Preparation method of agarose magnetic microspheres with uniform particle size

Abstract

The invention relates to the technical field of biological materials, in particular to a preparation method of uniform-particle-size agarose magnetic microspheres, which comprises the following steps: S1) mixing agarose, a surfactant, a magnetic core, inorganic salt and water, and stirring to form a homogeneous mixture; s2) under a stirring condition, heating the mixture to a certain temperature, keeping the temperature for a period of time, and stopping heating; and S3) after stopping heating, cooling to a certain temperature, and cooling with cold water for a period of time to obtain the agarose magnetic microspheres. A one-pot preparation method is adopted, a homogeneous system is formed by a water phase and an emulsion phase under a high-temperature condition, two phases are slowly formed along with the reduction of the temperature, an interface effect is generated, the microspheres are finally formed, and the whole process is performed in one reaction kettle, so that the particle size of the prepared magnetic agarose microspheres is more uniform.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a method for preparing agarose magnetic microspheres with uniform particle size. Background Art

[0002] Agarose magnetic microspheres are an important carrier tool, which are widely used in medical and molecular biology research. They have the characteristics of superparamagnetism, rapid magnetic responsiveness, rich hydroxyl functional groups and relatively concentrated particle size. The particle size distribution of magnetic agarose microspheres is about 30~100 μm, which is moderate and more suitable for the needs of biological inspection and purification experiments. Magnetic agarose microspheres have superparamagnetism, which enables them to be quickly separated from the suspension under a magnetic field, significantly accelerating the reliability of the operation and improving the reproducibility of the experiment. The surface of agarose is densely covered with hydroxyl groups, which allows them to be specially modified by cross-linking reagents to become agarose magnetic beads with special functions for coupling or separation and purification of different substances. The surface of agarose has the remarkable characteristic of low non-specific adsorption, which is particularly important in affinity chromatography. 6% agarose cross-linked microspheres have a smaller pore size, which can avoid non-specific adsorption caused by protein entering the pores to a certain extent. Agarose has excellent biocompatibility and is protein-friendly. Agarose has a porous structure that allows macromolecules such as proteins to pass quickly. Agarose has physical and chemical stability and a certain mechanical strength. Containing superparamagnetic particles, agarose microspheres have rapid magnetic responsiveness, which can greatly save operation time and facilitate automated operation. Therefore, it is an important carrier tool in medical and molecular biology research, and has a wide range of applications in cell separation, protein purification, nucleic acid separation and other fields.

[0003] Existing agarose magnetic microspheres are mainly prepared by an emulsification solidification method in which an emulsifier is added to an aqueous phase and an organic phase. The main steps are to heat and dissolve agarose and nano-magnetic cores to form an aqueous phase, then add them to an organic solvent containing an emulsifier, and after sufficient emulsification by mechanical stirring or ultrasound, cool and solidify to obtain agarose magnetic microspheres. The agarose magnetic microspheres prepared by the above method have a wide particle size distribution, and must be sieved to obtain the target particle size product, and a large amount of organic solvents and emulsifiers are required, wherein the recovery of the organic solvent and the repeated washing of the emulsifier both significantly increase the production cost. Therefore, it is necessary to develop a new preparation method to prepare agarose magnetic microspheres with a narrow particle size distribution, so as to overcome the shortcomings of the traditional preparation method and the application defects caused thereby. Summary of the invention

[0004] In view of this, the present invention provides a method for preparing agarose magnetic microspheres with uniform particle size. The "one-pot" preparation method is adopted, and a homogeneous system is formed between the water phase and the oil phase under heating conditions. As the temperature decreases, the two phases are gradually formed, and an interfacial effect is generated, finally forming microspheres. The whole process is carried out in a reaction kettle, so that the particle size of the prepared magnetic agarose microspheres is more uniform.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides a method for preparing agarose magnetic microspheres with uniform particle size, including the following steps: S1) Mix agarose, surfactant, magnetic core, inorganic salt and water to form a homogeneous mixture under stirring; S2) Under stirring conditions, heat the homogeneous mixture to a certain temperature, keep it warm for a period of time and then stop heating; S3) After stopping heating, cool it to a certain temperature, and then use cold water to cool it for a period of time to obtain agarose magnetic microspheres.

[0006] In S1, the mass ratio of agarose to water is (1~6):100.

[0007] In step S1, the surfactant is polyethylene glycol, and the average molecular weight of polyethylene glycol is 6000~75000Da;

[0008] The mass ratio of the surfactant to water is (10~30):100.

[0009] In S1, the magnetic core is magnetic Fe3O4, nano Fe or Fe3O4@SiO2;

[0010] The mass ratio of the magnetic core to water is (0.1~3):100.

[0011] In S1, the inorganic salt is one or more of sodium chloride, potassium chloride, and magnesium chloride;

[0012] The mass ratio of the inorganic salt to water is (2~7.5):100.

[0013] In S2, the heating temperature is 80~100°C.

[0014] In S2, the heat preservation time is 15~120 min.

[0015] In S3, after stopping heating, the temperature drops to the range of 70~80°C.

[0016] In S3, the temperature of the cold water for cooling is 0~20°C.

[0017] The cooling time is 30~60 min

[0018] The rotation speed of the stirring is 200~5000 rpm.

[0019] The present invention has the following beneficial effects:

[0020] The present invention mainly uses a one-pot method to obtain agarose magnetic microspheres after heating, cooling and solidification. Compared with the two-phase method reported in the literature, the experimental operation is greatly simplified, which is convenient for scale-up and industrial production. Moreover, the macromolecular polyethylene glycol surfactant used is easy to remove after preparing the microspheres and can be recycled, reducing the raw material cost to a certain extent. Specifically, its advantages are reflected in the following aspects:

[0021] 1. One-pot emulsification. Compared with the reported preparation methods, the present invention has the characteristics of simple operation and good repeatability, and tries to avoid the human error that may be caused by operation during the experiment. The whole preparation process does not involve the inflow of materials and the transfer of the reaction kettle. From heating and raising the temperature of the reaction kettle to finally solidifying into spheres, it is carried out in one reaction device.

[0022] 2. The state of the prepared microspheres is uniform. Compared with the two-phase method, the water and oil phases form a homogeneous system under high temperature conditions. As the temperature decreases, the two phases are slowly formed, generating an interfacial effect, so that the particle size of the prepared magnetic agarose microspheres is more uniform. While the particle size distribution of the agarose magnetic microspheres prepared by the two-phase method is relatively wide, and the target particle size product can only be obtained through screening.

[0023] 3. The post-treatment process is simple and not easy to cause pollution. The present invention uses polyethylene glycol, which can be easily removed by magnetic attraction after the preparation of microspheres due to its special properties and can be recycled, reducing the production cost to a certain extent. In the two-phase method, a large amount of organic solvents and emulsifiers are required, and the recovery of organic solvents and the repeated washing of emulsifiers both greatly increase the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the description of the embodiments or the prior art.

[0025] Figure 1 Micrograph of the agarose magnetic microspheres prepared in Example 1;

[0026] Figure 2 Micrograph of the agarose magnetic microspheres prepared in Example 2;

[0027] Figure 3 Micrograph of the agarose magnetic microspheres prepared in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention discloses a method for preparing agarose magnetic microspheres with uniform particle size. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The method and application of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0029] A method for preparing agarose magnetic microspheres includes the following steps: S1) Mix agarose, surfactant, magnetic core, inorganic salt, and water to form a homogeneous mixture under stirring; S2) Under stirring conditions, heat the mixture to a certain temperature, keep it warm for a period of time, and then stop heating; S3) After stopping heating, cool it to a certain temperature and then use cold water to cool it for a period of time to obtain agarose magnetic microspheres.

[0030] In some specific embodiments, in the mixture of the above preparation method, the mass ratio of agarose to water is (1 - 6):100; optionally, the mass ratio of agarose to water is 1:100, 2:100, 3:100, 4:100, 5:100, 6:100, or the range between any two of the above ratios.

[0031] In some specific embodiments, in the mixture of the above preparation method, the surfactant is polyethylene glycol, and the average molecular weight of polyethylene glycol is preferably 6000 - 75000 Da. Optionally, the average molecular weight of polyethylene glycol is 6000 Da, 20000 Da, 75000 Da, or the range between any two of the above values; the mass ratio of the surfactant to water is (10 - 30):100; optionally, the mass ratio of the surfactant to water is 10:100, 20:100, 30:100, or the range between any two of the above ratios.

[0032] In some specific embodiments, in the mixture of the above preparation method, the magnetic core is magnetic Fe3O4, nano Fe, or Fe3O4@SiO2; the mass ratio of the magnetic core to water is (0.1 - 3):100. Optionally, the mass ratio of the magnetic core to water is 0.1:100, 0.25:100, 1:100, 2:100, 3:100, or the range between any two of the above ratios.

[0033] In some specific embodiments, the inorganic salt in the mixture of the above preparation method is one or more of sodium chloride, potassium chloride, and magnesium chloride; the mass ratio of the inorganic salt to water is (2 to 7.5):100. Optionally, the mass ratio of the inorganic salt to water is 2:100, 3:100, 4:100, 5:100, 6:100, 7:100, 7.5:100, or the range between any two of the above ratios.

[0034] In some specific embodiments, in S2 of the above preparation method, the heating temperature is preferably 80 to 100 °C, more preferably 85 to 95 °C, and still more preferably 90 °C.

[0035] In some specific embodiments, the heat preservation time of the above preparation method is preferably 15 to 120 min, more preferably 30 to 60 min, and still more preferably 40 min.

[0036] In some specific embodiments, in the above preparation method, after stopping heating, the temperature drop range is preferably 60 to 80 °C; more preferably 65 to 75 °C, and still more preferably 70 °C.

[0037] In some specific embodiments, the rotation speed of the stirring in the above preparation method is preferably 200 to 5000 rpm; more preferably 500 to 2000 rpm, and still more preferably 1000 to 2000 rpm.

[0038] In some specific embodiments, the temperature of the cold water in the above preparation method is preferably 0 to 20 °C; more preferably 5 to 15 °C, and still more preferably 10 °C.

[0039] In some specific embodiments, the cooling time of the above preparation method is preferably 30 to 60 min; more preferably 40 to 50 min, and still more preferably 45 min.

[0040] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0041] The terms "comprising", "having", or "containing", including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0042] It should be understood that as long as the present application is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

[0043] The use of any and all examples or exemplary language such as "for example" or "including" in this document is merely intended to better illustrate the present application and does not limit the scope of the present application. Any language in this specification should not be construed as indicating that any unclaimed element is essential for the practice of the present application.

[0044] In addition, the numerical ranges and parameters used to define the present application are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0045] Unless otherwise specified, the raw materials, reagents, consumables, and instruments involved in the present invention are all ordinary commercially available products and can be purchased from the market.

[0046] The present invention will be further described below in conjunction with embodiments.

[0047] Example 1

[0048] Weigh 1 g of agarose, 30 g of PEG (6000 Da), 0.1 g of Fe3O4, and 2 g of NaCl into a 500 mL three-neck glass flask, add 100 mL of purified water, adjust the mechanical stirring speed to 200 rpm and stir evenly, set the temperature to 80 °C, and start heating. After the temperature rises to 80 °C, keep it at 80 °C for 15 min, turn off the heating. When the temperature of the water bath reaches 60 °C, remove the hot water and replace it with 0 °C cold water for cooling. Keep stable stirring for 30 min during the whole cooling process. After the cooling time ends, use a ring magnet to magnetically attract the encapsulated microspheres, wash them 5 times with purified water, and store them in 20% ethanol. Observe the morphology of the prepared agarose magnetic microspheres through an optical microscope, and measure the microsphere size with a measuring ruler. Most of them are within 60 μm ± 5 μm, and the particle size is relatively uniform, as Figure 1 shown.

[0049] Example 2

[0050] Weigh 2 g of agarose, 20 g of PEG (20000 Da), 2 g of Fe3O4@SiO2, and 5 g of KCl into a 500 mL three-necked glass flask. Add 100 mL of purified water, adjust the mechanical stirring speed to 2000 rpm and stir evenly. Set the temperature to 90 °C and start heating. After the temperature rises to 90 °C, maintain it at 90 °C for 60 min. Turn off the heating. When the temperature of the water bath drops to 70 °C, remove the hot water and replace it with 10 °C cold water for cooling. Keep stirring steadily for 60 min during the whole cooling process. After the cooling time ends, use a ring magnet to magnetically attract the encapsulated microspheres, wash them 5 times with purified water, and store them in 20% ethanol. Observe the morphology of the prepared agarose magnetic microspheres through an optical microscope, and measure the microsphere size with a ruler. Most of them are within 45 μm ± 5 μm, and the particle size is relatively uniform, as Figure 2 shown.

[0051] Example 3

[0052] Weigh 6 g of agarose, 10 g of PEG (75000 Da), 3 g of nano-Fe, and 7.5 g of MgCl into a 500 mL three-necked glass flask. Add 100 mL of purified water, adjust the mechanical stirring speed to 5000 rpm and stir evenly. Set the temperature to 100 °C and start heating. After the temperature rises to 100 °C, maintain it at 100 °C for 120 min. Turn off the heating. When the temperature of the water bath drops to 80 °C, remove the hot water and replace it with 20 °C cold water for cooling. Keep stirring steadily for 45 min during the whole cooling process. After the cooling time ends, use a ring magnet to magnetically attract the encapsulated microspheres, wash them 5 times with purified water, and store them in 20% ethanol. Observe the morphology of the prepared agarose magnetic microspheres through an optical microscope, and measure the microsphere size with a ruler. Most of them are within 40 μm ± 5 μm, and the particle size is relatively uniform, as Figure 3 shown.

[0053] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing agarose magnetic microspheres, characterized in that, The preparation process adopts a "one-pot" preparation method, which includes the following steps: S1) Mix agarose, surfactant, magnetic core, inorganic salt and water, and stir to form a homogeneous mixture; S2) Under stirring conditions, heat the homogeneous mixture to a certain temperature, keep it warm for a period of time, and then stop heating; S3) After stopping heating, cool it to a certain temperature, and then use cold water to cool it for a period of time to obtain agarose magnetic microspheres.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the agarose to the water in S1 is (1~6):

100.

3. The preparation method according to claim 1 or 2, characterized in that, The surfactant in S1 is polyethylene glycol, and the average molecular weight of the polyethylene glycol is 6000~75000 Da; The mass ratio of the surfactant to the water is (10~30):

100.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The magnetic core in S1 is magnetic Fe3O4, nano-Fe or Fe3O4@SiO2; The mass ratio of the magnetic core to the water is (0.1~3):

100.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The inorganic salt in S1 is one or more of sodium chloride, potassium chloride, and magnesium chloride; The mass ratio of the inorganic salt to the water is (2~7.5):

100.

6. The preparation method according to any one of claims 1 to 5, characterized in that, Heating the homogeneous mixture to a certain temperature in S2 means heating it to 80~100°C.

7. The preparation method according to any one of claims 1 to 6, characterized in that Keeping it warm for a period of time in S2 means keeping it warm for 15~120 min.

8. The preparation method according to any one of claims 1 to 7, characterized in that, Cooling it to a certain temperature in S3 means cooling it to 70~80°C.

9. The preparation method according to any one of claims 1 to 8, characterized in that, The temperature of the cold water in S3 is 0~20°C; Cooling it for a period of time means cooling it for 30~60 min.

10. The preparation method according to any one of claims 1 to 9, characterized in that, The rotation speed of the stirring is 200~5000 rpm.

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