WO2020010737A1

WO2020010737A1 - P-hydroxyphenylalanine test strip, preparation method and application

Info

Publication number: WO2020010737A1
Application number: PCT/CN2018/110240
Authority: WO
Inventors: 刘�东
Original assignee: 深圳华创生物医药科技有限公司
Priority date: 2018-07-11
Filing date: 2018-10-15
Publication date: 2020-01-16
Also published as: CN108872222B; CN108872222A

Abstract

Disclosed is a p-hydroxyphenylalanine test strip and a preparation method, comprising: a filter paper and tyrosinase and 4-aminoantipyrine loaded on the filter paper. The preparation method comprises: preparing a buffer solution, dissolving components which to be loaded on the filter paper in the buffer solution, applying a mixed solution to the filter paper by dipping or spraying, and then drying. The test strip may be used for preparing a test kit for p-hydroxyphenylalanine in urine. The test strip uses tyrosinase to detect the content of p-hydroxyphenylalanine, the components thereof are environmentally-friendly, no mercury, nickel or other heavy metal ions are contained therein, and environmental pollution will not be produced after use. In addition, the test strip is convenient to store and transport, and is convenient to use, and no waste liquid is produced.

Description

P-hydroxyphenylalanine detection test strip, preparation method and application thereof

Technical field

The invention belongs to the technical field of detection reagents, and particularly relates to a p-hydroxyphenylalanine detection test strip, a preparation method and application thereof.

Background technique

Early diagnosis and early treatment of tumors are the key to improve the cure rate of tumors. The diagnosis methods commonly used in clinical practice today include chest radiography, B-ultrasound, CT, and magnetic resonance imaging, which are often accompanied by puncture, blood draw, etc. that aggravate the patient's pain and may even cause cross infection, and are expensive, and more importantly, through these The tumors that can be detected by the method are generally in the middle and advanced stages, which brings great pain and economic burden to patients. Therefore, it is imperative to study a detection method that is simple and fast, low cost, high sensitivity and good repeatability.

Abnormal nucleotide metabolism in cancer cells will produce a monohydroxyphenol metabolite, in which the content of p-hydroxyphenylalanine is much higher than that of normal people, and this substance can be discharged through urine. Detecting the content of p-hydroxyphenylalanine can infer whether the human body has cancer, can detect tumors early, save lives, without additional expenses, and avoid patients' fear and pain.

At present, p-hydroxyphenylalanine urine detection reagents are developed using mercury ions and submercury ions. For example, the technical solutions in Chinese patents CN103323452A, CN104535565A, CN106706614A, and CN107490689A are based on this principle. The method has the following disadvantages: 1) the reagent uses the principle of coordination of amino acids and metal ions, uric acid in urine has a great interference at high concentrations, resulting in false negative results; 2) the detection method contains mercury ions, mercury The toxicity not only makes the preparation process a certain safety hazard, in addition, the detection of heavy metal ions such as mercury and nickel in waste liquid will also be harmful to the environment, so production and recycling require special treatment, increasing the cost of use; 3) strong acids such as sulfuric acid and nitric acid There are also many safety problems in the preparation process and the use process; 4) At present, the detection kits are packaged in ampoule liquid and then mixed with urine for detection. This packaging is large in volume, inconvenient to transport, has a lot of waste, and is used. It is not very convenient.

Summary of the invention

The invention provides a p-hydroxyphenylalanine detection test strip, a preparation method and application thereof, which are used to solve a series of problems caused by current p-hydroxyphenylalanine detection reagents containing mercury, strong acid and liquid detection reagents. .

In order to solve the above technical problem, the technical solution of the present invention is that the p-hydroxyphenylalanine detection test strip includes a filter paper and a tyrosinase loaded on the filter paper.

Optionally, the filter paper is further loaded with 4-aminoantipyrine.

Tyrosinase, also called polyphenol oxidase, catechol oxidase, etc., can oxidize colorless polyphenols to colored theaflavin, theaflavin and other substances. However, theaflavin theaflavin has a lighter color and no obvious color gradient. If it is used as a urine retrieval reagent, the sensitivity is insufficient, and 4-aminoantipyrine can deepen the color development effect, so that tyrosinase can be used as a Phenylalanine becomes possible.

The current p-hydroxyphenylalanine detection reagent is realized by combining with mercury and amalgam ions to form a colored precipitate. Therefore, the current p-hydroxyphenylalanine detection reagent cannot be made into a convenient, safe and convenient detection test. Note.

Optionally, the filter paper is Waterman brand, Xinhua, Cobetter filter paper.

Optionally, the filter paper is also loaded with a bright green dye. The addition of bright green makes the color change of the test strip more obvious, which is more conducive to the naked eye.

Optionally, the filter paper is further loaded with sucrose, albumin and Triton X-100 (polyethylene glycol octylphenyl ether). Tyrosinase also has a storage stability problem as a color developer. Appropriate amounts of sucrose, albumin and Triton X-100 can extend the shelf life of tyrosine and solve its problem of productization.

The present invention also provides a method for preparing the above-mentioned p-hydroxyphenylalanine test strip, which includes the following steps: configuring a buffer solution, dissolving a component to be supported on a filter paper in the buffer solution, and mixing the mixed solution It can be applied to the filter paper by soaking or spraying, and then dried.

Optionally, the buffer solution is an aqueous phosphate solution, and the pH value is 5.0 to 7.8.

Optionally, the phosphate is sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate dodecahydrate.

Optionally, the concentration of sodium dihydrogen phosphate monohydrate in the mixed solution is 15-25 mg / mL, and the concentration of disodium hydrogen phosphate dodecahydrate is 18-30 mg / mL.

Optionally, the concentration of the tyrosinase in the mixed solution is 50-2000 U / mL, and the concentration of the 4-aminoantipyrine is 1.2 g-1.5 mg / mL.

Optionally, the concentration of sucrose in the mixed solution is 0.02-0.1 g / mL, the concentration of albumin is 0.005-0.05 g / mL, and the concentration of Triton X-100 is 0.005-0.02 g / mL.

The invention also provides a kit for p-hydroxyphenylalanine in urine, which comprises a p-hydroxyphenylalanine detection test strip and a standard colorimetric card.

The determination of the standard color chart can be determined in advance by using the standard concentration test sample.

The technical solution provided by the present invention uses tyrosinase to detect the content of p-hydroxyphenylalanine, and successfully improves it into a urine test reagent strip that can be a product, and the detection reagent components are environmentally friendly, which no longer contains mercury and nickel. Heavy metal ions will not cause environmental pollution after use. In addition, test strips are convenient for storage and transportation, and also easy to use, and no waste liquid is generated.

detailed description

In order to facilitate understanding, the p-hydroxyphenylalanine detection test strip is described below with reference to the examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

All raw materials are ordinary chemical preparations of analytical grade, and the preparation process is also performed at normal temperature and pressure.

Example 1

1) Weigh sodium dihydrogen phosphate monohydrate (NaH ₂ PO ₄ · H ₂ O): 1.89g and disodium hydrogen phosphate dodecahydrate (Na ₂ HPO ₄ · 12H ₂ O): 2.26g mixed with water to make up to 100mL To obtain a phosphate buffer with a pH = 6.5;

2) Weigh 100KU dry tyrosinase powder and add it;

3) Then add 0.12g of 4-aminoantipyrine;

4) Then add 1.2g bovine serum albumin, 2.4g sucrose and 1.3g Triton X-100 to obtain a mixed solution, the concentration of sodium dihydrogen phosphate monohydrate in the mixed solution is 18.9mg / mL, hydrogen phosphate dodecahydrate The concentration of disodium is 22.6mg / mL, the concentration of tyrosinase is 1000U / mL, the concentration of 4-aminoantipyrine is 1.2mg / mL, the concentration of sucrose is 0.024g / mL, and the concentration of albumin is 0.012 The concentration of g / mL and Triton X-100 is 0.013g / mL;

5) Waterman filter paper is fully immersed in the mixed solution, and after being taken out, it is dried in an oven at 70 ° C. for 15 minutes; cut into small pieces and pasted on a plastic substrate.

Example 2

2) Weigh 70KU dry tyrosinase powder and add it;

3) Then add 0.15g of 4-aminoantipyrine;

4) Then add 200 μl of bright green aqueous solution (concentration: 0.1%):

5) Then add 1.2g bovine serum albumin, 2.9g sucrose and 1.1g Triton X-100 to obtain a mixed solution, the concentration of sodium dihydrogen phosphate monohydrate in the mixed solution is 18.9mg / mL, hydrogen phosphate dodecahydrate The concentration of disodium is 22.6mg / mL, the concentration of tyrosinase is 700U / mL, the concentration of 4-aminoantipyrine is 1.5mg / mL, the concentration of sucrose is 0.029g / mL, and the concentration of albumin is 0.012 The concentration of g / mL and Triton X-100 is 0.011 g / mL;

6) Spray the above mixed solution on Xinhua filter paper, remove it and place it in an oven for 15 minutes at 70 ° C; cut into small pieces and paste it on a plastic substrate.

Example 3 Effect Verification Test

3.1 Discoloration experiment

The p-hydroxyphenylalanine test strip prepared according to Example 1 or 2 was immersed in the test sample and taken out, or the sample to be tested was dropped on the test strip, and the color change results are shown in Table 1.

The test sample is a standard solution of p-hydroxyphenylalanine at a concentration of 0 mg / L, 120 mg / L, 240 mg / L, and 480 mg / L.

Table 1

It can be found from Table 1 that when the test sample containing p-hydroxyphenylalanine is dropped on the test strip provided by the present invention, the test strip changes color, and the higher the content, the higher the degree of discoloration, and the bright green test is added. Paper strips have richer color variations at different densities and are more distinctive.

Preparation method of standard color card

The p-hydroxyphenylalanine test paper prepared according to the examples is immersed in multiple standard solutions, such as the above four concentrations, and a color chart is prepared according to the color development of each test paper after 5 minutes. The four standard colors represent different Concentration of p-hydroxyphenylalanine.

The subsequent sample detection is compared with the standard color chart. If the sample test color is consistent with the standard color card, the corresponding concentration is the concentration of the test solution. If it is between two color levels, the p-hydroxyphenylalanine The concentration is between the two.

3.2 Stability experiment

Comparative Example 1 differs from Example 1 in that bovine serum albumin, sucrose and Triton X-100 were not added.

The test strips of Examples 1, 2 and Comparative Example 1 were stored in an oven at 50 ° C. for one week, and then tested separately. Comparative Example 1 could not develop color significantly at low concentrations, and Examples 1 and 2 were able to develop color normally.

3.3 Anti-interference experiment

A 250 mg / L solution of p-hydroxyphenylalanine in water was included, which contained 600 μmol / L of uric acid. The reagents in Examples 1 and 2 of this patent and CN104535565A were used to test. Examples 1 and 2 were able to develop color normally. The reagent of patent CN104535565A cannot develop color.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or equivalently replace some or all of the technical features, and These modifications or replacements do not leave the essence of the corresponding technical solutions outside the scope of the technical solutions of the embodiments of the present invention.

Claims

A p-hydroxyphenylalanine detection test strip, comprising a filter paper and a tyrosinase loaded on the filter paper.
The test strip for detecting p-hydroxyphenylalanine according to claim 1, wherein the filter paper is further loaded with 4-aminoantipyrine.
The test strip for detecting p-hydroxyphenylalanine according to claim 1, wherein the filter paper is further loaded with a bright green dye.
The test strip for detecting p-hydroxyphenylalanine according to claim 1, wherein the filter paper is further loaded with sucrose, albumin and Triton X-100.
The method for preparing a p-hydroxyphenylalanine test strip according to any one of claims 1-4, comprising the steps of: configuring a buffer solution, and dissolving a component to be loaded on a filter paper in the buffer solution In this method, the mixed solution is applied to the filter paper by soaking or spraying, and then dried.
The method for preparing a test strip of p-hydroxyphenylalanine according to claim 5, wherein the buffer solution is an aqueous phosphate solution and the pH value is 5.0-7.8.
The method for preparing a test strip of p-hydroxyphenylalanine according to claim 6, wherein the phosphate is sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate dodecahydrate.
The method for preparing a test strip of p-hydroxyphenylalanine according to claim 5, wherein the concentration of the tyrosinase in the mixed solution is 50-2000 U / mL, and the 4-aminoantipyramine The concentration of forest is 1.2g-1.5mg / mL.
The method for preparing a test strip of p-hydroxyphenylalanine according to claim 5, wherein the concentration of sucrose in the mixed solution is 0.02-0.1 g / mL, and the concentration of albumin is 0.005-0.05 g / mL, the concentration of Triton X-100 is 0.005-0.02g / mL.
A kit for p-hydroxyphenylalanine in urine, comprising a p-hydroxyphenylalanine test strip and a standard colorimetric card according to any one of claims 1-9.