles as essential components. It consists of a kneaded material, and in addition to the above-mentioned essential ingredients,
For example, an anionic, cationic, or nonionic surfactant for improving water wettability, color uniformity, etc. of the buffering agent and the test area layer is added as necessary. The organic solvent used in the ink composition is, for example, an organic solvent such as alcohols, ketones, aromatics, esters, hydrocarbons, etc., and a solvent that can dissolve the binder is selected and used. . Note that if the organic solvent used contains water, the enzyme activity in the ink composition will be rapidly lost, so it is preferable to use an organic solvent that has been dehydrated in advance. Among solvents, methanol has a tendency to cause a slight loss of enzyme activity, so it is preferable to avoid it if possible. The ink composition used to form the test area layer is made by pulverizing freeze-dried enzymes, indicators, buffers added as necessary, etc.
It can be easily obtained by grinding into particles of 25 μm or less and then dispersing and kneading the enzyme, indicator, water-absorbing fine particles, buffer, etc. in an organic solvent in which a binder is dissolved. This dispersion and kneading operation is carried out using a high-speed stirrer, sand mill, ball mill, homogenizer, three-roll mill, ultrasonic disperser, or the like. The test piece substrate on which the test zone layer is formed with the ink composition having the above-mentioned composition has no effect on the pathological components, enzymes, indicators, etc. contained in the above-mentioned body fluids, and ,
Any material may be used as long as it does not interfere with the body fluid test; for example, paper, synthetic paper, various plastic films, composites of two or more of these materials, etc. are used. To form a test area layer of an ink composition on a test strip substrate, known printing or coating,
Drying methods can be used. In particular, printing methods that allow printing with a relatively large coating amount and a constant coating amount, such as silk screen printing, intaglio printing, and gravure printing, are suitable. In addition, the coating amount of the ink composition in the inspection area layer is usually preferably 2 to 30 g (solid component)/m ² .

[Effect]

The test strip for body fluid component testing of the present invention has a test strip substrate and a test zone layer formed on the surface of the base, and the test zone layer includes an enzyme, an indicator, a binder, and a water-absorbing material. printing or coating an ink composition prepared by dispersing and kneading fine particles in an organic solvent;
It is formed by drying. The test piece for body fluid component testing of the present invention having the above-mentioned configuration has the enzyme dispersed in the ink composition when forming the test area layer in the test piece, which is difficult to change in quality and is stable. Good quality test pieces can be easily obtained because the ink composition can be easily stored and the ink composition can be easily handled during the production of test pieces for testing body fluid components. Although it is not clear why enzymes in the ink composition used to form the test area layer of the test strip for testing body fluid components of the present invention are resistant to deterioration and are stable over a long period of time, the ink composition Since the water-soluble enzyme exists in a dispersed state without being dissolved in the organic solvent, it is less susceptible to denaturation at the higher-order structure and active site of the protein that makes up the enzyme. Even if the site is denatured, this organic solvent is difficult to penetrate into the inside of the enzyme, and it is presumed that this protects the activity of the enzyme. The above assumption is also supported by the fact that the enzyme activity rapidly decreases in a mixed solvent of an organic solvent and water.

【Example】

Hereinafter, the specific structure of the test piece for body fluid component testing of the present invention will be explained based on manufacturing examples, and also,
The action of this test piece will be explained. Example 1 An ink composition [a] for glucose detection was prepared by finely dispersing a composition having the following composition using a homomixer. Glucose oxidase {Toyobo Co., Ltd., Grade
} ...0.5 parts by weight peroxidase {Toyobo Co., Ltd., Grade}
...0.1 part by weight 0-tolidine ...0.5 part by weight polyoxyethylene sorbitan monooleate
...1.0 parts by weight isobutylene/maleic anhydride copolymer {Clare Isobutylene Chemical, Isoban #60
...3.0 parts by weight n-butanol ...50.0 parts by weight Kaolin ...45.0 parts by weight A square with one side of 6 mm made of the obtained ink composition [a] was placed on a substrate for a test piece made of a white polystyrene film with a thickness of 300μ. After printing the pattern, the test area layer was formed by drying at 80° C. for 1 hour. Next, the white polystyrene film is cut into strips of approximately 6 mm x 100 mm, and the inspection area layer consisting of the square pattern printed portion is located at one end of the strip.
A test piece for testing body fluid components, which is an example product of the present invention, was obtained. Note that the mesh of the screen plate used in the printing was 100, and the total thickness of the resist and screen gauze was approximately 100μ. [Experiment 1] The obtained test piece for testing body fluid components was quickly immersed in urine containing a known glucose concentration, and the color tone was observed after 30 seconds. The results are shown in Table 1. Note that the color tones in Table 1 are based on JIS
It is based on the standard color standard based on Z8721.

[Table] From the results shown in Table 1, it is clear that urine containing a glucose concentration of 0.05% can be measured using the test piece for body fluid component testing of the Example product. Comparative Example 1 A solution [b] with the following composition was prepared. Glucose oxidase {Toyobo Co., Ltd., Grade
} ...0.6 parts by weight peroxidase {Toyobo Co., Ltd., Grade}
...0.15 parts by weight 0-tolidine dihydrochloride ...0.5 parts by weight Citric acid-sodium citrate buffer (PH = 5.5)
...80 parts by weight Gelatin aqueous solution (5% by weight) ...82.5 parts by weight Distilled water ...20 parts by weight Ethyl alcohol ...23.7 parts by weight The obtained impregnating solution [b] was filtered through filter paper (Toyo Roshi, No. 51) for about 10 minutes, and then dried at 50°C for 2 hours to obtain impregnated paper with a composition for body fluid component testing. Next, the impregnated paper was cut into squares with sides of 6 m, and then attached to one end of a strip of white polystyrene film approximately 6 mm x 100 mm using double-sided adhesive transfer tape (3M). A test piece for body fluid component testing was obtained for comparison. Comparative Example 2 An ink composition [c] having the following composition was prepared. Glucose oxidase {Toyobo Co., Ltd., Grade
} ...0.4 parts by weight peroxidase {Toyobo Co., Ltd., Grade}
...0.1 parts by weight 0-tolidine ...0.5 parts by weight Styrene/maleic acid copolymer resin solution {Seiko Kagaku Kogyo Co., Ltd., X-1202LL} ...30.0 parts by weight PH buffer solution (PH=7.0) ...20.0 Parts by weight Ethyl alcohol...15.8 parts Kaolin...15.0 parts by weight A square pattern with a side of 6 mm using the obtained ink composition [c] was printed on a test piece substrate made of a white polystyrene film with a thickness of 300μ. After that, it was dried at 45°C for 2 hours. Note that the mesh of the screen plate used in the printing was 100, and the total thickness of the resist and screen gauze was approximately 100μ. Next, the white polystyrene film is cut into strips of approximately 6 mm x 100 mm, and the inspection area layer consisting of the square pattern printed portion is located at one end of the obtained strip.
A test piece for body fluid component testing was obtained for comparison. [Experiment 2] The test pieces for body fluid component testing obtained in Example 1 and Comparative Examples 1 and 2 described above were quickly immersed in urine containing a known glucose concentration, and the respective preset judgment time elapsed. The color tone at the time was observed. The results are shown in Table 2. Note that when determining the color tone, the elapsed time at which the change in color tone due to glucose in each test piece was most noticeable was tested in advance, and that time was selected as the elapsed time required for the determination.

[Table] From Table 2 above, it is clear that the test using the test piece for body fluid component testing according to Example 1 has excellent quantitative properties and that the stability of the color tone after color development is good. It is. Example 2 After preparing the ink composition [a] described in Example 1, it was left to stand at 40°C for 1 to 7 days, and then the ink composition [a] was prepared according to the same procedure as the corresponding procedure of Example 1. A test piece for testing body fluid components, which is another example of the invention, was obtained. Comparative Example 3 After preparing the impregnating solution [b] described in Comparative Example 1 above, it was left to stand at 40°C for 1 to 7 days, and further, by the same procedure as the corresponding procedure of Comparative Example 1, A test piece for body fluid component testing was obtained for comparison. Comparative Example 4 After preparing the ink composition [c] described in Comparative Example 2, it was left to stand at 40°C for 1 to 7 days, and then a comparative example was prepared using the same procedure as the corresponding procedure of Comparative Example 2. A test piece for body fluid component testing was obtained. [Experiment 3] Each of the test pieces for body fluid component testing obtained in Example 2 and Comparative Examples 3 to 4 described above was quickly immersed in a 0.5% glucose solution, and the test pieces were respectively subjected to the experiment 2 described above.
The coloring density at the lapse of the corresponding determination time was measured using a reflection densitometer, and compared with the coloring density obtained in the previous [Experiment 2]. Table 3 shows the relative concentration (%) of each test piece for testing body fluid components when the coloring density obtained in [Experiment 2] is taken as 100.

[Table] *...It is impossible to prepare a test piece due to gelation of the ink. From the results shown in Table 3, the test piece for body fluid component testing according to Example 2 was not prepared immediately after the preparation of the ink composition. However, in Comparative Examples 3 and 4, the quality of the test piece remains unchanged compared to the test piece obtained by applying the impregnation solution and ink composition to the test piece substrate after adjusting the impregnation solution and ink composition. It is clear that as the storage time increases, the quality of the test specimens obtained deteriorates.

【Effect of the invention】

As is clear from the above Examples and Comparative Examples, the test piece for body fluid component testing of the present invention has a color change in the test area layer formed on the surface of the test piece.
Since it clearly changes depending on the content in the liquid to be tested, precise test results can be easily obtained. In addition, the test piece for body fluid component testing of the present invention has good storage stability of the ink composition prepared when forming the test area layer, and furthermore, the test area layer can be formed by ordinary printing means or coating means. Because it is formed by the above method, it is extremely easy to manufacture, and can be mass-produced on an industrial scale with good quality.

Claims (1)

[Claims] 1. A test piece for body fluid component testing that has a test piece substrate and a test area layer formed on the surface of the base, in which the test area layer contains an enzyme, an indicator, a binder, and water-absorbing fine particles in an organic solvent. 1. A test piece for testing body fluid components, characterized in that it is formed by printing or coating an ink composition dispersed and kneaded therein, and then drying.