A buffer and its application in improving stability of resazurin
By adding compounds with specific structures and buffer solutions to the RPMI-1640 culture medium, the problem of poor stability of razor cyanin under light was solved, and the accuracy and stability of colorimetric monitoring were achieved, with a color retention rate of 99.03%.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- AUTOBIO DIAGNOSTICS CO LTD
- Filing Date
- 2024-11-27
- Publication Date
- 2026-05-29
AI Technical Summary
The stability of *Razoria zebrina* under light conditions in RPMI-1640 culture medium is poor, leading to inaccurate results in microbial colorimetric monitoring.
Compounds with specific structures and buffer solutions, including osmotic regulators, pH buffers, and solubilizers, were added to the RPMI-1640 culture medium to form a buffer solution to improve the light stability of resazurin.
It significantly improves the stability of the azure blade under light conditions, maintains the color, extends the accuracy of colorimetric monitoring, and achieves a color retention rate of over 99.03%.
Smart Images
Figure BDA0005156635340000021 
Figure BDA0005156635340000071 
Figure BDA0005156635340000072
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more particularly to a buffer solution and its application in improving the stability of resazurin. Background Technology
[0002] Razor blue, also known as sodium azoride, is a chemical substance with the molecular formula C. 12 H6NNaO4 is a chromogenic solution commonly used in the study of biological materials. Most modern biotechnology utilizes cells as carriers; gene therapy, stem cell therapy, and cloning all take place within cells. Cell growth requires a specific nutrient environment, and the nutrient matrix used to maintain cell growth is called culture medium. This refers to all substances used for in vitro culture and preservation of cells for various purposes; essentially, it artificially simulates the nutrient environment of in vivo growth, enabling cells to grow and reproduce within this environment. It is the material basis for providing cell nutrition and promoting cell growth and proliferation. The main components of cell culture medium include: amino acids, water, vitamins, carbohydrates, inorganic ions, and other substances such as nucleic acid degradation products and hormones.
[0003] Resazurin is commonly used as a redox indicator in the analysis of bacterial activity and the proliferation of bacterial, yeast, or mammalian cells. It is frequently used to measure bacterial and eukaryotic cell viability, as a cell staining agent, and also as an acid-base indicator, with a pH range of 3.8 (orange) to 6.5 (deep purple). In the cytoplasm, resazurin can be reduced from its blue state to a pink, strongly fluorescent form called halogenin by various reductases; further reactions result in a colorless and non-fluorescent form of dihydrohalogenin.
[0004] Adding rezathoate to RPMI-1640 medium is often used to culture and monitor the metabolic responses of cells, bacteria, and fungi. After incubation, the presence of microorganisms or their growth and metabolism is detected by color changes (blue turning red or purple). However, this reaction system is highly sensitive to light, requiring strict light-protection during use. Otherwise, even a short period of light exposure can cause the blue to turn purple or red, affecting the colorimetric monitoring results. Therefore, a compound that enhances the stability of rezathoate is needed, allowing the RPMI-1640 medium containing rezathoate to maintain its blue color for an extended period under light conditions without turning red, thus accurately monitoring and reflecting the true growth and metabolism of microorganisms. Summary of the Invention
[0005] In view of this, the present invention provides a buffer solution and its application in improving the stability of resamaritan. Adding the buffer solution of the present invention to the reaction system of RPMI-1640 culture medium and resamaritan, or using the buffer solution of the present invention to prepare the resamaritan chromogenic solution, can significantly improve the light stability of the reaction system.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0007] Application of compounds with Formula I structure in improving the light stability of azuran.
[0008]
[0009] In Formula I, R1 is selected from H or -COCH2(OCH2CH2). n OCH3, where n is any integer between 4 and 12;
[0010] R2 is selected from H, -CH3, or -CH2CHOHCH2(OCH2CH2). m OCH3, where m is any integer between 4 and 12;
[0011] R3 is selected from H or -SO3H.
[0012] Specifically, the compound of Formula I is selected from one or more of the following: 2-hydroxy-4-monomethyl ether polyoxyethylene 6-ether glycidyl ether-benzophenone, 2-hydroxy-4-monomethyl ether polyoxyethylene 10-ether glycidyl ether-benzophenone, 2-monomethyl ether polyoxyethylene 5-ether acetate-4-hydroxy-benzophenone, 2-monomethyl ether polyoxyethylene 12-ether acetate-4-methyl ether-benzophenone, 5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid, 4-methoxy-2-hydroxybenzophenone, or 2,4-dihydroxybenzophenone.
[0013] This invention has revealed that other compounds with the core structure shown in Formula I have a weak or no effect on improving the light stability of resamarium. Among these, compounds with a weak effect include: 2-hydroxy-4-monomethyl ether polyoxyethylene 4-ether glycidyl ether-benzophenone, 2-hydroxy-4-monomethyl ether polyoxyethylene 12-ether glycidyl ether-benzophenone, etc. Ineffective compounds that do not improve the light stability of resamarium include: ethoxybenzone, 2,3,4,4'-tetrahydroxybenzophenone, hypericin, etc.
[0014] This invention provides a buffer solution for improving the stability of rezamidophos, comprising 0.09M-0.12M compounds of formula I.
[0015] In some embodiments, the buffer solution further includes an osmotic pressure regulator and / or a pH buffer and / or a solubilizer. Further, in addition to a 0.09M-0.12M compound of Formula I, the buffer solution includes components at the following concentrations: an osmotic pressure regulator of 0.5-2 g / L, and / or a pH buffer of 0.05-0.2M, and / or a solubilizer of 0.02 wt%-0.06 wt%. In some preferred embodiments, the buffer solution comprises a 0.09M-0.12M compound of Formula I, an osmotic pressure regulator of 0.5-2 g / L, a pH buffer of 0.05-0.2M, and a solubilizer of 0.02 wt%-0.06 wt%.
[0016] In this invention, the solvent of the buffer solution is water. Specifically, the buffer solution comprises water and components at the following concentrations: 0.09M-0.12M of a compound of formula I, 0.5-2 g / L of an osmotic pressure regulator, 0.05-0.2M of a pH buffer, and 0.02wt%-0.06wt% of a solubilizer. The concentrations of each component refer to their final concentrations in the buffer solution, wherein the final concentration of the compound of formula I is 0.09M-0.12M, the final concentration of the osmotic pressure regulator is 0.5-2 g / L, the final concentration of the pH buffer is 0.05-0.2M, and the final concentration of the solubilizer is 0.02wt%-0.06wt%.
[0017] In some implementations, the buffer solution comprises water and components at concentrations of:
[0018] The compound of formula I is 0.1 M, the osmotic pressure regulator is 0.5 g / L, the pH buffer is 0.1-0.2 M, and the solubilizer is 0.03 wt%-0.05 wt%.
[0019] In some embodiments, the osmotic pressure regulator includes at least one of sucrose, glucose, and sodium citrate; the pH buffer includes at least one pair of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, Tris-hydrochloric acid buffer, and sodium carbonate-sodium bicarbonate buffer; and the solubilizer includes at least one of polysorbate 20, polysorbate 60, and polysorbate 80.
[0020] In some embodiments, the buffer solution further includes a growth factor, which includes at least one of phenylalanine, biotin, riboflavin, and glutamine; the concentration of the growth factor is in the range of 0.1-0.5 g / L, preferably 0.3-0.5 g / L, and in some embodiments it may be 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L, or 0.5 g / L.
[0021] In some specific embodiments, the buffer solution of the present invention comprises water and components of the following concentrations:
[0022] Sucrose 0.5 g / L, sodium carbonate-sodium bicarbonate 0.1 M, biotin 0.2 g / L, 5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid 0.1 M and polysorbate 200.05 wt%;
[0023] Alternatively, sodium citrate 1 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.08 M, 4-methoxy-2-hydroxybenzophenone 0.09 M, polysorbate 60 0.03 wt% and phenylalanine 0.5 g / L;
[0024] Alternatively, glucose 1.5 g / L, disodium hydrogen phosphate (0.1 M), 2,4-dihydroxybenzophenone 0.12 M, and polysorbate 200.03 wt%.
[0025] In this invention, the resplenium is sodium resplenium salt, and in specific embodiments, the resplenium and resplenium indicator used are sodium resplenium salt. Studies have shown that adding the buffer solution of this invention to RPMI-1640 culture medium containing resplenium indicator, or using the buffer solution of this invention and resplenium to prepare a resplenium colorimetric solution, can achieve a color retention rate of over 99.03% within 5 hours of light exposure. This significantly reduces the impact of color changes caused by light exposure on the colorimetric monitoring experiment, indicating that the compound and its buffer solution of this invention can significantly improve the light stability of resplenium in the RPMI-1640 system.
[0026] This invention provides a method for improving the light stability of resplenium in a culture medium, comprising: adding the buffer solution described in this invention to RPMI-1640 culture medium containing resplenium;
[0027] Alternatively, it may include: dissolving resplenium in the buffer solution described in the present invention as a solvent to prepare a resplenium chromogenic solution, and then adding the resplenium chromogenic solution to RPMI-1640 medium.
[0028] In some embodiments, the amount of buffer solution added is 0.9-2.3 mL of the buffer solution described in this invention per liter of RPMI-1640 medium. In some specific embodiments, 0.9 mL, 1 mL, 1.2 mL, 1.5 mL, 1.8 mL, 2 mL, 2.2 mL, or 2.3 mL of the buffer solution described in this invention may be added per liter of RPMI-1640 medium.
[0029] By adding resamaritan and the buffer solution described in this invention using the two methods described above, the final culture system contains 1.4-100 mg of resamaritan (i.e., a final resamaritan concentration of 1.4-100 mg / L) and 0.9-2.3 mL of the buffer solution described in this invention per liter of culture system. In some specific embodiments of this invention, each 500 mL culture system contains 4 mg of resamaritan and 1.1 mL of the buffer solution described in this invention, i.e., each liter of culture system contains 8 mg of resamaritan and 2.2 mL of the buffer solution described in this invention.
[0030] RPMI-1640 culture medium has a complex nutrient composition. When resamaritan indicator is added to RPMI-1640 culture medium, the culture system changes from blue to red under light. However, this reaction system is prone to discoloration due to light exposure, so it must be protected from light. This invention addresses the light instability of resamaritan during use by providing a buffer solution. Adding this buffer solution to RPMI-1640 culture medium containing resamaritan indicator significantly improves the light stability of the culture system, maintaining its color for more than 5 hours under light (color retention rate reaches 99.03%). Attached Figure Description
[0031] Figures 1-1 to 1-5 The following are the comparison results of the OD values of the buffer solutions of formulations 1-1 to 1-5 in Example 4 and the control group under different light exposure times;
[0032] Figures 2-1 to 2-5 The following are the comparison results of the OD values of the buffer solutions of formulations 2-1 to 2-5 in Example 5 and the control group under different light exposure times;
[0033] Figures 3-1 to 3-4 The results show the comparison of OD values of the buffer solutions of formulations 3-1 to 3-4 in Example 6 and the control group under different light exposure times. Detailed Implementation
[0034] This invention provides a buffer solution and its application in improving the stability of resplenium. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that this invention uses RPMI-1640 medium as an example. In fact, when similar results occur in other mediums containing the active ingredient RPMI-1640, or when RPMI-1640 medium is directly mixed with other cell culture media, it is easy to conceive of implementing this invention. The color change problem in the resplenium colorimetric reaction system addressed by the buffer solution of this invention is due to a decrease in the stability of resplenium in the reaction system within a short period of time, rather than a color change caused by culture factors such as cell metabolism. If resplenium exhibits such color changes in other types of mediums, and the nature of the color change is the same as described above, it is also conceivable that the compounds and buffer solutions described in this invention can be used to improve the light stability of resplenium in the system. All similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.
[0035] The test materials used in this invention are all common commercial products and can be purchased on the market.
[0036] The present invention will be further illustrated below with reference to the embodiments:
[0037] Example 1: Buffer formulation of the present invention
[0038] Formula 1-1: 0.1M 5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid; solvent: water;
[0039] Formulas 1-2: sucrose 0.5 g / L, 5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid 0.1 M, solvent is water;
[0040] Formulas 1-3: sucrose 0.5 g / L, sodium carbonate-sodium bicarbonate 0.1 M, 5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid 0.1 M, solvent is water;
[0041] Formulas 1-4: sucrose 0.5 g / L, sodium carbonate-sodium bicarbonate 0.1 M, biotin 0.2 g / L, 5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid 0.1 M, solvent is water;
[0042] Formulas 1-5: sucrose 0.5 g / L, sodium carbonate-sodium bicarbonate 0.1 M, biotin 0.2 g / L, 5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid 0.1 M and polysorbate 200.05 wt%, solvent is water.
[0043] Example 2: Buffer formulation of the present invention
[0044] Formula 2-1: 0.09M 4-methoxy-2-hydroxybenzophenone, solvent is water;
[0045] Formula 2-2: Sodium citrate 1g / L, 4-methoxy-2-hydroxybenzophenone 0.09M, solvent is water;
[0046] Formulas 2-3: Sodium citrate 1g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.08M, 4-methoxy-2-hydroxybenzophenone 0.09M, solvent is water;
[0047] Formulas 2-4: Sodium citrate 1g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.08M, 4-methoxy-2-hydroxybenzophenone 0.09M, polysorbate 600.03wt%, solvent is water;
[0048] Formulas 2-5: Sodium citrate 1g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.08M, 4-methoxy-2-hydroxybenzophenone 0.09M, polysorbate 600.03wt%, phenylalanine 0.5g / L, solvent is water.
[0049] Example 3: Buffer formulation of the present invention
[0050] Formula 3-1: 0.12M 2,4-dihydroxybenzophenone, solvent is water;
[0051] Formula 3-2: 1.5 g / L glucose, 0.12 M 2,4-dihydroxybenzophenone, solvent is water;
[0052] Formula 3-3: 1.5 g / L glucose, 0.1 M disodium hydrogen phosphate, 0.1 M 2,4-dihydroxybenzophenone, solvent is water;
[0053] Formulas 3-4: 1.5 g / L glucose, 0.1 M disodium hydrogen phosphate, 0.1 M 2,4-dihydroxybenzophenone, 200.03 wt% polysorbate, and water as solvent.
[0054] Example 4
[0055] Take 500 ml of RPMI-1640 culture medium (homemade or commercially available), add 2 mL of rezinex chromogenic reagent (concentration 2 g / L), and then add 1 ml of any of the buffer solutions from Example 1; or prepare a rezinex indicator (rezinex concentration in the buffer solution is 4 g / L) using 1 mL of any of the buffer solutions from Example 1 and solid rezinex sodium salt, and add the prepared rezinex indicator to 500 ml of culture medium. The final result should be that each 500 ml of RPMI-1640 culture medium contains 1 mL of the buffer solution from Example 1. Finally, illuminate the entire system with an LED lamp (not less than 20W), and record the color and rate of change of the culture medium at 0 minutes, 10 minutes, 20 minutes, 40 minutes, and 5 hours of illumination. The color and rate of change are detected by a UV spectrophotometer using the blue OD value of the system. 600 The values are shown in Table 1. The color retention rate was calculated. The control group included 500 ml of RPMI-1640 culture medium (homemade or purchased) with 2 mL of resplenium chromogenic agent (the concentration of resplenium in the resplenium chromogenic agent is 2 g / L), without adding the buffer of the present invention.
[0056] The experimental group was given the buffer solution from Example 1 of this invention. Figures 1-1 to 1-5 The OD values of the control group and experimental group were measured under different light exposure times in Tables 1-1 to 1-5.
[0057] Table 1-1 Detection results of the control group and experimental group (Formula 1-1) under different light exposure times.
[0058]
[0059] Table 1-2 Detection results of control group and experimental group (formula 1-2) under different light exposure times
[0060]
[0061] Table 1-3 Detection results of control group and experimental group (formulas 1-3) under different light exposure times.
[0062]
[0063] Table 1-4 Detection results of control group and experimental group (formulas 1-4) under different light exposure times
[0064]
[0065] Table 1-5 Detection results of control group and experimental group (formulas 1-5) under different light exposure times.
[0066]
[0067] The results showed that the reaction system of *Razorazor* with RPMI-1640 culture medium was blue. The experimental group did not change color after 10 minutes of light exposure (laboratory LED light). The color retention rate was calculated using the formula: OD = (N / 10 / 20 / 30 / 40 / 300) minutes of light exposure. 600 Value / Light intensity 0 minutes OD 600 Value * 100%. The color retention rate was 98.54%, while the control group without buffer turned purplish-red, with a color retention rate of 61.40%. After 20 minutes of light exposure, the experimental group remained unchanged, with a color retention rate of 98.30%, while the control group without buffer turned red, with a color retention rate of 48.90%. After 30 minutes of light exposure, the experimental group remained unchanged, with a color retention rate of 98.18%, while the group without buffer turned red, with a color retention rate of 31.13%. After 40 minutes of light exposure, the system with buffer remained unchanged, maintaining a blue color, with a color retention rate of 98.66%, while the system without buffer turned red and became brighter, with a color retention rate of 22.18%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color for up to 5 hours without turning red, with a color retention rate of 99.03%. The reaction system without buffer turned red after 5 hours of light exposure, with a color retention rate decreasing to 17.89%.
[0068] The reaction system without the buffer solution of the present invention changed color after 10 minutes of light exposure. The reaction system with the buffer solution of the present invention remained unchanged at 5 hours after light exposure, and the photostability time was increased to 30 times the original time, effectively extending the photostability of the reaction system.
[0069] Example 5
[0070] Take 500 ml of RPMI-1640 culture medium (homemade or commercially available), add 2 mL of rezinex chromogenic reagent (concentration 2 g / L), and then add 1 ml of any of the buffer solutions from Example 2; or prepare rezinex indicator (concentration 4 g / L) using any of the buffer solutions from Example 2, and add 1 mL of the rezinex indicator to the culture medium. The final concentration should be 1 mL of buffer solution from Example 2 per 500 ml of RPMI-1640 culture medium. Illuminate the entire system with an LED lamp, and record the color of the culture medium at 0 minutes, 10 minutes, 20 minutes, 40 minutes, and 5 hours of illumination. Detect the blue OD value using a UV spectrophotometer. 600 The color retention rate was calculated, and the results are shown in Table 2. The control group did not add the buffer solution of the present invention, while the experimental groups were each added with one of the buffer solutions of Example 2 of the present invention. Figures 2-1 to 2-5 The OD values of the control group and experimental group were measured under different light exposure times in Tables 2-1 to 2-5.
[0071] Table 2-1 Detection results of the control group and experimental group (Formula 2-1) under different light exposure times.
[0072]
[0073] Table 2-2 Detection results of control group and experimental group (formula 2-2) under different light exposure times
[0074]
[0075] Table 2-3 Detection results of control group and experimental group (formula 2-3) under different light exposure times.
[0076]
[0077] Table 2-4 Detection results of control group and experimental group (formula 2-4) under different light exposure times
[0078]
[0079] Table 2-5 Detection results of control group and experimental group (formula 2-5) under different light exposure times
[0080]
[0081] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 99.40%, while the control group without buffer turned purplish-red, with a color retention rate of 66.71%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.88%, while the control group without buffer turned red, with a color retention rate of 39.04%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 98.32%, while the control group without buffer turned red, with a color retention rate of 18.92%. After 40 minutes, the system with buffer remained blue, with a color retention rate of 99.64%, while the system without buffer turned red and became brighter, with a color retention rate of 16.41%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.16%, while the system without buffer turned red, and the color retention rate decreased to 13.89%.
[0082] Example 6
[0083] Take 500 ml of RPMI-1640 culture medium (homemade or commercially available), add 2 mL of rezinex chromogenic reagent (rezinex concentration 2 g / L), and then add 1 ml of any of the buffer solutions from Example 3; or prepare a rezinex indicator using any of the buffer solutions from Example 3 (rezinex concentration 4 g / L), and add 1 mL of the rezinex indicator to the culture medium. The final concentration should be 1 mL of the buffer solution from Example 3 per 500 ml of RPMI-1640 culture medium. Illuminate the entire system with an LED lamp, and record the color of the culture medium at 0, 10, 20, 40, and 5 hours of illumination. Detect the blue OD value of the system using a UV spectrophotometer. 600 The color retention rate was calculated, and the results are shown in Tables 3-1 to 3-4. The control group did not add the buffer solution of the present invention, while the experimental groups were each added with the buffer solution of any one of the embodiments of Example 3 of the present invention. Figures 3-1 to 3-4 The OD values of the control group and experimental group were measured under different light exposure times in Tables 3-1 to 3-4.
[0084] Table 3-1 Detection results of the control group and experimental group (formula 3-1) under different light exposure times.
[0085]
[0086] Table 3-2 Detection results of control group and experimental group (formula 3-2) under different light exposure times
[0087]
[0088] Table 3-3 Detection results of control group and experimental group (formula 3-3) under different light exposure times.
[0089]
[0090] Table 3-4 Detection results of control group and experimental group (formula 3-4) under different light exposure times
[0091]
[0092] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 99.87%, while the control group without buffer turned purplish-red, with a color retention rate of 64.10%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.50%, while the control group without buffer turned red, with a color retention rate of 49.57%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.12%, while the control group without buffer turned red, with a color retention rate of 37.64%. After 40 minutes, the system with buffer remained blue, with a color retention rate of 99.62%, while the system without buffer turned red and became brighter, with a color retention rate of 26.34%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.50%, while the system without buffer turned red, reducing the color retention rate to 18.14%.
[0093] The above results show that the reaction system with the buffer solution of the present invention remains red for 5 hours after the photoreaction time is extended from 10 minutes to 5 hours, and the photostability time is increased to 30 times the original time, effectively extending the photostability of the reaction system.
[0094] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. Application of compounds with the structure of Formula I in improving the photostability of resazurin indicators. In Formula I, R1 is selected from H or -COCH2(OCH2CH2). n OCH3, in which n is any integer between 4 and 12; R2 is selected from H, -CH3, or -CH2CHOHCH2(OCH2CH2). m OCH3, where m is any integer between 4 and 12; R3 is selected from H or -SO3H.
2. The application according to claim 1, characterized in that, The compound of Formula I is selected from one or more of the following: 2-hydroxy-4-monomethyl ether polyoxyethylene 6-ether glycidyl ether-benzophenone, 2-hydroxy-4-monomethyl ether polyoxyethylene 10-ether glycidyl ether-benzophenone, 2-monomethyl ether polyoxyethylene 5-ether acetate-4-hydroxy-benzophenone, 2-monomethyl ether polyoxyethylene 12-ether acetate-4-methyl ether-benzophenone, 5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid, 4-methoxy-2-hydroxybenzophenone, and 2,4-dihydroxybenzophenone.
3. A buffer solution for improving the stability of rezuril, characterized in that, Compounds including those with a 0.09M-0.12M structure of formula I; In Formula I: R1 is selected from H or -COCH2(OCH2CH2) n OCH3, where n is any integer between 4 and 12; R2 is selected from H, -CH3, or -CH2CHOHCH2(OCH2CH2). m OCH3, where m is any integer between 4 and 12; R3 is selected from H or -SO3H.
4. The buffer solution according to claim 3, characterized in that, The compound of Formula I is selected from one or more of the following: 2-hydroxy-4-monomethyl ether polyoxyethylene 6-ether glycidyl ether-benzophenone, 2-hydroxy-4-monomethyl ether polyoxyethylene 10-ether glycidyl ether-benzophenone, 2-monomethyl ether polyoxyethylene 5-ether acetate-4-hydroxy-benzophenone, 2-monomethyl ether polyoxyethylene 12-ether acetate-4-methyl ether-benzophenone, 5-benzoyl-4-hydroxy-2-methoxybenzenesulfonic acid, 4-methoxy-2-hydroxybenzophenone, or 2,4-dihydroxybenzophenone.
5. The buffer solution according to claim 3, characterized in that, It also includes water and at least one of the following components: Osmotic pressure regulators and / or pH buffers and / or solubilizers.
6. The buffer solution according to claim 3, characterized in that, It also includes water and components at the following concentrations: Osmotic pressure regulator 0.5-2 g / L, and / or pH buffer 0.05-0.2M, and / or Solvent promoter: 0.02wt%-0.06wt%.
7. The buffer solution according to claim 5 or 6, characterized in that, The osmotic pressure regulator includes at least one of sucrose, glucose, and sodium citrate; the pH buffer includes at least one pair of disodium hydrogen phosphate – sodium dihydrogen phosphate, disodium hydrogen phosphate – potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate – sodium hydroxide buffer, Tris – hydrochloric acid buffer, and sodium carbonate – sodium bicarbonate buffer; the solubilizer includes at least one of polysorbate 20, polysorbate 60, and polysorbate 80.
8. The buffer solution according to any one of claims 3 to 7, characterized in that, It also includes growth factors; the growth factors include at least one of phenylalanine, biotin, riboflavin and glutamine; the concentration of the growth factors is 0.1-0.5 g / L, preferably 0.3-0.5 g / L.
9. A method for improving the stability of azurlan indicator in a culture medium, characterized in that, include: Add the buffer solution according to any one of claims 3 to 8 to RPMI-1640 medium containing rezinosa indicator; Alternatively, it may include: mixing the buffer solution according to any one of claims 3 to 8 with the resazu indicator to prepare a resazu colorimetric solution, and then adding the resazu colorimetric solution to RPMI-1640 medium.
10. The method according to claim 9, characterized in that, The buffer solution is added at a rate of 0.9-2.3 mL per liter of RPMI-1640 medium, according to any one of claims 3 to 8.