Folate receptor mediated cell staining solution as well as preparation method and application thereof
By preparing an acidic cell staining solution containing components such as methylene blue, and utilizing folic acid receptor-mediated and oxidative stress responses, the problem of insufficient accuracy in cervical cancer detection in existing technologies has been solved, achieving highly sensitive and accurate screening for different stages of cervical cancer.
Patent Information
- Application Number
- CN202511048649.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-29
- Publication Date
- 2025-11-07
AI Technical Summary
Existing methods for detecting cervical lesions, such as acetic acid visual examination and iodine staining visual examination, have low accuracy. The single colorimetric reaction mediated by folic acid receptors cannot effectively distinguish different stages of cervical cancer, resulting in insufficient sensitivity and accuracy of the test results.
An acidic cell staining solution containing methylene blue, ascorbic acid, folic acid, acetic acid, sodium acetate, propylene glycol, and sodium diphenylamine sulfonate was prepared. Different colorimetric reactions were used to distinguish different stages of cervical cancer. Methylene blue was introduced into cells via folic acid receptor-mediated staining, and combined with oxidative stress response, rapid and accurate screening was achieved.
The chromogenic solution can better distinguish different stages of cervical cancer, improve the sensitivity and accuracy of the test results, and has a simple preparation method, low cost, and good application prospects.
Smart Images
Figure CN120907928A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of cervical disease detection, and particularly relates to a folate receptor-mediated cell staining solution and a preparation method and application thereof. BACKGROUND
[0002] Cervical cancer is the second largest threat to women's health worldwide, second only to breast cancer, with more than 500,000 new cases each year, resulting in 250,000 deaths. In recent years, the incidence in China has increased and become younger, with about 150,000 new cases and 56,000 deaths in 2022. Cervical cancer is the only cancer that can be cured if detected early, but many patients miss the opportunity for treatment because they do not undergo screening. Cervical cancer develops slowly and has a clear precancerous stage, providing an opportunity for screening and treatment. Early screening and timely intervention are crucial to reducing the incidence and mortality rates and can significantly improve the cure rate.
[0003] Traditional cervical lesion detection relies on pathology and polymerase chain reaction technology, but cannot provide results in real time. Liquid-based cytology combined with human papillomavirus detection is costly, limiting their application in routine screening and timely diversion of outpatients and physical examinations. In economically underdeveloped areas, visual screening methods such as acetic acid visual inspection and iodine staining visual inspection are used for initial screening, but the accuracy is low. Folate receptor-mediated cervical special staining (FRD) is a new and convenient cervical lesion detection technology that takes advantage of the high expression of tumor cell folate receptors and oxidative stress, and uses the color reaction of methylene blue to achieve rapid and accurate screening. Different stages of tumor tissue show different color spectra, but screening based only on the single color reaction of methylene blue cannot well distinguish different stages of tumors, and the sensitivity and accuracy of the detection results cannot be guaranteed. SUMMARY
[0004] The technical problem solved by the present application is to provide a folate receptor-mediated cell staining solution, a preparation method and application thereof.
[0005] To solve the above technical problem, the present application discloses a folate receptor-mediated cell staining solution, a preparation method and application thereof.
[0006] The present application provides a folate receptor-mediated cell staining solution, which comprises the following components in percentage by weight: methylene blue 0.05-0.2%, ascorbic acid 0.3-1%, folate 0.3-1%, acetic acid 1-3%, sodium acetate 0.1-0.4%, propylene glycol 1-3%, and sodium diphenylamine sulfonate 0.1-0.5%, and the rest is water.
[0007] Preferably, the present application provides a folate receptor-mediated cell staining solution, which comprises the following components in percentage by weight: methylene blue 0.1%, ascorbic acid 0.3-1%, folate 0.3-1%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2%, and sodium diphenylamine sulfonate 0.3%, and the rest is water.
[0008] Further preferably, the cell staining solution comprises, by weight percentage, methylene blue 0.1%, ascorbic acid 0.3%, folic acid 1%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2%, sodium diphenylamine sulfonate 0.3%, and the rest is water; or comprises, by weight percentage, methylene blue 0.1%, ascorbic acid 0.5%, folic acid 1%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2%, sodium diphenylamine sulfonate 0.3%, and the rest is water; or comprises, by weight percentage, methylene blue 0.1%, ascorbic acid 1%, folic acid 0.3%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2%, sodium diphenylamine sulfonate 0.3%, and the rest is water; or comprises, by weight percentage, methylene blue 0.1%, ascorbic acid 1%, folic acid 1%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2%, sodium diphenylamine sulfonate 0.3%, and the rest is water.
[0009] More preferably, the cell staining solution comprises, by weight percentage, methylene blue 0.1%, ascorbic acid 0.5%, folic acid 1%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2%, sodium diphenylamine sulfonate 0.3%, and the rest is water, and the cell staining solution has the most sensitive and accurate color change behavior and the best storage performance, and still has excellent color change behavior after being stored at room temperature for 2 weeks.
[0010] In a second aspect, the present application provides a preparation method of the cell staining solution of the first aspect, comprising the following steps:
[0011] (1) weighing each component in the cell staining solution according to the weight ratio;
[0012] (2) mixing methylene blue and water uniformly; preferably, stirring to mix uniformly until the methylene blue is completely dissolved.
[0013] (3) adding ascorbic acid, acetic acid, sodium acetate, propylene glycol, sodium diphenylamine sulfonate and folic acid components into the solution obtained by mixing in step (2), and stirring to mix, to obtain the cell staining solution. Preferably, the ascorbic acid, acetic acid, sodium acetate, propylene glycol, sodium diphenylamine sulfonate and folic acid components are sequentially added into the solution obtained by mixing in step (2) in order, and stirred to mix, to obtain the cell staining solution, wherein only one component is added at a time, and the next component is added only after the previous component is added and stirred to mix. Further preferably, the ascorbic acid is added into the solution obtained by mixing in step (2), and stirred to mix until the color of the solution becomes light to obtain a first solution, the acetic acid is added into the first solution, and stirred to mix to obtain a second solution, the sodium acetate is added into the second solution, and stirred to mix to obtain a third solution, the propylene glycol is added into the third solution, and stirred to mix to obtain a fourth solution, the sodium diphenylamine sulfonate is added into the fourth solution, and stirred to mix to obtain a fifth solution, the folic acid is added into the fifth solution, and stirred to mix to obtain the cell staining solution. Further preferably, the cell staining solution is brownish yellow, and is stored in a brown bottle.
[0014] In the preparation method, each step is carried out at normal temperature and pressure.
[0015] In a third aspect, the present application provides the use of the cell staining solution of the first aspect in the preparation of a cervical cancer detection kit. Preferably, the cervical cancer detection kit is a cervical cancer staging detection kit. The kit can preliminarily detect and evaluate the progression of cervical cancer, has great application potential in the field of cervical cancer detection, and can make the cervical cancer detection have more significant color changes.
[0016] The present application simulates the oxidative microenvironment in cervical cancer cells by preparing H2O2 solutions with different concentrations. The staining solution has significantly different color changes for H2O2 solutions with different concentrations. The staining solution shows light green for the simulated early-stage cervical cancer H2O2 solution, shows blue or green for the simulated mid-stage cervical cancer H2O2 solution, and shows blue-violet for the simulated late-stage cervical cancer H2O2 solution.
[0017] Further preferably, the detection kit prepared from the cell staining solution is detected according to the following method: a cotton swab head is soaked in an H2O2 solution with a different concentration for about 10 seconds, and then the cotton swab head is immediately immersed in the prepared staining solution for about 10 seconds, and the color change of the cotton swab is observed within 30 seconds.
[0018] Advantages:
[0019] Compared with the prior art, the present application has the following significant advantages: it can better distinguish different stages of cervical cancer, has more significant color changes for cervical cancer detection, and has higher sensitivity and accuracy of detection results.
[0020] The application has great application potential in effectively detecting epithelial tissue tumor cells, and has good specificity, can well distinguish different stages of tumors, especially cervical cancer, the sensitivity and accuracy of the detection result are higher than existing detection reagents, and the preparation method is simple, the cost is low, and the application prospect is good. The preparation method of the application is simple, no three waste pollution is generated; no special treatment of medical waste is generated in use. BRIEF DESCRIPTION OF DRAWINGS
[0021] The above and / or other aspects of the present application will become more apparent by describing in detail the preferred embodiments thereof with reference to the attached drawings in which:
[0022] Figure 1 The cell staining solution prepared for Example 1.
[0023] Figure 2 The color reference chart of the cell staining solution prepared by the application for staining cervical cancer cells.
[0024] Figure 3 The discoloration of the cell staining solution prepared in Example 2 to different concentrations of H2O2, wherein a is 300nM, b is 1μM, c is 50mM, and d is 50nM.
[0025] Figure 4 The discoloration chart of different cells treated with different cell staining solutions within 30s, wherein a is Hela cells treated with the cell staining solution prepared in Example 2, b is Hela cells treated with the cell staining solution prepared in Comparative Example 1, c is 293T cells treated with the cell staining solution prepared in Example 2, and d is 293T cells treated with the cell staining solution prepared in Comparative Example 1. DETAILED DESCRIPTION
[0026] The technical solutions of the present application will be described in detail below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.
[0027] Example 1
[0028] The folate receptor-mediated cell staining solution in this embodiment is made of the following components by weight percentage: water 94.1%, methylene blue 0.1%, ascorbic acid 0.3%, folic acid 1%, acetic acid 2%, propylene glycol 2%, sodium acetate 0.2%, and sodium diphenylamine sulfonate 0.3%.
[0029] The folate receptor-mediated cell staining solution in this embodiment is prepared by the following preparation method, which specifically includes the following steps:
[0030] (1) methylene blue was stirred with water to complete dissolution at a preset weight percentage;
[0031] (2) ascorbic acid was added to the solution obtained in step (1) at a preset weight percentage, and stirred until the solution color became light;
[0032] (3) acetic acid was added to the solution obtained in step (2) at a preset weight percentage, and stirred to mix well;
[0033] (4) sodium acetate was added to the solution obtained in step (3) at a preset weight percentage, and stirred to mix well;
[0034] (5) propylene glycol was added to the solution obtained in step (4) at a preset weight percentage, and stirred to mix well;
[0035] (6) sodium diphenylamine sulfonate was added to the solution obtained in step (5) at a preset weight percentage, and stirred to mix well; (7) folic acid was added to the solution obtained in step (6) at a preset weight percentage, and stirred to mix well.
[0036] Each of the above steps was carried out at normal temperature and pressure.
[0037] Example 2
[0038] The folic acid receptor-mediated cell staining solution in this example was made from the following components at the following weight percentages: water 93.9%, methylene blue 0.1%, ascorbic acid 0.5%, folic acid 1%, acetic acid 2%, propylene glycol 2%, sodium acetate 0.2%, and sodium diphenylamine sulfonate 0.3%.
[0039] The preparation method of the folic acid receptor-mediated cell staining solution was the same as in Example 1.
[0040] Example 3
[0041] The folic acid receptor-mediated cell staining solution in this example was made from the following components at the following weight percentages: water 94.1%, methylene blue 0.1%, ascorbic acid 1%, folic acid 0.3%, acetic acid 2%, propylene glycol 2%, sodium acetate 0.2%, and sodium diphenylamine sulfonate 0.3%.
[0042] The preparation method of the folic acid receptor-mediated cell staining solution was the same as in Example 1.
[0043] Example 4
[0044] The folate receptor-mediated cell staining solution in this example is made from the following components in weight percentage: water 93.4%, methylene blue 0.1%, ascorbic acid 1%, folate 1%, acetic acid 2%, propylene glycol 2%, sodium acetate 0.2%, and sodium diphenylamine sulfonate 0.3%.
[0045] The preparation method of the folate receptor-mediated cell staining solution is the same as that of Example 1.
[0046] Comparative Example 1
[0047] The folate receptor-mediated cell staining solution in this example is made from the following components in weight percentage: water 93.4%, methylene blue 0.1%, ascorbic acid 1%, folate 1%, acetic acid 2%, propylene glycol 2%, sodium acetate 0.2%, and sodium diphenylamine sulfonate 0.3%.
[0048] The preparation method of the folate receptor-mediated cell staining solution is the same as that of Example 1.
[0049] The staining solutions prepared in Examples 1-4 and Comparative Example 1 above are all brownish yellow. Taking Example 1 as an example, the actual solution is shown in Figure 1 .
[0050] According to the literature, the redox potential in cervical cancer cells at different stages is closely related to the concentration of H2O2. The concentration of H2O2 in normal cervical cells is generally between 1-100 nM. The concentration of H2O2 in early stage (stage I) cervical cancer cells is relatively low, about 100-500 nM, and the redox potential is relatively stable. As the tumor progresses, the concentration of H2O2 increases, and the redox potential also changes accordingly. The concentration of H2O2 in intermediate stage (stages II-III) cervical cancer cells is about 500 nM-1.5 μM. The concentration of H2O2 in advanced stage (stage IV) cervical cancer cells can reach 1-2 μM, and even in more malignant tumors, the concentration of H2O2 can be as high as 100 mM, and the redox potential is significantly increased, reflecting a stronger oxidative stress state. This change may be related to the metabolic reprogramming and changes in antioxidant capacity of the cells.
[0051] In view of the experimental conditions of this study, and for the convenience of research, the present application simulates the oxidative microenvironment in cervical cancer cells at different stages by preparing H2O2 solutions of different concentrations. Specifically, 300 nM, 1 μM, 50 mM, and 50 nM H2O2 solutions are prepared in 10 mM phosphate buffer at pH 6.5, respectively, to simulate early stage (stage I) cervical cancer cells, intermediate stage (stages II-III) cervical cancer cells, advanced stage (stage IV) cervical cancer cells, and normal cervical cells.
[0052] The cell staining solution prepared from each of Examples 1-4 and Comparative Example 1 was tested according to the following method. A cotton swab was taken from the individual package, and the tip of the cotton swab was immersed in an H2O2 solution of different concentration for about 10 seconds. Then, the tip of the cotton swab was immediately immersed in the prepared staining solution for about 10 seconds. The cotton swab was taken out and the color change of the cotton swab was observed within 30 seconds. The color change of the cotton swab after 30 seconds was not considered as a judgment basis.
[0053] As shown in Table 1, according to the color chart in Figure 2 , it can be seen from the color change that, under the condition of the staining solution prepared from Comparative Example 1, the color change of the H2O2 solution of different concentration was only 2 kinds (light green and blue). Similarly, under the condition of the staining solution prepared from Examples 1-4, the color change of the H2O2 solution of different concentration was 3 kinds (light green, blue / green, and blue / purple), which could distinguish the H2O2 solution of different concentration more clearly. It can be seen from the color change time that the color change time of the staining solution prepared from Example 2 was shorter, and the color change was faster. The color change of the staining solution prepared from Example 2 to simulate different cervical cancer cells is shown in Figure 3 . In addition, it can be seen from Table 1 that the H2O2 solution as a control group did not change color within 30 seconds. It can be seen from the above results that the color change behavior of the staining solution prepared from Example 2 is the best.
[0054] In addition, the storage stability of the staining solution prepared from Examples 1-4 and Comparative Example 1 was also tested. The prepared staining solution was placed in a brown glass bottle and stored at room temperature for 2 weeks, and then the performance was tested. The results showed that the color change behavior of the staining solution prepared from Examples 2-4 was basically unchanged, but the staining solution prepared from Comparative Example 1 and Example 1 did not change color and lost the color change behavior. It can be seen that the staining solution prepared from Examples 2-4 has excellent storage stability.
[0055] After comprehensive evaluation, the staining solution prepared from Example 2 showed the most excellent performance. In the field of cervical cancer screening, the staining solution has great application potential and is expected to make important contributions to improving the accuracy and reliability of screening.
[0056] Table 1 Color change of H2O2 of different concentration under different staining solutions
[0057]
[0058]
[0059] In the present application, DMEM medium was also used to culture cervical cancer cells Hela cells and human kidney epithelial cells 293T cells, and the specific culture conditions were 37℃, 5% CO2, and 95% humidity.
[0060] The cell staining solution prepared in the above Example 2 and Comparative Example 1 was respectively taken to test Hela cells and 293T cells, and the specific testing method was as follows: a cotton swab was taken out from the single package bag, the cotton swab head was first used to wipe 3-5 circles on the surface of the culture dish containing cells to pick up cells, and then the cotton swab head with cells was immediately immersed in the prepared staining solution for about 10s, the cotton swab was taken out and the color change of the cotton swab was observed within 30s, and the color of the cotton swab after 30s could not be used as a judgment basis. Figure 4 It can be seen that, according to the color change, under the condition of the staining solution prepared in Example 2, the Hela cells presented green, and the 293T cells as a control presented yellow, and under the condition of the staining solution prepared in Comparative Example 1, the Hela cells presented part of light green, and the 293T cells as a control still presented yellow.
[0061] The present application provides a folate receptor-mediated cell staining solution, a preparation method and application ideas and methods, and there are many methods and ways to realize the technical scheme, the above-mentioned is only the preferred embodiment of the present application, it should be pointed out that, for ordinary skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application. The components not explicitly described in the present embodiment can be realized by the existing technology.
Claims
1. A folate receptor-mediated cell staining solution, characterized by, By weight percentage, it comprises the following components: methylene blue 0.05%~0.2%, ascorbic acid 0.3%~1%, folic acid 0.3%~1%, acetic acid 1%~3%, sodium acetate 0.1%~0.4%, propylene glycol 1%~3% and sodium diphenylamine sulfonate 0.1%~0.5%, and the rest is water. 2. The cell staining solution of claim 1, wherein By weight percentage, it comprises the following components: methylene blue 0.1%, ascorbic acid 0.3%~1%, folic acid 0.3%~1%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2% and sodium diphenylamine sulfonate 0.3%, and the rest is water.
3. The cell staining solution of claim 1, wherein By weight percentage, it comprises the following components: methylene blue 0.1%, ascorbic acid 0.3%, folic acid 1%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2% and sodium diphenylamine sulfonate 0.3%, and the rest is water.
4. The cell staining solution of claim 1, wherein By weight percentage, it comprises the following components: methylene blue 0.1%, ascorbic acid 0.5%, folic acid 1%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2% and sodium diphenylamine sulfonate 0.3%, and the rest is water.
5. The cell staining solution of claim 1, wherein By weight percentage, it comprises the following components: methylene blue 0.1%, ascorbic acid 1%, folic acid 0.3%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2% and sodium diphenylamine sulfonate 0.3%, and the rest is water.
6. The cell staining solution of claim 1, wherein By weight percentage, it comprises the following components: methylene blue 0.1%, ascorbic acid 1%, folic acid 1%, acetic acid 2%, sodium acetate 0.2%, propylene glycol 2% and sodium diphenylamine sulfonate 0.3%, and the rest is water.
7. The method for preparing a cell staining solution according to any one of claims 1 to 6, characterized by, It comprises the following steps: (1) Each component in the cell staining solution is weighed according to the weight ratio; (2) Methylene blue is mixed with water; (3) Ascorbic acid, acetic acid, sodium acetate, propylene glycol, sodium diphenylamine sulfonate and folic acid components are added to the solution obtained by mixing in step (2), and stirred and mixed to obtain the cell staining solution.
8. The method of claim 7, wherein, In step (3), ascorbic acid, acetic acid, sodium acetate, propylene glycol, sodium diphenylamine sulfonate and folic acid components are sequentially added to the solution obtained by mixing in step (2), one component at a time, and the next component is added only after the previous component is stirred and mixed.
9. The use of the cell staining solution of any one of claims 1~6 in the preparation of a cervical cancer detection kit.
10. Use according to claim 9, characterized in that, The cervical cancer detection kit is a cervical cancer staging detection kit.