JPH0623744B2 - Test piece for nitrite detection - Google Patents

Description

Description: BACKGROUND OF THE INVENTION (1) Technical Field The present invention relates to a test strip for detecting nitrite in body fluids, particularly urine.

(2) Prior art and its problems Recently, rapid diagnostic agents have become remarkably important in medical practice and clinical examinations.
So-called test pieces using a water-absorbent substrate are often used. In this test piece, a water-absorbent substrate (often paper) is impregnated with a drug necessary for the detection reaction, and when it is immersed in the body fluid to be measured, there is a substance to be detected. Shows a color reaction, and its presence can be easily confirmed.

Measurement of nitrite in body fluids has become a very important measurement item in medical diagnosis.

Nitrite is produced by a nitrate reducing activity of certain bacteria such as Escherichia coli, para-Escherichia coli, Salmonella typhi, Salmonella typhi, Staphylococcus and Enterococcus.

Therefore, the detection of nitrite, especially nitrite in urine, is important for diagnosing infections caused by pathogenic bacteria and microorganisms to the human body.

Then, the principle of detection of nitrite is that a nitrite is reacted with a diazotizable amine under acidic conditions to form a diazo compound, and the diazo compound is reacted with a coupling agent to form an azo group of the red chromophore. Form a diazo compound having -N = N-. The density of this color has a sufficient correlation with the density of nitrite ions, and is a measure of the amount of nitrite.

There are many test pieces for detecting nitrite as described above, for example, an amine capable of being diazotized on an absorption carrier, a coupling component with a diazonium salt, and a solid organic acid. When detecting bacteria that form nitrates, 1,2,3,4-
Some contain tetrahydro-7,8-benzoquinoline or a derivative thereof (Japanese Patent Publication No. Sho 49-27719).

However, the conventional test piece has a problem that it is easily affected by heat, light, humidity (moisture), etc. and has poor stability.

II Object of the Invention An object of the present invention is to provide a test piece for detecting nitrite, which is not easily affected by heat, light, humidity (moisture), etc. and has improved stability.

What achieves the above-mentioned object is a water-absorbent substrate, a diazotizable amine and 1,2,3,4-tetrahydro-7,
It is a test piece for detecting nitrite which carries a solid inorganic acid together with a coupling agent which is 8-benzoquinoline or a derivative thereof.

Further, the solid inorganic acid is boric acid, molybdic acid,
It is preferably at least one inorganic acid selected from the group consisting of metaphosphoric acid. Furthermore, the 1,2,3,4-tetrahydro-7,8-benzoquinoline or a derivative thereof is
It is preferably 2,3,4-tetrahydro-7,8-benzoquinoline hydrochloride. In addition, the diazotizable amine,
It is preferably an aromatic primary amine. Further, the aromatic primary amine is preferably sulfanilamide. Moreover, it is preferable that the water-absorbent substrate is paper.

III Detailed Description of the Invention The test piece for detecting nitrite of the present invention comprises a diazotizable amine and 1,2,3,4-tetrahydro-7,8-benzoquinoline or a derivative thereof as a water-absorbent substrate. A test piece supporting a solid inorganic acid together with a coupling agent, which is used to detect and measure nitrite in a sample liquid by using a diazo coupling reaction. The diazotizable amine contained in the test piece of the present invention acts with nitrite contained in the sample liquid under acidic conditions to form a diazo compound, and aromatic primary amines are used. In particular, arsanilic acid and sulfanilamide can be preferably used.

Further, the coupling agent contained in the test piece of the present invention,
1,2,3,4-tetrahydro-7,8-benzoquinoline or a derivative thereof, for example, 1,2,3,4-tetrahydro-7,8-benzoquinoline, 1,2,3,4-tetrahydro -7,8-benzo (h) quinaldine, 3-hydroxy-N-methyl-1,2,3,4-tetrabenzo (h) quinoline, 3-acetoxy-N-methyl-1,2,3,4-tetrabenzo (h) quinoline, N-methyl-1,2,3,4-tetrabenzo (h) quinoline, 1,2,3,4-tetrahydro-7,8-benzoquinoline hydrochloride, etc., especially 1,2, 3,4-tetrahydro-
7,8-benzoquinoline hydrochloride is preferred.

In addition, a solid inorganic acid is used as the acid that provides acidic conditions in the reaction between the nitrite and the diazotizable amine contained in the test piece of the present invention. In the present invention, by using this inorganic acid, stability against heat, light, and humidity could be improved. As a solid inorganic acid,
Boric acid, iodic acid, molybdic acid, tungstic acid, titanic acid, vanadium acid, selenic acid, metaphosphoric acid and the like can be used, preferably boric acid, molybdic acid, at least one inorganic selected from the group consisting of metaphosphoric acid It is an acid, and metaphosphoric acid is particularly preferable. The inorganic acid needs to be contained to such an extent that a pH value required for the grease reaction can be obtained, and if the content is small, the color reaction takes time.

Further, when the test piece is immersed in the sample liquid, a wetting agent may be contained in the test piece so that the sample liquid is uniformly wetted.
As the wetting agent, sodium lauryl sulfate, sodium dodecyl sulfate, sodium tetradodecyl sulfate, sodium alkylbenzenesulfonate such as sodium dodecylbenzenesulfonate, and a surfactant such as dialkylsulfosuccinate such as sodium diheptylsulfosuccinate are preferably used. it can.

Further, the test piece preferably contains a viscous agent in order to prevent the drug contained in the water-absorbent substrate from flowing out. As the viscous agent, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene glycol, polyacrylic acid salt, polyacrylamide, poly (hydroxyethyl methacrylate), poly (hydroxyethyl acrylate), polymers such as carboxymethyl cellulose, gelatin, gum arabic, etc. are used. In particular, polyvinylpyrrolidone is preferable.

The drug is dissolved in a solvent such as benzene, toluene, xylene, methanol, ethanol, isopropanol, acetone, methyl ethyl ketone, chloroform, carbon tetrachloride, ethyl acetate, water, etc., and the solution is impregnated into a water-absorbent substrate and dried. By doing so, the base material is loaded with these drugs.

Further, as the water-absorbent substrate used in the test piece of the present invention,
There are paper such as filter paper, non-woven fabric made of glass fiber or plastic material, water-absorbent plastic sheet, etc., which do not react with or dissolve in the drug contained in the test piece, its solvent and sample liquid, and water-absorbent As long as it has It is preferably paper. next,
The present invention will be described in more detail with reference to examples.

[Example 1] Solution A 1,2,3,4-tetrahydro-7,8-benzoquinoline hydrochloride 0.1 g Metaphosphoric acid 2.0 g Polyvinylpyrrolidone 10 g Distilled water 100 ml Solution B sulfanilamide 0.09 g Ethyl acetate 100 ml A on filter paper After impregnating the solution, it was dried and further impregnated with solution B and dried to prepare a test piece of the present invention. The reason for dividing into A liquid and B liquid is that the respective reagents have different solubilities in the solvent. Further, the reason why the solution A is first impregnated and dried and then the solution B is impregnated and dried is to avoid the decrease in stability caused by the contact between the diazotizable amine and the coupling agent.

The color of the prepared test piece was white.

[Example 2] Liquid A 1,2,3,4-tetrahydro-7,8-benzoquinoline hydrochloride 0.1 g Boric acid 1.5 g Polyvinylpyrrolidone 10 g Distilled water 100 ml Liquid B sulfanilamide 0.09 g Ethyl acetate 100 ml As in Example 1, the filter paper was impregnated with the liquid A, dried, and then impregnated with the liquid B and dried to prepare a test piece of the present invention.

The color of the prepared test piece was white.

[Example 3] Solution A 1,2,3,4-tetrahydro-7,8-benzoquinoline hydrochloride 0.1 g Molybdic acid 1.0 g Polyvinylpyrrolidone 10 g Distilled water 100 ml Solution B sulfanilamide 0.09 g Ethyl acetate 100 ml As in Example 1, the filter paper was impregnated with the liquid A, dried, and then impregnated with the liquid B and dried to prepare a test piece of the present invention.

The color of the prepared test piece was white.

[Comparative Example 1] Liquid A 1,2,3,4-tetrahydro-7,8-benzoquinoline hydrochloride 0.1 g Tartaric acid 3.7 g Polyvinylpyrrolidone 10 g Distilled water 100 ml Liquid B sulfanilamide 0.09 g Ethyl acetate 100 ml Liquid A on filter paper Was impregnated, dried, and then impregnated with solution B and dried to prepare a test piece of Comparative Example 1.

The color of the prepared test piece was white.

[Comparative Example 2] A test piece of Comparative Example 2 was prepared in the same manner as in Comparative Example 1 except that 4.7 g of citric acid was used instead of tartaric acid in Comparative Example 1.

[Comparative Example 3] A test piece of Comparative Example 3 was prepared using the same material as Comparative Example 1 except that 3.3 g of malic acid was used instead of tartaric acid of Comparative Example 1.

[Comparative Example 4] A test piece of Comparative Example 4 was prepared in the same manner as in Comparative Example 1 except that 3.1 g of oxalic acid was used instead of tartaric acid in Comparative Example 1.

The color of the test pieces of Comparative Examples 2 to 4 was white as in Comparative Example 1.

The following experiments were conducted using the test pieces of the above-mentioned Examples and Comparative Examples.

[Experiment 1] Each of the prepared test pieces was placed in a constant temperature device at 60 ° C., and the color change and the change in performance of the test piece itself were observed after 1 day, 2 days, 3 days, and 6 days.
I checked about the day after.

Regarding changes in performance, normal human urine with negative nitrite concentration, normal human urine with nitrite added to a concentration of 0.2 μg / ml, and normal human urine with nitrite added to a concentration of 1.5 μg / ml were used. Each test piece was immersed in urine.

The results for the color change of the test piece itself are shown in Table 1, and the results for the change in the performance of the test piece are shown in Tables 2 (a) to 2 (c).

[Experiment 2] Each of the created test pieces was used for indoor light (illuminance: 500 Lx, ultraviolet intensity: 0.0
The color change and performance change of the test piece itself were observed at 09 μw / cm ² ).

The color change was compared with that stored at room temperature in the dark, and the change in performance was 1 min, 3 min,
It carried out about 7 minutes, 10 minutes, 30 minutes, and 60 minutes.

Regarding changes in performance, normal human urine with negative nitrite concentration, normal human urine with nitrite added to a concentration of 0.2 μg / ml, and normal human urine with nitrite added to a concentration of 1.5 μg / ml were used. Each test piece was immersed in urine.

The results regarding the color change of the test piece itself are shown in Table 3, and the results regarding the change in the performance of the test piece are shown in Tables 4 (a) to 4 (c).

[Experiment 3] Each of the prepared test pieces was placed in a thermo-hygrostat at a temperature of 40 ° C. and a relative humidity of 75%, and the color change and the change in performance of the test piece itself were 1
The day, three days, six days, and nine days were examined.

The change in performance was performed by immersing each test piece in human normal urine with a negative nitrite concentration and human normal urine to which nitrite was added at a concentration of 1.5 μg / ml.

The results for the color change of the test piece itself are shown in Table 5, and the results for the change in the performance of the test piece are shown in Table 6 (a) and Table 6 (b).

From the results of the above experiment, it was found that the test piece according to the example of the present invention using the inorganic acid is more stable to temperature, light and humidity than the test piece according to the comparative example using the organic acid.

IV Specific Effect of the Invention The test piece for nitrite detection of the present invention comprises a diazotizable amine and 1,2,3,4-tetrahydro-7,8-benzoquinoline or a derivative thereof as a water-absorbent substrate. Since a solid inorganic acid is supported together with the coupling agent, it is not easily affected by heat, light, moisture, etc., has high stability, and can detect nitrite in body fluids reliably and easily. And measurements can be made.

In particular, if the solid inorganic acid is metaphosphoric acid, the coupling agent is 1,2,3,4-tetrahydro-7,8-benzoquinoline hydrochloride, and the diazotizable amine is sulfanilamide. , Will be better.

Claims (6)

[Claims] 1. A diazotizable amine is provided on the absorbent substrate,
A test piece for detecting nitrite, comprising a coupling agent, which is 1,2,3,4-tetrahydro-7,8-benzoquinoline or a derivative thereof, and a solid inorganic acid. 2. The test piece for detecting nitrite according to claim 1, wherein the solid inorganic acid is at least one inorganic acid selected from the group consisting of boric acid, molybdic acid and metaphosphoric acid. . 3. The 1,2,3,4-tetrahydro-7,
8-benzoquinoline or its derivative is 1, 2, 3,
The test piece for detecting nitrite according to claim 1 or 2, which is 4-tetrahydro-7,8-benzoquinoline hydrochloride. 4. The diazotizable amine is an aromatic primary
The test piece for detecting nitrite according to any one of claims 1 to 3, which is a primary amine. 5. The test piece for detecting nitrite according to claim 4, wherein the aromatic primary amine is sulfanilamide. 6. The test piece for detecting nitrite according to any one of claims 1 to 5, wherein the water-absorbent substrate is paper.