CN113702363B - Folic acid staining solution and preparation method thereof - Google Patents
Folic acid staining solution and preparation method thereof Download PDFInfo
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- CN113702363B CN113702363B CN202110843642.8A CN202110843642A CN113702363B CN 113702363 B CN113702363 B CN 113702363B CN 202110843642 A CN202110843642 A CN 202110843642A CN 113702363 B CN113702363 B CN 113702363B
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Classifications
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- G—PHYSICS
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- 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
- G01N2021/775—Indicator and selective membrane
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Abstract
The invention belongs to the technical field of cancerous cell detection liquid, and in particular relates to folic acid staining liquid which comprises the following components in percentage by mass: 50% -70% of aqueous solution of leaf acid and 10% -20% of methylene blue solution; wherein: the folic acid aqueous solution comprises folic acid, deionized water, water-soluble azone and saccharifying enzyme; the methylene blue solution comprises methylene blue, deionized water, citric acid and dimethyl sulfoxide, a certain amount of water-soluble azone is added into the folic acid aqueous solution, the permeability of the folic acid aqueous solution in cells is effectively promoted, the permeability of folic acid staining solution is effectively improved, reduced methylene blue is easy to enter cells to be oxidized and developed, the accuracy of detection results is ensured, meanwhile, the detection time is shortened to a large extent, and the detection mode is simplified to a large extent.
Description
Technical Field
The invention relates to the technical field of cancerous cell detection solutions, in particular to folic acid staining solution and a preparation method thereof.
Background
Cancer is one of the leading causes of death in the global population, and the improvement of the presence of tumor cells is of great importance for cancer treatment, and internationally, similar tumor screening technologies, such as tumor marker inspection and identification, are the mainstream screening means. Tumor marker screening refers to tumor screening by biochemical properties of tumor cells and their metabolic abnormalities, i.e., substances that are qualitatively or quantitatively altered in body fluids, exclusions and tissues of tumor patients, such as carcinoembryonic antigens, tumor antigens, carbohydrate antigens, and the like. The tumor marker is mainly used for the clinical discovery of primary tumors, the screening of tumor high risk groups, the differential diagnosis of benign and malignant tumors, the judgment of the development degree of the tumors, the observation and evaluation of the treatment effect of the tumors, the prediction of tumor recurrence and prognosis and the like. However, detection periods similar to tumor screening techniques (such as tumor marker detection and identification) are long, precise instruments and special operation techniques are required, and particularly operators are required to have abundant experience and high cost.
Folate receptor 1 (FOLR 1), also known as folate receptor alpha or folate binding protein, is an N-glycosylated protein expressed on the cytoplasmic membrane. FOLR1 has a high affinity for folic acid and several reduced folic acid derivatives. FOLR1 mediates the delivery of physiological folate, i.e., 5-methyltetrahydrofolate, into the cell interior. FOLR1 is overexpressed in most ovarian cancers as well as many uterine, endometrial, pancreatic, renal, lung and breast cancers, whereas FOLR1 expression on normal tissues is limited to the parietal membrane of epithelial cells in the proximal tubules of the kidney, alveolar lung cells of the lung, bladder, testes, choroid plexus and thyroid. This expression pattern of FOLR1 makes it a desirable target for FOLR 1-directed cancer therapies.
Based on the specificity of folic acid receptor, a new technology for diagnosing canceration, namely folic acid receptor mediated special staining, appears on the market at present. The technology utilizes two characteristics of tumor cells, namely high expression of cell surface folic acid receptor and strong oxidative stress reaction in cells, realizes rapid and accurate screening by means of chromogenic reaction of methylene blue, but has limited permeability of the existing folic acid staining solution, so that reduced methylene blue is difficult to enter cells to be oxidized and developed, and the accuracy of detection results cannot be ensured.
Disclosure of Invention
The invention aims to provide folic acid staining solution and a preparation method thereof so as to solve the technical problems mentioned in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions: a folic acid staining solution comprises the following components in percentage by mass: 50% -70% of aqueous solution of leaf acid and 10% -20% of methylene blue solution;
Wherein: the folic acid aqueous solution comprises folic acid, deionized water, water-soluble azone and saccharifying enzyme;
the methylene blue solution comprises methylene blue, deionized water, citric acid and dimethyl sulfoxide.
Preferably, the folic acid aqueous solution comprises the following components in percentage by mass: 40-50% of folic acid, 20-30% of deionized water, 10-15% of water-soluble azone and 5-30% of saccharifying enzyme.
Preferably, the methylene blue solution comprises the following components in percentage by mass: 25-35% of methylene blue, 10-20% of deionized water, 3-6% of citric acid and 39-62% of dimethyl sulfoxide.
Preferably, the folic acid staining solution also comprises peptidoglycan and compound amino acids.
Preferably, the folic acid staining solution comprises the following components in percentage by mass: 60% of folic acid aqueous solution, 20% of methylene blue solution, 10% of peptidoglycan and 10% of compound amino acid.
Preferably, the folic acid aqueous solution comprises the following components in percentage by mass: 45% of folic acid, 20% of deionized water, 15% of water-soluble azone and 20% of saccharifying enzyme.
Preferably, the methylene blue solution comprises the following components in percentage by mass: 30% of methylene blue, 15% of deionized water, 5% of citric acid and 50% of dimethyl sulfoxide.
A preparation method of folic acid staining solution comprises the following steps:
step 1: preparation of aqueous solutions of phyllonic acid
Uniformly mixing folic acid, deionized water, water-soluble azone and saccharifying enzyme according to the corresponding parts by weight for standby;
Step 2: methylene blue solution
Uniformly mixing methylene blue, deionized water, citric acid and dimethyl sulfoxide according to the corresponding parts by weight for standby;
step 3: and (3) uniformly mixing the two solutions prepared in the step (1) and the step (2), adding peptidoglycan and compound amino acid into the mixture, and uniformly stirring the mixture to prepare folic acid staining solution.
A folic acid staining solution is applied, a cotton wool tip is dipped in the folic acid staining solution and is uniformly smeared on the surface of tissues or cells, the color of the cotton wool tip and the change of the colors of the tissues and the cells are observed, and whether the tested tissue cell areas or cells have lesions is judged according to whether the colors are blue or not.
The beneficial effects of the invention are as follows: the folic acid dye solution and the preparation method thereof provided by the invention have the advantages that a certain amount of water-soluble azone is added into the folic acid aqueous solution, so that the permeability of the folic acid aqueous solution in the cells is effectively promoted, the permeability of the folic acid dye solution is effectively improved, the reduced methylene blue is easy to enter the cells to be oxidized and developed, the accuracy of the detection result is ensured, the detection time is greatly shortened, and the detection mode is greatly simplified;
the starch generates glucose from a non-reducing terminal water alpha-1.4 glucosidic bond under the action of saccharifying enzyme, so that the dyeing liquid has an antioxidation effect;
The effect of the citric acid in the staining solution is to accelerate the updating of cutin, which is helpful for the peeling of folic acid receptors on the cell surface, further shortens the detection time, and meanwhile, the citric acid plays the roles of preservative and antistaling agent;
dimethyl sulfoxide has the characteristics of high polarity, high boiling point, good thermal stability, aprotic property and water miscibility, and is mainly used as a solvent in a dyeing liquid;
The peptidoglycan is a substance special for prokaryotic cells, plays an important physiological function, and peptidoglycan molecules are interwoven into a grid shape to form a compact net sleeve structure with strong mechanical property, so that the cells are effectively protected from being damaged by the outside;
the compound amino acid comprises lysine, tryptophan, methionine, threonine, alanine, leucine and the like, and mainly plays a role in resisting oxidization of the dyeing liquid.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
step 1: preparation of aqueous solutions of phyllonic acid
Uniformly mixing 40% of folic acid, 20% of deionized water, 10% of water-soluble azone and 30% of saccharifying enzyme according to the corresponding parts by weight for standby;
Step 2: methylene blue solution
Uniformly mixing 25% of methylene blue, 10% of deionized water, 3% of citric acid and 62% of dimethyl sulfoxide according to the corresponding parts by weight for standby;
step 3: and (2) uniformly mixing the two solutions prepared in the step (1) and the step (2), wherein the folic acid aqueous solution accounts for 50% and the methylene blue solution accounts for 10%, adding 20% of peptidoglycan and 20% of compound amino acid into the mixture, and uniformly stirring the mixture to prepare the folic acid staining solution.
Dipping folic acid staining solution with a cotton wool tip, uniformly smearing on the surface of a tissue or a cell, observing the color of the cotton wool tip and the color change of the tissue and the cell, and judging whether the tested tissue cell area or cell has lesions according to whether the color is blue.
Example 2:
step 1: preparation of aqueous solutions of phyllonic acid
Uniformly mixing 42% of folic acid, 22% of deionized water, 11% of water-soluble azone and 25% of saccharifying enzyme according to the corresponding parts by weight for standby;
Step 2: methylene blue solution
Uniformly mixing 28% of methylene blue, 12% of deionized water, 4% of citric acid and 56% of dimethyl sulfoxide according to the corresponding parts by weight for standby;
Step 3: and (2) uniformly mixing the two solutions prepared in the step (1) and the step (2), wherein the folic acid aqueous solution accounts for 55% and the methylene blue solution accounts for 12%, adding 15% of peptidoglycan and 18% of compound amino acid into the mixture, and uniformly stirring to prepare the folic acid staining solution.
Dipping folic acid staining solution with a cotton wool tip, uniformly smearing on the surface of a tissue or a cell, observing the color of the cotton wool tip and the color change of the tissue and the cell, and judging whether the tested tissue cell area or cell has lesions according to whether the color is blue.
Example 3:
step 1: preparation of aqueous solutions of phyllonic acid
Uniformly mixing 45% of folic acid, 25% of deionized water, 12% of water-soluble azone and 18% of saccharifying enzyme according to the corresponding parts by weight for standby;
Step 2: methylene blue solution
Uniformly mixing 30% of methylene blue, 15% of deionized water, 5% of citric acid and 50% of dimethyl sulfoxide according to the corresponding parts by weight for standby;
Step 3: and (2) uniformly mixing the two solutions prepared in the step (1) and the step (2), wherein the folic acid aqueous solution accounts for 60% and the methylene blue solution accounts for 20%, adding the peptidoglycan 10% and the compound amino acid 10% into the mixture, and uniformly stirring the mixture to prepare the folic acid staining solution.
Dipping folic acid staining solution with a cotton wool tip, uniformly smearing on the surface of a tissue or a cell, observing the color of the cotton wool tip and the color change of the tissue and the cell, and judging whether the tested tissue cell area or cell has lesions according to whether the color is blue.
Example 4:
step 1: preparation of aqueous solutions of phyllonic acid
Uniformly mixing 48% of folic acid, 28% of deionized water, 14% of water-soluble azone and 10% of saccharifying enzyme according to the corresponding parts by weight for standby;
Step 2: methylene blue solution
Uniformly mixing 32% of methylene blue, 18% of deionized water, 5% of citric acid and 45% of dimethyl sulfoxide according to the corresponding parts by weight for standby;
Step 3: and (2) uniformly mixing the two solutions prepared in the step (1) and the step (2), wherein the folic acid aqueous solution accounts for 65% and the methylene blue solution accounts for 18%, adding 7% of peptidoglycan and 10% of compound amino acid into the mixture, and uniformly stirring to prepare the folic acid staining solution.
Dipping folic acid staining solution with a cotton wool tip, uniformly smearing on the surface of a tissue or a cell, observing the color of the cotton wool tip and the color change of the tissue and the cell, and judging whether the tested tissue cell area or cell has lesions according to whether the color is blue.
Example 5:
step 1: preparation of aqueous solutions of phyllonic acid
Uniformly mixing 50% of folic acid, 30% of deionized water, 15% of water-soluble azone and 5% of saccharifying enzyme according to the corresponding parts by weight for standby;
Step 2: methylene blue solution
Uniformly mixing 35% of methylene blue, 20% of deionized water, 6% of citric acid and 39% of dimethyl sulfoxide according to the corresponding parts by weight for standby;
Step 3: and (2) uniformly mixing the two solutions prepared in the step (1) and the step (2), wherein the folic acid aqueous solution accounts for 70% and the methylene blue solution accounts for 15%, adding 7% of peptidoglycan and 8% of compound amino acid into the mixture, and uniformly stirring to prepare the folic acid staining solution.
Dipping folic acid staining solution with a cotton wool tip, uniformly smearing on the surface of a tissue or a cell, observing the color of the cotton wool tip and the color change of the tissue and the cell, and judging whether the tested tissue cell area or cell has lesions according to whether the color is blue.
The folic acid staining solutions prepared in examples 1 to 5 were used as follows:
although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A folic acid staining solution is characterized in that: the composition comprises the following components in percentage by mass: 50% -70% of aqueous solution of leaf acid and 10% -20% of methylene blue solution;
Wherein: the folic acid aqueous solution comprises folic acid, deionized water, water-soluble azone and saccharifying enzyme;
the methylene blue solution comprises methylene blue, deionized water, citric acid and dimethyl sulfoxide.
2. A folic acid staining solution according to claim 1, characterised in that: the aqueous solution of the leaf acid comprises the following components in percentage by mass: 40-50% of folic acid, 20-30% of deionized water, 10-15% of water-soluble azone and 5-30% of saccharifying enzyme.
3. A folic acid staining solution according to claim 2, characterised in that: the methylene blue solution comprises the following components in percentage by mass: 25-35% of methylene blue, 10-20% of deionized water, 3-6% of citric acid and 39-62% of dimethyl sulfoxide.
4. A folic acid staining solution according to any of claims 1-3, characterised in that: the folic acid staining solution also comprises peptidoglycan and compound amino acid.
5. A folic acid staining solution according to claim 4, wherein: the composition comprises the following components in percentage by mass: 60% of folic acid aqueous solution, 20% of methylene blue solution, 10% of peptidoglycan and 10% of compound amino acid.
6. The folic acid staining solution according to claim 5, wherein: the aqueous solution of the leaf acid comprises the following components in percentage by mass: 45% of folic acid, 20% of deionized water, 15% of water-soluble azone and 20% of saccharifying enzyme.
7. The folic acid staining solution according to claim 5, wherein: the methylene blue solution comprises the following components in percentage by mass: 30% of methylene blue, 15% of deionized water, 5% of citric acid and 50% of dimethyl sulfoxide.
8. A preparation method of folic acid staining solution is characterized in that: the preparation method comprises the following steps:
step 1: preparation of aqueous solutions of phyllonic acid
Uniformly mixing folic acid, deionized water, water-soluble azone and saccharifying enzyme according to the corresponding parts by weight for standby;
Step 2: methylene blue solution
Uniformly mixing methylene blue, deionized water, citric acid and dimethyl sulfoxide according to the corresponding parts by weight for standby;
step 3: and (3) uniformly mixing the two solutions prepared in the step (1) and the step (2), adding peptidoglycan and compound amino acid into the mixture, and uniformly stirring the mixture to prepare folic acid staining solution.
9. The method for preparing folic acid staining solution according to claim 8, comprising the following steps: the method is characterized in that: the folic acid staining solution prepared by the preparation method is applied, a cotton swab is used for dipping the folic acid staining solution, the solution is uniformly smeared on the surface of tissues or cells, the color of the cotton swab and the change of the colors of the tissues and the cells are observed, and whether the tested tissue cell areas or cells have lesions is judged according to whether the colors are blue or not.
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| WO2013003507A1 (en) * | 2011-06-27 | 2013-01-03 | Morphotek, Inc. | Multifunctional agents |
| CA2860484A1 (en) * | 2011-12-29 | 2013-07-04 | Wenguang YAN | Composition of detection agents for epithelial tumour cells and preparation method therefor |
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| WO2013003507A1 (en) * | 2011-06-27 | 2013-01-03 | Morphotek, Inc. | Multifunctional agents |
| CA2860484A1 (en) * | 2011-12-29 | 2013-07-04 | Wenguang YAN | Composition of detection agents for epithelial tumour cells and preparation method therefor |
| CN104483472A (en) * | 2014-12-19 | 2015-04-01 | 武汉大学 | Diagnostic reagent for cervical cancer cell detection and preparation method thereof |
| CN108871911A (en) * | 2018-07-06 | 2018-11-23 | 辽宁钰航生物医疗科技有限公司 | A kind of folic acid composite and the preparation method and application thereof |
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