Preparation method of magnetic polystyrene composite microspheres for cell sorting

Abstract

The invention provides a preparation method of magnetic polystyrene composite microspheres for cell sorting, and relates to the field of polymer composites.The preparation method comprises the steps that 1, an oil phase is prepared; step 2, pre-emulsifying: under the ultrasonic condition of an ultrasonic cleaning machine, dropwise adding the oil phase into a water phase containing a certain emulsifier, and continuously stirring by using a mechanical stirrer to obtain uniformly distributed brownish black emulsion; placing the obtained emulsion in an ice bath, and carrying out ultrasonic fine emulsification by using an ultrasonic cell crusher to obtain a stable fine emulsion; step 3, pouring the miniemulsion obtained in the step 2 into a three-neck flask, and pre-introducing nitrogen for a second time while stirring at a first stirring rotating speed; then at a certain reaction temperature, adding an initiator and a functional monomer for polymerization, and continuously reacting for a first time; and 4, carrying out magnetic separation on the magnetic composite microspheres obtained in the step 3. The magnetic polystyrene microspheres prepared by the method have the characteristics of adjustable particle size, controllable magnetic content, good monodispersity and the like.

Description

Technical Field

[0001] The invention relates to the technical field of polymer composite materials, in particular to a method for preparing magnetic polystyrene composite microspheres for cell sorting. Background Art

[0002] Cell sorting is an important biotechnology that uses specific markers to distinguish target cells from other cells, thereby achieving efficient separation and purification of a single population of cells. In recent years, magnetic composite microspheres have attracted widespread attention in the field of cell sorting due to their unique physicochemical properties. Ideal magnetic composite microspheres should have good monodispersity, high magnetic field responsiveness, a functionalized surface, and excellent biocompatibility. However, in the prior art, the dispersion of magnetic nanoparticles in the polymer matrix is ​​poor, resulting in insufficient overall magnetic content of the microspheres and a complex preparation process, which limits their application in cell sorting. Therefore, it is of great significance to develop an efficient, simple, and high-magnetic-content preparation method for magnetic polystyrene composite microspheres. Summary of the Invention

[0003] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a method for preparing magnetic polystyrene composite microspheres for cell sorting. Through the specific selection of raw materials and the miniemulsion preparation process, the final magnetic polystyrene microspheres have the characteristics of adjustable particle size and excellent monodispersity performance, which can be used as an important application tool for cell sorting.

[0004] To solve the above technical problems, the present invention discloses a method for preparing magnetic polystyrene composite microspheres for cell sorting, comprising:

[0005] Step 1: Preparation of oil phase;

[0006] Step 2: Pre-emulsification: Under ultrasonic conditions of an ultrasonic cleaning machine, the oil phase is added dropwise to the aqueous phase containing a certain emulsifier, and continuously stirred with a mechanical stirrer to obtain a uniformly distributed brown-black emulsion; the obtained emulsion is then placed in an ice bath and ultrasonically fine-emulsified using an ultrasonic cell disruptor to obtain a stable fine emulsion;

[0007] Step 3: Pour the miniemulsion obtained in step 2 into a three-necked flask, stir at a first stirring speed, and pre-flow nitrogen for a second time; then, add an initiator and a functional monomer at a certain reaction temperature to carry out polymerization, and continue the reaction for a first time;

[0008] Step 4: Perform magnetic separation on the magnetic composite microspheres obtained in step 3 to obtain monodisperse magnetic polystyrene composite microspheres.

[0009] Furthermore, the raw materials for preparing the magnetic polystyrene microspheres of the present invention include a combination of oil-soluble magnetic Fe3O4 nanoparticles, an emulsifier, an initiator, a cross-linking agent, a styrene monomer, and a functional monomer. By selecting and matching the above raw materials, the prepared magnetic polystyrene microspheres have the characteristics of adjustable particle size and excellent monodispersity.

[0010] Further, including:

[0011] In the step 2, the pre-emulsification process must be carried out simultaneously with ultrasound and stirring. The power of the ultrasonic cleaning machine is 80W, the speed of the mechanical stirrer is 350-450r / min, and the stirring time is 30min. Finally, an ultrasonic cell disruptor must be used for ultrasonic fine emulsification. The ultrasonic power is 200-600W and the ultrasonic time is 10-20min.

[0012] Furthermore, the emulsifier is one of sodium lauryl sulfate, sodium lauryl sulfonate, sodium dodecylbenzene sulfonate or sodium stearate, and the concentration of the emulsifier is 0.02 to 0.08 wt%;

[0013] The reaction temperature is 65-85° C.; the initiator is at least one of azobisisobutyronitrile, azobisisoheptylnitrile, benzoyl peroxide, ammonium persulfate, potassium persulfate, or sodium persulfate; the functional monomer is at least one of acrylic acid, methacrylic acid, methyl methacrylate, crotonic acid, itaconic acid, and maleic anhydride;

[0014] The first duration is 18 to 24 hours, and the second duration is half an hour.

[0015] Furthermore, based on 100 g of dispersion medium, the amount of magnetic fluid is 1 to 6 g, the concentration of magnetic fluid is 20 to 70 wt%, the amount of styrene is 5 to 12 g, the amount of crosslinker is 0.05 to 0.15 g, the amount of emulsifier is 0.15 to 1 g, the amount of initiator is 0.02 to 0.1 g, and the amount of functional monomer is 0.05 to 3 g.

[0016] Furthermore, the oil phase is obtained by mixing magnetic fluid, styrene and divinylbenzene in a certain ratio (the ratio of magnetic fluid to styrene is 1:1-5); the preparation process of the magnetic fluid (oil-soluble magnetic Fe3O4 nanoparticles) includes:

[0017] Step 11: Mix the ferric salt and ferrous salt solutions in a certain proportion and stir;

[0018] Step 12: adding an appropriate amount of alkaline substance (alkaline solution) as a precipitant to the solution obtained in step 11, and preparing magnetic Fe3O4 nanoparticles by chemical coprecipitation at a certain temperature and pH;

[0019] Step 13: Surface modification with a surfactant to obtain oil-soluble Fe₃O₄ is then performed in situ in n-octane at a specific ratio to obtain a magnetic fluid of the corresponding concentration. The magnetic fluid is then washed 2-3 times with anhydrous ethanol and deionized water by magnetic separation, and then in situ in n-octane at a specific ratio to obtain a magnetic fluid of the corresponding concentration.

[0020] Furthermore, the particle size of the magnetic Fe3O4 nanoparticles is 10-20 nm;

[0021] The iron salt is one of ferric sulfate, ferric chloride, and ferric nitrate; the ferrous salt is one of ferrous sulfate, ferrous chloride, ferrous carbonate, ferrous oxide, and ferrous hydroxide; the molar ratio of the iron salt to the ferrous salt is 2:1; the alkaline substance is one of NH3H2O, NaOH, or KOH; and the surfactant is one or more of oleic acid, undecylenic acid, and sodium oleate.

[0022] Furthermore, the oil-soluble magnetic Fe3O4 nanoparticles must be dispersed in situ in n-octane in a wet state, that is, they must not be dried or freeze-dried.

[0023] Furthermore, in step 12, the reaction temperature is 70-85° C.; the iron salt concentration is 1-2M; the ferrous salt concentration is 0.5-1M; the alkaline solution concentration is 25-28wt%; and the surfactant concentration is 2.5-5wt%.

[0024] Furthermore, before the brown-black emulsion of the current type is ultrasonically fine-emulsified using the current ultrasonic cell disruptor to obtain a stable fine emulsion, a target input power determination process is performed, which includes:

[0025] Step 201: Acquire historical ultrasonic data of the current ultrasonic cell disruptor in the most recent historical period, the historical ultrasonic data including: historical polydispersity index and historical average particle size;

[0026] Step 202: determining the coefficient of difference and coefficient of variation of the polydispersity index, and the coefficient of difference and coefficient of variation of the average particle size of the current ultrasonic cell disruptor based on step 201;

[0027] Step 203: When each coefficient determined in step 202 is less than or equal to the corresponding preset value, the target input power of the current type of brown-black emulsion is determined to be: the input power corresponding to the median of the required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion in the input power-standard ultrasonic power fitting curve of the current ultrasonic cell disruptor;

[0028] Step 204: When any coefficient determined in step 203 is greater than the corresponding preset value;

[0029] Obtaining the required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion corresponding to the required input power range in the input power-standard ultrasonic power fitting curve of the current ultrasonic cell disruptor, and determining the first input power for ultrasonic fine emulsification of the current type of brown-black emulsion;

[0030] Step 205: Obtaining the required parameters for ultrasonic fine emulsification of the current type of brown-black emulsion and the standard polydispersity index and standard average particle size of the current type of brown-black emulsion at the corresponding first input power, wherein the required parameters include: the required range of the polydispersity index and the required range of the average particle size;

[0031] Step 206: Based on steps 202 and 205, determine the comprehensive adaptability of each first input power corresponding to the current type of brown-black emulsion, and determine the average value of several first input powers with a comprehensive adaptability equal to 2 and the smallest as the target input power of the current type of brown-black emulsion.

[0032] Furthermore, determining the first input power of ultrasonic fine emulsification of the current type of brown-black emulsion includes:

[0033] Step 2041: obtaining a second input power-standard polydispersity index curve and a second input power-standard average particle size curve corresponding to the required input power range for ultrasonic fine emulsification of the current type of brown-black emulsion;

[0034] Step 2042: Divide the second input power-standard polydispersity index curve into a plurality of segments 1, wherein the difference between the maximum dispersion index and the minimum dispersion index of each segment 1 is smaller than the first difference;

[0035] Step 2043: Divide the second input power-standard average particle size curve into a plurality of segments 2, wherein the difference between the maximum average and minimum average particle sizes in each segment 2 is less than the second difference;

[0036] Step 2044: Each second input power segment of the required input power range for ultrasonic fine emulsification of the current type of brown-black emulsion is segmented according to the larger value of the number of segments one and the number of segments two in the corresponding segment, and finally multiple second input power segments are obtained, and the median value of each second input power segment is determined as the first input power.

[0037] Furthermore, the process of determining the target speed of the mechanical stirring process of the oil phase and the water phase of the current batch and the current type includes:

[0038] Step 2001: Obtain the target Reynolds number range for the oil phase and water phase mechanical stirring process of the current type;

[0039] Step 2002: determining an initial stirring speed based on the median of the target Reynolds number range for the oil phase and water phase mechanical stirring process of the current class, the average density and average viscosity of the oil phase and water phase, and a Reynolds number model;

[0040] Step 2003: performing a stirring test on the oil phase test material of the current type and the water phase test material of the current type at the initial stirring speed, and determining the stirring time-equivalent density curve and the stirring time-equivalent viscosity curve during the stirring test, and marking the density non-uniformity coefficient and the viscosity non-uniformity coefficient corresponding to the stirring time;

[0041] Step 2004: Segmenting the stirring time-equivalent density curve by stirring time to obtain segment three, and segmenting the stirring time-equivalent viscosity curve by stirring time to obtain segment four, wherein the stirring time periods corresponding to the segments three and four are the same, and the difference between the maximum equivalent density and the minimum equivalent density of the segment three in the same stirring time period is less than the third difference, and the difference between the maximum equivalent viscosity and the minimum equivalent viscosity of the segment four in the same stirring time period is less than the fourth difference;

[0042] Step 2005: Based on the target Reynolds number range of the oil phase and water phase mechanical stirring process of the current type, the density non-uniformity coefficient and the viscosity non-uniformity coefficient corresponding to the stirring time of each segment three, determine the stirring speed range corresponding to each segment three, determine the segment three groups consisting of several consecutive adjacent segment threes, determine the theoretical stirring speed of each segment three group as the stirring speed existing in each corresponding segment three, and when there are multiple corresponding theoretical stirring speeds for the current segment three group, determine the target stirring speed according to the difference state of the current segment three group and the segment three groups adjacent to it.

[0043] Furthermore, determining the target stirring speed according to the difference between the current segmented three groups and the three segmented groups before and after them includes:

[0044] Obtain the theoretical stirring speed of the segmented three groups before the current segmented three groups, the theoretical stirring speed of the current segmented three groups, and the theoretical stirring speed of the segmented three groups after the current segmented three groups. According to the principle of minimizing the third absolute difference between the theoretical stirring speed of the segmented three groups before the current segmented three groups and the theoretical stirring speed of the current segmented three groups, and the fourth absolute difference between the theoretical stirring speed of the current segmented three groups and the theoretical stirring speed of the segmented three groups after the current segmented three groups, select the target stirring speed of the shared three groups before the current segmented three groups, the target stirring speed of the current segmented three groups, and the target stirring speed of the segmented three groups after the current segmented three groups.

[0045] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] 1. The raw materials for preparing the magnetic polystyrene composite microspheres provided by the present invention include a combination of oil-soluble magnetic Fe₃O₄ nanoparticles, an emulsifier, an initiator, a crosslinking agent, a styrene monomer, and a functional monomer. By selecting and combining these raw materials, the resulting magnetic polystyrene microspheres exhibit adjustable particle size, controllable magnetic content, and excellent monodispersity.

[0048] 2. The oil-soluble magnetic Fe3O4 nanoparticles used in the present invention have a size of 10-20 nm. The octane phase magnetic fluid obtained by in-situ dispersion can be uniformly dispersed in styrene, overcoming the problem that magnetic Fe3O4 nanoparticles are difficult to disperse in polymer materials. It can be used to prepare magnetic polystyrene composite microspheres of different sizes.

[0049] 3. The magnetic polystyrene composite microspheres provided by the present invention have a dispersed structure, which makes the magnetic particles in the microspheres more evenly distributed, maintains more consistent magnetic responsiveness during operation, and effectively avoids leakage and oxidation of magnetic particles.

[0050] 4. The size of the magnetic polystyrene composite microspheres provided by the present invention can be adjusted between 50-200 nm, and the magnetic content can also be freely adjusted between 30-70 wt % (see attached Figure 10 ), and its subsequent use will be wider.

[0051] 5. The present invention can achieve functionalization of the surface of the composite microspheres by adjusting the amount of functional monomers, and can subsequently be coupled with biological antibodies for applications such as cell sorting. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0053] Figure 1 is a flow chart of the method of the present invention;

[0054] Figure 2 is a particle size distribution diagram of the oil-soluble magnetic Fe3O4 nanoparticles in Example 1 of the present invention;

[0055] Figure 3 This is a contact angle test graph of the oil-soluble magnetic Fe3O4 nanoparticles in Example 1 of the present invention (contact angle test graph of the oil-soluble magnetic Fe3O4 nanoparticles (a) unmodified (b) modified);

[0056] Figure 4 This is a diagram showing the actual effect of the oil-soluble magnetic Fe3O4 nanoparticles in Example 1 of the present invention;

[0057] Figure 5is a particle size distribution diagram of the magnetic polystyrene composite microspheres in Example 1 of the present invention;

[0058] Figure 6 are SEM and TEM images of the magnetic polystyrene composite microspheres in Example 1 of the present invention;

[0059] Figure 7 is a VSM diagram of the oil-soluble magnetic Fe3O4 nanoparticles and magnetic polystyrene composite microspheres in Example 1 of the present invention at room temperature;

[0060] Figure 8 is a particle size distribution diagram of the magnetic polystyrene composite microspheres in Example 2 of the present invention;

[0061] Figure 9 is a SEM image of the magnetic polystyrene composite microspheres in Example 2 of the present invention;

[0062] Figure 10 is a SEM image of the magnetic polystyrene composite microspheres in Example 3 of the present invention;

[0063] Figure 11 This is the thermogravimetric diagram of the magnetic polystyrene composite microspheres of the present invention;

[0064] Figure 12 This is a structural diagram of the homemade magnetic frame used in the present invention;

[0065] Figure 13 This is a purity chart of CD3 cell separation in Example 4 of the present invention. DETAILED DESCRIPTION

[0066] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0067] In addition, in the present invention, descriptions such as "first" and "second" are only used for descriptive purposes, and do not specifically refer to the order or sequence, nor are they used to limit the present invention. They are only used to distinguish components or operations described with the same technical terms, and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions and technical features between the various embodiments can be combined with each other, but this must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0068] Combine Figure 1 , the present invention provides the following embodiments:

[0069] Example 1: This example provides a method for preparing dispersed magnetic polystyrene composite microspheres, comprising the following steps:

[0070] Step 1: Preparation of oil-soluble magnetic Fe3O4 nanoparticles: Weigh 15g of FeSO4·7H2O and 25g of FeCl3·6H2O and dissolve them in 50mL of water to form an iron salt solution. The solution is then added to a 250mL three-necked flask and stirred at 350r / min under nitrogen. After heating to 70°C, 50mL of 25% ammonia solution is added and the reaction is continued at 450r / min for half an hour. 2g of oleic acid and 3g of undecylenic acid are then added and the reaction is continued for 30min. The solution is then heated to 85°C and kept in the reaction for 30min. After cooling, the solution is repeatedly washed three times with deionized water and anhydrous ethanol. Finally, the wet magnetic Fe3O4 particles are directly in situ dispersed in n-octane to form an oily magnetic fluid with a concentration of 40w%.

[0071] Step 2: Preparation of a magnetic miniemulsion: 10g of styrene, 0.1g of divinylbenzene, and 4g of a 40wt% magnetic fluid were weighed and ultrasonically dispersed to form the oil phase. 0.5g of sodium lauryl sulfate was then dissolved in 100g of deionized water to form the aqueous phase. The aqueous phase was poured into a 250mL three-necked flask and, under ultrasonic cleaning, the oil phase was added dropwise. Stirring was continued at 400 rpm for 30 minutes to obtain a uniform brown-black emulsion. Ultrasonic miniemulsification was then performed using an ultrasonic cell disruptor at 200W for 10 minutes to obtain a stable miniemulsion.

[0072] Step 3: Preparation of Magnetic Polystyrene Composite Microspheres: Pour the miniemulsion into a three-necked flask and pre-flow nitrogen for half an hour while stirring at 350 rpm. Then, add 0.05g of potassium persulfate and 0.5g of methacrylic acid in a 70°C water bath to initiate polymerization. Continue the reaction for 20 hours. After the reaction is complete, discard the supernatant by magnetic absorption and repeatedly wash with deionized water 3-5 times.

[0073] Step 4: Magnetic separation is performed on the magnetic composite microspheres obtained in step 3 to obtain monodisperse magnetic polystyrene composite microspheres. Figure 13 Magnetic separation was performed using the homemade magnetic stand shown;

[0074] See attached figure: Figure 2 is a particle size distribution diagram of the oil-soluble magnetic Fe3O4 nanoparticles in Example 1 of the present invention; Figure 3 This is a contact angle test graph of the oil-soluble magnetic Fe3O4 nanoparticles in Example 1 of the present invention (contact angle test graph of the oil-soluble magnetic Fe3O4 nanoparticles (a) unmodified (b) modified); Figure 4 This is a diagram showing the actual effect of the oil-soluble magnetic Fe3O4 nanoparticles in Example 1 of the present invention; Figure 5 is a particle size distribution diagram of the magnetic polystyrene composite microspheres in Example 1 of the present invention; Figure 6 are SEM and TEM images of the magnetic polystyrene composite microspheres in Example 1 of the present invention; Figure 7 is a VSM diagram of the oil-soluble magnetic Fe3O4 nanoparticles and magnetic polystyrene composite microspheres in Example 1 of the present invention at room temperature;

[0075] The particle size distribution of magnetic particles and magnetic composite microspheres was measured using a Malvern nanoparticle size analyzer. Figure 2 、 Figure 5 As shown, the average particle size of the magnetic particles is 23.47 nm, the PDI is 0.09, and the average particle size of the magnetic polystyrene composite microspheres is 146.7 nm, the PDI is 0.09, showing a monodisperse distribution.

[0076] The magnetic polystyrene composite microspheres prepared by the above reaction were characterized by scanning electron microscopy and transmission electron microscopy. Figure 6 As shown in the figure, the microspheres are dispersed and have an average size of about 100 nm. The dried magnetic particles and magnetic composite microsphere powders were tested using a vibrating sample magnetometer. Figure 7 As shown, no remanence or coercive force was detected in the magnetic particles and magnetic composite microsphere powder, showing superparamagnetism, and the magnetic saturation intensity of the magnetic nanoparticles was 60.25emu / g, and the magnetic saturation intensity of the magnetic composite microsphere powder was 28.82emu / g, showing good magnetic responsiveness.

[0077] Example 2:

[0078] Step 1: Preparation of oil-soluble magnetic Fe3O4 nanoparticles: Weigh 15g FeSO4·7H2O and 25g FeCl3·6H2O and dissolve them in 50mL water to form an iron salt solution. The solution is then added to a 250mL three-necked flask and stirred at 350r / min under nitrogen protection. After heating to 70°C, 50mL ammonia solution (25%) is added and the reaction is continued at 450r / min for half an hour. 2g oleic acid and 3g undecylenic acid are then added and the reaction is continued for 30min. The reaction is then heated to 85°C and kept incubated for 30min. After cooling, the solution is repeatedly washed three times with deionized water and anhydrous ethanol. Finally, the wet magnetic Fe3O4 particles are directly in situ dispersed in n-octane and made into an oily magnetic fluid with a concentration of 50w%.

[0079] Step 2: Preparation of a magnetic miniemulsion: 5g of styrene, 0.1g of divinylbenzene, and 4g of a 50wt% magnetic fluid were weighed and ultrasonically dispersed to form the oil phase. 0.5g of sodium lauryl sulfate was then dissolved in 100g of deionized water to form the aqueous phase. The aqueous phase was poured into a 250mL three-necked flask and, under ultrasonic cleaning, the oil phase was added dropwise. Stirring was continued at 400 rpm for 30 minutes to obtain a uniform brown-black emulsion. Ultrasonic miniemulsification was then performed using an ultrasonic cell disruptor at 300W for 10 minutes to obtain a stable miniemulsion.

[0080] Step 3: Preparation of Magnetic Polystyrene Composite Microspheres: Pour the miniemulsion into a three-necked flask and pre-flow nitrogen for half an hour while stirring at 350 rpm. Then, add 0.05g of potassium persulfate and 1g of acrylic acid in a 70°C water bath to initiate polymerization. Continue the reaction for 20 hours. After the reaction is complete, discard the supernatant by magnetic absorption and repeatedly wash with deionized water 3-5 times.

[0081] Step 4: Perform magnetic separation on the magnetic composite microspheres obtained in step 3 to obtain monodisperse magnetic polystyrene composite microspheres.

[0082] The particle size distribution of magnetic composite microspheres was measured using a Malvern nanoparticle size analyzer. Figure 8 As shown in Figure 2, the average particle size of the magnetic polystyrene composite microspheres is 93.67 nm, and the PDI is 0.04282, showing a monodisperse distribution. The magnetic polystyrene composite microspheres were characterized by scanning electron microscopy, as shown in Figure 2. Figure 9 As shown, the microspheres are spherical with uniform size, and the average size is about 80 nm.

[0083] Example 3:

[0084] Step 1: Preparation of oil-soluble magnetic Fe3O4 nanoparticles: Weigh 30g FeSO4·7H2O and 50g FeCl3·6H2O and dissolve them in 100mL water to form an iron salt solution. The solution is then added to a 500mL three-necked flask and stirred at 350r / min under nitrogen protection. After heating to 70°C, 100mL ammonia solution (25%) is added and the reaction is continued at 450r / min for half an hour. 8g oleic acid is then added and the reaction is continued for 30min. The solution is then heated to 85°C and kept in the reaction for 30min. After cooling, the solution is repeatedly washed three times with deionized water and anhydrous ethanol. Finally, the wet magnetic Fe3O4 particles are directly in situ dispersed in n-octane and made into an oily magnetic fluid with a concentration of 20w%.

[0085] Step 2: Preparation of a magnetic miniemulsion: Weigh 10g of styrene, 0.05g of benzoyl peroxide, 0.1g of divinylbenzene, and 4g of a 20wt% magnetic fluid and ultrasonically disperse them to form the oil phase. Dissolve 0.5g of sodium lauryl sulfate in 100g of deionized water to form the aqueous phase. Pour the aqueous phase into a 250mL three-necked flask and add the oil phase dropwise under ultrasonic cleaning. Stir continuously at 400 rpm for 30 minutes to obtain a uniform brown-black emulsion. The resulting suspension is then placed in an ice bath and ultrasonically mini-emulsified using an ultrasonic cell disruptor at 400W for 10 minutes to obtain a stable miniemulsion.

[0086] Step 3: Preparation of Magnetic Polystyrene Composite Microspheres: Pour the miniemulsion into a three-necked flask and pre-flow nitrogen for half an hour while stirring at 350 rpm. Then, add 1 g of methacrylic acid and allow polymerization to proceed in a 70°C water bath for 20 hours. After the reaction, discard the supernatant by magnetic absorption and repeatedly wash with deionized water 3-5 times.

[0087] Step 4: Perform magnetic separation on the magnetic composite microspheres obtained in step 3 to obtain monodisperse magnetic polystyrene composite microspheres.

[0088] The average particle size of the obtained magnetic polystyrene composite microspheres was 127.3 nm, and the PDI was 0.051, showing a monodisperse distribution. The magnetic polystyrene composite microspheres were characterized by scanning electron microscopy. Figure 10 As shown, the microspheres are spherical with uniform size, and the average size is about 95 nm.

[0089] Example 4:

[0090] This embodiment provides a method for preparing magnetic polystyrene composite microspheres for cell sorting based on embodiment 2, comprising the following steps:

[0091] (1) Washing: Take 1 mg of magnetic polystyrene composite microspheres in a 2 mL centrifuge tube and wash twice with deionized water.

[0092] (2) Activation: Add 0.1 mL of EDC solution (10 mg / mL), then add 0.1 mL of NHS solution (10 mg / mL), add 0.8 mL of deionized water to 1 mL, mix with 1 mg of magnetic microspheres, and place on a shaker at 37°C and 200 rpm for 30 min. After the reaction, perform magnetic separation, discard the supernatant, and wash three times with ultrapure water for later use.

[0093] (3) Coupling: Add 1 mL of boric acid buffer (pH = 6.6) to the activated magnetic microspheres, and then add 12.5, 25, 37.5, and 50 μg of CD3 antibody, respectively. After gently shaking to mix, place in a shaker at 37°C and 200 rpm for 1 h.

[0094] (4) Blocking: Add 1 mL of boric acid buffer (pH = 6.6) containing 1% BSA and continue to react in a shaker at 37°C and 200 rpm for 1 hour.

[0095] (5) Resuspension: After the blocking reaction is completed, perform magnetic separation, discard the supernatant, wash three times with washing buffer, add resuspension buffer and resuspend to 1 mg / mL, and store in a refrigerator at 4°C.

[0096] The coupling efficiency of CD3 antibodies on the surface of magnetic polystyrene composite microspheres was compared by varying the amount of CD3 antibody used, with the results shown in Table 1. This coupling method enabled the assembly of an appropriate amount of CD3 antibodies on the surface of magnetic composite microspheres, and subsequent cell sorting of PBMCs from human peripheral blood yielded CD3 cells with a purity of 95%.

[0097] Table 1 Coupling efficiency of CD3 antibody with different dosages of magnetic polystyrene microspheres

[0098]

[0099] The beneficial effects of the above technical solution are:

[0100] 1. The raw materials for preparing the magnetic polystyrene composite microspheres provided by the present invention include a combination of oil-soluble magnetic Fe₃O₄ nanoparticles, an emulsifier, an initiator, a crosslinking agent, a styrene monomer, and a functional monomer. By selecting and combining these raw materials, the resulting magnetic polystyrene microspheres exhibit adjustable particle size, controllable magnetic content, and excellent monodispersity.

[0101] 2. The oil-soluble magnetic Fe3O4 nanoparticles used in the present invention have a size of 10-20 nm. The octane phase magnetic fluid obtained by in-situ dispersion can be uniformly dispersed in styrene, overcoming the problem that magnetic Fe3O4 nanoparticles are difficult to disperse in polymer materials. It can be used to prepare magnetic polystyrene composite microspheres of different sizes.

[0102] 3. The magnetic polystyrene composite microspheres provided by the present invention have a dispersed structure, which makes the magnetic particles in the microspheres more evenly distributed, maintains more consistent magnetic responsiveness during operation, and effectively avoids leakage and oxidation of magnetic particles.

[0103] 4. The size of the magnetic polystyrene composite microspheres provided by the present invention can be adjusted between 50-200 nm, and the magnetic content can also be freely adjusted between 30-70 wt % (see attached Figure 11 ), and its subsequent use will be wider.

[0104] 5. The present invention can achieve functionalization of the surface of the composite microspheres by adjusting the amount of functional monomers, and can subsequently be coupled with biological antibodies for applications such as cell sorting.

[0105] Example 5, based on any one of Examples 1-4,

[0106] Before the brown-black emulsion of the current type (the oil phase and water phase of the brown-black emulsion of the same type have the same formula and preparation process) is ultrasonically fine-emulsified using the current ultrasonic cell disruptor to obtain a stable fine emulsion, a target input power determination process is performed, which includes:

[0107] Step 201: Obtain historical ultrasonic data for the current ultrasonic cell disruptor within a recent historical period. This historical ultrasonic data includes: historical polydispersity index (PDI) and historical average particle size. Ultrasonic cell disruptors typically have built-in data storage (or an external data recording device), which records operating parameters and test results for each use. To obtain data for the "recent historical period," access the device's "Data Query / Export" module, select the target period (e.g., the last hour of usage records), and extract the polydispersity index (PDI) and average particle size measured by particle size detection equipment (e.g., dynamic light scattering) after each ultrasonic treatment of the brown-black emulsion. These data are then compiled into a historical dataset.

[0108] Step 202: determining the coefficient of difference and coefficient of variation of the polydispersity index, and the coefficient of difference and coefficient of variation of the average particle size of the current ultrasonic cell disruptor based on step 201;

[0109] Step 203: When each coefficient determined in step 202 is less than or equal to the corresponding preset value (greater than 0 and less than 0.2), the target input power of the current type of brown-black emulsion is determined as: the input power corresponding to the median of the required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion in the input power-standard ultrasonic power fitting curve of the current ultrasonic cell disruptor;

[0110] The input power-standard ultrasonic power fitting curve of the current ultrasonic cell disruptor is determined by testing a qualified and recently used current ultrasonic cell disruptor (or an ultrasonic cell disruptor of the same type as the current ultrasonic cell disruptor);

[0111] During the formal preparation process of each type of brown-black emulsion using a current ultrasonic disruptor with a relatively short usage time (determined according to the efficiency attenuation state of the current ultrasonic disruptor, the shorter the usage time, the smaller the efficiency attenuation), the ultrasonic disruptor is operated at an input power corresponding to the input power-standard ultrasonic power fitting curve of the current ultrasonic disruptor, the median value of the ultrasonic power (output power of the ultrasonic cell disruptor) required for ultrasonic fine emulsification of each type of brown-black emulsion;

[0112] Step 204: When any coefficient determined in step 203 is greater than the corresponding preset value;

[0113] Obtaining the required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion corresponding to the required input power range in the input power-standard ultrasonic power fitting curve of the current ultrasonic cell disruptor, and determining the first input power for ultrasonic fine emulsification of the current type of brown-black emulsion (the required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion corresponding to the required input power range in the input power-standard ultrasonic power fitting curve of the current ultrasonic cell disruptor);

[0114] The required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion can be determined based on experiments using a qualified and recently used current ultrasonic cell disruptor (or an ultrasonic cell disruptor similar to the current ultrasonic cell disruptor) before batch production of the current type of brown-black emulsion. The required ultrasonic power range is an ultrasonic power range that can achieve effective droplet refinement (particle size meeting product standards, such as reaching the nanometer level with a narrow distribution) while ensuring emulsion stability (no stratification and stable performance during storage / use period).

[0115] Step 205: Obtaining the required parameters for ultrasonic fine emulsification of the current type of brown-black emulsion and the standard polydispersity index and standard average particle size of the current type of brown-black emulsion at the corresponding first input power, wherein the required parameters include: the required range of the polydispersity index and the required range of the average particle size;

[0116] The standard polydispersity index and standard average particle size of the brown-black emulsion of the current type at the corresponding first input power are determined based on the test data of the required ultrasonic power range process of the above-mentioned brown-black emulsion of the current type;

[0117] Step 206: Based on steps 202 and 205, determine the comprehensive adaptability of each first input power corresponding to the current type of brown-black emulsion, and determine the average of several first input powers (e.g., 2-5) with a comprehensive adaptability equal to 2 as the target input power for the current type of brown-black emulsion. If the target input power cannot be determined based on step 206, the ultrasonic cell disruptor needs to be repaired or replaced.

[0118] Determine the first input power of the current type of brown-black emulsion ultrasonic fine emulsification including:

[0119] Step 2041: Obtain a second input power-standard polydispersity index curve corresponding to the required input power range for ultrasonic fine emulsification of the current type of brown-black emulsion (determined based on fitting test data of the required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion) and a second input power-standard average particle size curve (determined based on fitting test data of the required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion, the polydispersity index and average particle size obtained by test being the standard polydispersity index and standard average particle size);

[0120] The second input power is determined according to a preset parameter selection rule within the corresponding required input power range (e.g., it can be determined as an integer, or selected according to a preset required input power interval);

[0121] Step 2042: Divide the second input power-standard polydispersity index curve into a plurality of segments 1, wherein the difference between the maximum dispersion index and the minimum dispersion index of each segment 1 is smaller than the first difference;

[0122] Step 2043: Divide the second input power-standard average particle size curve into a plurality of segments 2, wherein the difference between the maximum average and minimum average particle sizes in each segment 2 is less than the second difference;

[0123] Step 2044: Each second input power segment of the required input power range for ultrasonic fine emulsification of the current type of brown-black emulsion is segmented according to the larger value of the number of segments one and the number of segments two in the corresponding segment, and finally multiple second input power segments are obtained, and the median value of each second input power segment is determined as the first input power.

[0124] in:

[0125]

[0126] P1 is the coefficient of difference of the polydispersity index of ultrasonic cell disruptors; P 10 is the coefficient of variation of the polydispersity index of the ultrasonic cell disruptor; H i is the actual multi-dispersion index of the i-th historical period; H i0 is the theoretical polydispersity index of the i-th historical period (the particle size of the brown-black emulsion corresponding to the i-th historical period is determined by ultrasonic microemulsification using a qualified and recently used ultrasonic cell disruptor. The polydispersity index is the variance of all detected particle sizes divided by the square of the average of all detected particle sizes); H i-1 is the actual multi-dispersion index of the i-1th historical period; H (i-1)0 is the theoretical multi-dispersion index of the i-1th historical period; t iis the ultrasound duration of the i-th historical period; the i-th historical period is after the i-1-th historical period; M is the number of historical ultrasound data in the most recent historical period (excluding the historical period with abnormal ultrasonic cell disruptor);

[0127]

[0128] P2 is the coefficient of variation of the average particle size of ultrasonic cell disruptors; P 20 is the coefficient of variation of the average particle size of the ultrasonic cell disruptor; d i is the actual average particle size in the i-th historical period; d i0 is the theoretical average particle size of the i-th historical period (determined by ultrasonically mini-emulsifying the brown-black emulsion corresponding to the i-th historical period using a qualified and recently used ultrasonic cell disruptor, and then performing particle size testing on the obtained mini-emulsion (the average value of multiple tests can be taken); d i with d i0 The input power of ultrasonic cell disruptor is the same); d i-1 is the actual average particle size in the i-1th historical period; d (i-1)0 is the theoretical average particle size in the i-1th historical period; t i is the ultrasound duration of the i-th historical period;

[0129] G k =G k1 +G k2 ;

[0130]

[0131]

[0132] Among them, G k1 is the polydispersity index adaptation of the kth first input power; G k2 is the average particle size adaptation of the kth first input power; G k is the comprehensive adaptability of the kth first input power; t1 is the target ultrasonic time range of the current type of brown-black emulsion; H k1 is the standard polydispersity index at the kth first input power; d k1 is the standard average particle size at the kth first input power;

[0133] when The required range of polydispersity index for brown-black emulsions belonging to the current class is The value is 1, otherwise The value is 0; when The average particle size requirement range of the brown-black emulsion belongs to the current category, then The value is 1, otherwise The value is 0.

[0134] The beneficial effects of the above technical solution are:

[0135] By mining historical data (polydispersity index, average particle size, etc.) and combining it with the coefficient of difference and coefficient of variation to quantify the status of the device (ultrasonic cell disruptor), we can accurately match the ultrasonic power. For example, based on the stability of the device's historical operation, we can dynamically adjust the input power to ensure that key indicators such as particle size distribution (polydispersity index) and average particle size remain within the target range after ultrasonic fine emulsification of brown-black emulsions, thereby improving the consistency of emulsion quality.

[0136] The concept of comprehensive adaptability is introduced. By combining the polydispersity index and average particle size with the target ultrasonic time range, the input power is segmented and screened. Dynamically calculating adaptability through a formula allows the power selection to better meet the actual needs of the emulsion, avoiding over- or under-emulsification due to inappropriate power, and ensuring the stability of the miniemulsion.

[0137] Quantitatively determine device status: Utilize the coefficient of difference and coefficient of variation to distinguish between stable and fluctuating device states, and select a targeted power determination strategy (directly using a standard curve or recalibration). This avoids redundant recalibration when device performance is good, while enabling timely adjustments when device status fluctuates, fully utilizing device performance and extending the device's useful life.

[0138] A standard curve is constructed based on testing of qualified, recently used equipment, and historical data is subsequently used to monitor equipment status. This curve dynamically correlates with the equipment's lifecycle, reflecting real-time performance changes. This provides a reliable benchmark for precise power control and mitigates fluctuations in emulsification performance caused by equipment aging and wear.

[0139] Parameter traceability and reuse: Historical data, standard curves, and fitness calculations are used to deeply correlate ultrasonic microemulsification process parameters (power, time, etc.) with emulsion quality indicators (particle size, dispersibility), forming a standardized and traceable process system. Subsequent production of similar emulsions can directly reuse verified parameters, improving process development efficiency and ensuring product consistency during large-scale production.

[0140] Before batch production, we determine the "required ultrasonic power range" through testing. Combined with segmented power verification and adaptability screening, we lock in the appropriate power range in advance. This avoids the risk of batch rejection due to improper power during large-scale production, reduces production costs, and improves production stability.

[0141] Example 6, based on any one of Examples 1-5, the process of determining the target rotational speed of the mechanical stirring process of the oil phase and the water phase of the current batch and the current type includes:

[0142] Step 2001: Obtain the target Reynolds number range for the oil phase and water phase mechanical stirring process of the current type;

[0143] Step 2002: determining an initial stirring speed based on the median of the target Reynolds number range for the oil phase and water phase mechanical stirring process of the current class, the average density and average viscosity of the oil phase and water phase, and a Reynolds number model;

[0144] Step 2003: Perform a stirring test on the oil phase test material of the current type and the water phase test material of the current type at the initial stirring speed, and determine the stirring time-equivalent density curve and the stirring time-equivalent viscosity curve during the stirring test, and mark the density unevenness coefficient and viscosity unevenness coefficient corresponding to the stirring time; during the stirring test, perform density / viscosity testing multiple times, for example, after reaching a certain stirring time Z, sample and test density (to obtain equivalent density U and density unevenness coefficient W) and viscosity (to obtain equivalent viscosity Y and viscosity unevenness coefficient Q), then the stirring time A corresponds to equivalent density U, equivalent viscosity Y, density unevenness coefficient W, and viscosity unevenness coefficient Q;

[0145] Step 2004: Segment the stirring time-equivalent density curve by stirring time to obtain segment three, and segment the stirring time-equivalent viscosity curve by stirring time to obtain segment four. Segments three and four correspond to the same stirring time period (for example, if the number of segments three is 3 and the number of segments two is 2 in a certain time period, the stirring time period is divided by the number of segments three), and the difference between the maximum equivalent density and the minimum equivalent density of segment three in the same stirring time period is less than the third difference, and the difference between the maximum equivalent viscosity and the minimum equivalent viscosity of segment four in the same stirring time period is less than the fourth difference;

[0146] Step 2005: Based on the target Reynolds number range of the oil phase and water phase mechanical stirring process of the current type, the density non-uniformity coefficient and the viscosity non-uniformity coefficient corresponding to the stirring time of each segment three, determine the stirring speed range corresponding to each segment three, determine the segment three groups consisting of several consecutive adjacent segment threes, determine the theoretical stirring speed of each segment three group as the stirring speed existing in each corresponding segment three (selected from the stirring speed range corresponding to each segment three of the corresponding segment three groups), when there are multiple corresponding theoretical stirring speeds for the current segment three group, determine the target stirring speed according to the difference state of the current segment three group and the segment three groups adjacent to it.

[0147] Determining the target stirring speed based on the difference between the current segmented three groups and the three adjacent segmented groups before and after them includes:

[0148] Obtain the theoretical stirring speed of the segmented three groups before the current segmented three groups, the theoretical stirring speed of the current segmented three groups, and the theoretical stirring speed of the segmented three groups after the current segmented three groups. According to the principle of minimizing the third absolute difference between the theoretical stirring speed of the segmented three groups before the current segmented three groups and the theoretical stirring speed of the current segmented three groups, and the fourth absolute difference between the theoretical stirring speed of the current segmented three groups and the theoretical stirring speed of the segmented three groups after the current segmented three groups, select the target stirring speed of the shared three groups before the current segmented three groups, the target stirring speed of the current segmented three groups, and the target stirring speed of the segmented three groups after the current segmented three groups.

[0149] The Reynolds number model is:

[0150]

[0151] Among them, R e is the Reynolds number; R is the average density of the system (the system of oil phase and water phase mixture) (which can be calculated using the existing formula for determining the average density of the oil phase and water phase mixture); V is the stirring speed of the stirring paddle (r / min); D is the diameter of the stirring paddle; S is the average viscosity of the system (which can be calculated using the existing formula for determining the average viscosity of the oil phase and emulsifier mixture);

[0152] The average density of the system can be:

[0153] The current mass ratio of the oil-like phase × the current density of the oil-like phase + the current mass ratio of the water-like phase × the current density of the water-like phase;

[0154] The current mass ratio of the oil-like phase is:

[0155]

[0156] ρ is the equivalent density corresponding to the current density detection process; N is the total number of first density detection points among all density detection points detected in the current density detection process; the first absolute difference between the density detection value of the first density detection point and the average density after the oil phase and water phase of the current class are evenly mixed is greater than the first preset absolute difference; ρ m is the density detection value of the mth first density detection point in the current density detection process; ρ0 is the density detection value corresponding to the maximum value of the first absolute difference among all the first density detection points in the current density detection process; ε1 and ε2 are density weight 1 and density weight 2, respectively (the values ​​are greater than 0 and less than 1, and the sum of density weight 1 and density weight 2 is 1, which can be 0.4 and 0.6, respectively);

[0157] The density non-uniformity coefficient of the current density detection process is the ratio of the standard deviation of the density detection values ​​of all density detection points detected by the current density detection process to the equivalent density corresponding to the current density detection process;

[0158]

[0159] μ is the equivalent viscosity of the current viscosity test process; L is the total number of first viscosity test points among all viscosity test points tested in the current viscosity test process; the second absolute difference between the viscosity test value of the first viscosity test point and the average viscosity after the oil phase and water phase of the current class are evenly mixed is greater than the second preset absolute difference; μ n is the viscosity detection value of the nth first viscosity detection point in the current viscosity detection process; μ0 is the viscosity detection value corresponding to the maximum value of the second absolute difference among all the first viscosity detection points in the current viscosity detection process; ε3 and ε4 are viscosity weight three and viscosity weight four, respectively (the value is greater than 0 and less than 1, and the sum of density weight three and density weight four is 1, which can be 0.4 and 0.6 respectively);

[0160] The viscosity non-uniformity coefficient of the current viscosity detection process is the ratio of the standard deviation of the viscosity detection values ​​of all viscosity detection points detected in the current viscosity detection process to the equivalent viscosity of the current viscosity detection process;

[0161] Based on the target Reynolds number range of the oil phase and water phase mechanical stirring process of the current type, the density non-uniformity coefficient and viscosity non-uniformity coefficient corresponding to the stirring time of each segment three, the stirring speed range corresponding to each segment three is determined, specifically:

[0162]

[0163] Among them, S0 is the equivalent viscosity of the current segment three; R0 is the equivalent density of the current segment three;

[0164] K1 is the density unevenness coefficient of the current segment three; K2 is the density unevenness coefficient of the current segment three; R e1 is the lower limit of the target Reynolds number range for the mechanical stirring process of the oil phase and the water phase of the current type; Re2 is the upper limit of the target Reynolds number range for the mechanical stirring process of the oil phase and the water phase of the current type; K1 is the density unevenness coefficient of the current segment three; K1' is the density correction coefficient corresponding to the density unevenness coefficient of the current segment three (the larger the density unevenness coefficient, the larger the density correction coefficient, and the density correction coefficient can be greater than or equal to 0 and less than 1); K2 is the viscosity unevenness coefficient of the current segment three; K2' is the viscosity correction coefficient corresponding to the viscosity unevenness coefficient of the current segment three (the larger the viscosity unevenness coefficient, the larger the viscosity correction coefficient, and the viscosity correction coefficient can be greater than or equal to 0 and less than 1);

[0165] Directly link the correction coefficient to the unevenness coefficient and fit the functional relationship through experimental data. Through the "unevenness coefficient-speed compensation" experiment, fit the functional relationship (such as exponential function) between the correction coefficient (speed compensation ratio) and the unevenness coefficient;

[0166] The beneficial effects of the above technical solution are:

[0167] By deeply binding the Reynolds number to parameters such as system density, viscosity, and impeller diameter, and combining them with the density and viscosity inhomogeneity coefficients, the stirring speed can be transformed from "empirical setting" to "quantitative derivation." For example, when stirring oil and water phases, the speed range can be dynamically adjusted based on fluctuations in the actual physical properties of the system (such as changes in density and viscosity caused by uneven mixing) to ensure the stirring effect (such as emulsion dispersion and uniformity).

[0168] Segmented, refined control: The equivalent density and viscosity curves are segmented by mixing time, and the speed range is calculated for each segment to adapt to the dynamic changes in system properties over time during mixing (such as the gradual stabilization of viscosity due to emulsification reactions). Compared to the traditional "fixed speed throughout the entire process," this avoids problems such as uneven mixing in the early stages and excessive shearing in the later stages, improving product quality consistency.

[0169] Data-driven standardization improves process stability and repeatability: From acquiring the target Reynolds number range, to determining the initial speed, segmented verification, and finally to theoretical speed screening, a complete data closed loop is formed. Each parameter (such as the density and viscosity non-uniformity coefficients) is quantified through experimental testing (such as the standard deviation of density / viscosity test points), transforming the mixing process from "relying on operational experience" to "based on data standards," facilitating process reuse across different batches and equipment.

[0170] Adaptive Speed ​​Difference: By comparing the theoretical speed differences between the three segments, the target speed is determined, automatically adapting to system fluctuations. Even with subtle fluctuations in oil and water phase characteristics, the speed can be adjusted through the differential compensation mechanism to ensure stable final product performance.

[0171] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing magnetic polystyrene composite microspheres for cell sorting, characterized by: include: Step 1: Preparation of oil phase; Step 2: Pre-emulsification: Under ultrasonic conditions of an ultrasonic cleaning machine, the oil phase is added dropwise to the aqueous phase containing a certain emulsifier, and continuously stirred with a mechanical stirrer to obtain a uniformly distributed brown-black emulsion; the obtained emulsion is then placed in an ice bath and ultrasonically fine-emulsified using an ultrasonic cell disruptor to obtain a stable fine emulsion; Step 3: Pour the miniemulsion obtained in step 2 into a three-necked flask, stir at a first stirring speed, and pre-flow nitrogen for a second time; then, add an initiator and a functional monomer at a certain reaction temperature to carry out polymerization, and continue the reaction for a first time; Step 4: Magnetic separation of the magnetic composite microspheres obtained in step 3.

2. The method for preparing magnetic polystyrene composite microspheres for cell sorting according to claim 1, characterized in that: include: In the step 2, the pre-emulsification process must be carried out simultaneously with ultrasound and stirring. The power of the ultrasonic cleaning machine is 80W, the speed of the mechanical stirrer is 350-450r / min, and the stirring time is 30min. Finally, an ultrasonic cell disruptor must be used for ultrasonic fine emulsification. The ultrasonic power is 200-600W and the ultrasonic time is 10-20min.

3. The method for preparing magnetic polystyrene composite microspheres for cell sorting according to claim 1, wherein: The emulsifier is one of sodium lauryl sulfate, sodium lauryl sulfonate, sodium dodecylbenzene sulfonate or sodium stearate; The reaction temperature is 65-85° C.; the initiator is at least one of azobisisobutyronitrile, azobisisoheptylnitrile, benzoyl peroxide, ammonium persulfate, potassium persulfate, or sodium persulfate; the functional monomer is at least one of acrylic acid, methacrylic acid, methyl methacrylate, crotonic acid, itaconic acid, and maleic anhydride; The first duration is 18 to 24 hours; the second duration is half an hour.

4. The method for preparing magnetic polystyrene composite microspheres for cell sorting according to claim 1, wherein: The oil phase is obtained by mixing magnetic fluid, styrene and divinylbenzene in a certain proportion; the preparation process of the magnetic fluid includes: Step 11: Mix the ferric salt and ferrous salt solutions in a certain proportion; Step 12: adding an appropriate amount of alkaline substance as a precipitant to the solution obtained in step 11, and preparing magnetic Fe3O4 nanoparticles by chemical coprecipitation at a certain temperature and pH; Step 13: Surface modification is performed under the action of a surfactant to obtain oil-soluble Fe3O4, which is then in situ dispersed in n-octane in a certain proportion to obtain a magnetic fluid of corresponding concentration.

5. The method for preparing magnetic polystyrene composite microspheres for cell sorting according to claim 4, characterized in that: Based on 100g of dispersion medium, the amount of magnetic fluid is 1 to 6g, the concentration of magnetic fluid is 20 to 70wt%, the amount of styrene is 5 to 12g, the amount of crosslinking agent is 0.05 to 0.15g, the amount of emulsifier is 0.15 to 1g, the amount of initiator is 0.02 to 0.1g, and the amount of functional monomer is 0.05 to 3g.

6. The method for preparing magnetic polystyrene composite microspheres for cell sorting according to claim 4, characterized in that: The particle size of the magnetic Fe3O4 nanoparticles is 10-20nm; The iron salt is one of ferric sulfate, ferric chloride, and ferric nitrate; the ferrous salt is one of ferrous sulfate, ferrous chloride, ferrous carbonate, ferrous oxide, and ferrous hydroxide; the molar ratio of the iron salt to the ferrous salt is 2:1; the alkaline substance is one of NH3H2O, NaOH, or KOH; and the surfactant is one or more of oleic acid, undecylenic acid, and sodium oleate. The oil-soluble magnetic Fe3O4 nanoparticles must be dispersed in situ in n-octane in a wet state, that is, they must not be dried or freeze-dried.

7. The method for preparing magnetic polystyrene composite microspheres for cell sorting according to claim 1, characterized in that: Before the brown-black emulsion of the current type is ultrasonically fine-emulsified using the current ultrasonic cell disruptor to obtain a stable fine emulsion, the target input power determination process is performed, which includes: Step 201: Acquire historical ultrasonic data of the current ultrasonic cell disruptor in the most recent historical period, the historical ultrasonic data including: historical polydispersity index and historical average particle size; Step 202: determining the coefficient of difference and coefficient of variation of the polydispersity index, and the coefficient of difference and coefficient of variation of the average particle size of the current ultrasonic cell disruptor based on step 201; Step 203: When each coefficient determined in step 202 is less than or equal to the corresponding preset value, the target input power of the current type of brown-black emulsion is determined to be: the input power corresponding to the median of the required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion in the input power-standard ultrasonic power fitting curve of the current ultrasonic cell disruptor; Step 204: When any coefficient determined in step 203 is greater than the corresponding preset value; Obtaining the required ultrasonic power range for ultrasonic fine emulsification of the current type of brown-black emulsion corresponding to the required input power range in the input power-standard ultrasonic power fitting curve of the current ultrasonic cell disruptor, and determining the first input power for ultrasonic fine emulsification of the current type of brown-black emulsion; Step 205: Obtaining the required parameters for ultrasonic fine emulsification of the current type of brown-black emulsion and the standard polydispersity index and standard average particle size of the current type of brown-black emulsion at the corresponding first input power, wherein the required parameters include: the required range of the polydispersity index and the required range of the average particle size; Step 206: Based on steps 202 and 205, determine the comprehensive adaptability of each first input power corresponding to the current type of brown-black emulsion, and determine the average value of several first input powers with a comprehensive adaptability equal to 2 and the smallest as the target input power of the current type of brown-black emulsion.

8. The method for preparing magnetic polystyrene composite microspheres for cell sorting according to claim 7, characterized in that: Determine the first input power of the current type of brown-black emulsion ultrasonic fine emulsification including: Step 2041: obtaining a second input power-standard polydispersity index curve and a second input power-standard average particle size curve corresponding to the required input power range for ultrasonic fine emulsification of the current type of brown-black emulsion; Step 2042: Divide the second input power-standard polydispersity index curve into a plurality of segments 1, wherein the difference between the maximum dispersion index and the minimum dispersion index of each segment 1 is smaller than the first difference; Step 2043: Divide the second input power-standard average particle size curve into a plurality of segments 2, wherein the difference between the maximum average and minimum average particle sizes in each segment 2 is less than the second difference; Step 2044: Each second input power segment of the required input power range for ultrasonic fine emulsification of the current type of brown-black emulsion is segmented according to the larger value of the number of segments one and the number of segments two in the corresponding segment, and finally multiple second input power segments are obtained, and the median value of each second input power segment is determined as the first input power.

9. The method for preparing magnetic polystyrene composite microspheres for cell sorting according to claim 1, characterized in that: The process of determining the target speed for the mechanical stirring of the oil and water phases of the current batch and type includes: Step 2001: Obtain the target Reynolds number range for the oil phase and water phase mechanical stirring process of the current type; Step 2002: determining an initial stirring speed based on the median of the target Reynolds number range for the oil phase and water phase mechanical stirring process of the current class, the average density and average viscosity of the oil phase and water phase, and a Reynolds number model; Step 2003: performing a stirring test on the oil phase test material of the current type and the water phase test material of the current type at the initial stirring speed, and determining the stirring time-equivalent density curve and the stirring time-equivalent viscosity curve during the stirring test, and marking the density non-uniformity coefficient and the viscosity non-uniformity coefficient corresponding to the stirring time; Step 2004: Segmenting the stirring time-equivalent density curve by stirring time to obtain segment three, and segmenting the stirring time-equivalent viscosity curve by stirring time to obtain segment four, wherein the stirring time periods corresponding to the segments three and four are the same, and the difference between the maximum equivalent density and the minimum equivalent density of the segment three in the same stirring time period is less than the third difference, and the difference between the maximum equivalent viscosity and the minimum equivalent viscosity of the segment four in the same stirring time period is less than the fourth difference; Step 2005: Based on the target Reynolds number range of the oil phase and water phase mechanical stirring process of the current type, the density non-uniformity coefficient and the viscosity non-uniformity coefficient corresponding to the stirring time of each segment three, determine the stirring speed range corresponding to each segment three, determine the segment three groups consisting of several consecutive adjacent segment threes, determine the theoretical stirring speed of each segment three group as the stirring speed existing in each corresponding segment three, and when there are multiple corresponding theoretical stirring speeds for the current segment three group, determine the target stirring speed according to the difference state of the current segment three group and the segment three groups adjacent to it.

10. The method for preparing magnetic polystyrene composite microspheres for cell sorting according to claim 9, characterized in that: Determining the target stirring speed based on the difference between the current segmented three groups and the three adjacent segmented groups before and after them includes: Obtain the theoretical stirring speed of the segmented three groups before the current segmented three groups, the theoretical stirring speed of the current segmented three groups, and the theoretical stirring speed of the segmented three groups after the current segmented three groups. According to the principle of minimizing the third absolute difference between the theoretical stirring speed of the segmented three groups before the current segmented three groups and the theoretical stirring speed of the current segmented three groups, and the fourth absolute difference between the theoretical stirring speed of the current segmented three groups and the theoretical stirring speed of the segmented three groups after the current segmented three groups, select the target stirring speed of the shared three groups before the current segmented three groups, the target stirring speed of the current segmented three groups, and the target stirring speed of the segmented three groups after the current segmented three groups.

Images