CN117804876A - Folic acid receptor mediated epithelial tissue cell staining solution and preparation method thereof - Google Patents
Folic acid receptor mediated epithelial tissue cell staining solution and preparation method thereof Download PDFInfo
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Abstract
The invention relates to folic acid receptor mediated epithelial tissue cell staining solution and a preparation method thereof, belonging to the technical field of cell staining, and comprising the following raw materials: modified folic acid, methylene blue, sodium hydroxide, tween 80, ascorbic acid, carboxymethyl chitosan, glucose, neutral red, glacial acetic acid, hydrogen peroxide solution and sterile deionized water. The novel human epithelial tissue tumor cell staining solution is prepared from the components of modified folic acid, methylene blue, tween 80, carboxymethyl chitosan and the like, and has good water solubility by modifying the folic acid and forming sodium salt. The hydrophilic functional groups are grafted on the chitosan, so that the hydrophilicity of the chitosan is improved, the use of solvents in the traditional dyeing liquid is avoided, the irritation generated when the dyeing liquid is applied to a human body is reduced, and the uncomfortable feeling is reduced. The prepared staining solution has the characteristics of simple operation, high identification rate, no stimulation in use, good stability, long storage time and the like.
Description
Technical Field
The invention relates to the technical field of cell staining preparation, in particular to folic acid receptor mediated epithelial tissue cell staining solution and a preparation method thereof.
Background
Cervical cancer is the most common disease of gynaecology, is secondary to breast cancer in female harm, and is the only malignancy known to be the cause of morbidity at present. The pathogenesis of the Human Papilloma Virus (HPV) is mainly transmitted by Human Papilloma Virus (HPV) in an epidermis contact mode, and continuous HPV infection can integrate the HPV with DNA of cervical epithelial cells, transcribe to generate oncogenic proteins, cause abnormal hyperplasia of the cervical epithelial cells and cause canceration. Cervical lesions are precancerous lesions of the cervix, early diagnosis of cervical lesions and administration of appropriate treatment can reduce the incidence of cervical cancer.
The traditional cervical disease detection can not obtain results at the time of cervical inspection by means of pathological inspection and PCR technology to make preliminary judgment, and liquid-based cytology (TCT) combined with HPV detection has high price, so that the conventional screening and timely diversion of outpatient and physical examination patients are inconvenient. At present, a plurality of staining methods aiming at tumor cells can detect the benign and malignant degrees of the cells, and the common staining methods include acetic acid whitening reaction, iodine experiments, acridine orange fluorescent staining and the like, and have different advantages, such as volatile solvents, slower reaction, high sensitivity, low specificity and the like.
The folic acid receptor mediated cervical special staining method (FRD) is a living cell staining diagnosis technology applied to clinical human epithelial tissue tumor cells in recent years, and consists of folic acid, derivatives thereof, methylene blue and the like, and the principle is that by utilizing the two characteristics that folic acid receptors on the surfaces of tumor cells are highly expressed and the inside of the cells are in a high oxidative stress state, folic acid derivatives are used for wrapping reduced methylene blue (yellow brown) to prepare a living cell stain, after the stain is ingested by tumor cells, the folic acid derivatives participate in metabolism, the reduced methylene blue is oxidized and then escapes out of the cells, and a doctor can directly judge whether abnormal lesions exist on the cervix or not in real time by observing and detecting the color change of the staining solution before and after detection. Methylene blue has a high affinity for tumor cells and melanin, and its redox properties cause methylene blue to exhibit different color-changing response spectra in the redox state of tumor tissue. However, the existing FRD staining solution has limited stability and low recognition rate, and the reduced methylene blue exposed in the air is easily oxidized and developed, so that the production process and the accuracy of the detection result are affected, and the product quality is uneven; in addition, the dye needs to be smeared on human tissues, and common solvents of the dye solution such as dimethyl sulfoxide (DMSO), propylene glycol and the like have certain toxic effects, wherein the DMSO can act on hydrophobic groups of proteins to cause protein denaturation, has vascular toxicity and hepatorenal toxicity, and can burn skin and cause skin to have stinging.
For this reason, a folic acid receptor mediated epithelial tissue cell staining solution and a preparation method thereof are needed to improve clinical use.
Disclosure of Invention
The invention aims to provide folic acid receptor mediated epithelial tissue cell staining solution and a preparation method thereof, which aim to solve the problems in the background technology. The novel human epithelial tissue tumor cell staining solution is prepared from the components of modified folic acid, methylene blue, tween 80, carboxymethyl chitosan and the like, and has good water solubility by modifying the folic acid and forming sodium salt. The hydrophilic functional groups are grafted on the chitosan, so that the hydrophilicity of the chitosan is improved, the use of solvents such as dimethyl sulfoxide, propylene glycol and the like in the traditional dyeing liquid is avoided, the irritation generated when the dyeing liquid is applied to a human body is reduced, and the uncomfortable feeling is reduced. The prepared staining solution has the characteristics of simple operation, high identification rate, no stimulation in use, good stability, long storage time and the like.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a folic acid receptor mediated epithelial tissue cell staining solution comprises the following raw materials in parts by mass:
0.1-12 parts of modified folic acid, 0.1-2 parts of methylene blue, 0.05-14 parts of sodium hydroxide, 5-20 parts of tween 80, 2-20 parts of ascorbic acid, 0.1-12 parts of carboxymethyl chitosan, 5-30 parts of glucose, 0.02-0.5 part of neutral red, 0.02-0.1 part of glacial acetic acid, a plurality of parts of hydrogen peroxide solution and 30 parts of sterile deionized water.
Further, the cell staining solution comprises the following raw materials in parts by mass:
2-8 parts of modified folic acid, 0.5-1.3 parts of methylene blue, 3-10 parts of sodium hydroxide, 5-20 parts of tween 80, 2-20 parts of ascorbic acid, 0.1-12 parts of carboxymethyl chitosan, 10-25 parts of glucose, 0.2-0.3 part of neutral red, 0.03-0.06 part of glacial acetic acid, a plurality of parts of hydrogen peroxide solution and 30 parts of sterile deionized water.
Further, the cell staining solution comprises the following raw materials in parts by mass:
6.0 parts of modified folic acid, 1.0 parts of methylene blue, 6.5 parts of sodium hydroxide, 13.0 parts of tween 80, 10.0 parts of ascorbic acid, 6.0 parts of carboxymethyl chitosan, 20 parts of glucose, 0.23 part of neutral red, 0.05 part of glacial acetic acid, a plurality of parts of hydrogen peroxide solution and 30 parts of sterile deionized water.
Further, the modified folic acid is prepared by the following steps:
a1, adding 0.585g of 0.1 mol/L2-morpholinoethanesulfonic acid and 0.0876g of 0.05mol/L NaCl into 30mL of sterile deionized water, and regulating the pH value of the system to 5.1 by using NaOH solution to obtain a 2-morpholinoethanesulfonic acid buffer solution for later use;
a2, dissolving folic acid, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), NHS (N-hydroxysuccinimide) and dopamine hydrochloride in 10mL 2-morpholinoethanesulfonic acid buffer solution, and fully stirring for 24 hours at room temperature in an inert gas atmosphere to obtain the modified folic acid.
In the reaction process, an EDC/NHS system is utilized to activate carboxyl in folic acid and react with amino on dopamine hydrochloride to generate an amide bond, so that catechol groups on dopamine hydrochloride are grafted at the tail end of folic acid.
Further, in the step A2, the mass ratio of folic acid to dopamine hydrochloride is (0.1-1.0) g: (0.1-2.0) g.
A preparation method of folic acid receptor mediated epithelial tissue cell staining solution comprises the following preparation steps:
s1, weighing a formula part of methylene blue, adding the methylene blue into sterile deionized water, uniformly stirring at room temperature, adding part of Tween 80, wherein the addition amount is 6% of the mass of the formula part, continuously stirring until the mixture is uniform, adding components of ascorbic acid, carboxymethyl chitosan and glucose, uniformly stirring, and standing for 15 minutes to obtain a methylene white water solution;
s2, adding modified folic acid, residual Tween 80 and sodium hydroxide into sterile deionized water, stirring uniformly at room temperature, stirring with the methylene white water solution obtained in the step S1 at a speed of 30rpm, dropwise adding glacial acetic acid in the stirring process to adjust the pH value of the system to 5.8-6.8, dropwise adding 5% hydrogen peroxide while shaking until blue, blackish green or purple black in the system disappear, then adding neutral red, and continuing stirring uniformly to obtain a dyeing liquid;
and S3, packaging the staining solution obtained in the step S2 by using an integrated secretion pretreatment sampling container sterilized by an ultraviolet sterilizer, and sealing and preserving by using an aluminum foil bag to obtain the folic acid receptor mediated epithelial tissue cell staining solution.
In the reaction process, methylene blue is used for preparing a methylene white solution in a system, and then the methylene white solution is mixed with components such as modified folic acid and the like to prepare a staining solution; the carboxyl at the upper end of the modified folic acid is easier to react, so sodium hydroxide and the modified folic acid are used to form sodium folate, so that the modified folic acid is easy to dissolve in water. Since catechol groups have reducibility and are easy to turn methylene white into blue, a hydrogen peroxide strong oxidant is used to increase the oxidation degree of methylene in methylene blue, and the catechol is mostly oxidized into catecholquinone, so that the concentration of the catechol groups in the system is reduced, and the methylene white is prevented from being reduced.
Further, in step S1, the mass ratio of methylene blue to tween 80 is 1: (3-5).
Compared with the prior art, the invention has the following beneficial effects:
in the technical scheme of the invention, the novel human epithelial tissue tumor cell staining solution is prepared by using components such as modified folic acid, methylene blue, tween 80, carboxymethyl chitosan and the like, and has good water solubility by modifying folic acid and forming sodium salt. The hydrophilic functional groups are grafted on the chitosan, so that the hydrophilicity of the chitosan is improved, the use of solvents such as dimethyl sulfoxide, propylene glycol and the like in the traditional dyeing liquid is avoided, the irritation generated when the dyeing liquid is applied to a human body is reduced, and the uncomfortable feeling is reduced. The prepared staining solution has the characteristics of simple operation, high identification rate, no stimulation in use, good stability, long storage time and the like;
the folic acid is modified by using dopamine hydrochloride, so that the surface of the folic acid contains catechol groups. Dopamine hydrochloride is a substance with low allergy risk, can be quickly metabolized when applied to human tissues, has higher safety, and catechol groups have higher polarity, so that the solubility of the catechol groups in a sterile deionized water system can be increased, the effect of better compatibility with methylene blue components and quicker approaching to tumor cells is achieved, and part of oxidized catechol groups can be combined with active amine thioalkyl groups in proteins on the surfaces of the tumor cells in the form of catecholquinone in a Michael addition mode, so that the proteins are denatured, the effect of fixing the cells is achieved, the dissolution of the tumor cells is prevented, and the more accurate detection effect is achieved;
methylene blue is oxidized into imine groups on the methylene blue in a sodium hydroxide system to form a methylene white solution; the carboxyl at the upper end of the modified folic acid is easier to react, so sodium hydroxide and the modified folic acid are used to form sodium folate, so that the modified folic acid is easy to dissolve in water. Since catechol groups have reducibility and are easy to turn methylene white into blue, a hydrogen peroxide strong oxidant is used to increase the oxidation degree of methylene in methylene blue, and the catechol is mostly oxidized into catecholquinone, so that the concentration of the catechol groups in the system is reduced, and the methylene white is prevented from being reduced. In addition, the Tween 80 component used in the formula belongs to an amphiphilic surfactant, has strong solubilisation, can effectively improve the dissolution of folic acid and methylene blue, promotes the combination and enhances the stability of oxidation-reduction reaction. The catechol group remained in the system in the storage process can protect methylene blue white, avoid the environmental oxidation, and improve the storage stability and the use stability;
after the chitosan is modified by carboxymethylation, the water solubility is improved, and particularly, the solubility in neutral and alkaline solutions is obviously enhanced, so that the chitosan has the characteristics of film formation, thickening, moisture preservation, chelation and the like. Catechol and catechol quinone groups in the system can have good compatibility with carboxymethyl chitosan through hydrogen bond interaction, and the carboxymethyl chitosan and modified folic acid can be contacted with tumor cells with stronger hydrophilicity by utilizing hydrophilic groups contained in the carboxymethyl chitosan and the modified folic acid in the dyeing process, so that the permeability of the dyeing liquid on cell membranes is increased. And normal cells have natural hydrophobicity, so that nonspecific accumulation of non-cancerous epithelial cells on dye can be reduced, and the accuracy of the dye can be improved. In addition, as the organic macromolecule Tween 80 can improve the water flux of cell membranes, can promote the more complete combination of the staining solution and tumor cells, and has better detection effect on the epithelial cells in the N/A tumor stage.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below 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, 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.
The reagents used in the following examples and comparative examples were analytical grade, dopamine hydrochloride was supplied by Shanghai Ala Biochemical technologies Co., ltd, with CAS number 62-31-7.
Example 1, modified folic acid was prepared by the following steps:
a1, adding 0.585g of 0.1 mol/L2-morpholinoethanesulfonic acid and 0.0876g of 0.05mol/L NaCl into 30mL of sterile deionized water, and regulating the pH value of the system to 5.1 by using NaOH solution to obtain a 2-morpholinoethanesulfonic acid buffer solution for later use;
a2, 0.1g folic acid, 0.192g EDC, 0.115. 0.115g N-hydroxysuccinimide and 0.1g dopamine hydrochloride were dissolved in 10mL 2-morpholinoethanesulfonic acid buffer at room temperature and N 2 Fully stirring for 24 hours in the gas atmosphere to obtain the modified folic acid.
Example 2, modified folic acid was prepared by the following steps:
a1, adding 0.585g of 0.1 mol/L2-morpholinoethanesulfonic acid and 0.0876g of 0.05mol/L NaCl into 30mL of sterile deionized water, and regulating the pH value of the system to 5.1 by using NaOH solution to obtain a 2-morpholinoethanesulfonic acid buffer solution for later use;
a2, 0.66g folic acid, 0.192g EDC, 0.115. 0.115g N-hydroxysuccinimide and 0.95g dopamine hydrochloride were dissolved in 10mL 2-morpholinoethanesulfonic acid buffer at room temperature and N 2 Fully stirring for 24 hours in the gas atmosphere to obtain the modified folic acid.
Example 3, modified folic acid was prepared by the following steps:
a1, adding 0.585g of 0.1 mol/L2-morpholinoethanesulfonic acid and 0.0876g of 0.05mol/L NaCl into 30mL of sterile deionized water, and regulating the pH value of the system to 5.1 by using NaOH solution to obtain a 2-morpholinoethanesulfonic acid buffer solution for later use;
a2, 1g folic acid, 0.192g EDC, 0.115. 0.115g N-hydroxysuccinimide and 2g dopamine hydrochloride were dissolved in 10mL 2-morpholinoethanesulfonic acid buffer at room temperature and N 2 Fully stirring for 24 hours in the gas atmosphere to obtain the modified folic acid.
Example 4, a folate receptor mediated epithelial tissue cell staining solution, comprising the following raw materials in parts by mass:
0.1 part of modified folic acid prepared in example 1, 0.1 part of methylene blue, 0.05 part of sodium hydroxide, 5 parts of tween 80, 2 parts of ascorbic acid, 0.1 part of carboxymethyl chitosan, 5 parts of glucose, 0.02 part of neutral red, 0.02 part of glacial acetic acid, a plurality of hydrogen peroxide solutions and sterile deionized water.
The preparation method comprises the following steps:
s1, weighing raw materials in parts by weight, adding methylene blue into 10 parts of sterile deionized water, uniformly stirring at room temperature, adding 0.3 part of Tween 80, continuously stirring until the components are uniform, adding ascorbic acid, carboxymethyl chitosan and glucose, uniformly stirring, and standing for 15 minutes to obtain methylene white water solution;
s2, adding the modified folic acid, the rest 4.7 parts of Tween 80 and sodium hydroxide prepared in the embodiment 1 into 30 parts of sterile deionized water, stirring uniformly at room temperature, stirring and mixing with the methylene white water solution obtained in the step S1 at a speed of 30rpm, dropwise adding glacial acetic acid in the stirring process to adjust the pH value of the system to 5.8, dropwise adding 5% hydrogen peroxide while shaking until blue, blackish green or purplish black in the system disappear, then adding neutral red, and continuing stirring uniformly to obtain a dyeing liquid;
and S3, packaging the staining solution obtained in the step S2 by using an integrated secretion pretreatment sampling container sterilized by an ultraviolet sterilizer, and sealing and preserving by using an aluminum foil bag to obtain the folic acid receptor mediated epithelial tissue cell staining solution.
The difference between example 5 and this example and example 4 is that the cell staining solution comprises the following raw materials in parts by mass:
6.0 parts of modified folic acid prepared in example 2, 1.0 part of methylene blue, 6.5 parts of sodium hydroxide, 13.0 parts of tween 80, 10.0 parts of ascorbic acid, 6.0 parts of carboxymethyl chitosan, 20 parts of glucose, 0.23 part of neutral red, 0.05 part of glacial acetic acid, a plurality of parts of hydrogen peroxide solution and sterile deionized water; in the step S1, the mass ratio of the methylene blue to the Tween 80 is 1:4, a step of; in the step S2, glacial acetic acid is used for regulating the pH value of the system to 6.3.
The difference between example 6 and this example and example 4 is that the cell staining solution comprises the following raw materials in parts by mass:
12 parts of modified folic acid prepared in example 3, 2 parts of methylene blue, 14 parts of sodium hydroxide, 20 parts of tween 80, 20 parts of ascorbic acid, 12 parts of carboxymethyl chitosan, 30 parts of glucose, 0.5 part of neutral red, 0.1 part of glacial acetic acid, a plurality of parts of hydrogen peroxide solution and sterile deionized water; in the step S1, the mass ratio of the methylene blue to the Tween 80 is 1:5, a step of; in the step S2, glacial acetic acid is used for regulating the pH value of the system to 6.8.
Comparative example 1
This comparative example differs from example 5 in that folic acid was not modified in this comparative example and no hydrogen peroxide component was added to the formulation.
Comparative example 2
The difference between this comparative example and example 5 is that no tween 80 component was added to the formulation in this comparative example.
Comparative example 3
The difference between this comparative example and example 5 is that the carboxymethyl chitosan component was not added to the formulation in this comparative example.
The cell staining solutions prepared in examples 4 to 6 and comparative examples 1 to 3 were now subjected to storage stability test. The staining solution samples prepared in examples 4 to 6 and comparative examples 1 to 3 were stored in a sealed manner in a shade at a dry place for 48 hours, followed by observing the change in appearance of the staining solution and detecting the change in pH therein to judge the storage stability of the cell staining solution. Five readings were taken for each sample and averaged, and the test results are shown in table 1 below.
TABLE 1 storage stability test of cell staining solutions prepared in examples 4 to 6 and comparative examples 1 to 3
。
As is clear from the results in Table 1, the cell staining solutions prepared in examples 4 to 6 all had good storage stability, and the pH was not substantially changed. From the results in comparative example 1, it was found that methylene blue having a reducing property in the dyeing liquid had been oxidized, the dyeing liquid showed blue color, and the pH value was lowered due to decomposition of methylene blue. From the results in comparative examples 2 and 3, it is understood that the use of tween 80 and carboxymethyl chitosan components has a synergistic effect with the modified folic acid in improving the storage stability of the dye solution.
The cell staining solutions prepared in examples 4 to 6 and comparative examples 1 to 3 were now subjected to detection accuracy tests. The test was performed by 420 clinical cases excluding cervical surface bleeding or a tendency to bleed, a history of cervical surgery or hysterectomy, pregnancy and other history of tumor disease. The cases were divided into 6 groups, and were detected using the staining solution detection kits prepared in examples 4 to 6 and comparative examples 1 to 3, and were detected again using the HPV E6/E7 detection kit after the detection was completed, the HPV E6/E7 detection kit and sampling consumables were provided by hao-strage corporation in the united states, the cervical surface was gently rotated 5 times using a sterile cervical brush, and then the brush head was broken off and placed in a special preservation solution, sealed and sent to a laboratory, and detected according to the kit instructions, and if any of the cases positive in the detection was examined by colposcopy, and a multipoint pathological biopsy was taken under the mirror, such as a colposcopy dissatisfaction while cervical scraping was performed, and the pathological diagnosis results were using a CIN naming system, including: n/a (no abnormal lesions or inflammation), CIN1 (mild atypical hyperplasia), CIN2 (moderate atypical hyperplasia), CIN3 (severe atypical hyperplasia and carcinoma in situ), CA (invasive carcinoma), and were judged positive in CIN2 and above.
The cell staining solution detection method comprises the following steps:
1. placing a vaginal dilator to expose the cervical orifice;
2. tearing the aluminum foil bag, taking out the integrated secretion pretreatment sampling device, opening the container, and completely submerging the epithelial tissue staining solution into the lower cotton swab sleeve;
3. after the solution completely flows out, the cotton swab fully absorbs the solution, then forcibly smearing the solution on the surface of the epithelial tissue near the cervix for 3-5 circles, slightly pressing the tail pipe to supplement the cotton swab solution in the smearing process, and finally pressing the cotton swab at the cervical opening for 3-5 seconds;
4. immediately remove the swab, immediately observe and record the color in the control colorimetric card, and dry the excess fluid remaining in the epithelial tissue with a dry swab.
The detection results were recorded, and the positive predictive value=the number of true positive cases/(the number of true positive cases+the number of false positive cases) ×100%, and the negative predictive value=the number of true negative cases/(the number of true negative cases+the number of false negative cases) ×100%. The test results are shown in table 2 below.
TABLE 2 detection accuracy test of stain kit in examples 4-6 and comparative examples 1-3
As is clear from the results in Table 2, the cell staining solutions prepared in examples 4 to 6 were excellent in the accuracy of detecting CIN2, CIN3 and CA patients. As is clear from the results of comparative examples 1 to 3 and HPV E6/E7, the detection accuracy of the detection solution for N/A patients was not very high, and the deviation was severe. Under the synergistic effect of folic acid modification, tween 80 and carboxymethyl chitosan components, the characteristics of high hydrophilicity of inflammatory cells and tumor cells and the characteristics of high protein expression level can be utilized, the combination with cell proteins is promoted, and the detection accuracy of N/A patients is higher.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing is merely illustrative and explanatory of the invention, as various modifications and additions may be made to the particular embodiments described, or in a similar manner, by those skilled in the art, without departing from the scope of the invention or exceeding the scope of the invention as defined in the claims.
Claims (7)
1. A folic acid receptor mediated epithelial tissue cell staining solution is characterized by comprising the following raw materials in parts by mass:
0.1-12 parts of modified folic acid, 0.1-2 parts of methylene blue, 0.05-14 parts of sodium hydroxide, 5-20 parts of tween 80, 2-20 parts of ascorbic acid, 0.1-12 parts of carboxymethyl chitosan, 5-30 parts of glucose, 0.02-0.5 part of neutral red, 0.02-0.1 part of glacial acetic acid, a plurality of parts of hydrogen peroxide solution and 30 parts of sterile deionized water.
2. The folic acid receptor mediated epithelial tissue cell staining solution according to claim 1, wherein the staining solution comprises the following raw materials in parts by mass:
2-8 parts of modified folic acid, 0.5-1.3 parts of methylene blue, 3-10 parts of sodium hydroxide, 5-20 parts of tween 80, 2-20 parts of ascorbic acid, 0.1-12 parts of carboxymethyl chitosan, 10-25 parts of glucose, 0.2-0.3 part of neutral red, 0.03-0.06 part of glacial acetic acid, a plurality of parts of hydrogen peroxide solution and 30 parts of sterile deionized water.
3. The folic acid receptor mediated epithelial tissue cell staining solution according to claim 1, wherein the staining solution comprises the following raw materials in parts by mass:
6.0 parts of modified folic acid, 1.0 parts of methylene blue, 6.5 parts of sodium hydroxide, 13.0 parts of tween 80, 10.0 parts of ascorbic acid, 6.0 parts of carboxymethyl chitosan, 20 parts of glucose, 0.23 part of neutral red, 0.05 part of glacial acetic acid, a plurality of parts of hydrogen peroxide solution and 30 parts of sterile deionized water.
4. The folate receptor mediated epithelial tissue cell staining solution of claim 1, wherein the modified folic acid is prepared by:
a1, adding 2-morpholinoethanesulfonic acid and NaCl into sterile deionized water, and regulating the pH value of the system to 5.1 by using a NaOH solution to obtain a 2-morpholinoethanesulfonic acid buffer solution for later use;
a2, dissolving folic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride in A2-morpholinoethanesulfonic acid buffer solution, and fully stirring for 24 hours at room temperature in an inert gas atmosphere to obtain the modified folic acid.
5. The folate receptor mediated epithelial tissue cell staining solution according to claim 1, wherein in the A2 step, the mass ratio of folic acid to dopamine hydrochloride is (0.1-1.0) g: (0.1-2.0) g.
6. A method for preparing a folate receptor mediated epithelial tissue cell staining solution according to any of claims 1 to 5, comprising the steps of:
s1, weighing and adding methylene blue serving as a formula part into sterile deionized water, stirring uniformly at room temperature, adding part of tween 80, continuously stirring uniformly, adding ascorbic acid, carboxymethyl chitosan and glucose components, stirring uniformly, and standing to obtain a methylene white water solution;
s2, adding modified folic acid, residual Tween 80 and sodium hydroxide into sterile deionized water, stirring uniformly at room temperature, stirring and mixing with the methylene white water solution obtained in the step S1, dropwise adding glacial acetic acid in the stirring process to adjust the pH value of the system to 5.8-6.8, dropwise adding hydrogen peroxide while shaking until blue, blackish green or purple black in the system disappear, then adding neutral red, and continuing stirring uniformly to obtain a dyeing liquid;
and S3, packaging the staining solution obtained in the step S2 by using an integrated secretion pretreatment sampling container sterilized by an ultraviolet sterilizer, and sealing and preserving by using an aluminum foil bag to obtain the folic acid receptor mediated epithelial tissue cell staining solution.
7. The method for preparing a folate receptor mediated epithelial tissue cell staining solution according to claim 6, wherein in step S1, the mass ratio of methylene blue to tween 80 is 1: (3-5).
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