CN108872222B - Test strip for detecting p-hydroxyphenylalanine, preparation method and application - Google Patents
Test strip for detecting p-hydroxyphenylalanine, preparation method and application Download PDFInfo
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- CN108872222B CN108872222B CN201810757561.4A CN201810757561A CN108872222B CN 108872222 B CN108872222 B CN 108872222B CN 201810757561 A CN201810757561 A CN 201810757561A CN 108872222 B CN108872222 B CN 108872222B
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- filter paper
- hydroxyphenylalanine
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7756—Sensor type
- G01N2021/7759—Dipstick; Test strip
Abstract
The invention belongs to the technical field of detection reagents, and particularly relates to a p-hydroxyphenylalanine detection test strip and a preparation method thereof. The filter paper comprises filter paper and tyrosinase and 4-aminoantipyrine loaded on the filter paper. The preparation method comprises the steps of preparing a buffer solution, dissolving the components to be loaded on the filter paper in the buffer solution, applying the mixed solution on the filter paper through a soaking or spraying method, and drying the filter paper. The test strip can be used for preparing a kit of p-hydroxyphenylalanine in urine. According to the technical scheme provided by the invention, the content of the p-hydroxyphenylalanine is detected by adopting tyrosinase, and the p-hydroxyphenylalanine is successfully improved into a urine detection reagent strip which can be a product, the detection reagent components are environment-friendly, heavy metal ions such as mercury and nickel are not contained any more, and the environment pollution cannot be caused after the detection reagent strip is used.
Description
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
Early diagnosis and early treatment of tumors are key to improving the cure rate of tumors. The current clinical commonly used diagnosis methods include chest X-ray, B-ultrasonic, CT, nuclear magnetic resonance and the like, are usually accompanied with puncture, blood drawing and the like to aggravate the pain of patients and even possibly cause cross infection, are expensive, and more importantly, the tumors which can be detected by the methods are generally in middle and late stages, thereby bringing great pain and economic burden to the patients. Therefore, the research of a detection method which is simple and rapid in operation, low in cost, high in sensitivity and good in repeatability is imperative.
Abnormal nucleotide metabolism of cancer cells produces a monohydroxyphenol metabolite in which the content of p-hydroxyphenylalanine is much higher than that of normal persons, and which can be excreted through urine. The detection of the content of the p-hydroxyphenylalanine can infer whether a human body has cancer, can discover the tumor as soon as possible, save life, avoid additional expenditure and avoid fear and pain of patients.
At present, the p-hydroxyphenylalanine urine detection reagent is developed by mercury ions and mercurous ions, and for example, the technical schemes in Chinese patents CN103323452A, CN104535565A, CN106706614A and CN107490689A are all based on the principle. The method has the following defects: 1) the reagent adopts the coordination principle of amino acid and metal ions, so that uric acid and the like in urine generate great interference under the condition of high concentration, and a false negative result is caused; 2) the detection method contains mercury ions, the toxicity of mercury not only causes certain potential safety hazard in the preparation process, but also causes harm to the environment due to the detection of heavy metal ions such as mercury, nickel and the like in the waste liquid, so that special treatment is needed for production and recovery, and the use cost is increased; 3) the use of strong acids such as sulfuric acid and nitric acid also has many safety problems in the preparation process and the use process; 4) at present, all detection kits adopt ampoule bottles for liquid packaging and then are mixed with urine for detection, and the packaging has the disadvantages of large volume, inconvenient transportation, more wastes and inconvenient use.
Disclosure of Invention
The invention provides a p-hydroxyphenylalanine detection test strip as well as a preparation method and application thereof, which are used for solving a series of problems caused by the fact that the existing p-hydroxyphenylalanine detection reagents all contain mercury, strong acid and liquid detection reagents.
In order to solve the technical problems, the technical scheme of the invention is as follows: the p-hydroxyphenylalanine detection test strip comprises filter paper and tyrosinase loaded on the filter paper.
Optionally, the filter paper is also loaded with 4-aminoantipyrine.
Tyrosinase, also called polyphenol oxidase, catechol oxidase, etc., can oxidize colorless polyphenols into colored thearubigins, theaflavins, etc. But thearubigin has lighter color and unobvious color gradient, and has insufficient sensitivity if used as a urine retrieval reagent, while 4-aminoantipyrine can deepen the color development effect, so that tyrosinase can be used as p-hydroxyphenylalanine.
The existing p-hydroxyphenylalanine detection reagent is realized by forming colored precipitates by combining with mercury and mercurous ions, so that the existing p-hydroxyphenylalanine detection reagent cannot be made into a detection test strip which is convenient to carry, safe and simple.
Optionally, the filter paper is a whatman, newwak, cobert filter paper.
Optionally, the filter paper is also loaded with a bright green dye. The addition of the bright green makes the color change of the test strip more obvious, and is more beneficial to the judgment and reading by naked eyes.
Optionally, the filter paper is also loaded with sucrose, albumin and Triton X-100 (polyethylene glycol octyl phenyl ether). Tyrosinase also has a storage stability problem as a color developing agent, and the shelf life of tyrosine can be prolonged by using a proper amount of sucrose, albumin and Triton X-100, so that the problem of commercialization of tyrosine is solved.
The invention also provides a preparation method of the p-hydroxyphenylalanine detection test strip, which comprises the following steps: preparing a buffer solution, dissolving the components to be loaded on the filter paper in the buffer solution, applying the mixed solution on the filter paper through a soaking or spraying method, and then drying the filter paper.
Optionally, the buffer solution is a phosphate aqueous solution, and the pH value is 5.0-7.8.
Optionally, the phosphate salts are sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate dodecahydrate.
Optionally, the concentration of sodium dihydrogen phosphate monohydrate in the mixed solution is 15-25mg/mL, and the concentration of disodium hydrogen phosphate dodecahydrate is 18-30 mg/mL.
Optionally, the concentration of tyrosinase in the mixed solution is 50-2000U/mL, and the concentration of 4-aminoantipyrine is 1.2g-1.5 mg/mL.
Optionally, the concentration of sucrose in the mixed solution is 0.02-0.1g/mL, the concentration of albumin is 0.005-0.05g/mL, and the concentration of Triton X-100 is 0.005-0.02 g/mL.
The invention also provides a kit for detecting the p-hydroxyphenylalanine in urine, which comprises a p-hydroxyphenylalanine detection test strip and a standard colorimetric card.
The standard colorimetric card is determined in advance by using a standard concentration test sample.
According to the technical scheme provided by the invention, the content of the p-hydroxyphenylalanine is detected by adopting tyrosinase, and the p-hydroxyphenylalanine is successfully improved into a urine detection reagent strip which can be a product, the detection reagent components are environment-friendly, heavy metal ions such as mercury and nickel are not contained any more, and the environment pollution cannot be caused after the detection reagent strip is used.
Detailed Description
For the convenience of understanding, the p-hydroxyphenylalanine assay strip is described in the following examples, which are intended to illustrate the invention and not to limit the scope of the invention.
All raw materials are ordinary chemical preparations of analytical grade, and the preparation process is also carried out at normal temperature and normal pressure.
Example 1
1) Weighing sodium dihydrogen phosphate monohydrate (NaH)2PO4·H2O): 1.89g and disodium hydrogen phosphate dodecahydrate (Na)2HPO4·12H2O): 2.26g of the mixture was added with water to a constant volume of 100mL, and a phosphate buffer solution was obtained, the pH of which was 6.5;
2) weighing 100KU tyrosinase dry powder, and adding into the powder;
3) then 0.12g of 4-aminoantipyrine is added;
4) then 1.2g of bovine serum albumin, 2.4g of sucrose and 1.3g of Triton X-100 were added to obtain a mixed solution in which the concentration of sodium dihydrogen phosphate monohydrate was 18.9mg/mL, the concentration of disodium hydrogen phosphate dodecahydrate was 22.6mg/mL, the concentration of tyrosinase was 1000U/mL, the concentration of 4-aminoantipyrine was 1.2mg/mL, the concentration of sucrose was 0.024g/mL, the concentration of albumin was 0.012g/mL and the concentration of Triton X-100 was 0.013 g/mL;
5) fully soaking the Whatman filter paper into the mixed solution, taking out the Whatman filter paper, and placing the Whatman filter paper in an oven to dry for 15 minutes at 70 ℃; cutting into small pieces and adhering to the plastic substrate.
Example 2
1) Weighing sodium dihydrogen phosphate monohydrate (NaH)2PO4·H2O): 1.89g and disodium hydrogen phosphate dodecahydrate (Na)2HPO4·12H2O): 2.26g of the mixture was added with water to a constant volume of 100mL, and a phosphate buffer solution was obtained, the pH of which was 6.5;
2) weighing 70KU tyrosinase dry powder, and adding into the powder;
3) then 0.15g of 4-aminoantipyrine is added;
4) then 200. mu.l of a bright green aqueous solution (concentration 0.1%) were added:
5) then adding 1.2g of bovine serum albumin, 2.9g of sucrose and 1.1g of Triton X-100 to obtain a mixed solution, wherein the concentration of sodium dihydrogen phosphate monohydrate in the mixed solution is 18.9mg/mL, the concentration of disodium hydrogen phosphate dodecahydrate 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, the concentration of albumin is 0.012g/mL and the concentration of Triton X-100 is 0.011 g/mL;
6) spraying the mixed solution on Xinhua filter paper, taking out the Xinhua filter paper, and then placing the Xinhua filter paper in a drying oven to dry for 15 minutes at 70 ℃; cutting into small pieces and adhering to the plastic substrate.
Example 3 Effect verification test
3.1 color change experiment
The p-hydroxyphenylalanine strip prepared according to example 1 or 2 was immersed in a test sample and taken out, or the test sample was dropped on the strip, and the discoloration results are shown in table 1.
The test sample is p-hydroxyphenylalanine standard solution with the concentration of 0mg/L, 120mg/L,240mg/L and 480 mg/L.
TABLE 1
Table 1 shows that when a test sample containing p-hydroxyphenylalanine is dripped on the test strip provided by the invention, the test strip changes color, the content is higher, the color change degree is higher, and the brilliant green test strip is added, so that the color change of each concentration is richer, and the distinctiveness is stronger.
Preparation method of standard colorimetric card
P-hydroxyphenylalanine test strips prepared according to the examples were immersed in a plurality of standard solutions, for example, four concentrations as described above, respectively, and colorimetric cards were prepared according to the color development of each test strip after 5 minutes, the four standard colors representing the concentrations of p-hydroxyphenylalanine at different concentrations.
And (3) comparing the subsequent sample detection with a standard colorimetric card, wherein if the color of the sample to be detected is consistent with that of the standard colorimetric card, the corresponding concentration is the concentration of the liquid to be detected, and if the color is between two color levels, the concentration of the p-hydroxyphenylalanine is between the two color levels.
3.2 stability test
Comparative example 1 differs from example 1 in that no bovine serum albumin, sucrose and Triton X-100 were added.
The test strips of examples 1, 2 and comparative example 1 were stored in an oven at 50 ℃ for one week and then tested separately, with comparative example 1 not developing significant color at low concentrations and examples 1 and 2 developing normal color.
3.3 anti-interference experiment
250mg/L of p-hydroxyphenylalanine aqueous solution containing 600 mu mol/L of uric acid is prepared, and the reagents in examples 1 and 2 and patent CN104535565A in the patent are respectively used for testing, wherein the examples 1 and 2 can normally develop color, and the reagent in patent CN104535565A cannot develop color.
Finally, it should be noted that: the above examples are only for illustrating the technical solutions of the present invention, and are not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: it is to be understood that modifications may be made to the technical solutions described in the foregoing embodiments, or some or all of the technical features may be equivalently replaced, and such modifications or replacements may not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (4)
1. The p-hydroxy phenylalanine detection test strip is characterized by comprising filter paper and tyrosinase, 4-aminoantipyrine, brilliant green dye, sucrose, albumin and Triton X-100 loaded on the filter paper, wherein the components are dissolved in a prepared buffer solution, the buffer solution is a phosphate aqueous solution, the pH value is 5.0-7.8, and a mixed solution is applied to the filter paper by soaking or spraying, wherein the concentration of the tyrosinase in the mixed solution is 50-2000U/mL, the concentration of the 4-aminoantipyrine is 1.2-1.5 mg/mL, the concentration of the sucrose is 0.02-0.1g/mL, the concentration of the albumin is 0.005-0.05g/mL, and the concentration of the Triton X-100 is 0.005-0.02 g/mL.
2. The method for preparing the p-hydroxyphenylalanine detection test strip of claim 1 is characterized by comprising the following steps: preparing a buffer solution, dissolving the components to be loaded on the filter paper in the buffer solution, applying the mixed solution on the filter paper by soaking or spraying, and then drying the filter paper.
3. The method for preparing a p-hydroxyphenylalanine detection test strip according to claim 2, wherein the phosphate is sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate dodecahydrate.
4. A kit for detecting p-hydroxyphenylalanine in urine, which is characterized by comprising the p-hydroxyphenylalanine detection test strip and a standard colorimetric card according to claim 1.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810757561.4A CN108872222B (en) | 2018-07-11 | 2018-07-11 | Test strip for detecting p-hydroxyphenylalanine, preparation method and application |
| PCT/CN2018/110240 WO2020010737A1 (en) | 2018-07-11 | 2018-10-15 | P-hydroxyphenylalanine test strip, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201810757561.4A CN108872222B (en) | 2018-07-11 | 2018-07-11 | Test strip for detecting p-hydroxyphenylalanine, preparation method and application |
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| CN108872222A CN108872222A (en) | 2018-11-23 |
| CN108872222B true CN108872222B (en) | 2020-05-19 |
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| CN110006884A (en) * | 2019-04-08 | 2019-07-12 | 深圳华创生物医药科技有限公司 | A kind of urine sulfhydryl compound mercury-free test strip and preparation method |
| CN111208130B (en) * | 2020-03-17 | 2022-07-29 | 福建师范大学 | Test strip for rapidly detecting tyrosinase in serum and preparation method and application thereof |
| CN111665241B (en) * | 2020-06-12 | 2023-03-28 | 苏州良辰生物仪器试剂有限公司 | Tyrosine detection test strip and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1446925A (en) * | 2003-03-24 | 2003-10-08 | 肖洪武 | Single stable colorimetric reagent of enzyme in liquid state and its application |
| KR20170124401A (en) * | 2016-05-02 | 2017-11-10 | (주)큐브바이오 | Enzyme Compositions for Detecting Cancer Biomarker Tyrosine |
| KR101707123B1 (en) * | 2016-05-18 | 2017-02-15 | (주)큐브바이오 | Diagnostic Kit for Detecting Cancer Existence of in the Toilet bowl and Cancer Self-diagnostic System having the same |
| KR101702601B1 (en) * | 2016-05-30 | 2017-02-06 | (주)큐브바이오 | Portable Diagnostic Kit for Detecting Cancer Existence Using Enzyme Composition |
| KR101702596B1 (en) * | 2016-06-15 | 2017-02-06 | (주)큐브바이오 | Self-detectable Diagnostic Kit for Detecting Cancer Existence Using Enzyme Composition |
| CN106645120B (en) * | 2016-11-21 | 2019-01-15 | 江苏华鸣生物科技有限公司 | Detect the kit and preparation method thereof of single hydroxyl phenolic metabolism object in urine |
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| WO2020010737A1 (en) | 2020-01-16 |
| CN108872222A (en) | 2018-11-23 |
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