CN108872221B - Lyophilized powder for detecting p-hydroxyphenylalanine and preparation method and application thereof - Google Patents
Lyophilized powder for detecting p-hydroxyphenylalanine and preparation method and application thereof Download PDFInfo
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- CN108872221B CN108872221B CN201810757087.5A CN201810757087A CN108872221B CN 108872221 B CN108872221 B CN 108872221B CN 201810757087 A CN201810757087 A CN 201810757087A CN 108872221 B CN108872221 B CN 108872221B
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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 freeze-dried powder for detecting p-hydroxyphenylalanine, a preparation method and application thereof. The freeze-dried powder contains tyrosinase and 4-aminoantipyrine. The preparation method comprises the steps of preparing a buffer solution, dissolving other components in the buffer solution, freezing the mixed solution at-80 ℃, and drying by using a freeze dryer. The freeze-dried powder can be used for preparing a kit of p-hydroxy phenylalanine in urine. According to the technical scheme provided by the invention, the tyrosinase is adopted to detect the concentration condition of the p-hydroxy-phenylalanine, and the p-hydroxy-phenylalanine is successfully improved into the urine detection freeze-dried powder which can be used as 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 is used.
Description
Technical Field
The invention belongs to the technical field of detection reagents, and particularly relates to freeze-dried powder for detecting p-hydroxyphenylalanine, a preparation method and application thereof.
Background
Early diagnosis and early treatment of tumors are key to improving the cure rate of tumors. The existing clinical commonly used diagnosis methods include chest X-ray, B-ultrasonic, CT, nuclear magnetic resonance and the like, are usually means for aggravating the pain of patients and even possibly causing cross infection along with puncture, blood drawing and the like, are expensive, and more importantly, the tumors detected by the methods are generally in middle and late stages, so that the cure rate is greatly reduced.
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 freeze-dried powder for detecting p-hydroxyphenylalanine, 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 freeze-dried powder for detecting the p-hydroxyphenylalanine contains tyrosinase.
Optionally, the freeze-dried powder also contains 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-hydroxy phenylalanine detection reagent is prepared into freeze-dried powder which is convenient to carry, safe and simple and convenient because the detection is completed in a strong acid solution through mercury and mercurous ions.
Optionally, the lyophilized powder further contains a brilliant 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 freeze-dried powder also contains 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 the freeze-dried powder for detecting the p-hydroxyphenylalanine, which comprises the following steps: preparing a buffer solution, dissolving other components in the buffer solution, freezing the mixed solution at-80 ℃, and drying by using a freeze dryer.
Alternatively, the mixed solution is divided into ampoules to be frozen and dried, and the amount of the mixed solution in each ampoule is 100. mu.L.
Optionally, the buffer solution is a phosphate aqueous solution, and the pH value is 5.0-8.0.
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 10-20mg/mL, and the concentration of disodium hydrogen phosphate dodecahydrate is 40-50 mg/mL.
Optionally, the concentration of tyrosinase in the mixed solution is 50-2000U/mL, and the concentration of 4-aminoantipyrine is 1 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 the freeze-dried powder for detecting the p-hydroxyphenylalanine 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, tyrosinase is adopted to detect the concentration condition of p-hydroxyphenylalanine, and the concentration condition of p-hydroxyphenylalanine is successfully improved into urine detection freeze-dried powder which can be used as 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 is used.
Detailed Description
For the convenience of understanding, the lyophilized powder for p-hydroxyphenylalanine assay is described below with reference to examples, which are intended 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 carried out at normal temperature and normal pressure.
Example 1
1) Weighing sodium dihydrogen phosphate monohydrate (NaH)2PO4·H2O): 1.08g and disodium hydrogen phosphate dodecahydrate (Na)2HPO4·12H2O): 4.37g of the mixture was added with water to a constant volume of 100mL, and a phosphate buffer solution was obtained, wherein the pH thereof was 6.8;
2) weighing 50KU tyrosinase dry powder, and adding into the powder;
3) then 0.1g of 4-aminoantipyrine is added;
4) then adding 1.1g of bovine serum albumin, 2.8g 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 10.8mg/mL, the concentration of disodium hydrogen phosphate dodecahydrate in the mixed solution is 43.7mg/mL, the concentration of tyrosinase is 500U/mL, the concentration of 4-aminoantipyrine is 1mg/mL, the concentration of sucrose is 0.028g/mL, the concentration of albumin is 0.011g/mL and the concentration of Triton X-100 is 0.011 g/mL;
5) the mixed solution was dispensed into an ampoule in an amount of 100. mu.L per bottle, and then the ampoule was frozen at-80 ℃ for 24 hours and then freeze-dried in a freeze-dryer for 24 hours.
Example 2
1) Weighing sodium dihydrogen phosphate monohydrate (NaH)2PO4·H2O): 1.08g and disodium hydrogen phosphate dodecahydrate (Na)2HPO4·12H2O): 4.37g of the mixture was added with water to a constant volume of 100mL, and a phosphate buffer solution was obtained, wherein the pH thereof was 6.8;
2) weighing 50KU tyrosinase dry powder, and adding into the powder;
3) then 0.1g of 4-aminoantipyrine is added;
4) then 200. mu.l of a bright green aqueous solution (concentration 0.1%) were added:
5) then adding 1.1g of bovine serum albumin, 2.8g 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 10.8mg/mL, the concentration of disodium hydrogen phosphate dodecahydrate in the mixed solution is 43.7mg/mL, the concentration of tyrosinase is 500U/mL, the concentration of 4-aminoantipyrine is 1mg/mL, the concentration of sucrose is 0.028g/mL, the concentration of albumin is 0.011g/mL and the concentration of Triton X-100 is 0.011 g/mL;
6) the mixed solution was dispensed into an ampoule in an amount of 100. mu.L per bottle, and then the ampoule was frozen at-80 ℃ for 24 hours and then freeze-dried in a freeze-dryer for 24 hours.
Example 3 Effect verification test
3.1 color change experiment
The test sample is p-hydroxyphenylalanine standard solution with the concentration of 0mg/L, 120mg/L,240mg/L and 480 mg/L.
Add 500. mu.L of the test sample to the ampoule of example 1 or 2 and wait for 5 minutes for the liquid to change color. The sample of example 3 was developed by mixing with the test sample, and the results of color change are shown in Table 1.
TABLE 1
As can be found from Table 1, after the freeze-dried powder provided by the invention is added into a test sample containing p-hydroxyphenylalanine, the sample changes color, the content is higher, the color change degree is higher, and the brilliant green freeze-dried powder is added, so that the color change of each concentration is more abundant, and the distinctiveness is stronger.
Preparation method of standard colorimetric card
A plurality of standard solutions were added to the p-hydroxyphenylalanine lyophilized powder ampoules prepared in the examples, for example, at the four concentrations described above, and colorimetric cards were prepared according to the color development in each ampoule after 5 minutes, with the four standard colors representing the p-hydroxyphenylalanine concentrations 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 lyophilized powders of examples 1 and 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 a color at a low concentration and examples 1 and 2 developing a color normally.
3.3 anti-interference experiment
250mg/L of p-hydroxyphenylalanine aqueous solution is prepared, wherein the solution contains 600 mu mol/L of uric acid, and the lyophilized powder in examples 1 and 2 and the reagent in patent CN104535565A in the patent are respectively adopted, wherein 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. A freeze-dried powder for detecting para hydroxybenzene alanine is characterized in that the freeze-dried powder contains tyrosinase, 4-aminoantipyrine, brilliant green dye, sucrose, albumin and Triton X-100; the freeze-dried powder is obtained by freeze-drying a mixed solution of the components dissolved in a buffer solution, the buffer solution is a phosphate aqueous solution, and the pH value is 5.0-8.0; the concentration of tyrosinase in the mixed solution is 50-2000U/mL, the concentration of 4-aminoantipyrine is 1mg/mL, the concentration of sucrose 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.
2. The preparation method of the lyophilized powder for detecting p-hydroxyphenylalanine according to claim 1, which is characterized by comprising the following steps: preparing a buffer solution, dissolving other components in the buffer solution, freezing the mixed solution at-80 ℃, and drying by using a freeze dryer.
3. The method for preparing lyophilized powder for p-hydroxyphenylalanine assay according to claim 2, wherein the mixed solution is divided into ampoules to be frozen and dried, and the amount of the mixed solution in each ampoule is 100. mu.L.
4. A kit for detecting p-hydroxyphenylalanine in urine, which is characterized by comprising freeze-dried powder for detecting p-hydroxyphenylalanine in claim 1 and a standard colorimetric card.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810757087.5A CN108872221B (en) | 2018-07-11 | 2018-07-11 | Lyophilized powder for detecting p-hydroxyphenylalanine and preparation method and application thereof |
| PCT/CN2018/110241 WO2020010738A1 (en) | 2018-07-11 | 2018-10-15 | Lyophilized powder for detecting p-hydroxyphenylalanine, preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810757087.5A CN108872221B (en) | 2018-07-11 | 2018-07-11 | Lyophilized powder for detecting p-hydroxyphenylalanine and preparation method and application thereof |
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| CN108872221A CN108872221A (en) | 2018-11-23 |
| CN108872221B true CN108872221B (en) | 2020-05-22 |
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| CN117110256B (en) * | 2023-05-29 | 2024-04-19 | 兰州大学第一医院 | Urine tyrosine detection reagent and detection method based on N-GQDs fluorescence quenching principle |
Citations (5)
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| 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 |
| 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 |
| CN106645120A (en) * | 2016-11-21 | 2017-05-10 | 江苏华鸣生物科技有限公司 | Kit for detecting monohydroxyphenol metabolite in urine and preparation method of kit |
| KR20170124401A (en) * | 2016-05-02 | 2017-11-10 | (주)큐브바이오 | Enzyme Compositions for Detecting Cancer Biomarker Tyrosine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1446925A (en) * | 2003-03-24 | 2003-10-08 | 肖洪武 | Single stable colorimetric reagent of enzyme in liquid state and its application |
| CN105606605B (en) * | 2016-01-08 | 2019-09-20 | 张凤华 | A kind of Hydroxyphenylalanineurine urine detection reagent box and preparation method |
| KR101685091B1 (en) * | 2016-05-17 | 2016-12-12 | (주)큐브바이오 | High Resolution Window Diagnostic Kit for Detecting Cancer Existence |
| CN106706614B (en) * | 2016-11-21 | 2019-03-22 | 江苏华鸣生物科技有限公司 | Detect the kit and preparation method thereof of para hydroxybenzene alanine in urine |
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- 2018-07-11 CN CN201810757087.5A patent/CN108872221B/en active Active
- 2018-10-15 WO PCT/CN2018/110241 patent/WO2020010738A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106645120A (en) * | 2016-11-21 | 2017-05-10 | 江苏华鸣生物科技有限公司 | Kit for detecting monohydroxyphenol metabolite in urine and preparation method of kit |
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| CN108872221A (en) | 2018-11-23 |
| WO2020010738A1 (en) | 2020-01-16 |
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