Water-based fluorescent color paste for carbon-based biological experiment and preparation method of water-based fluorescent color paste

Abstract

The invention relates to water-based fluorescent color paste for a carbon-based biological experiment and a preparation method of the water-based fluorescent color paste. According to the water-based fluorescent color paste and the preparation method thereof, the technical problem that an existing water-based color paste lacks applicability, stability, biocompatibility and safety is solved, quality control and performance test are carried out on a finished product through the operation procedures of mixing, stirring, high-temperature sterilization, high-speed stirring, high-pressure homogenization, vacuum degassing, sterile filling and the like, and the complete preparation method is formed. The preparation method is simple, raw materials are easy to obtain, and the method is suitable for large-scale production, has a wide market prospect and can be widely applied to the application fields of a biological experiment technology, a fluorescent material preparation technology, a water-based color paste preparation technology and the like.

Description

Technical Field

[0001] The invention relates to the technical fields of biological experiments, fluorescent material preparation and water-based color paste preparation, and in particular to a water-based fluorescent color paste for carbon-based biological experiments and a preparation method thereof. Background Art

[0002] In the field of biological experimental technology, water-based colorants are a commonly used reagent for labeling and detecting biomolecules. These colorants are usually composed of water-soluble dyes or fluorophores, which can be easily dissolved in water and can bind to various biomolecules such as proteins, nucleic acids and carbohydrates. In addition, the field of fluorescent material preparation technology also involves the use of water-based colorants, because fluorescent materials often need to be dyed or labeled with colorants for easy observation and analysis.

[0003] Existing water-based colorants are mainly composed of water-soluble dyes or fluorophores, which are dispersed in water by physical or chemical methods to form a stable colorant system. This colorant can be used to label and detect biomolecules, thereby realizing the observation and analysis of biological samples. In addition, some researchers have improved the stability and sensitivity of colorants by changing the types of dyes or fluorophores, or adjusting the formula of colorants, thereby improving the efficiency and accuracy of biological experiments.

[0004] However, there are still some problems with the existing water-based colorants in practical applications. First, the existing colorants are often only applicable to specific biomolecules, and may not be able to effectively label and detect other types of biomolecules. Secondly, the existing colorants may have a problem of decreased stability during long-term storage or high temperature environment, which will affect the results of biological experiments. In addition, the existing colorants may cause damage to biological samples during use, affecting the accuracy of experimental results. Repeated excitation of dye molecules or the use of ultra-strong loss light not only damages the dye itself, but is also a fatal blow to living biological samples. During the imaging process, the damage to samples such as living cells and tissues is called phototoxicity. Phototoxicity is a common phenomenon, which mainly comes from the energy transfer between the highly active species in the triplet excited state of the chromophore and the triplet oxygen molecules in the ground state of the environment through collision, thereby generating singlet oxygen and other reactive oxygen species. These reactive oxygen species react with biological macromolecules such as lipids, proteins, and DNA to destroy their normal physiological structure and function. The problem of phototoxicity undoubtedly puts forward a new dimension of requirements for fluorescent dyes. In addition to requiring fluorescent dyes to have anti-bleaching photostability, they are also required to have low phototoxicity to samples to match the biocompatibility of living samples. Therefore, the solution to reduce the phototoxicity of fluorescent dyes is to reduce the components of the triplet excited state of fluorescence. Reducing the lifetime of the triplet state and pulling the triplet state dye back to the ground state as much as possible is the only solution strategy. In order to increase the photostability of dye molecules, deoxygenation is usually used to inhibit the reaction of highly active triplet excited state dye molecules with oxygen. Therefore, how to develop a new type of water-based color paste to solve these problems in the prior art is a problem that needs to be solved urgently in this field. Summary of the invention

[0005] The first purpose of the present invention is to solve the technical problem that the stability of the existing water-based color paste may decrease during long-term storage or in a high-temperature environment, thereby affecting the results of biological experiments.

[0006] The second purpose of the present invention is to solve the technical problem that the existing water-based color paste can only be applied to specific biological molecules and may not be able to effectively mark and detect other types of biological molecules.

[0007] The third purpose of the present invention is to solve the technical problem that the existing water-based color paste may cause damage to biological samples during use, thereby affecting the accuracy of experimental results.

[0008] The first objective of the present invention is achieved through the following technical scheme: developing a new type of water-based fluorescent color paste, which is composed of a variety of water-soluble dyes or fluorophores, has good light stability and thermal stability, and can remain stable under various environmental conditions, thereby ensuring the accuracy of biological experimental results.

[0009] A water-based fluorescent color paste for carbon-based biological experiments. The main raw materials of the water-based fluorescent color paste and their mass fractions are as follows: 3-5 parts of water-soluble dye, 27-45 parts of deionized water, 3-5 parts of moisturizer, and the finished water-based fluorescent color paste is a neutral reagent.

[0010] Preferably, the water-soluble dye comprises: eosin Y, rhodamine B, and fluorescein, and the mass ratio is 1:1:1.

[0011] Furthermore, the eosin Y stain is an acidic water-soluble reagent dye, which dissociates into negatively charged anions in water, and combines with positively charged cations of the amino groups of proteins to stain the cytoplasm. The cytoplasm, red blood cells, muscles, connective tissues, eosinophilic granules, etc. are stained red or pink to varying degrees, forming a sharp contrast with the blue nucleus, and can achieve specific localization of alkaline organelles;

[0012] Furthermore, the Rhodamine B dye is an alkaline water-soluble reagent dye that can achieve specific localization of acidic organelles. Rhodamine B is applied to all living cells, which broadens the application scope of the dye, and its spatial conformation helps to improve the oxidative quenching effect and reduce phototoxicity;

[0013] Furthermore, the fluorescein dye is fluorescent yellow, easily soluble in water, has strong green fluorescence, has a certain cell membrane permeability, and can be used to locate living cells and dead cells in carbon-based biological experiments;

[0014] Furthermore, the finished aqueous fluorescent color paste is a neutral reagent prepared by mixing an aqueous phase liquid of eosin Y, rhodamine B, and fluorescein water-soluble dye in a mass ratio of 1:1:1, which reduces damage to biological samples and improves the safety and reliability of biological experiments.

[0015] Furthermore, the mass ratio of the water-soluble dye eosin Y, rhodamine B and fluorescein is 1:1:1, and the pH of the aqueous phase is adjusted to be neutral to protect the dispersibility and stability of the effective ingredient of the fluorescent dye.

[0016] Preferably, the aqueous fluorescent color paste is prepared by adding 5-15% of glycerol by mass of the dye solution into the water-soluble mixed dye solution as a humectant to slow down the fluorescence quenching of the fluorescent dye in the aqueous fluorescent color paste and improve the stability of the fluorescent dye.

[0017] The second object of the present invention is achieved through the following technical solution: designing a new color paste structure that can combine with various types of biological molecules (such as proteins, nucleic acids and carbohydrates), thereby solving the problem that existing water-based color pastes can only be applied to specific biological molecules.

[0018] Preferably, the aqueous fluorescent color paste comprises: 1 part of rhodamine B, 1 part of fluorescein, 1 part of eosin Y, 27 parts of deionized water, and 3 parts of glycerol, which are obtained by mixing, stirring, high temperature sterilization, high pressure homogenization, degassing, and aseptic filling. The finished aqueous fluorescent color paste is subjected to quality control and performance testing, including performance indicators such as color vividness, brightness, light resistance, weather resistance, and stability, to ensure that it meets the requirements of carbon-based biological experiments. Detailed instructions for use and operating guidelines are provided to help experimenters use and store the color paste correctly to extend its service life and reduce waste.

[0019] The third object of the present invention is achieved through the following technical solutions: optimizing the preparation process of the color paste, by improving the selection, ratio and preparation conditions of raw materials, so that the prepared color paste has better biocompatibility and will not cause damage to biological samples, thereby improving the accuracy of the experimental results.

[0020] A method for preparing an aqueous fluorescent color paste for carbon-based biological experiments, comprising the following specific process steps:

[0021] Step 1: Select three water-soluble dyes, namely rhodamine B, fluorescein and eosin Y, and mix them in a mass ratio of 1:1:1 to obtain a mixed dye;

[0022] Step 2: Add the mixed dye into deionized water and stir evenly to obtain a dye solution with a concentration of 5-15%;

[0023] Step 3: Heat the dye solution at 70-90°C for 1-3 hours for high temperature sterilization;

[0024] Step 4: Cool the sterilized dye solution to room temperature, then add glycerin as a moisturizer in an amount of 5-15% of the dye solution mass;

[0025] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000-1500 rpm for 30-60 minutes to mix them evenly;

[0026] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0027] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0028] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0029] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0030] Preferably, the dye solution is heated at 80° C. for 2 hours for high temperature sterilization to prevent the influence of bacteria and other microorganisms on the dye purity during the dye preparation, sample addition, mixing and stirring process.

[0031] Preferably, the dye solution is cooled to room temperature after sterilization, and then glycerol is added as a humectant in an amount of 10% of the mass of the dye solution.

[0032] Preferably, after the humectant is added to the dye solution, the solution is stirred in a high-speed stirrer at a speed of 1000 rpm for 30 minutes to make the mixture uniform.

[0033] Preferably, the high-speed mixer comprises a base with a tank body fixedly connected to the surface, a power chamber fixedly connected to the top of the tank body, an air flow chamber matched with the power chamber fixedly connected to the top of the tank body located on the right side of the power chamber, a motor fixedly connected to the inside of the power chamber, a stirring shaft is arranged inside the tank body, a plurality of stirring paddles for convenient stirring are fixedly connected to the side wall of the stirring shaft near the lower end, the upper end of the stirring shaft is fixedly connected to the lower end rotating shaft of the motor through a coupling, a fan is fixedly connected in the air flow chamber, a power shaft for rotating the fan is arranged at the upper end of the fan, the upper end rotating shaft of the motor and the power shaft are connected by a transmission belt, and the tank body An air pipe is arranged inside, and the upper end of the air pipe passes through the upper wall of the tank body and is connected with the fan air outlet. An air intake pipe is arranged inside the tank body on the left side of the air pipe, and the upper end of the air intake pipe passes through the upper wall of the tank body and is connected with the fan air inlet. A feed pipe is arranged on the left side of the tank body, and a water injection pipe is arranged near the right end. The feed pipe and the water injection pipe penetrate into the tank body wall and are fixedly connected to the tank body. A discharge pipe is arranged near the lower end of the side of the tank body, and the discharge pipe passes through the tank wall and is fixedly connected to the tank body. The power chamber and the air flow chamber have the same height, and are connected through a transmission hole. A gap is left between the outer wall of the drive belt and the inner wall of the transmission hole.

[0034] The high-speed mixer of the present invention fixes an air flow chamber on the right side of the power chamber, transmits the power of the motor to the fan through a transmission belt, drives the fan to rotate while the motor is working, and blows air into the stirring material through an air pipe through the fan, thereby improving the activity of the stirring material, reducing the resistance of the stirring paddle, thereby reducing the power consumption of the motor, improving the quality of the coating, and improving the stirring efficiency. The high-speed mixer is used in the present invention to deepen the stirring degree and make the dye solution mixed evenly, so as to prevent the solution from stratifying during the stirring process, affecting the uniformity and stability of the dye.

[0035] Preferably, after the dye solution is evenly mixed, it is homogenized in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the relative colorability of the color paste.

[0036] Preferably, the dye solution is put into a vacuum degasser for degassing after homogenization to remove air dissolved in the dye solution, prevent oxidation of the dye, increase the color vividness of the dye, and enhance the stability of the finished dye product.

[0037] Preferably, the finished water-based fluorescent color paste is subjected to quality control and performance testing, including performance indicators such as color vividness, brightness, light resistance, weather resistance, and stability, to ensure that it meets the requirements of carbon-based biological experiments, so as to form guiding operating instructions and suggestions for the staff who produce and use the product.

[0038] In summary, the present invention has the following beneficial effects:

[0039] 1. Wide application: The water-based fluorescent color paste of the present invention can not only effectively mark and detect common biomolecules such as proteins, nucleic acids and carbohydrates, but also be applicable to other types of biomolecules, and has a wider range of applications;

[0040] 2. High stability: The water-based fluorescent color paste of the present invention adopts a special preparation method, so that it can still maintain good stability under long-term storage or high temperature environment, and will not affect the results of biological experiments;

[0041] 3. High safety: The water-based fluorescent color paste of the present invention causes little damage to biological samples during use and will not affect the accuracy of experimental results, thereby improving the safety and reliability of biological experiments;

[0042] 4. High sensitivity: The aqueous fluorescent color paste of the present invention optimizes the types of dyes or fluorophores and adjusts the formula of the color paste to make it have higher sensitivity and can more accurately mark and detect biological molecules, thereby improving the efficiency and accuracy of biological experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a schematic diagram of the structure of a high-speed mixer;

[0044] Figure 2 It is a schematic diagram showing the comparison of product performance of Examples 1-8 and Comparative Examples 1-3.

[0045] In the figure: 1. base, 2. tank body, 3. power chamber, 4. air flow chamber, 5. motor, 6. stirring shaft, 7. stirring paddle, 8. coupling, 9. fan, 10. power shaft, 11. transmission belt, 12. air pipe, 13. air inlet pipe, 14. feed pipe, 15. water injection pipe, 16. discharge pipe, 17. transmission hole. DETAILED DESCRIPTION

[0046] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

[0047] Embodiment 1:

[0048] Step 1: Select three water-soluble dyes, namely, 1 part of eosin Y, 1 part of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0049] Step 2: Add the mixed dye to 27 parts of deionized water and stir evenly;

[0050] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0051] Step 4: Cool the sterilized dye solution to room temperature and then add 3 parts of glycerin as a moisturizer;

[0052] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0053] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0054] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0055] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0056] Step 9: According to GB / T 21473-2008 "Color Pastes for Coloring Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments;

[0057] The high-speed mixer used in step five comprises: a base 1 with a tank body 2 fixedly connected on the surface, a power chamber 3 fixedly connected to the top of the tank body 2, an air flow chamber 4 matched with the power chamber 3 fixedly connected to the top of the tank body 2 located on the right side of the power chamber 3, a motor 5 fixedly connected inside the power chamber 3, a stirring shaft 6 is arranged inside the tank body 2, a plurality of stirring paddles 7 for convenient stirring are fixedly connected to the side wall of the stirring shaft 6 near the lower end, the upper end of the stirring shaft 6 is fixedly connected to the lower end shaft of the motor 5 through a coupling 8, a fan 9 is fixedly connected in the air flow chamber 4, a power shaft 10 for rotating the fan 9 is arranged on the upper end of the fan 9, the upper end shaft of the motor 5 is connected to the power shaft 10 through a transmission belt 11, and an air transmission shaft 10 is arranged inside the tank body 2. Tube 12, the upper end of the air pipe 12 passes through the upper wall of the tank body 2 and is connected to the air outlet of the fan 9, the inside of the tank body 2 is provided with an air intake pipe 13 on the left side of the air intake pipe 12, the upper end of the air intake pipe 13 passes through the upper wall of the tank body 2 and is connected to the air inlet of the fan 9, a feed pipe 14 is provided on the left side of the tank wall of the tank body 2, a water injection pipe 15 is provided on the right side of the tank wall of the tank body 2, the feed pipe 14 and the water injection pipe 15 are fixedly connected to the tank wall of the tank body 2, a discharge pipe 16 is provided at the lower end of the tank wall of the tank body 2, the discharge pipe 16 passes through the tank wall and is fixedly connected to the tank body 2; the power chamber 3 and the air flow chamber 4 have the same height, the power chamber 3 and the air flow chamber 4 are connected through a transmission hole 17, and a gap is left between the outer wall of the transmission belt 11 and the inner wall of the transmission hole 17.

[0058] By fixing an air flow cavity on the right side of the power cavity, the power of the motor is transmitted to the fan through a transmission belt, the fan is driven to rotate while the motor is working, and the air is blown into the mixing material through an air pipe by the fan; the structure of the high-speed mixer is as follows: a tank body with a power cavity is fixedly connected to the top of the base surface, a power cavity with an internal motor and an air flow cavity are fixedly connected to the top of the tank body, a stirring shaft is arranged inside the tank body, and a plurality of stirring paddles for convenient stirring are fixedly connected to the side wall near the lower end, the upper end of the stirring shaft is fixedly connected to the rotating shaft at the lower end of the motor through a coupling, a fan is fixedly connected in the air flow cavity, and the fan A power shaft for rotating the fan is provided at the upper end of the fan, and the upper end shaft of the motor is connected to the power shaft by a transmission belt. The upper end of the air pipe inside the tank body passes through the upper wall of the tank body and is connected to the air outlet of the fan. An air intake pipe is provided on the left side of the air pipe, and the upper end of the air intake pipe passes through the upper wall of the tank body and is connected to the air inlet of the fan. Feed pipes and water injection pipes are provided at both ends of the upper part of the tank surface, which penetrate into the tank wall and are fixedly connected to the tank body. A discharge pipe is provided at the lower end of the tank body, which penetrates into the lower wall of the tank body and is fixedly connected to the tank body. The power chamber and the air flow chamber of the same height are connected through the transmission hole, and a gap is left between the outer wall of the transmission belt and the inner wall of the transmission hole.

[0059] Embodiment 2:

[0060] Step 1: Select three water-soluble dyes, namely, 1 part of eosin Y, 1 part of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0061] Step 2: Add the mixed dye to 36 parts of deionized water and stir evenly;

[0062] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0063] Step 4: Cool the sterilized dye solution to room temperature and then add 3 parts of glycerin as a moisturizer;

[0064] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0065] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0066] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0067] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0068] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0069] Embodiment 3:

[0070] Step 1: Select three water-soluble dyes, namely, 1 part of eosin Y, 1 part of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0071] Step 2: Add the mixed dye to 45 parts of deionized water and stir evenly;

[0072] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0073] Step 4: Cool the sterilized dye solution to room temperature and then add 3 parts of glycerin as a moisturizer;

[0074] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0075] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0076] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0077] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0078] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0079] Embodiment 4:

[0080] Step 1: Select three water-soluble dyes, namely, 1 part of eosin Y, 1 part of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0081] Step 2: Add the mixed dye to 27 parts of deionized water and stir evenly;

[0082] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0083] Step 4: Cool the sterilized dye solution to room temperature and then add 4 parts of glycerin as a moisturizer;

[0084] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0085] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0086] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0087] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0088] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0089] Embodiment 5:

[0090] Step 1: Select three water-soluble dyes, namely, 1 part of eosin Y, 1 part of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0091] Step 2: Add the mixed dye to 27 parts of deionized water and stir evenly;

[0092] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0093] Step 4: Cool the sterilized dye solution to room temperature and then add 5 parts of glycerin as a moisturizer;

[0094] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0095] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0096] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0097] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0098] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0099] Embodiment 6:

[0100] Step 1: Select three water-soluble dyes, namely, 2 parts of eosin Y, 1 part of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0101] Step 2: Add the mixed dye to 27 parts of deionized water and stir evenly;

[0102] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0103] Step 4: Cool the sterilized dye solution to room temperature and then add 3 parts of glycerin as a moisturizer;

[0104] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0105] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0106] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0107] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0108] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0109] Embodiment 7:

[0110] Step 1: Select three water-soluble dyes, namely, 1 part of eosin Y, 2 parts of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0111] Step 2: Add the mixed dye to 27 parts of deionized water and stir evenly;

[0112] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0113] Step 4: Cool the sterilized dye solution to room temperature and then add 3 parts of glycerin as a moisturizer;

[0114] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0115] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0116] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0117] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0118] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0119] Embodiment 8:

[0120] Step 1: Select three water-soluble dyes, namely, 2 parts of eosin Y, 2 parts of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0121] Step 2: Add the mixed dye to 27 parts of deionized water and stir evenly;

[0122] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0123] Step 4: Cool the sterilized dye solution to room temperature and then add 3 parts of glycerin as a moisturizer;

[0124] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0125] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0126] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0127] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0128] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0129] Comparative Example 1:

[0130] Step 1: Select three water-soluble dyes, namely, 1 part of eosin Y, 1 part of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0131] Step 2: Add the mixed dye to 27 parts of deionized water and stir evenly;

[0132] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0133] Step 4: Cool the sterilized dye solution to room temperature without adding moisturizer;

[0134] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0135] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0136] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0137] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0138] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0139] Comparative Example 2:

[0140] Step 1: Select three water-soluble dyes, namely, 1 part of eosin Y, 1 part of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0141] Step 2: Add the mixed dye to 27 parts of deionized water and stir evenly;

[0142] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0143] Step 4: Cool the sterilized dye solution to room temperature and then add 3 parts of glycerin as a moisturizer;

[0144] Step 5: Stir the dye solution with moisturizer in a blender at low speed for 30 minutes to make it evenly mixed;

[0145] Step 6: homogenize the mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste;

[0146] Step 7: Degas the homogenized dye solution in a vacuum degasser to remove air dissolved in the dye solution and prevent oxidation of the dye;

[0147] Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0148] Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0149] Comparative Example 3:

[0150] Step 1: Select three water-soluble dyes, namely, 1 part of eosin Y, 1 part of rhodamine B, and 1 part of fluorescein, and mix them to obtain a mixed dye;

[0151] Step 2: Add the mixed dye to 27 parts of deionized water and stir evenly;

[0152] Step 3: Heat the dye solution at 80°C for 2 hours for high temperature sterilization;

[0153] Step 4: Cool the sterilized dye solution to room temperature and then add 3 parts of glycerin as a moisturizer;

[0154] Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000 rpm for 30 minutes to mix it evenly;

[0155] Step 6: Degas the homogenized dye solution in a vacuum degasser to remove the air dissolved in the dye solution and prevent the dye from oxidizing;

[0156] Step 7: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste;

[0157] Step 8: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

[0158] From the data results of the product performance comparison table of Examples 1-8 and Comparative Examples 1-3, it can be seen that the hue difference, chroma difference and lightness difference of Examples 1-8 of the present invention are smaller than those of Comparative Examples 1-3, indicating that the product of the present invention has better color fastness and better coloring stability; the relative tinting strength and moisture retention of each product in Examples 1-8 are greater than those of Comparative Examples 1-3, indicating that the present product has high tinting strength and moisture retention, and has greater storage advantages; the fluorescent reflectivity of each product in Examples 1-8 is greater than that of Comparative Examples 1-3, indicating that the present product is easier to mark in carbon-based biological experiments; the viscosity of each product in Examples 1-8 is uniform, the values ​​are close, and they are greater than those of Comparative Examples 1-3, proving that the product provided by the present invention has better stability and reproducibility; the weather resistance, acid resistance and alkali resistance of the present product have significant advantages.

[0159] Due to the advanced nature of the technical solution, it can be widely used in application fields such as biological experiment technology, fluorescent material preparation technology, and water-based color paste preparation technology. First of all, the water-based fluorescent color paste of the present invention can effectively solve the problem that the color paste in the prior art can only be applied to specific biological molecules, improves the versatility of the color paste, and is conducive to promotion and application. Secondly, the color paste of the present invention has good stability, and there will be no problem of decreased stability even in long-term storage or high temperature environment. This has important practical value for research institutions that need to preserve biological samples for a long time. In addition, the color paste of the present invention causes little damage to biological samples during use, which is conducive to improving the accuracy of experimental results and has an important role in promoting the development of the field of biological experiment technology. Finally, the preparation method of the color paste of the present invention is simple, the raw materials are easily available, it is suitable for large-scale production, and has broad market prospects.

Claims

1. A water-based fluorescent color paste for carbon-based biological experiments, characterized in that: The main raw materials of the water-based fluorescent color paste and their mass fractions are as follows: 3-5 parts of water-soluble dyes, 27-45 parts of deionized water, and 3-5 parts of moisturizers; the water-soluble dyes include: eosin Y, rhodamine B and fluorescein; the finished water-based fluorescent color paste is a neutral reagent.

2. The water-based fluorescent color paste according to claim 1, characterized in that: The water-soluble dyes include: eosin Y, rhodamine B, and fluorescein, and the mass ratio is (1-2):(1-2):1; The eosin Y stain is an acidic water-soluble reagent dye, which dissociates into negatively charged anions in water, combines with positively charged cations of the amino groups of proteins to stain the cytoplasm, and achieves specific localization of alkaline organelles by staining the cytoplasm, red blood cells, muscles, connective tissue, eosinophilic granules, etc. red, and the cell nucleus blue; The Rhodamine B dye is an alkaline water-soluble reagent dye used for the specific localization of acidic organelles. Rhodamine B is applied to all living cells, which broadens the scope of application of the dye. The spatial conformation of Rhodamine is used to improve the oxidative quenching effect and reduce phototoxicity. The fluorescein dye is fluorescent yellow, easily soluble in water, and has strong green fluorescence. It is used to locate living cells and dead cells in carbon-based biological experiments based on certain characteristics of cell membrane permeability.

3. The water-based fluorescent color paste according to claim 1, characterized in that: The amount of the moisturizing agent added is 5-15% of the mass of the dye solution, and the moisturizing agent used is glycerol.

4. A method for preparing a water-based fluorescent color paste for carbon-based biological experiments, used to prepare the water-based fluorescent color paste according to any one of claims 1 to 3, characterized in that: The specific process steps of the preparation method are: Step 1: Select three water-soluble dyes, namely, eosin Y, rhodamine B, and fluorescein, and mix them in a mass ratio of (1-2):(1-2):1 to obtain a mixed dye; Step 2: Add the mixed dye into deionized water and stir evenly to obtain a dye solution with a concentration of 5-15%; Step 3: Heat the dye solution at 70-90°C for 1-3 hours for high temperature sterilization; Step 4: Cool the sterilized dye solution to room temperature, then add glycerin as a moisturizer in an amount of 5-15% of the dye solution mass; Step 5: Stir the dye solution with the moisturizer in a high-speed mixer at a speed of 1000-1500 rpm for 30-60 minutes to mix them evenly; Step 6: homogenizing the uniformly mixed dye solution in a high-pressure homogenizer at a pressure of 650-800 MPa; Step 7: Degas the homogenized dye solution in a vacuum degasser; Step 8: Filling the degassed dye solution in a sterile environment to obtain a finished water-based fluorescent color paste; Step 9: According to GB / T 21473-2008 "Color Pastes for Color Adjustment Systems", the finished water-based fluorescent color paste is subjected to quality control and performance testing (the specific indicators are carried out according to the methods in the appendix of the above standard), including color vividness, brightness, light resistance, weather resistance, stability and other performance indicators, to ensure that it meets the requirements of carbon-based biological experiments.

5. The method for preparing a water-based fluorescent color paste for carbon-based biological experiments according to claim 4, characterized in that: The dye solution is heated at 80° C. for 2 hours for high temperature sterilization to prevent the influence of bacteria and other microorganisms on the purity of the dye during the dye preparation, sample addition, mixing and stirring process.

6. The method for preparing a water-based fluorescent color paste for carbon-based biological experiments according to claim 4, characterized in that: After the humectant is added to the dye solution, the solution is stirred in a high-speed stirrer at a speed of 1000 rpm for 30 minutes to make the mixture uniform.

7. The high-speed mixer according to claim 4 or 6, characterized in that: include: The surface of the tank body (2) is fixedly connected to a base (1), the top of the tank body (2) is fixedly connected to a power chamber (3), the top of the tank body (2) is located on the right side of the power chamber (3) and is fixedly connected to an air flow chamber (4) matched with the power chamber (3), the power chamber (3) is fixedly connected to a motor (5), a stirring shaft (6) is arranged inside the tank body (2), a plurality of stirring paddles (7) for convenient stirring are fixedly connected near the lower end of the side wall of the stirring shaft (6), the upper end of the stirring shaft (6) is fixedly connected to the lower end of the motor (5) through a coupling (8), a fan (9) is fixedly connected inside the air flow chamber (4), a power shaft (10) for rotating the fan (9) is arranged at the upper end of the fan (9), the upper end of the motor (5) and the power shaft (10) are connected through a transmission belt (11), an air supply pipe (12) is arranged inside the tank body (2), and the air supply pipe (12) is fixedly connected to the lower end of the side wall of the stirring shaft (6). 2) the upper end of which passes through the upper wall of the tank body (2) and is connected to the air outlet of the fan (9); an air inlet pipe (13) is arranged inside the tank body (2) on the left side of the air delivery pipe (12); the upper end of the air inlet pipe (13) passes through the upper wall of the tank body (2) and is connected to the air inlet of the fan (9); a feed pipe (14) is arranged on the left side of the tank wall of the tank body (2); a water injection pipe (15) is arranged on the right end of the tank wall of the tank body (2); the feed pipe (14) and the water injection pipe (15) pass through the tank body (2) the tank wall is fixedly connected to the tank body (2), a discharge pipe (16) is provided at the lower end of the tank wall of the tank body (2), and the discharge pipe (16) passes through the tank wall of the tank body (2) and is fixedly connected to the tank body (2); the power chamber (3) and the air flow chamber (4) are at the same height, the power chamber (3) and the air flow chamber (4) are connected via a transmission hole (17), and a gap is left between the outer wall of the transmission belt (11) and the inner wall of the transmission hole (17).

8. The method for preparing a water-based fluorescent color paste for carbon-based biological experiments according to claim 4, characterized in that: After the dye solution is evenly mixed, it is homogenized in a high-pressure homogenizer at a pressure of 650-800 MPa to make the size of the dye particles more uniform and improve the stability of the color paste.

9. The method for preparing a water-based fluorescent color paste for carbon-based biological experiments according to claim 4, characterized in that: The dye solution is put into a vacuum degasser for degassing after homogenization.

10. The method for preparing a water-based fluorescent color paste for carbon-based biological experiments according to claim 1 or 2, characterized in that: The water-soluble dyes include: eosin Y, rhodamine B, and fluorescein, and the mass ratio is 1:1:1.

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