Method for quantitatively determining substrate concentration, substrate concentration kit for quantitative determination, and test paper

Abstract

To provide a method for quantitatively determining substrate concentration in which a stable measurement result is obtained regardless of a subjective view of a measurer, a substrate concentration kit for quantitative determination, and test paper.SOLUTION: In a method for quantitatively determining substrate concentration in a test sample of the present invention, oxidase of the substrate is allowed to react with the substrate to create hydrogen peroxide, hydrogen ion is generated by autocatalytic reaction of the hydrogen peroxide and sulfite, a color of a pH indicator is changed by pH reduction by increasing the hydrogen ion, and substrate concentration is quantitatively determined on the basis of a time of color change of the pH indicator.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for quantifying a substrate concentration, a kit for quantifying a substrate concentration, and a test paper. [Background technology]

[0002] A reaction using an oxidase is employed to quantify a substance to be measured in a specimen. Specifically, an oxidase that oxidizes the substance to be measured is allowed to act to generate hydrogen peroxide, which is then detected to quantify the substance to be measured. Hydrogen peroxide can be detected by a method using peroxidase, a method using catalase, a method using a hydrogen peroxide electrode, an oxygen electrode, or the like. In the method using peroxidase, a method using a chromogen and colorimetric quantification of the generated pigment is used widely because it is rapid and simple (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 5646323 Summary of the Invention [Problem to be solved by the invention]

[0004] In conventional methods for quantifying the concentration of a substance to be measured, such as a substrate, using oxidase, a change in color intensity was observed. For example, a chromogenic substrate for peroxidase turns blue-green in the presence of hydrogen peroxide. The measurer visually observes the change in color intensity, and the substrate concentration is determined based on the result. For this reason, the measurement result is subjective to the measurer, making it difficult to obtain stable measurement results. Therefore, an object of the present invention is to provide a method for quantifying a substrate concentration, a kit for quantifying a substrate concentration, and a test paper, which are capable of obtaining stable measurement results without being influenced by the subjectivity of the measurer. [Means for solving the problem]

[0005] In order to achieve the above object, the inventors conducted extensive research and discovered that the pH of the autocatalytic reaction between sulfite and hydrogen peroxide changes rapidly until the end of the reaction, and found that an autocatalytic reaction can be combined with the determination of substrate concentration using an oxidase reaction. By visualizing the pH change in the autocatalytic reaction, the substrate concentration can be determined without being influenced by the subjectivity of the measurer, and stable measurement results can be obtained.

[0006] That is, the present invention is a method for quantifying a substrate concentration in a test sample, which comprises reacting an oxidase of the substrate with the substrate to generate hydrogen peroxide, generating hydrogen ions by an autocatalytic reaction between the hydrogen peroxide and sulfite, changing the color of a pH indicator by a decrease in pH caused by an increase in the hydrogen ions, and quantifying the substrate concentration based on the time it takes for the color of the pH indicator to change.

[0007] The present invention also relates to a substrate concentration quantification kit for quantifying the concentration of a substrate in a test sample, the kit comprising: a solution obtained by adding an oxidase of the substrate to a test sample containing the substrate; and a test paper having a liquid-permeable carrier carrying a sulfite and a pH indicator.

[0008] Furthermore, the present invention relates to a test paper comprising a liquid-permeable carrier carrying a sulfite and a pH indicator. Effect of the Invention

[0009] According to the present invention, it is possible to provide a method for quantifying a substrate concentration, a kit for quantifying a substrate concentration, and a test paper, which are capable of obtaining stable measurement results without being influenced by the subjectivity of the measurer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will be described in detail below. The quantitative method of the present invention is used to quantify the concentration of a substrate in a test sample. The test sample is not particularly limited as long as it contains a component that reacts with an oxidase, and examples of the test sample include biological samples (blood, urine, saliva, tears, etc.) and food and beverage products (nutritional drinks, amino acid drinks, etc.). Examples of the substrate include amino acids, particularly glutamic acid, etc. The quantitative method of the present invention is particularly effectively used for quantifying glutamic acid.

[0011] In the method of the present invention, first, as shown below, a substrate is reacted with an oxidase for this substrate to generate hydrogen peroxide by an oxidase-catalyzed reaction.

[0012] [ka]

[0013] For example, when glutamic acid is used as a substrate, the following reaction occurs using glutamate oxidase as an oxidase.

[0014] [ka]

[0015] Hydrogen ions are then generated by a self-catalytic reaction between the hydrogen peroxide thus produced and a sulfite, such as sodium sulfite or potassium sulfite.

[0016] [ka]

[0017] At the start of the autocatalytic reaction, the pH is about 9 to 11, but after a certain induction time, it drops rapidly to about pH 5, and the color of the pH indicator changes due to the drop in pH. The substrate concentration is quantified based on the time it takes for the pH indicator to change color. Examples of pH indicators include bromothymol blue (BTB) and litmus, which have a color change range of about pH 7. Bromothymol blue (BTB) is preferred because the colors before and after the color change are complementary, making it easy to detect the change in color tone. The induction time refers to the time from when the oxidase is reacted with the substrate to when the color of the test sample changes, and can be confirmed by measuring with a stopwatch or the like.

[0018] The rapid color change of the pH indicator at the end of the reaction can be easily observed by visual inspection. The time required for the color change of the pH indicator is measured, and the substrate concentration is quantified based on the measured time. The length of the induction time depends on the substrate concentration. Therefore, the substrate concentration can be quantified by measuring the time until the pH indicator changes color with a stopwatch.

[0019] Alternatively, the time may be measured while observing the color change using the camera function of a mobile device such as a tablet. In this case, it is possible to automatically calculate the substrate concentration. Specifically, a dedicated application reads the intensity of color information (y, u, v) of the image at a specified position in the video recording of the device. The y value is color information related to brightness, the u value is color information related to blue intensity, and the v value is color information related to red intensity.

[0020] When BTB is used as a pH indicator, the induction time is measured by starting the time count when the color intensity of the u value of the sample image becomes 120 or more, and then ending the time count when the u value becomes 120 or less. In addition, the concentration vs. time function is recorded in the terminal from the results of measurements using standard solutions of several concentrations, and the concentration of the sample is automatically calculated and displayed by applying this function.

[0021] In the present invention, an autocatalytic reaction that causes a rapid pH change is combined with an enzyme-catalytic reaction to visualize the pH change, making it possible to quantify the substrate concentration without being influenced by the subjective judgment of the measurer. In addition, since the induction time changes depending on the substrate concentration, the reaction time can be measured and the substrate concentration can be calculated from the length of the reaction time.

[0022] To quantify the substrate concentration by the method of the present invention, the kit for quantifying the substrate concentration of the present invention can be used. The kit for determining the concentration of a substrate of the present invention will be described below.

[0023] The kit for quantifying a substrate concentration of the present invention includes a predetermined solution and the test paper of the present invention. The solution can be prepared by adding an oxidase of the substrate to a test sample containing the substrate. In this solution, hydrogen peroxide is generated by an oxidase-catalyzed reaction. Examples of the test sample and the substrate include those mentioned above. The concentration of the oxidase in the solution can be, for example, 10 to 1,000,000 U / L.

[0024] The test paper of the present invention is a liquid-permeable carrier carrying a sulfite and a pH indicator. Examples of the sulfite and pH indicator include those mentioned above. As the liquid-permeable carrier, a filter paper or a porous membrane made of cellulose, glass fiber, or the like can be used. The shape and size of the carrier are not particularly limited, and it can be any shape and size, for example, a small piece of cellulose fiber filter paper measuring 5 mm x 5 mm x 1 mm can be used.

[0025] The test paper of the present invention can be produced by impregnating a carrier with a dyeing solution containing a sulfite and a pH indicator, and drying the carrier. The solvent for the dyeing solution can be a mixture of water and alcohol. The volume ratio of water to alcohol (water:alcohol) is preferably 90:10 to 10:90, more preferably 80:20 to 60:40. As the water, ultrapure water, pure water, etc. can be used. As the alcohol, methanol, ethanol, isopropyl alcohol, etc. can be selected.

[0026] The concentration of the sulfite in the staining solution is preferably about 1 to 20 mM, more preferably about 5 to 10 mM. The concentration of the pH indicator in the staining solution is preferably about 0.01 to 2 mM, more preferably about 0.1 to 0.5 mM.

[0027] The staining solution can be obtained, for example, by adding 0.063 to 0.126 g of sodium sulfite and 0.006 to 0.031 g of BTB to a solvent consisting of a mixture of 60 to 80 mL of ultrapure water and 20 to 40 mL of methanol, and mixing them uniformly.

[0028] The staining solution is placed in a glass beaker or stainless steel tank, and a carrier such as paper is immersed in the staining solution for about 0.01 to 1 minute to impregnate the carrier with the staining solution. The carrier is then dried at 35 to 80°C for 5 to 30 minutes using a blower dryer or drum dryer, to produce the test paper of the present invention carrying the sulfite and pH indicator.

[0029] The amount of sulfite carried on the test paper is 28.6 to 572.0 g / m 3 is preferable, and 143.0 to 286.0 g / m 3 The amount of the pH indicator carried in the test paper is more preferably 1.3 to 283.8 g / m 3 is preferable, and 13.6 to 68.1 g / m 3 The amount of each component supported on the test paper can be adjusted, for example, by the concentration of the staining solution, the thickness of the liquid-permeable carrier, the staining time, etc.

[0030] The test paper of the present invention is immersed in a solution of a test sample to which an oxidase has been added for about 1 to 30 seconds, and then removed. The temperature of the solution is not particularly limited and can be about 20 to 30°C. The immersion time in the solution is preferably about 1 to 10 seconds, and more preferably about 1 to 2 seconds. Excess solution is shaken off, and the induction time is measured using a stopwatch or the like. The induction time is measured in advance using a solution with a known substrate concentration.

[0031] As a result of the autocatalytic reaction between hydrogen peroxide produced in the solution and sulfite supported on the test paper, hydrogen ions are produced and the pH drops rapidly. The color of the pH indicator changes with the drop in pH, so the substrate concentration can be quantified based on the time for this color change. By using the substrate concentration quantification kit of the present invention, it is now possible to quantitate the substrate concentration without being influenced by the subjective judgment of the measurer. EXAMPLES

[0032] The present invention will be specifically described below with reference to examples. The materials, contents, mixing ratios, etc. shown in the following examples can be appropriately changed without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.

[0033] First, the following components were mixed uniformly to prepare an enzyme-containing liquid. The oxidase used here was glutamate oxidase. Ultrapure water 5mL Na2SO3(50mM) 1mL Oxidase (8000U / L) 1mL (initial pH 8.9-9)

[0034] The following components were blended and mixed uniformly to prepare a substrate-containing solution. The substrate used here was glutamic acid. Five substrate concentrations were used, ranging from 0 to 100 mL. Hydrogen peroxide was added to the substrate-containing solution to promote the reaction, but this is not necessarily required. Substrate (x mM) 1mL H2O2 (100 mM) 1 mL

[0035] The enzyme-containing solution was added with 1 mL of 0.2 mM BTB as a pH indicator. Each substrate-containing solution was added to the enzyme-containing solution, and the color change of BTB was observed. The induction time was measured using a stopwatch. The measured induction times are summarized in the table below together with the substrate concentration x.

[0036] [Table 1]

[0037] At each substrate concentration, a color change from blue to yellow was observed in the BTB after a certain induction period.

[0038] Next, the substrate concentration was quantified using the substrate concentration quantification kit of the present invention, which contains a predetermined solution and a test paper. The solution was prepared by adding glutamate oxidase as an oxidase to a test sample containing 50 mM glutamic acid as a substrate and leaving it for 10 minutes. The concentration of the oxidase in the solution was 4000 U / L.

[0039] The staining solution prepared according to the formula shown in the table below was used to prepare the test paper. Qualitative filter paper No. 2 (300 mm x 300 mm) made by Toyo Roshi Co., Ltd. was used as the carrier, and the carrier was impregnated with the staining solution and dried to prepare the test paper of the present invention.

[0040] [Table 2]

[0041] Specifically, the dye solution was placed in a metal tray, and the carrier was immersed in the tray for 5 seconds to impregnate the carrier with the dye solution. The carrier impregnated with the dye solution was then removed and dried at 60°C for 15 minutes using a Toyo Seisakusho blower dryer to remove the solvent. In this way, the test paper of the present invention was obtained, in which the sulfite and the pH indicator were supported on the qualitative filter paper as a carrier.

[0042] The obtained test paper was immersed in a solution of the test sample to which oxidase had been added, and the color change of the test paper was visually observed. As a result, it was confirmed that the color of the test paper changed from light blue to yellow-green after about 5 minutes of induction time.

[0043] By quantifying the substrate concentration in a test sample using the present invention, it has become possible to obtain stable measurement results that are not influenced by the subjective judgment of the person making the measurement.

Claims

1. 1. A method for quantifying a substrate concentration in a test sample, comprising the steps of: The substrate is reacted with an oxidase to generate hydrogen peroxide, generating hydrogen ions by a self-catalytic reaction between the hydrogen peroxide and a sulfite; The color of the pH indicator is changed by the decrease in pH due to the increase in hydrogen ions, The concentration of the substrate is quantified based on the time for the color change of the pH indicator. A method for quantifying a substrate concentration, comprising:

2. 2. The method for quantifying a substrate concentration according to claim 1, wherein the substrate is an amino acid.

3. 3. The method for quantifying a substrate concentration according to claim 2, wherein the amino acid is glutamic acid.

4. 4. The method for quantifying a substrate concentration according to claim 1, wherein the sulfite is sodium sulfite.

5. 4. The method for quantifying a substrate concentration according to claim 1, wherein the pH indicator has a color change range around pH 7.

6. 6. The method for quantifying a substrate concentration according to claim 5, wherein the pH indicator is bromothymol blue.

7. The quantification of the substrate concentration includes: The time required for the color change of the pH indicator is measured, and the measurement is performed based on the measured time. The method for quantifying a substrate concentration according to any one of claims 1 to 3.

8. A kit for quantifying a substrate concentration in a test sample, comprising: A solution obtained by adding an oxidase for a substrate to a test sample containing the substrate; A test paper in which a sulfite and a pH indicator are supported on a liquid-permeable carrier. A kit for quantifying a substrate concentration, comprising:

9. 9. The kit for quantifying a substrate concentration according to claim 8, wherein the substrate is an amino acid.

10. 10. The kit for quantifying a substrate concentration according to claim 9, wherein the amino acid is glutamic acid.

11. 11. The kit for quantifying a substrate concentration according to claim 8, wherein the sulfite is sodium sulfite.

12. The kit for quantifying a substrate concentration according to any one of claims 8 to 10, wherein the pH indicator has a color change range around pH 7.

13. 13. The kit for quantifying a substrate concentration according to claim 12, wherein the pH indicator is bromothymol blue.

14. A method for quantifying a substrate concentration, comprising using the kit for quantifying a substrate concentration according to any one of claims 8 to 10.

15. A test paper comprising a liquid-permeable carrier carrying a sulfite and a pH indicator.

16. 16. The test paper according to claim 15, wherein the sulfite is sodium sulfite.

17. 17. The test paper according to claim 15, wherein the pH indicator has a color change range around pH 7.

18. 18. The test paper of claim 17, wherein the pH indicator is bromothymol blue.

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