Preparation method of shell-controllable magnetic agarose gel microspheres

Abstract

The application discloses a preparation method of a shell-controllable magnetic agarose gel microsphere. First, a first agarose solution is added into a mixed oil phase under high-speed stirring to be emulsified, then the emulsion is stirred and cooled, and is solidified in an ice-water bath, and is broken and washed to obtain agarose microspheres; the agarose microspheres are placed in an equipment to be in a suspended state by blowing air, and are sprayed to adhere to a layer of agarose solution; then magnetic powder suspension is sprayed to adhere to a layer of magnetic particles; then agarose solution is sprayed to adhere to a layer of agarose solution again, air is blown to guide the agarose magnetic microspheres into water to obtain magnetic agarose gel microspheres; wherein, the spraying step is repeated according to needs. The prepared agarose gel microspheres can control the magnetic separation time and the epoxy group density of the agarose magnetic microspheres, and can significantly improve the acid and alkali resistance, oxidation resistance and resistance to other solvents, and improve the biocompatibility of the magnetic core.

Description

Technical Field

[0001] This invention relates to the field of gel microsphere preparation, and more particularly to a method for preparing agarose gel microsphere with controllable shell magnetic coating. Background Technology

[0002] Magnetic microspheres are a versatile material widely used in the biomedical field. Their core provides separation capabilities, while the outer biopolymer functional groups provide carrier functionality. They have broad application prospects in enzyme immobilization, immunoassay, cell separation, targeted drug delivery, and chemical analysis. Agarose is a linear, water-soluble natural polysaccharide extracted from seaweed. It is a polymer composed of alternating 1,3-linked β-D-galactopyranose and 1,4-linked 3,6-dehydrated α-L-galactopyranose residues. Agarose gel, as a matrix, has the following advantages: it is rich in hydroxyl groups, which, after chemical activation, can couple various ligands; its porous network structure facilitates the diffusion of macromolecules within the beads, thereby increasing the effective density of ligands; it has good hydrophilicity, allowing biomolecules to easily approach and interact with ligands without inactivating them; and it is uncharged, exhibiting minimal non-specific adsorption capacity. Therefore, it is an excellent inert support for separation media.

[0003] Currently, the preparation methods for agarose magnetic microspheres are quite limited, mainly employing methods such as ultrasound, mechanical stirring, and homogenization emulsification. Patent CN1376730A discloses a method for preparing magnetic agarose composite microspheres, obtaining them by adding alkaline solution dropwise to a mixed solution of agarose and ferrous chloride under mechanical stirring. The initial state of this mixed solution is a homogeneous system; as magnetic particles precipitate, a layer of agarose is adsorbed on the particle surface, but it lacks sufficient sphericity, resulting in microspheres with a wide particle size distribution and non-spherical shape. Patent CN1316450A discloses a method for preparing magnetic agarose microspheres using an oil-water two-phase method, directly dispersing rubidium iron boron particles in an agarose solution, adding them to the oil phase under stirring to form water-in-oil droplets, and obtaining agarose magnetic microspheres after cooling, cross-linking solidification, and washing. While micron-sized rubidium-iron-boron alloy particles are embedded within agarose to form magnetic agarose microspheres, improving their resistance to acids, alkalis, and oxidation, the large particle size and high density make them prone to sedimentation and magnetization in magnetic fields, leading to remanence and agglomeration. Patent CN104587977A discloses a method for preparing surface-carboxylated modified agarose magnetic microspheres. This method involves first preparing W / O type agarose microspheres, activating cross-linking, adsorbing iron ions, and then precipitating with ammonia to produce agarose microspheres containing magnetic particles. However, because iron salt ions freely diffuse into the interior of the agarose microspheres or adsorb onto their surface, the number of magnetic particles loaded on the microspheres is relatively small, resulting in a slow magnetic response during application. Furthermore, when different types of ligands need to be attached to the surface of the agarose magnetic microspheres, a high density of epoxy groups is often required. Therefore, the epoxy group density is particularly important for expanding the applications of agarose magnetic microspheres. Currently, agarose magnetic microspheres prepared using the emulsification and curing method require the addition of a high concentration of magnetic cores to enhance their high magnetic responsiveness. This results in a thinner agarose shell, reducing the hydroxyl content of the agarose and thus decreasing the epoxy density.

[0004] Ideal agarose magnetic microspheres and their production method should possess the following characteristics: 1) The magnetic particles should be superparamagnetic with no remanence; 2) The microspheres should have high magnetic content and fast magnetic response; 3) The microspheres should have good chemical stability, be resistant to acids, alkalis, and oxidation, and exhibit no magnetic particle or iron ion precipitation or contamination; 4) The microspheres should have a narrow particle size distribution and be monodisperse; 5) The agarose magnetic microspheres should have a high epoxy group density; 6) The process should be simple, the production cost low, the reproducibility good, and the process easy to scale up. Based on the preparation methods reported in existing patents, it is clear that none of the existing methods can produce agarose magnetic microspheres that meet the requirements of ideal microspheres, which limits their widespread application in medical and biological separation and purification. Therefore, it is necessary to provide a method for preparing agarose magnetic microspheres to address these issues. Summary of the Invention

[0005] This invention improves upon the traditional method of coating magnetic particles with agarose by changing the traditional direct coating method to an alternating core-shell coating method. The aim is to provide a method for preparing magnetic agarose gel microspheres with controllable shell layers.

[0006] To achieve the above objectives, the present invention provides a method for preparing controllable shell magnetic agarose gel microspheres, characterized by comprising the following steps:

[0007] S1. Preparation of micron-sized agarose gel microspheres with core material:

[0008] The first agarose solution was added to the mixed oil phase of Span 80 and liquid paraffin under high-speed stirring and emulsified under high-speed stirring. After emulsification, the emulsion was cooled to room temperature under stirring and then solidified in an ice-water bath. After solidification, the emulsion was demulsified. The lower precipitate was washed multiple times with distilled water to obtain the first agarose microspheres.

[0009] S2. Preparation of controllable magnetic agarose gel microspheres with shells:

[0010] The obtained first agarose microspheres are placed in the equipment, and a first airflow is introduced to suspend the first agarose microspheres. Then, a second agarose solution is sprayed into the reaction chamber to coat the surface of the first agarose microspheres with a layer of agarose solution. A magnetic powder suspension is sprayed into the reaction chamber to coat the surface of the first agarose microspheres with another layer of magnetic particles. A third agarose solution is then sprayed into the reaction chamber to coat the outermost surface of the agarose microspheres with another layer of agarose solution. A second airflow is then introduced to introduce the agarose magnetic microspheres into deionized water to obtain magnetic agarose gel microspheres. The process of spraying the first agarose microspheres with the second agarose solution, the magnetic powder suspension, and the second agarose solution is repeated as needed.

[0011] Furthermore, in step S1, the concentration of the first agarose solution is 2–6 w / v%; and / or

[0012] The Span80 concentration is 1–5 w / v%.

[0013] Furthermore, in step S1, the water-to-oil ratio in the emulsified system is 1:10 to 9:10; and / or

[0014] The high-speed stirring emulsification includes both upward stirring emulsification and downward stirring emulsification; wherein the upward stirring emulsification rate is 16000 rpm to 24000 rpm, and the downward stirring emulsification rate is 800 rpm to 1600 rpm; and / or

[0015] The emulsification temperature is 70–100°C, and the time is 5–30 minutes.

[0016] Furthermore, in step S1, the stirring rate during curing is 800 rpm to 1200 rpm; and / or

[0017] The particle size of the first agarose microsphere is 1–10 μm.

[0018] Furthermore, in step S2, the concentration of the second agarose solution is 0.1 w / v% to 0.5 w / v%; and / or

[0019] The concentration of the magnetic powder suspension is 5 w / v% to 20 w / v%; and / or

[0020] The concentration of the third agarose solution is 2 w / v% to 6 w / v%.

[0021] Further, in step S2, the magnetic powder in the magnetic powder suspension is at least one of iron(III) oxide powder and iron(II) oxide powder; and / or

[0022] The magnetic powder has a particle size of 10–100 nm and is in the form of rods, polyhedra, and hollow spheres; and / or

[0023] The magnetic saturation strength of the magnetic powder is greater than 80 emu / g.

[0024] Furthermore, in step S2, the temperature of the first airflow is 60–100°C; and / or

[0025] The spray temperature of the second agarose solution is 60–100°C; and / or

[0026] The spray temperature of the magnetic powder suspension is 90–120°C; and / or

[0027] The spray temperature of the third agarose solution is 80–100°C; and / or

[0028] The temperature of the second airflow is 0–25°C.

[0029] In step S2 of this invention, by repeatedly spraying the second agarose solution, the magnetic powder suspension, and the third agarose solution once, the particle size of the agarose magnetic microspheres increases by 20–40 μm. Therefore, the number of repetitions can be selected according to requirements.

[0030] The present invention can prepare agarose magnetic microspheres with a particle size of 20-150 μm and a magnetic separation time of less than 10 s.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] This invention utilizes superparamagnetic Fe3O4 or Fe2O3 particles with high saturation magnetization as the magnetic material, improving upon the poor magnetic response performance or remanence issues of magnetic agarose microspheres prepared by previous techniques. By introducing a chemically inert, hydrophilic, and biocompatible shell layer onto the surface of the magnetic Fe3O4 or Fe2O3, the acid and alkali resistance, oxidation resistance, and resistance to other solvents of the magnetic agarose are significantly improved. Simultaneously, the biocompatibility of the magnetic core is improved, ensuring good biocompatibility throughout the entire magnetic agarose microsphere. By controlling the core-shell thickness and number of layers, the magnetic separation time and epoxy group density of the agarose magnetic microspheres can be adjusted. Attached Figure Description

[0033] Figure 1 This is the result graph obtained from Comparative Example 1.

[0034] Figure 2 This is the result graph obtained from Comparative Example 2.

[0035] Figure 3 This is the result graph obtained from Comparative Example 3.

[0036] Figure 4 This is a result diagram obtained from Example 1.

[0037] Figure 5 This is a result diagram obtained from Example 2.

[0038] Figure 6 This is a result diagram obtained from Example 3.

[0039] Figure 7 This is a result diagram obtained from Example 4.

[0040] Figure 8 This is a result diagram obtained from Example 5.

[0041] Figure 9 This is a result diagram obtained from Example 6.

[0042] Figure 10 This is a result diagram obtained from Example 7.

[0043] Figure 11 This is a flowchart illustrating the preparation process of the controllable magnetic agarose gel microspheres of the present invention. Detailed Implementation

[0044] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0045] A method for preparing controllable shell magnetic agarose gel microspheres, characterized by comprising the following steps:

[0046] S1. Preparation of micron-sized agarose gel microspheres with core material:

[0047] The first agarose solution was added to the mixed oil phase of Span 80 and liquid paraffin under high-speed stirring and emulsified under high-speed stirring. After emulsification, the emulsion was cooled to room temperature under stirring and then solidified in an ice-water bath. After solidification, the emulsion was demulsified. The lower precipitate was washed multiple times with distilled water to obtain the first agarose microspheres.

[0048] S2. Preparation of controllable magnetic agarose gel microspheres with shells:

[0049] The obtained first agarose microspheres are placed in the equipment, and a first airflow is introduced to suspend the first agarose microspheres. Then, a second agarose solution is sprayed into the reaction chamber to coat the surface of the first agarose microspheres with a layer of agarose solution. A magnetic powder suspension is sprayed into the reaction chamber to coat the surface of the first agarose microspheres with another layer of magnetic particles. A third agarose solution is then sprayed into the reaction chamber to coat the outermost surface of the agarose microspheres with another layer of agarose solution. A second airflow is then introduced to introduce the agarose magnetic microspheres into deionized water to obtain magnetic agarose gel microspheres. The process of spraying the first agarose microspheres with the second agarose solution, the magnetic powder suspension, and the second agarose solution is repeated as needed.

[0050] Furthermore, in step S1, the concentration of the first agarose solution is 2–6 w / v%; and / or

[0051] The Span80 concentration is 1–5 w / v%.

[0052] Furthermore, in step S1, the water-to-oil ratio in the emulsified system is 1:10 to 9:10; and / or

[0053] The high-speed stirring emulsification includes both upward stirring emulsification and downward stirring emulsification; wherein the upward stirring emulsification rate is 16000 rpm to 24000 rpm, and the downward stirring emulsification rate is 800 rpm to 1600 rpm; and / or

[0054] The emulsification temperature is 70–100°C, and the time is 5–30 minutes.

[0055] Furthermore, in step S1, the stirring rate during curing is 800 rpm to 1200 rpm; and / or

[0056] The particle size of the first agarose microsphere is 1–10 μm.

[0057] Furthermore, in step S2, the concentration of the second agarose solution is 0.1 w / v% to 0.5 w / v%; and / or

[0058] The concentration of the magnetic powder suspension is 5 w / v% to 20 w / v%; and / or

[0059] The concentration of the third agarose solution is 2 w / v% to 6 w / v%.

[0060] Further, in step S2, the magnetic powder in the magnetic powder suspension is at least one of iron(III) oxide powder and iron(II) oxide powder; and / or

[0061] The magnetic powder has a particle size of 10–100 nm and is in the form of rods, polyhedra, and hollow spheres; and / or

[0062] The magnetic saturation strength of the magnetic powder is greater than 80 emu / g.

[0063] Furthermore, in step S2, the temperature of the first airflow is 60–100°C; and / or

[0064] The spray temperature of the second agarose solution is 60–100°C; and / or

[0065] The spray temperature of the magnetic powder suspension is 90–120°C; and / or

[0066] The spray temperature of the third agarose solution is 80–100°C; and / or

[0067] The temperature of the second airflow is 0–25°C.

[0068] The flowchart for the preparation of the shell-controllable magnetic agarose gel microspheres of the present invention is shown below. Figure 11 The following examples are combined with Figure 11 conduct.

[0069] The following method was used to determine the epoxy group content of magnetic microspheres in the following experiment: 7.5 g of agarose magnetic microspheres were weighed into a wide-mouth conical flask, 4 mL of 5M sodium hydroxide solution and 6 mL of deionized water were added, and the mixture was shaken and mixed. Then, 4 mL of epichlorohydrin and 4 mL of 1,4-dioxane were added sequentially. The flask was sealed and placed in a shaker at 30°C for a certain period of time for activation. After activation, the flasks were removed, washed with deionized water, and dried. 1.0 g of the cross-linked activated microsphere sample was weighed, 2-3 drops of phenolphthalein were added, and 3 mL of 1.3 mol / L sodium thiosulfate solution was added. The mixture was reacted in a shaker for 30 min, and titrated with 0.01 mol / L hydrochloric acid. The data were recorded. The calculation formula is Q(μmol / g) = c 盐酸 (v0-v1) / m 磁球 *1000.

[0070] Magnetic separation time of magnetic beads: Accurately weigh 0.1g of sample powder, add 10mL of water to a 10mL glass bottle, disperse it fully by ultrasound, place a magnet vertically on one side of the glass bottle, and calculate the time it takes for the solution to go from turbid to clear.

[0071] Comparative Example 1: A method for preparing agarose magnetic microspheres

[0072] Includes the following steps:

[0073] S1: Add agarose to 40 mL of distilled water to form a suspension, and dissolve the agarose suspension by heating to form a homogeneous solution with a concentration of 4% (w / v); use 200 mL of liquid paraffin as the oil phase, and add 1.5% (w / v) Span 80 to the oil phase to obtain a mixed oil phase; add the above agarose solution to the above mixed oil phase;

[0074] 6g of polyhedral magnetic powder was dispersed in 20mL of distilled water. The magnetic powder was then slowly poured into an agarose solution (obtained by adding the agarose solution to the mixed oil phase). The emulsification temperature was adjusted to 80℃, and emulsification was carried out at a stirring rate of 1200rpm for 20min. After emulsification, the emulsion was cooled to room temperature at 500rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain agarose magnetic microspheres with a final size of approximately 100μm. Figure 1 ).

[0075] The magnetic separation time of the magnetic microspheres was measured to be 42 s, and the epoxy group content of the magnetic microspheres was 60 μmol / g.

[0076] Comparative Example 2: A method for preparing agarose magnetic microspheres

[0077] Includes the following steps:

[0078] Agarose was added to 40 mL of distilled water to form a suspension. The agarose suspension was then dissolved by heating to form a homogeneous solution with a concentration of 4% (w / v). Using 200 mL of liquid paraffin as the oil phase, 1.5% (w / v) Span 80 was added to the oil phase to obtain a mixed oil phase. The agarose solution was then added to the mixed oil phase.

[0079] 15g of polyhedral magnetic powder was dispersed in 20mL of distilled water. The magnetic powder was then slowly poured into an agarose solution (obtained by adding agarose solution to a mixed oil phase). The emulsification temperature was adjusted to 80℃, and emulsification was carried out at a stirring rate of 1200rpm for 20min. After emulsification, the emulsion was cooled to room temperature at 500rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain agarose magnetic microspheres with a final size of approximately 100μm. Figure 2 ).

[0080] The magnetic separation time of the magnetic microspheres was measured to be 8.5 s, and the epoxy group content of the magnetic microspheres was 24 μmol / g.

[0081] Comparative Example 3: A method for preparing agarose magnetic microspheres with controllable shells

[0082] Includes the following steps:

[0083] S1. Agarose is added to distilled water to form a suspension. The agarose suspension is dissolved by heating to form a homogeneous solution with a concentration of 2% (w / v). Liquid paraffin is used as the oil phase. 1% (w / v) Span 80 is added to the oil phase to obtain a mixed oil phase. The agarose solution is then added to the mixed oil phase.

[0084] The emulsification temperature was adjusted to 70℃, and emulsification was carried out at a lower stirring rate of 800 rpm and an upper stirring rate of 16000 rpm for 5 minutes. After emulsification, the emulsion was cooled to room temperature at 800 rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain the final product, 1 μm agarose microspheres.

[0085] S2. Place 1μm agarose microspheres in the device and introduce an airflow at 60℃ to suspend the agarose microspheres. Spray a 0.1% (w / v) agarose solution 1 into the reaction chamber at a spray temperature of 60℃ to coat the surface of the agarose microspheres with a layer of agarose solution. Spray a 5% (w / v) rod-shaped iron oxide suspension with a magnetic saturation intensity of 82 emu / g, a length of 100nm, and a concentration of ferric oxide into the reaction chamber at a spray temperature of 90℃ to coat the surface of the agarose microspheres with magnetic particles. Spray a 2% (w / v) agarose solution 2 into the reaction chamber at a spray temperature of 80℃ to coat the surface of the agarose magnetic microspheres with a layer of agarose solution. Adjust the airflow temperature to 25℃ and collect the agarose magnetic microspheres in deionized water to obtain agarose magnetic microspheres with a particle size of approximately 20μm. Figure 3 ).

[0086] The magnetic separation time of the magnetic microspheres was measured to be 42 s, and the epoxy group content of the magnetic microspheres was 66 μmol / g.

[0087] Example 1: A method for preparing agarose magnetic microspheres with controllable shells.

[0088] Includes the following steps:

[0089] S1. Agarose was added to distilled water to form a suspension. The agarose suspension was then dissolved by heating to obtain a homogeneous solution with a concentration of 2% (w / v). Liquid paraffin was used as the oil phase, and 1% (w / v) Span 80 was added to the oil phase. The agarose solution was then added to the mixed oil phase. The emulsification temperature was adjusted to 70°C, and emulsification was carried out under the conditions of lower stirring at 800 rpm and upper stirring at 16000 rpm for 5 minutes. After emulsification, the emulsion was cooled to room temperature at 800 rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain the final product, 1 μm agarose microspheres.

[0090] S2. Place 1μm agarose microspheres in the device and introduce an airflow at 60℃ to suspend the agarose microspheres. Spray a 0.1% (w / v) agarose solution 1 into the reaction chamber at a spray temperature of 60℃ to coat the surface of the agarose microspheres with a layer of agarose solution. Spray a 10% (w / v) rod-shaped iron oxide suspension with a magnetic saturation intensity of 82 emu / g, a length of 100nm, and a concentration of 10% (w / v) into the reaction chamber at a spray temperature of 90℃ to coat the surface of the agarose microspheres with magnetic particles. Spray a 2% (w / v) agarose solution 2 into the reaction chamber at a spray temperature of 80℃ to coat the surface of the agarose magnetic microspheres with a layer of agarose solution. Adjust the airflow temperature to 25℃ and collect the agarose magnetic microspheres in deionized water to obtain agarose magnetic microspheres with a particle size of approximately 20μm. Figure 4 ).

[0091] The magnetic separation time of the magnetic microspheres was measured to be less than 10 s, and the epoxy group content of the magnetic microspheres was 80 μmol / g.

[0092] Example 2: A method for preparing agarose magnetic microspheres with controllable shells.

[0093] Includes the following steps:

[0094] S1. Agarose was added to distilled water to form a suspension. The agarose suspension was then dissolved by heating to obtain a homogeneous solution with a concentration of 2% (w / v). Liquid paraffin was used as the oil phase, and 1% (w / v) Span 80 was added to the oil phase. The agarose solution was then added to the mixed oil phase. The emulsification temperature was adjusted to 70°C, and emulsification was carried out under the conditions of lower stirring at 800 rpm and upper stirring at 16000 rpm for 5 minutes. After emulsification, the emulsion was cooled to room temperature at 800 rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain the final product, 1 μm agarose microspheres.

[0095] S2. Place 1μm agarose microspheres in the equipment and introduce an airflow at a temperature of 60℃ to suspend the agarose microspheres. Spray a 0.1% (w / v) agarose solution 1 into the reaction chamber at a spray temperature of 60℃ to coat the surface of the agarose microspheres with a layer of agarose solution. Spray a rod-shaped iron oxide suspension with a magnetic saturation intensity of 82 emu / g, a particle size of 20nm, and a concentration of 10% (w / v) into the reaction chamber at a spray temperature of 90℃ to coat the surface of the agarose microspheres with magnetic particles. Spray a 2% (w / v) agarose solution 2 into the reaction chamber at a spray temperature of 80℃ to coat the surface of the agarose magnetic microspheres with a layer of agarose solution.

[0096] A rod-shaped iron oxide suspension with a magnetic saturation intensity of 82 emu / g, a particle size of 20 nm, and a concentration of 5% (w / v) was sprayed into the reaction chamber. The spray temperature of the magnetic fluid was 90℃, causing the magnetic particles to re-adhere to the surface of the agarose microspheres. A 2% (w / v) agarose solution was then sprayed into the reaction chamber at a spray temperature of 80℃, causing a layer of agarose solution to adhere to the surface of the agarose magnetic microspheres. The gas flow temperature was adjusted to 25℃, and the agarose magnetic microspheres were introduced into deionized water for collection, yielding particles with a size of approximately 45 μm. Figure 5 ).

[0097] The magnetic separation time of the magnetic microspheres was less than 10 s, and the epoxy group content of the magnetic microspheres was 76 μmol / g.

[0098] Example 3: A method for preparing agarose magnetic microspheres with controllable shells.

[0099] Includes the following steps:

[0100] S1. Agarose was added to distilled water to form a suspension. The agarose suspension was dissolved by heating to obtain a homogeneous solution with a concentration of 4% (w / v). Liquid paraffin was used as the oil phase, and 2.5% (w / v) Span 80 was added to the oil phase. The agarose solution was added to the mixed oil phase. The emulsification temperature was adjusted to 80℃, and emulsification was carried out under the conditions of lower stirring emulsification rate of 1000 rpm and upper stirring emulsification rate of 18000 rpm for 10 min. After emulsification, the emulsion was cooled to room temperature at 900 rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain the finished 6μm agarose microspheres.

[0101] S2. Place 6μm agarose microspheres in the equipment and introduce an airflow at 80℃ to suspend the agarose microspheres. Spray a 0.2% (w / v) agarose solution 1 into the reaction chamber at a spray temperature of 80℃ to coat the surface of the agarose microspheres with a layer of agarose solution. Spray a 15% (w / v) suspension of polyhedral iron oxide with a magnetic saturation intensity of 85 emu / g, a particle size of 30nm, into the reaction chamber at a spray temperature of 100℃ to coat the surface of the agarose microspheres with magnetic particles. Spray a 4% (w / v) agarose solution 2 into the reaction chamber at a spray temperature of 90℃ to coat the surface of the agarose magnetic microspheres with a layer of agarose solution. Adjust the airflow temperature to 20℃ and collect the agarose magnetic microspheres in deionized water to obtain agarose magnetic microspheres with a particle size of 30μm. Figure 6 ).

[0102] The magnetic separation time of the magnetic microspheres was measured to be less than 10 s, and the epoxy group content of the magnetic microspheres was 72 μmol / g.

[0103] Example 4: A method for preparing agarose magnetic microspheres with controllable shells.

[0104] Includes the following steps:

[0105] S1. Agarose was added to distilled water to form a suspension. The agarose suspension was dissolved by heating to obtain a homogeneous solution with a concentration of 4% (w / v). Liquid paraffin was used as the oil phase, and 2.5% (w / v) Span 80 was added to the oil phase. The agarose solution was added to the mixed oil phase. The emulsification temperature was adjusted to 80℃, and emulsification was carried out under the conditions of lower stirring emulsification rate of 1000 rpm and upper stirring emulsification rate of 18000 rpm for 10 min. After emulsification, the emulsion was cooled to room temperature at 900 rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain the finished 6μm agarose microspheres.

[0106] S2. Place 6μm agarose microspheres in the equipment and introduce an airflow at 80℃ to suspend the agarose microspheres. Spray a 0.2% (w / v) agarose solution 1 into the reaction chamber at a spray temperature of 80℃ to coat the surface of the agarose microspheres with a layer of agarose solution. Spray a 10% (w / v) polyhedral iron oxide suspension with a magnetic saturation intensity of 85 emu / g, a particle size of 30nm, and a concentration of 10% (w / v) into the reaction chamber at a spray temperature of 100℃ to coat the surface of the agarose microspheres with magnetic particles. Spray a 4% (w / v) agarose solution 2 into the reaction chamber at a spray temperature of 90℃ to coat the surface of the agarose magnetic microspheres with a layer of agarose solution.

[0107] A polyhedral iron(III) oxide suspension with a magnetic saturation intensity of 85 emu / g, a particle size of 30 nm, and a concentration of 10% (w / v) was sprayed into the reaction chamber at a magnetic fluid spray temperature of 100℃, causing magnetic particles to re-adhere to the surface of the agarose microspheres. A 4% (w / v) agarose solution was then sprayed into the reaction chamber at a spray temperature of 90℃, causing a layer of agarose solution to adhere to the surface of the agarose magnetic microspheres. The gas flow temperature was adjusted to 20℃, and the agarose magnetic microspheres were collected by introducing them into deionized water, yielding agarose magnetic microspheres with a particle size of approximately 60 μm. Figure 7 ).

[0108] The magnetic separation time of the magnetic microspheres was measured to be less than 10 s, and the epoxy group content of the magnetic microspheres was 66 μmol / g.

[0109] Example 5: A method for preparing agarose magnetic microspheres with controllable shells.

[0110] Includes the following steps:

[0111] S1. Agarose was added to distilled water to form a suspension. The agarose suspension was then dissolved by heating to obtain a homogeneous solution with a concentration of 6% (w / v). Liquid paraffin was used as the oil phase, and 4% (w / v) Span 80 was added to the oil phase. The agarose solution was then added to the mixed oil phase. The emulsification temperature was adjusted to 90℃, and emulsification was carried out under the conditions of a lower stirring emulsification rate of 1200 rpm and an upper stirring emulsification rate of 24000 rpm for 30 minutes. After emulsification, the emulsion was cooled to room temperature at 1000 rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain the final product, 10 μm agarose microspheres.

[0112] S2. Place 10μm agarose microspheres in the device and introduce an airflow at 90℃ to suspend the agarose microspheres. Spray a 0.5% (w / v) agarose solution 1 into the reaction chamber at a spray temperature of 90℃ to coat the surface of the agarose microspheres with a layer of agarose solution. Spray a 20% (w / v) hollow spherical iron oxide suspension with a magnetic saturation intensity of 90 emu / g, a particle size of 100nm, into the reaction chamber at a spray temperature of 110℃ to coat the surface of the agarose microspheres with magnetic particles. Spray a 6% (w / v) agarose solution 2 into the reaction chamber at a spray temperature of 100℃ to coat the surface of the agarose magnetic microspheres with a layer of agarose solution. Adjust the airflow temperature to 10℃ and collect the agarose magnetic microspheres in deionized water to obtain a particle size of 40μm. Figure 8 ).

[0113] The magnetic separation time of the magnetic microspheres was measured to be less than 10 s, and the epoxy group content of the magnetic microspheres was 68 μmol / g.

[0114] Example 6: A method for preparing agarose magnetic microspheres with controllable shells.

[0115] Includes the following steps:

[0116] S1. Agarose was added to distilled water to form a suspension. The agarose suspension was then dissolved by heating to obtain a homogeneous solution with a concentration of 6% (w / v). Liquid paraffin was used as the oil phase, and 4% (w / v) Span 80 was added to the oil phase. The agarose solution was then added to the mixed oil phase. The emulsification temperature was adjusted to 90℃, and emulsification was carried out under the conditions of a lower stirring emulsification rate of 1200 rpm and an upper stirring emulsification rate of 24000 rpm for 30 minutes. After emulsification, the emulsion was cooled to room temperature at 1000 rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain the final product, 10 μm agarose microspheres.

[0117] S2. Place 10μm agarose microspheres in the equipment and introduce an airflow at a temperature of 90℃ to suspend the agarose microspheres. Spray a 0.5% (w / v) agarose solution 1 into the reaction chamber at a spray temperature of 90℃ to coat the surface of the agarose microspheres with a layer of agarose solution. Spray a 5% (w / v) hollow spherical iron oxide suspension with a magnetic saturation intensity of 90 emu / g, a particle size of 100nm, and a concentration of 5% (w / v) into the reaction chamber at a spray temperature of 110℃ to coat the surface of the agarose microspheres with magnetic particles. Spray a 6% (w / v) agarose solution 2 into the reaction chamber at a spray temperature of 100℃ to coat the surface of the agarose magnetic microspheres with a layer of agarose solution.

[0118] A 20% (w / v) concentration of hollow spherical iron oxide suspension with a magnetic saturation intensity of 90 emu / g, a particle size of 100 nm, and a concentration of 20% was sprayed into the reaction chamber. The spray temperature of the magnetic fluid was 110℃, causing magnetic particles to adhere to the surface of the agarose microspheres. A 6% (w / v) agarose solution was then sprayed into the reaction chamber at a spray temperature of 100℃, causing a layer of agarose solution to adhere to the surface of the agarose magnetic microspheres. The gas flow temperature was adjusted to 10℃, and the agarose magnetic microspheres were collected by introducing them into deionized water, obtaining agarose magnetic microspheres with a particle size of 80 μm. Figure 9 ).

[0119] The magnetic separation time of the magnetic microspheres was measured to be less than 10 s, and the epoxy group content of the magnetic microspheres was 77 μmol / g.

[0120] Example 7: A method for preparing agarose magnetic microspheres with controllable shells.

[0121] Includes the following steps:

[0122] S1. Agarose was added to distilled water to form a suspension. The agarose suspension was then dissolved by heating to obtain a homogeneous solution with a concentration of 6% (w / v). Liquid paraffin was used as the oil phase, and 4% (w / v) Span 80 was added to the oil phase. The agarose solution was then added to the mixed oil phase. The emulsification temperature was adjusted to 90℃, and emulsification was carried out under the conditions of a lower stirring emulsification rate of 1200 rpm and an upper stirring emulsification rate of 24000 rpm for 30 minutes. After emulsification, the emulsion was cooled to room temperature at 1200 rpm and then solidified in an ice-water bath for a period of time. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain the final product, 10 μm agarose microspheres.

[0123] S2. Place 10μm agarose microspheres in the equipment and introduce an airflow at 100℃ to suspend the agarose microspheres. Spray a 0.5% (w / v) agarose solution 1 into the reaction chamber at a spray temperature of 100℃ to coat the surface of the agarose microspheres with a layer of agarose solution. Spray a 5% (w / v) hollow spherical ferric oxide suspension with a magnetic saturation intensity of 82 emu / g, a particle size of 50nm, and a concentration of 5% (w / v) into the reaction chamber at a spray temperature of 120℃ to coat the surface of the agarose microspheres with magnetic particles. Spray a 6% (w / v) agarose solution 2 into the reaction chamber at a spray temperature of 100℃ to coat the surface of the agarose magnetic microspheres with a layer of agarose solution.

[0124] First repetition: A rod-shaped iron oxide suspension with a magnetic saturation intensity of 85 emu / g, a length of 150 nm, and a concentration of 10% (w / v) was sprayed into the reaction chamber at a magnetic fluid spray temperature of 110°C, causing magnetic particles to adhere to the surface of the agarose microspheres. Then, a 6% (w / v) agarose solution 2 was sprayed into the reaction chamber at a spray temperature of 100°C, causing a layer of agarose solution to adhere to the surface of the agarose magnetic microspheres.

[0125] Second repetition: A polyhedral iron(III) oxide suspension with a magnetic saturation intensity of 90 emu / g, a particle size of 100 nm, and a concentration of 10% (w / v) was sprayed into the reaction chamber at a magnetic fluid spray temperature of 110°C, causing magnetic particles to adhere to the surface of the agarose microspheres. A 6% (w / v) agarose solution was then sprayed into the reaction chamber at a spray temperature of 100°C, causing a layer of agarose solution to adhere to the surface of the agarose magnetic microspheres. The airflow temperature was adjusted to 0°C, allowing the agarose solution on the surface of the agarose magnetic microspheres to solidify rapidly, resulting in agarose magnetic microspheres with a particle size of approximately 150 μm. Figure 10 ).

[0126] The magnetic separation time of the magnetic microspheres was measured to be less than 10 s, and the epoxy group content of the magnetic microspheres was 62 μmol / g.

[0127] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for preparing shell-controllable magnetic agarose gel microspheres, characterized in that, Includes the following steps: S1. Preparation of micron-sized agarose gel microspheres with core material: The first agarose solution was added to the mixed oil phase of Span 80 and liquid paraffin under high-speed stirring and emulsified under high-speed stirring. After emulsification, the emulsion was cooled to room temperature under stirring and then solidified in an ice-water bath. After solidification, demulsification was performed. The lower precipitate was washed multiple times with distilled water to obtain the first agarose microspheres. S2. Preparation of controllable magnetic agarose gel microspheres with shells: The obtained first agarose microspheres were placed in the equipment, and the first airflow was introduced to suspend the first agarose microspheres. Then, the second agarose solution was sprayed into the reaction chamber to make the surface of the first agarose microspheres adhere to a layer of agarose solution. A magnetic powder suspension is sprayed into the reaction chamber of the equipment, causing a layer of magnetic particles to adhere to the surface of the first agarose microspheres. Then, a third agarose solution is sprayed into the reaction chamber, causing a layer of agarose solution to adhere to the outermost surface of the agarose microspheres. A second airflow is then introduced to guide the agarose magnetic microspheres into deionized water, thus obtaining magnetic agarose gel microspheres. The process of spraying the first agarose microspheres with the magnetic powder suspension and the third agarose solution can be repeated multiple times.

2. The preparation method according to claim 1, characterized in that, In step S1, the concentration of the first agarose solution is 2–6 w / v %; and / or The Span 80 concentration is 1–5 w / v.

3. The preparation method according to claim 1, characterized in that, In step S1, the water-to-oil ratio in the emulsified system is 1:10 to 9:10; and / or The high-speed stirring emulsification includes both upward stirring emulsification and downward stirring emulsification; wherein the upward stirring emulsification rate is 16000 rpm to 24000 rpm, and the downward stirring emulsification rate is 800 rpm to 1600 rpm; and / or The emulsification temperature is 70–100°C, and the time is 5–30 min.

4. The preparation method according to claim 1, characterized in that, In step S1, the stirring rate for curing is 800 rpm to 1200 rpm; and / or The particle size of the first agarose microsphere is 1–10 μm.

5. The preparation method according to claim 1, characterized in that, In step S2, the concentration of the second agarose solution is 0.1 w / v% to 0.5 w / v%; and / or The concentration of the magnetic powder suspension is 5 w / v% to 20 w / v%; and / or The concentration of the third agarose solution is 2 w / v% to 6 w / v.

6. The preparation method according to claim 1, characterized in that, In step S2, the magnetic powder in the magnetic powder suspension is at least one of iron(III) oxide powder and iron(II) oxide powder; and / or The magnetic powder has a particle size of 10–100 nm and is in the form of rods, polyhedra, and hollow spheres; and / or The magnetic saturation strength of the magnetic powder is greater than 80 emu / g.

7. The preparation method according to claim 1, characterized in that, In step S2, the temperature of the first airflow is 60–100°C; and / or The spray temperature of the second agarose solution is 60–100°C; and / or The spray temperature of the magnetic powder suspension is 90–120°C; and / or The spray temperature of the third agarose solution is 80–100°C; and / or The temperature of the second airflow is 0–25°C.

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