A urine tyrosine detection system, a detection method and a detection kit

Abstract

The application discloses a urine tyrosine detection system, a detection method and a detection kit. The detection system comprises a buffer solution, 4-aminoantipyrine, horseradish peroxidase and hydrogen peroxide, and the pH of the detection system is 6.5-7.8. After the sample to be detected is placed in the detection system and reacts at room temperature, the quantitative analysis of tyrosine can be realized by observing the color change of the reaction solution or testing the absorbance value of the reaction solution at 485 nm. Compared with the traditional Millon reaction and the multi-enzyme reaction system, the reagent used in the application has no toxicity and corrosion, does not cause hidden troubles to human health and environmental safety, is simple to operate, does not need special instruments and has low cost, and is conducive to realizing 'home health detection'.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biochemical detection, and particularly relates to a urine tyrosine detection system, a detection method and a detection kit. BACKGROUND

[0002] Tyrosine, also known as 4-hydroxy-phenylalanine, is a semi-essential amino acid in the human body. Tyrosine is an important precursor substance for the synthesis of adrenaline, dopamine and melanin in the body. Lack of tyrosine in the human body can cause diseases such as dementia and Parkinson's disease, but excessive tyrosine can cause hyperthyroidism of the body. Current research shows that a variety of malignant cancers including liver cancer, gastric cancer and cervical cancer can metabolize high concentrations of tyrosine during the production process. The content of tyrosine in the urine of a normal human body is about 1-1.30 mmol / L –1 , and the content of urine tyrosine of a patient during the occurrence of cancer is as high as 2.50 mmol / L –1 . Therefore, urine tyrosine can be used as a tumor marker for early screening and detection of cancer, which can help patients gain valuable treatment time and play a very important role in cancer treatment.

[0003] Millon reaction occurs chelation reaction of the phenolic group in tyrosine by mercury salt, producing brick red product. Chinese patent CN201510962013.1 discloses a reagent for detecting tyrosine tumor marker in human urine, which is prepared from mercury nitrate solution, mercury sulfate solution, nickel nitrate solution, cobalt sulfate solution. CN201510537446.2 discloses a urine detection reagent for p-hydroxyphenylalanine tyrosine, which is composed of H + , Hg + , Ni 2+ , NO3 – , phosphomolybdic acid, etc. Millon reaction requires high concentration of mercury salt to participate in the reaction, and these reagents have toxicity, volatility, accumulation, etc. They not only cause damage to the digestive system and nervous system of the human body, but also cause serious pollution to the environment. At present, many tyrosine detection reagents based on Millon have been used for early screening of cancer. The detection reagents and color development reaction of these products need to be carried out in ampoules. However, the discarded reagents still have potential harm to human health and environmental protection, which is not conducive to wide promotion.

[0004] To reduce the harm caused by Millon reagent, some strategies based on various biological enzymes are used to detect the content of tyrosine in samples. For example, Chinese Patent CN202010175482.X discloses a tyrosine detection kit, which is composed of tyrosine hydroxylase, polyphenol oxidase, tetrahydrobiopterin, sodium periodate, enzyme stabilizer and the like. There are some problems in this strategy, such as the complexity of by-products produced by the cascade reaction of multiple enzymes, which is easy to interfere with the color reaction; the need for a protective agent to stabilize the activity of the enzyme; high cost and the like. Therefore, it is of great significance to develop an environment-friendly, simple-to-operate, easy-to-store and transport urine tyrosine detection method. SUMMARY

[0005] In view of the problems in the prior art, the present application aims to provide a method suitable for semi-quantitative and quantitative detection of tyrosine in urine, which is low in cost, environment-friendly and simple to operate.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] The present application first provides a urine tyrosine detection system, which comprises a buffer solution, 4-aminoantipyrine, horseradish peroxidase (HRP) and hydrogen peroxide, wherein the pH of the detection system is 6.5-7.8.

[0008] As a preferred, in the above-mentioned urine tyrosine detection system, the buffer solution is PBS buffer.

[0009] As a preferred, in the above-mentioned urine tyrosine detection system, the concentration of horseradish peroxidase is 15-25 U / mL, the concentration of 4-aminoantipyrine is 0.15-0.50 g / L, and the concentration of hydrogen peroxide is 0.50-2.50 mmol / L.

[0010] The principle of the above-mentioned detection system for detecting tyrosine in urine is that under neutral or weak alkaline conditions, HRP / H2O2 catalyzes the coupling reaction of 4-aminoantipyrine and the phenolic group of tyrosine, thereby producing red quinone substances.

[0011] The present application also provides a method for detecting tyrosine in urine by using the above-mentioned detection system, which specifically comprises:

[0012] (1) A series of standard solutions with known tyrosine concentrations are first prepared, and the standard solutions are added to the urine tyrosine detection system, shaken and mixed, and then reacted at room temperature (25-37℃) for 3-5 min, after which a standard colorimetric card is prepared or a standard working curve is drawn by determining the color change of the reaction solution or testing the absorbance value of the reaction solution at 485 nm;

[0013] (2) adding the urine sample to be detected into the urine tyrosine detection system in step (1), and after reaction under the same conditions, observing the color of the reaction solution or the absorbance value of the test reaction solution at 485 nm, and then calculating the content of tyrosine in the urine sample to be detected according to the standard colorimetric card or the standard working curve.

[0014] In the method for detecting tyrosine in urine, the color change of the reaction solution can be observed by naked eyes or by RGB analysis software on a terminal. Since the presence of tyrosine can produce red quinone substances in the detection system, when the color of the reaction solution is red, it indicates that the urine sample to be detected contains tyrosine.

[0015] In the method for detecting tyrosine in urine, the absorbance value of the test reaction solution at 485 nm can be measured by an ultraviolet spectrophotometer or an enzyme label instrument, and the detection method is suitable for the detection of the concentration of tyrosine in batch samples.

[0016] The application also provides a urine tyrosine detection kit, which comprises a buffer solution or a buffer powder with a pH of 6.5-7.8, 4-aminoantipyrine, horseradish peroxidase, and hydrogen peroxide.

[0017] It can be understood that the above-mentioned substances can be independently packaged in the kit, or part of the substances can be directly configured into a mixed system that can exist stably for a long time.

[0018] In the urine tyrosine detection kit, relevant detection consumables such as a dropper, a reaction tube, a colorimetric card, and a urine cup can also be included.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] 1) The urine tyrosine detection system provided by the application is safe, does not involve toxic and corrosive reagents, and does not cause hidden dangers to human health and environmental safety.

[0021] 2) The detection reaction involved in the detection system is simple and can be completed at room temperature, realizing one-step detection, which is simple to operate and does not require special instruments, and can meet the daily detection of urine tyrosine in families. Meanwhile, the detection system can also meet the high-throughput detection of tyrosine in multiple urine samples in hospitals.

[0022] 3) The detection reagents required by the application are easy to obtain and low in price, and the prepared detection kit is easy to store and transport, and only needs to be protected from light, without the need for cold storage and transportation, which is suitable for industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1Reaction result comparison chart of the sample to be detected containing different amino acids in Example 1 of the present application in the detection system provided by the present application;

[0024] Figure 2 Reaction result comparison chart of the sample to be detected containing different tyrosine concentrations in Example 2 of the present application in the detection system provided by the present application;

[0025] Figure 3 Absorbance change chart of the sample to be detected containing different tyrosine concentrations in Example 2 of the present application after reacting with the detection system provided by the present application. DETAILED DESCRIPTION

[0026] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects more clearly understood, the present application will be further described in detail below in combination with the drawings and examples. Obviously, the described examples are only some of the embodiments of the present application, not all.

[0027] In the following examples, if not specifically stated, they are all conventional methods; the reagents and materials, if not specifically stated, can be obtained from commercial channels.

[0028] Example 1

[0029] (1) Prepare PBS buffer powder with pH 7.20; weigh 100 U of HRP dry powder, dissolve in 1 mL of deionized water solution, called A liquid; weigh 0.034 g of H2O2, dissolve in 100 mL of deionized water solution, called B liquid; weigh 0.050 g of 4-aminoantipyrine, dissolve in 25 mL of deionized water solution, called C liquid.

[0030] (2) Weigh 0.010 g of PBS buffer powder and add to a centrifuge tube; then the tyrosine detection reagents A, B and C are divided into each centrifuge tube at 0.020 mL; then 0.020 mL of 50 mmol / L Na2HPO4 is added to the above centrifuge tube, respectively. –1 Tyrosine, proline, serine, aspartic acid, hydroxyproline, ornithine, alanine, threonine, glycine, cysteine, cystine, lysine, phenylalanine, leucine, valine, tryptophan, isoleucine, glutamine, glutamic acid, arginine, histidine; place at room temperature (25-37℃) for 3-5 min, observe the color change.

[0031] In the above reaction system, the final concentration of HRP is 25 U / mL, the final concentration of H2O2 is 2.5 mmol / L, and the final concentration of 4-aminoantipyrine is 0.50 g / L.

[0032] The photos of the sample to be detected containing different amino acids after reacting with the detection system are as followsFigure 1 As shown, only the test sample containing tyrosine turned the detection system red, while other centrifuge tubes remained pale yellow. This indicates that the detection system is selective and specific for tyrosine.

[0033] Example 2

[0034] (1) Same as Example 1.

[0035] (2) Weigh 0.010 g of PBS buffer powder and add it to a centrifuge tube; then dispense the tyrosine detection reagents A, B, and C into centrifuge tubes at a rate of 0.020 mL each; then add 0.020 mL of 0, 0.050, 0.10, 0.25, 0.50, 1, 2, 2.50, 3, 5, 10, and 12 mmol L to the centrifuge tubes respectively. –1 Tyrosine. React at room temperature (25-37℃) for 3-5 minutes, observe the color change, and use a UV spectrophotometer to detect the absorbance value of the test solution at 485nm.

[0036] The above-mentioned samples after reaction with the detection system are shown in the following images. Figure 2 As shown, the color of the liquid in the centrifuge tube changes significantly as the concentration of tyrosine in the sample increases, gradually turning red. Therefore, in routine home testing, semi-quantitative analysis of tyrosine in urine can be achieved by directly observing the color change with the naked eye or by using a colorimetric card.

[0037] The absorbance values ​​of the mixtures obtained after the reaction of the above-mentioned samples with the detection system at different wavelengths are as follows: Figure 3 As shown in Figure A: the absorbance value at 480 nm gradually increases with the increase of tyrosine concentration in the sample. Further analysis of the absorbance value at 480 nm versus the sample concentration yielded the following graph: Figure 3 The working curve shown in Figure B has the standard equation: Y = 0.25X + 0.21, R 2 =0.97. The limit of detection was calculated to be 0.25 mmol / L (S / N = 3), which is much lower than the tyrosine level in the urine of patients with abnormal tyrosine metabolism (2.50 mmol / L).

[0038] In summary, the urine tyrosine detection method provided by this invention has excellent selectivity for tyrosine, and the detection limit is much lower than the tyrosine level in the urine of patients with abnormal tyrosine metabolism. More importantly, this invention can clearly distinguish whether the tyrosine content is abnormal with the naked eye, making it suitable for home testing.

[0039] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A urine tyrosine detection reaction solution, characterized in that, The detection reaction solution comprises a buffer solution, 4-aminoantipyrine, horseradish peroxidase and hydrogen peroxide, the pH of the detection reaction solution is 6.5-7.8, the concentration of the horseradish peroxidase is 15-25 U / mL, the concentration of the 4-aminoantipyrine is 0.15-0.50 g / L, and the concentration of the hydrogen peroxide is 0.50-2.50 mmol / L.

2. The urine tyrosine detection reaction solution according to claim 1, characterized by, The buffer solution is a PBS buffer solution.

3. A urine tyrosine test kit, characterized by, The detection reaction solution further comprises a 4-aminoantipyrine stock solution, a horseradish peroxidase stock solution and a hydrogen peroxide stock solution.

4. The urine tyrosine test kit according to claim 3, characterized by, The detection consumables are further included.

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