Preparation method of streptavidin polymer microspheres

Abstract

The invention provides a preparation method of streptavidin polymer microspheres, and relates to the technical field of polymer composites.The preparation method comprises the following steps that carboxyl polystyrene microspheres are prepared from target amounts of styrene, functional monomers, a mixed solvent, an initiator and a dispersing agent; activating the prepared carboxyl polystyrene microspheres; carrying out covalent coupling on the activated carboxyl polystyrene microspheres and streptavidin; the streptavidin physically attached to the carboxyl polystyrene microspheres coupled with the streptavidin is stripped, and streptavidin polymer microspheres are obtained; the prepared streptavidin polymer microspheres are sealed and stored; compared with a traditional chemical coupling method, the preparation method disclosed by the invention has the advantages that a cross-linking agent does not need to be additionally added, the cost is reduced, the process can be simplified, the process is easy to control, and the prepared streptavidin polymer microspheres are uniform in particle size, controllable in size, considerable in surface carboxyl content, good in stability and high in activity.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer composites, and specifically to a method for preparing streptavidin polymer microspheres. Background Art

[0002] Efficient solid-phase carrier materials have always been the focus of research in the fields of biomedical detection, diagnosis, and biological separation. Traditional carrier materials (such as agarose microspheres or silica particles) face significant challenges when immobilizing streptavidin: agarose microspheres have low mechanical strength and uneven pore size distribution, and are prone to rupture under high-speed centrifugation or fluid shear force; silica substrates have high non-specific adsorption, which affects the specificity of complex biological sample detection. Polystyrene microspheres have become a widely used solid-phase carrier due to their good chemical stability, easy surface modification, and low cost.

[0003] Currently, in order to achieve specific biomolecule recognition and separation functions, it is often necessary to modify the surface of polystyrene microspheres to connect them with biomolecules having specific binding ability. Streptavidin, as a protein with extremely high affinity for biotin, plays an important role in biomolecule detection and separation. Immobilizing streptavidin on the surface of polystyrene microspheres to prepare streptavidin polystyrene microspheres can utilize the specific binding between streptavidin and biotin to achieve efficient capture and separation of biotin-labeled biomolecules.

[0004] However, there are many problems with existing methods for preparing streptavidin polystyrene microspheres. The traditional physical adsorption method is simple to operate, but the binding stability of streptavidin on the surface of polystyrene microspheres is poor, and it is prone to fall off during subsequent applications, resulting in poor detection and separation effects. Although the chemical coupling method can improve the binding stability, the reaction conditions are relatively harsh, and it is easy to damage the biological activity of streptavidin, affecting its specific binding ability with biotin. In addition, the streptavidin polystyrene microspheres prepared by existing methods also have deficiencies in terms of particle size uniformity and dispersibility, which limit their application in some fields with high requirements for microsphere quality. Summary of the Invention

[0005] The present invention provides a method for preparing streptavidin polymer microspheres to solve the above-mentioned technical problems. The traditional physical adsorption method is simple to operate, but the binding stability of streptavidin on the surface of polystyrene microspheres is poor, and it is easy to fall off during subsequent applications, resulting in poor detection and separation effects. Although the chemical coupling method can improve the binding stability, the reaction conditions are relatively harsh, which is likely to damage the biological activity of streptavidin and affect its specific binding ability with biotin. In addition, the streptavidin polystyrene microspheres prepared by the existing methods also have deficiencies in terms of particle size uniformity, dispersibility, etc., which limits their application in some fields with high requirements for the quality of microspheres.

[0006] To solve the above technical problems, the present invention discloses a method for preparing streptavidin polymer microspheres, which includes the following steps: Step 1: Prepare carboxyl polystyrene microspheres by using a target amount of styrene, functional monomer, mixed solvent, initiator, and dispersant.

[0007] Step 2: Activate the prepared carboxyl polystyrene microspheres.

[0008] Step 3: Covalently couple the activated carboxyl polystyrene microspheres with streptavidin.

[0009] Step 4: Peel off the physically attached streptavidin in the carboxyl polystyrene microspheres coupled with streptavidin to obtain streptavidin polymer microspheres.

[0010] Step 5: Block and store the prepared streptavidin polymer microspheres.

[0011] Preferably, the specific method of Step 1 is as follows:

[0012] Step 11: Use ethanol and deionized water as the mixed solvent, dissolve a target amount of dispersant into the target amount of mixed solvent, then transfer it into a three-necked flask equipped with a condenser, nitrogen balloon, and mechanical stirrer, stir and disperse evenly at 300 rpm, and then pass nitrogen to remove oxygen for 15 min.

[0013] Step 12: Mix a target amount of initiator with a target amount of styrene evenly, then add it into the above three-necked flask, heat up to 70 °C and keep the temperature constant for reaction for 30 min.

[0014] Step 13: Continue to add a target amount of functional monomer into the above three-necked flask, continuously pass nitrogen, and continue to stir and react at 300 rpm for 10.5 h to obtain a microsphere emulsion.

[0015] Step 14: Centrifuge and wash the obtained microsphere emulsion to obtain carboxyl polystyrene microspheres.

[0016] Preferably, the mixed solvent includes ethanol and deionized water, and the mass ratio of ethanol to deionized water is 6-11:1-5, preferably 8-11:1-3. The dispersant includes any one or a combination of polyvinylpyrrolidone, alkylphenol polyoxyethylene ether, polyethylene glycol, and polyvinyl alcohol. The initiator includes any one or a combination of potassium persulfate, azobisisobutyronitrile, and benzoyl peroxide. The functional monomer includes any one or a combination of methacrylic acid, acrylic acid, and methyl methacrylate.

[0017] Preferably, in step 1, the target mass ratio of styrene, functional monomer, ethanol, deionized water, initiator, and dispersant is 1:0.004-0.04:3-5.5:0.5-2.5:0.01-0.05:0.05-0.25.

[0018] Preferably, the specific method of step 2 is as follows:

[0019] Step 21: Take the carboxyl polystyrene microsphere stock solution in a centrifuge tube, and then place the centrifuge tube in a high-speed centrifuge for centrifugal separation to remove the preservation solution of the carboxyl polystyrene microsphere stock solution;

[0020] Step 22: Gently shake and mix the carboxyl polystyrene microsphere stock solution after removing the preservation solution, pipette 10 mg of the carboxyl polystyrene microsphere stock solution into a 4 ml EP tube, add 1 ml of 0.01 M phosphate buffer solution, gently shake and wash, and then place it in a centrifuge for centrifugal washing three times to separate the carboxyl polystyrene microspheres and remove the supernatant;

[0021] Step 23: Add 100 μL of freshly prepared 10 mg / ml NHS and 100 μL of freshly prepared 10 mg / ml EDC solution to the carboxyl polystyrene microspheres, and incubate at 37 °C with rotation for 2 hours for activation;

[0022] Step 24: After activation, centrifuge and separate the carboxyl polystyrene microspheres, and add 1 ml of 0.01 M phosphate buffer solution to wash the carboxyl polystyrene microspheres three times to remove the activator.

[0023] Preferably, the specific method of step 3 is as follows:

[0024] Step 31: Place the activated carboxyl polystyrene microspheres in step 2 in 1 ml of coupling buffer, add the target amount of streptavidin, and incubate at room temperature with rotation for at least 3 hours, then separate the microspheres and remove the supernatant;

[0025] Step 32: Add 1 ml of 0.01 M phosphate buffer solution to wash the microspheres three times;

[0026] Step 33: Add 500 μL of 0.01 M phosphate buffer solution to resuspend the microspheres.

[0027] Preferably, the coupling buffer is prepared as follows: Take 1.067 g of MES reagent and add it to 100 ml of deionized water. Then add an appropriate amount of potassium hydroxide solution to make the pH value of the above mixed solution 5.5, obtaining the coupling buffer. After sterilization, it is stored for standby at 4°C. The target mass of streptavidin added with 1 ml of the coupling buffer is 400 μg to 600 μg.

[0028] Preferably, the specific method of step 4 is as follows:

[0029] Step 41: Put the carboxyl polystyrene microspheres conjugated with streptavidin into a dialysis bag (5), immerse the dialysis bag (5) in 500 ml of 0.01 M phosphate buffer for dialysis, and stir magnetically for 24 h, changing the dialysis solution every 4 hours during this period;

[0030] Step 42: Place the dialyzed microspheres in 1 ml of the coupling buffer, then add 1 mg of streptavidin and incubate again. After incubation, wash the microspheres three times with 1 ml of 0.01 M phosphate buffer.

[0031] Preferably, the specific method of step 5 is as follows:

[0032] Step 51: Gently mix the microspheres washed in step 42 with 1000 μL of the blocking buffer, incubate on a thermostatic shaker for 45 minutes to carry out the blocking reaction;

[0033] Step 52: After the blocking reaction, centrifuge to separate and remove the supernatant, and collect the streptavidin polymer microspheres;

[0034] Step 53: Add 1000 μL of the preservation solution to the streptavidin polymer microspheres for preservation.

[0035] Preferably, the blocking buffer includes 0.01 M phosphate buffer and 1% BSA, and the preservation solution includes 0.1% to 0.5% Proclin-300, 0.1% to 1% BSA, and 0.01 M phosphate buffer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The 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 to the present invention. In the drawings:

[0037] Figure 1 is the process flow chart of the present invention;

[0038] Figure 2 is the particle size distribution diagram of the streptavidin polymer microspheres prepared by the present invention;

[0039] Figure 3SEM image of streptavidin polymer microspheres prepared according to the present invention;

[0040] Figure 4 Graph showing the measurement results of the coupling effect of streptavidin on the streptavidin polymer microspheres prepared according to the present invention;

[0041] Figure 5 Schematic diagram of the carboxyl group content determination by conductometric titration of carboxyl polymer microspheres prepared according to the present invention;

[0042] Figure 6 Schematic diagram of the structure of the dialysis device of the present invention.

[0043] In the figure: 1, base; 2, magnetic stirrer; 3, dialysis cup; 4, rotor; 5, dialysis bag; 6, fixing clip; 7, rotating sleeve; 8, pulling rope; 9, guiding block; 10, worm gear; 11, rope winding wheel; 12, worm; 13, first support block; 14, second support block. Detailed implementation manners

[0044] The following describes the preferred embodiments of the present invention 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.

[0045] In addition, in the present invention, descriptions such as "first" and "second" are only for descriptive purposes, and do not particularly refer to the order or sequence. Nor are they used to limit the present invention. They are merely 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 quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In addition, the technical solutions and technical features between various embodiments can be combined with each other, but it must be based on the fact that those skilled in the art can implement them. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0046] The present invention provides the following embodiments

[0047] Example 1

[0048] The embodiment of the present invention provides a method for preparing streptavidin polymer microspheres, as Figures 1 - 5 shown, including the following steps:

[0049] Step 1: Prepare carboxyl polystyrene microspheres using a target amount of styrene, functional monomer, mixed solvent, initiator, and dispersant;

[0050] Step 2: Activate the prepared carboxyl polystyrene microspheres;

[0051] Step 3: Covalently couple the activated carboxyl polystyrene microspheres with streptavidin;

[0052] Step 4: Strip the physically attached streptavidin from the carboxyl polystyrene microspheres conjugated with streptavidin to obtain streptavidin polymer microspheres;

[0053] Step 5: Block and store the prepared streptavidin polymer microspheres.

[0054] The beneficial effects of the above technical solution are as follows:

[0055] In the present invention, carboxyl polystyrene microspheres are conjugated with streptavidin to prepare streptavidin polymer microspheres. Compared with traditional chemical coupling methods, no additional cross-linking agent is required, which reduces the cost, simplifies the process, and the process is easy to control. The prepared streptavidin polymer microspheres have uniform particle size, controllable size, considerable surface carboxyl content, good stability, and strong activity, solving the problems of the traditional physical adsorption method that is simple to operate, but the binding stability of streptavidin on the surface of polystyrene microspheres is poor, and it is easy to fall off during subsequent applications, resulting in poor detection and separation effects. Although the chemical coupling method can improve the binding stability, the reaction conditions are relatively harsh, which is likely to damage the biological activity of streptavidin and affect its specific binding ability with biotin. In addition, the streptavidin polystyrene microspheres prepared by existing methods also have deficiencies in terms of particle size uniformity and dispersibility, restricting their application in some fields with high requirements for microsphere quality.

[0056] Example 2

[0057] On the basis of Example 1, as Figures 1 - 5 shown, the specific method of Step 1 is as follows:

[0058] Step 11: Using ethanol and deionized water as a mixed solvent, dissolve the target amount of dispersant into the target amount of mixed solvent, then transfer it into a three-necked flask equipped with a condenser, nitrogen balloon, and mechanical stirrer, stir and disperse evenly at 300 rpm, and then pass nitrogen to remove oxygen for 15 min;

[0059] Step 12: Mix the target amount of initiator and the target amount of styrene evenly, then add them into the above three-necked flask, heat up to 70 °C and keep the temperature constant for reaction for 30 min;

[0060] Step 13: Continue to add the target amount of functional monomer into the above three-necked flask, continuously pass nitrogen, and continue to stir and react at 300 rpm for 10.5 h to obtain a microsphere emulsion;

[0061] Step 14: Centrifuge and wash the obtained microsphere emulsion to obtain carboxyl polystyrene microspheres;

[0062] The mixed solvent includes ethanol and deionized water, and the mass ratio of ethanol to deionized water is 6-11:1-5, preferably 8-11:1-3. The dispersant includes any one or a combination of polyvinylpyrrolidone, alkylphenol polyoxyethylene ether, polyethylene glycol, and polyvinyl alcohol. The initiator includes any one or a combination of potassium persulfate, azobisisobutyronitrile, and benzoyl peroxide. The functional monomer includes any one or a combination of methacrylic acid, acrylic acid, and methyl methacrylate;

[0063] In step 1, the target mass ratio of styrene, functional monomer, ethanol, deionized water, initiator, and dispersant is 1:0.004-0.04:3-5.5:0.5-2.5:0.01-0.05:0.05-0.25;

[0064] In step 1, the preferred target mass ratio of styrene, functional monomer, ethanol, deionized water, initiator, and dispersant is 1:0.02-0.04:4-5.5:0.5-1.5:0.01-0.03:0.2-0.25.

[0065] The beneficial effects of the above technical solutions are:

[0066] Weigh 2 g of polyvinylpyrrolidone and dissolve it in 53 g of absolute ethanol and 12 g of deionized water. Then transfer it to a three-necked flask equipped with a condenser, nitrogen balloon, and mechanical stirrer. Stir and disperse evenly at 300 rpm, and then purge with nitrogen for 15 min to remove oxygen. Mix 0.1 g of azobisisobutyronitrile with 10 g of styrene evenly and add it to the above three-necked flask. Then heat up to 70 °C and keep the temperature constant for reaction for 30 min. Subsequently, add 0.04 g of acrylic acid, continue to purge with nitrogen, and continue to stir and react at 300 rpm for 10.5 h to obtain a microsphere emulsion. Centrifuge and wash the obtained emulsion to obtain a carboxyl polystyrene microsphere stock solution.

[0067] Example 3

[0068] On the basis of Example 1, as Figures 1 - 5 shown, the specific method of step 2 is as follows:

[0069] Step 21: Take the carboxyl polystyrene microsphere stock solution in a centrifuge tube, and then place the centrifuge tube in a high-speed centrifuge for centrifugal separation to remove the preservation liquid of the carboxyl polystyrene microsphere stock solution;

[0070] Step 22: Gently shake and mix the carboxyl polystyrene microsphere stock solution after removing the preservation liquid. Pipette 10 mg of the carboxyl polystyrene microsphere stock solution into a 4 ml EP tube, add 1 ml of 0.01 M phosphate buffer solution, gently shake and wash, and then place it in a centrifuge for centrifugal washing three times to separate the carboxyl polystyrene microspheres and remove the supernatant;

[0071] Step 23: Add 100 μL of freshly prepared 10 mg / ml NHS and 100 μL of freshly prepared 10 mg / ml EDC solution to the carboxyl polystyrene microspheres, and incubate with rotation at 37 °C for 2 hours for activation.

[0072] Step 24: After activation, centrifuge the carboxyl polystyrene microspheres to separate them, and add 1 ml of 0.01 M phosphate buffer solution to wash the carboxyl polystyrene microspheres three times to remove the activator.

[0073] The beneficial effects of the above technical solution are as follows:

[0074] If directly activating the carboxyl polystyrene microspheres after their preparation, there is no need to perform the step of removing the preservation solution of the carboxyl polystyrene microspheres in Step 21. Directly take the microsphere stock solution and gently shake it to mix evenly. In Step 24, place the mixed solution containing the carboxyl polystyrene microspheres after incubation in Step 23 into a high-speed centrifuge to centrifuge and separate the carboxyl polystyrene microspheres, and then add 1 ml of 0.01 M phosphate buffer solution to wash the carboxyl polystyrene microspheres three times.

[0075] Example 4

[0076] Based on Example 1, as Figures 1 - 5 shown, the specific method of Step 3 is as follows:

[0077] Step 31: Place the activated carboxyl polystyrene microspheres in Step 2 into 1 ml of coupling buffer, add the target amount of streptavidin, incubate with rotation at room temperature for at least 3 hours, and separate the microspheres to remove the supernatant.

[0078] Step 32: Add 1 ml of 0.01 M phosphate buffer solution to wash the microspheres three times.

[0079] Step 33: Add 500 μL of 0.01 M phosphate buffer solution to resuspend the microspheres.

[0080] The preparation method of the coupling buffer is as follows: Take 1.067 g of MES reagent and add it to 100 ml of deionized water, and then add an appropriate amount of potassium hydroxide solution to make the pH value of the above mixed solution 5.5 to obtain the coupling buffer. After sterilization, store it at 4 °C for standby. The target mass of streptavidin added to 1 ml of the coupling buffer is 400 μg - 600 μg.

[0081] The beneficial effects of the above technical solution are as follows:

[0082] In step 31, the method for separating the microspheres and removing the supernatant is to place the mixture containing carboxyl polystyrene microspheres conjugated with streptavidin in a centrifuge tube, then place the centrifuge tube in a centrifuge for centrifugation to separate and remove the supernatant. The target masses of streptavidin in 1 ml of coupling buffer can be selected as 400 μg, 500 μg, and 600 μg. For example, Figure 4 , sample 1 is the sample where the target mass of streptavidin in 1 ml of coupling buffer added in step 31 is 500 μg, sample 2 is the sample where the target mass of streptavidin in 1 ml of coupling buffer added in step 31 is 600 μg, and sample 3 is the sample where the target mass of streptavidin in 1 ml of coupling buffer added in step 31 is 400 μg. Figure 2 , Figure 3 and Figure 5 are all sample 1, that is, the experimental diagram of the streptavidin polymer microspheres prepared corresponding to the case where the target mass of streptavidin in 1 ml of coupling buffer added in step 31 is 500 μg.

[0083] Example 5

[0084] Based on Example 1, as Figures 1 - 5 shown, the specific method for step 4 is as follows:

[0085] Step 41: Put the carboxyl polystyrene microspheres conjugated with streptavidin into dialysis bag 5, immerse dialysis bag 5 in 500 ml of 0.01 M phosphate buffer for dialysis, and stir magnetically for 24 h, changing the dialysis solution every 4 hours during this period;

[0086] Step 42: Place the dialyzed microspheres in 1 ml of coupling buffer, then add 1 mg of streptavidin and incubate again. After incubation, wash the microspheres three times with 1 ml of 0.01 M phosphate buffer;

[0087] The specific method for step 5 is as follows:

[0088] Step 51: Gently mix the microspheres washed in step 42 with 1000 μL of blocking buffer, incubate on a thermostatic shaker for 45 minutes to carry out the blocking reaction;

[0089] Step 52: After the blocking reaction, centrifuge to separate and remove the supernatant to collect the streptavidin polymer microspheres;

[0090] Step 53: Add 1000 μL of preservation solution to the streptavidin polymer microspheres for preservation;

[0091] The blocking buffer includes 0.01 M phosphate buffer and 1% BSA, and the preservation solution includes 0.1% - 0.5% Proclin - 300, 0.1% - 1% BSA, and 0.01 M phosphate buffer.

[0092] The beneficial effects of the above technical solution are as follows:

[0093] After the closed reaction in step 52, the mixture containing streptavidin polymer microspheres is placed in a centrifuge tube, and then the centrifuge tube is placed in a centrifuge for centrifugal separation to remove the supernatant. The components of the blocking buffer include phosphate buffer and BSA, where the content of BSA in the blocking buffer is 1%. The components of the storage solution include phosphate buffer, Proclin-300, and BSA, where the content of Proclin-300 in the storage solution is 0.1% - 0.5%, and the content of BSA is 0.1% - 1%.

[0094] Example 6

[0095] Based on Example 5, as Figure 6 shown, it includes a dialysis device. The dialysis device includes a base 1. A magnetic stirrer 2 is installed at the lower end of the base 1. A dialysis cup 3 is provided at the upper end of the magnetic stirrer 2. A rotor 4 is provided at the lower side inside the dialysis cup 3. A dialysis bag 5 is correspondingly provided inside the dialysis cup 3. A fixing clip 6 is installed at the upper end of the dialysis bag 5. The upper end of the fixing clip 6 is rotatably connected to a rotating sleeve 7. The rotating sleeve 7 is fixedly connected to a pull rope 8. The pull rope 8 is fixedly connected to a guide block 9. The guide block 9 is slidably connected to the side end of the base 1. The pull rope 8 is wound around a rope winding wheel 11. The rope winding wheel 11 is fixedly connected to a worm gear 10 through a rotating shaft. The worm gear 10 meshes with a worm 12. The worm 12 is rotatably arranged on a first support block 13. The rotating shaft is rotatably arranged on a second support block 14. Both the first support block 13 and the second support block 14 are fixedly arranged at the upper end of the base 1.

[0096] The beneficial effects of the above technical solution are as follows:

[0097] When the carboxyl polystyrene microspheres conjugated with streptavidin are put into the dialysis bag 5 for dialysis, first use the fixing clip 6 to fix the dialysis bag 5, then rotate the worm 12, the worm 12 drives the worm gear 10 to rotate, the worm gear 10 drives the rope winding wheel 11 to rotate, and the rope winding wheel 11 gradually loosens the pulling rope 8. Under the action of gravity, the dialysis bag 5 gradually moves into the dialysis cup 3. During this process, the pulling rope 8 drives the guiding block 9 to slide along the side end of the base 1. The dialysis cup 3 is filled with dialysis fluid to completely immerse the dialysis bag 5 in the dialysis fluid. Then control the magnetic stirrer 2 to work. The magnetic stirrer 2 drives the rotor 4 to rotate to stir the dialysis fluid, accelerating the flow rate of the dialysis fluid and improving the dialysis efficiency. When the dialysis fluid flows during the stirring process, it will generate a certain force on the dialysis bag 5, causing the dialysis bag 5 to twist or even rotate. At this time, the dialysis bag 5 can drive the fixing clip 6 to rotate, reducing the force between the dialysis bag 5 and the fixing clip 6, avoiding the fixing clip 6 being stationary and fixed, so that the dialysis bag 5 twists too much and is damaged. And the rotation of the dialysis bag 5 can also improve the dialysis efficiency. When the fixing clip 6 rotates, it can drive the part of the pulling rope 8 close to the rotating sleeve 7 to twist slightly, playing a buffering role. The setting of the guiding block 9 avoids the twisting of the pulling rope 8 at the connection part with it, thereby avoiding the twisting of the pulling rope 8 between the guiding block 9 and the rope winding wheel 11, resulting in the pulling rope 8 falling off from the rope winding wheel 11. The worm gear 10 and the worm 12 have a self-locking effect. Therefore, only by rotating the worm 12 can the worm gear 10 rotate. When the worm 12 is not controlled, the worm gear 10 remains stationary to avoid the deflection of the worm gear 10 during the stirring process.

[0098] Example 7

[0099] On the basis of Example 6, it further includes a control method for the magnetic stirrer 2 during the magnetic stirring process, which is specifically as follows:

[0100] Velocity sensor 1: Velocity sensor 1 is installed on the upper side of the dialysis cup 3 and is at the same horizontal level as the upper end of the dialysis bag 5. It is used to detect the flow velocity of the dialysis fluid corresponding to the upper end of the dialysis bag 5;

[0101] Velocity sensor 2: Velocity sensor 2 is installed on the lower side of the dialysis cup 3 and is at the same horizontal level as the lower end of the dialysis bag 5. It is used to detect the flow velocity of the dialysis fluid corresponding to the lower end of the dialysis bag 5;

[0102] Rotation speed sensor: The rotation speed sensor is installed on the fixing clip 6 and is used to detect the rotation speed of the fixing clip 6;

[0103] Controller; The controller is electrically connected to the velocity sensor 1, the velocity sensor 2, the rotation speed sensor, and the magnetic stirrer 2;

[0104] The controller controls the operation of the magnetic stirrer 2 based on the detection values of the velocity sensor 1, the velocity sensor 2, and the rotation speed sensor, including the following steps:

[0105] Step 1: The controller calculates the theoretical force of the dialysis fluid on the dialysis bag 5 during the magnetic stirring process according to the detection values of the first speed sensor and the second speed sensor and formula (1);

[0106]

[0107] where F is the theoretical force of the dialysis fluid on the dialysis bag 5 during the magnetic stirring process, K is the dynamic viscosity of the dialysis fluid, A is the surface area of the dialysis bag 5, V 2 is the detection value of the second speed sensor, V 1 is the detection value of the first speed sensor, and H is the vertical distance between the second speed sensor and the first speed sensor;

[0108] Step 2: The controller calculates the theoretical centrifugal force on the dialysis bag 5 during the magnetic stirring process according to the detection value of the rotational speed sensor and formula (2);

[0109]

[0110] where G is the theoretical centrifugal force on the dialysis bag 5 during the magnetic stirring process, M is the total weight of the dialysis bag 5 after putting the carboxyl polystyrene microspheres conjugated with streptavidin into the dialysis bag 5, R is the rotation radius of the dialysis bag 5, and N is the detection value of the rotational speed sensor;

[0111] Step 3: The controller calculates the theoretical maximum strain of the dialysis bag 5 during the magnetic stirring process according to the theoretical force of the dialysis fluid on the dialysis bag 5 calculated in Step 1, the theoretical centrifugal force on the dialysis bag 5 calculated in Step 2 and formula (3). The controller compares the theoretical maximum strain of the dialysis bag 5 during the magnetic stirring process with the maximum allowable strain of the dialysis bag 5. If the calculated theoretical maximum strain of the dialysis bag 5 is greater than the maximum allowable strain of the dialysis bag 5, the controller controls the magnetic stirrer 2 to reduce the rotational speed of the rotor 4;

[0112]

[0113] where X is the theoretical maximum strain of the dialysis bag 5 during the magnetic stirring process, MAX is the maximum value, E is the elastic modulus of the dialysis bag 5, S is the contact area between the dialysis bag 5 and the clamping end of the fixed clamp 6, and C is the corresponding contact area between the dialysis bag 5 and the side edge of the fixed clamp 6.

[0114] The beneficial effects of the above technical solution are:

[0115] The control method of the magnetic stirrer 2 during the magnetic stirring process includes: installing a first speed sensor on the upper side of the dialysis cup 3 and keeping it at the same horizontal level as the upper end of the dialysis bag 5, which is used to detect the flow rate of the dialysis fluid corresponding to the upper end of the dialysis bag 5; installing a second speed sensor on the lower side of the dialysis cup 3 and keeping it at the same horizontal level as the lower end of the dialysis bag 5, which is used to detect the flow rate of the dialysis fluid corresponding to the lower end of the dialysis bag 5; installing a rotational speed sensor on the fixed clamp 6, which is used to detect the rotational speed of the fixed clamp 6; the controller is electrically connected to the first speed sensor, the second speed sensor, the rotational speed sensor, and the magnetic stirrer 2; the controller calculates the theoretical force of the dialysis fluid on the dialysis bag 5 during the magnetic stirring process according to the detection values of the first speed sensor and the second speed sensor and formula (1); is the velocity gradient of the dialysis fluid during the stirring process (the change in the velocity of the dialysis fluid perpendicular to the flow direction of the dialysis fluid. Since the rotor 4 is on the lower side of the dialysis fluid, the flow rate of the dialysis fluid on the lower side is greater than that on the upper side); the controller calculates the theoretical centrifugal force received by the dialysis bag 5 during the magnetic stirring process according to the detection value of the rotational speed sensor and formula (2), where the rotation radius of the dialysis bag 5 is the maximum distance between the contact center part of the dialysis bag 5 and the fixed clamp 6 and the side end of the dialysis bag 5 in the left - right horizontal direction. The side ends of the dialysis bag 5 include the left end and the right end of the dialysis bag 5. Generally, the middle part of the upper end of the dialysis bag 5 is in contact with and clamped by the fixed clamp 6, and the distances from the left and right side ends of the dialysis bag 5 to the fixed clamp 6 are the same. Therefore, half of the horizontal distance between the left and right ends of the dialysis bag 5 is the rotation radius;

[0116] Since the rotation radii of the dialysis bag 5 and the fixed clamp 6 are different, the centrifugal forces of the dialysis bag 5 and the fixed clamp 6 are different. During the process of the dialysis bag 5 driving the fixed clamp 6 to rotate, there is a relative centrifugal force between the dialysis bag 5 and the fixed clamp 6, and the dialysis bag 5 has a tendency to break away from the fixed clamp 6. Assuming that the fixed clamp 6 firmly clamps the dialysis bag 5 and ignoring the centrifugal force of the fixed clamp 6, the force exerted by the fixed clamp 6 on the dialysis bag 5 during the rotation of the dialysis bag 5, that is, G, is determined. And since the dialysis bag 5 is driven to rotate by the dialysis fluid, when the dialysis bag 5 drives the fixed clamp 6 to rotate, it will also exert a torsional force on the fixed clamp 6, that is, F - G. During the torsion process, the dialysis bag 5 will contact the side edge of the fixed clamp 6 (that is, the side of the clamping end face of the fixed clamp 6). The controller calculates the theoretical maximum strain of the dialysis bag 5 during the magnetic stirring process according to the theoretical force of the dialysis fluid on the dialysis bag 5 calculated in step 1, the theoretical centrifugal force received by the dialysis bag 5 during the magnetic stirring process calculated in step 2, and formula (3). The controller compares the theoretical maximum strain of the dialysis bag 5 during the magnetic stirring process with the maximum allowable strain of the dialysis bag 5. If the calculated theoretical maximum strain of the dialysis bag 5 is greater than the maximum allowable strain of the dialysis bag 5, the controller controls the magnetic stirrer 2 to reduce the rotational speed of the rotor 4, reduce the force received by the dialysis bag 5, and avoid excessive deformation and damage of the dialysis bag 5, thereby affecting the dialysis step.

[0117] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing streptavidin polymer microspheres, characterized in that: The following steps are involved: Step 1: preparing carboxyl polystyrene microspheres using target amounts of styrene, functional monomers, mixed solvents, initiators and dispersants; Step 2: activating the prepared carboxyl polystyrene microspheres; Step 3: covalently coupling the activated carboxyl polystyrene microspheres with streptavidin; Step 4: stripping the physically attached streptavidin from the carboxyl polystyrene microspheres coupled with streptavidin to obtain streptavidin polymer microspheres; Step 5: The prepared streptavidin polymer microspheres are sealed and stored.

2. The method for preparing streptavidin polymer microspheres according to claim 1, characterized in that: The specific method of step 1 is as follows: Step 11: Using ethanol and deionized water as a mixed solvent, dissolve the target amount of dispersant into the target amount of the mixed solvent, and then transfer it into a three-necked flask equipped with a condenser, a nitrogen balloon and a mechanical stirrer, stir and disperse evenly at 300 rpm, and then introduce nitrogen to deoxygenate for 15 minutes; Step 12: Evenly mix the target amount of initiator and the target amount of styrene, then add them into the above three-necked flask, heat to 70°C and react at constant temperature for 30 minutes; Step 13: Continue to add the target amount of functional monomers into the three-necked flask, continue to flow nitrogen, and continue to stir and react at 300 rpm for 10.5 hours to obtain a microsphere emulsion; Step 14: Centrifuge and wash the obtained microsphere emulsion to obtain carboxyl polystyrene microspheres.

3. The method for preparing streptavidin polymer microspheres according to claim 2, characterized in that: The mixed solvent includes ethanol and deionized water, the mass ratio of ethanol to deionized water is 6-11:1-5, preferably 8-11:1-3, the dispersant includes any one or more combinations of polyvinyl pyrrolidone, alkylphenol polyoxyethylene ether, polyethylene glycol, and polyvinyl alcohol, the initiator includes any one or more combinations of potassium persulfate, azobisisobutyronitrile, and benzoyl peroxide, and the functional monomer includes any one or more combinations of methacrylic acid, acrylic acid, and methyl methacrylate.

4. The method for preparing streptavidin polymer microspheres according to claim 2, characterized in that: The target mass ratio of styrene, functional monomer, ethanol, deionized water, initiator and dispersant in step 1 is 1: 0.004-0.04: 3-5.5: 0.5-2.5: 0.01-0.05: 0.05-0.

25.

5. The method for preparing streptavidin polymer microspheres according to claim 1, characterized in that: The specific method of step 2 is as follows: Step 21: taking the carboxyl polystyrene microsphere stock solution into a centrifuge tube, and then placing the centrifuge tube in a high-speed centrifuge for centrifugal separation to remove the storage solution of the carboxyl polystyrene microsphere stock solution; Step 22: Gently shake and mix the carboxyl polystyrene microsphere stock solution after removing the preservation solution, pipette 10 mg of the carboxyl polystyrene microsphere stock solution into a 4 ml EP tube, add 1 ml of 0.01 M phosphate buffer and gently shake to wash, then centrifuge and wash three times to separate the carboxyl polystyrene microspheres and remove the supernatant; Step 23: Add 100uL of freshly prepared 10mg / ml NHS and 100uL of freshly prepared 10mg / ml EDC solution to the carboxyl polystyrene microspheres and incubate with rotation at 37°C for 2 hours for activation; Step 24: After activation, the carboxyl polystyrene microspheres were centrifuged and 1 ml of 0.01 M phosphate buffer was added to wash the carboxyl polystyrene microspheres three times to remove the activator.

6. The method for preparing streptavidin polymer microspheres according to claim 1, characterized in that: The specific method of step 3 is as follows: Step 31: Place the activated carboxyl polystyrene microspheres in step 2 in 1 ml of coupling buffer, add the target amount of streptavidin, incubate with rotation at room temperature for at least 3 hours, and separate the microspheres and remove the supernatant; Step 32: Add 1 ml of 0.01 M phosphate buffer to wash the microspheres three times; Step 33: Add 500 μL of 0.01 M phosphate buffer to resuspend the microspheres.

7. The method for preparing a high-efficiency streptavidin polymer microsphere according to claim 6, characterized in that: The preparation method of coupling buffer is as follows: 1.067g MES reagent is added to 100ml deionized water, and then an appropriate amount of potassium hydroxide solution is added to make the pH value of the mixed solution 5.5 to obtain coupling buffer, which is sterilized and stored at 4°C for future use. The target mass of streptavidin added with 1ml of coupling buffer is 400μg to 600μg.

8. The method for preparing streptavidin polymer microspheres according to claim 1, characterized in that: The specific method of step 4 is: Step 41: placing the streptavidin-coupled carboxyl polystyrene microspheres into a dialysis bag (5), immersing the dialysis bag (5) in 500 ml of 0.01 M phosphate buffer for dialysis, and magnetically stirring for 24 hours, during which the dialysate is replaced every 4 hours; Step 42: The dialyzed microspheres are placed in 1 ml of coupling buffer, and then 1 mg of streptavidin is added and incubated again. After incubation, the microspheres are washed three times with 1 ml of 0.01 M phosphate buffer.

9. The method for preparing streptavidin polymer microspheres according to claim 8, characterized in that: The specific method of step 5 is: Step 51: Gently mix the microspheres washed in step 42 with 1000 μL of blocking buffer and incubate on a constant temperature shaker for 45 minutes to perform a blocking reaction; Step 52: After the blocking reaction, centrifuge to remove the supernatant and collect the streptavidin polymer microspheres; Step 53: Add 1000 μL of preservation solution to the streptavidin polymer microspheres for preservation.

10. The method for preparing a high-efficiency streptavidin polymer microsphere according to claim 9, characterized in that: The blocking buffer includes 0.01M phosphate buffer and 1% BSA, and the preservation solution includes 0.1% to 0.5% Proclin-300, 0.1% to 1% BSA and 0.01M phosphate buffer.

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