Preparation method of streptavidin magnetic beads

Abstract

The invention provides a preparation method of streptavidin magnetic beads, and relates to the technical field of preparation of polymer composites.The preparation method comprises the steps that S1, PS / SiO2 composite microspheres are prepared; s2, carboxyl magnetic beads are prepared on the basis of the PS / SiO2 composite microspheres prepared in the step S1; s3, activating the carboxyl magnetic beads; s4, carrying out covalent coupling on the carboxyl magnetic beads and streptavidin; step S5, stripping of the physically combined streptavidin; and S6, sealing and storing the streptavidin magnetic beads. The streptavidin magnetic beads prepared through the method are controllable in particle size, superparamagnetic, good in stability and high in activity, compared with a traditional method, a cross-linking agent does not need to be additionally added, the process is effectively simplified, the cost is reduced, the streptavidin magnetic beads have excellent performance in the using process of a chemiluminescence platform, and the process is easy to amplify and beneficial to control.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer composite material preparation, in particular to a method for preparing streptavidin magnetic beads. The streptavidin magnetic composite microspheres (streptavidin magnetic beads) of the present invention are applied to magnetic particle chemiluminescence immunoassay. Background Art

[0002] Magnetic microspheres are a new type of multifunctional material. Their research began in the 1970s and have been widely used in biomedicine, environmental engineering, biochemical separation and other fields in recent years.

[0003] Due to its excellent superparamagnetic properties, it can easily achieve solid-liquid separation under the action of an external magnetic field. It has broad application prospects in the fields of protein purification, immobilized enzymes, nucleic acid extraction, cell separation and immunoassay. Therefore, it is particularly important to modify its surface to form functionalized magnetic microspheres with higher biocompatibility, higher sensitivity and good specificity.

[0004] In this context, the surface of magnetic microspheres was modified with different types of polymers and biomolecules, and the resulting functionalized magnetic microspheres were evaluated and characterized using chemiluminescence immunoassays to provide appropriate reference for the application of magnetic microspheres. Biomolecules were modified on the surface of magnetic microspheres using streptavidin and antibodies to prepare biofunctionalized magnetic microspheres.

[0005] Biotin magnetic composite microspheres (streptavidin magnetic beads) are one of the most widely used types of magnetic beads in the field of biological detection. The specific binding of streptavidin and biotin has a good effect on amplifying luminescent signals, so they are highly used in the in vitro diagnostic market. Compared with products such as carboxyl magnetic beads directly linked to antibodies, which require the optimization of reagent systems for different projects, biotin magnetic composite microspheres (streptavidin magnetic beads) eliminate the tedious optimization step of magnetic bead coupling antibodies and are directly used as one of the components of the detection reagent, effectively shortening the development cycle of different product systems.

[0006] At present, the preparation of mass-produced magnetic composite microspheres (streptavidin magnetic beads) with stable performance, small batch-to-batch differences and controllable costs is an important means to achieve the widespread application of domestic magnetic composite microspheres (streptavidin magnetic beads). Summary of the Invention

[0007] In response to the deficiencies in the prior art and actual needs, the present invention provides streptavidin magnetic beads and a preparation method thereof. The present invention couples prepared carboxyl magnetic beads with streptavidin to prepare streptavidin magnetic beads. At the same time, combined with a specific process, the prepared streptavidin magnetic beads have strong activity and high stability, which is conducive to process control.

[0008] To solve the above technical problems, the present invention discloses a method for preparing streptavidin magnetic beads, comprising:

[0009] Step S1: Preparation of PS / SiO2 composite microspheres;

[0010] Step S2: preparing carboxyl magnetic beads based on the PS / SiO2 composite microspheres prepared in step S1;

[0011] Step S3: Activation of carboxyl magnetic beads;

[0012] Step S4: covalent coupling of carboxyl magnetic beads and streptavidin;

[0013] Step S5: stripping of physically bound streptavidin;

[0014] Step S6: Blocking and storage of streptavidin magnetic beads.

[0015] Preferably, the preparation method of PS / SiO2 composite microspheres in step S1 includes:

[0016] Step S11: using styrene as a monomer and ethanol and deionized water as a mixed solvent, reacting under the action of an initiator to produce polystyrene microspheres;

[0017] Step S12: using the polystyrene microspheres obtained in step S11, tetraethyl orthosilicate is used to synthesize core-shell PS / SiO2 microspheres in the presence of ammonia water, wherein the core-shell PS / SiO2 microspheres are the PS / SiO2 composite microspheres;

[0018] Furthermore, in step S11, the mass ratio of ethanol to deionized water is (20-55): (10-30), preferably (40-55): (10-15). In a preferred embodiment, g is preferably used as the unit, that is, the mass ratio of ethanol to deionized water is (20-55) g: (10-30) g, preferably (40-55) g: (10-15) g.

[0019] Furthermore, in step S11, the initiator is one or more of potassium persulfate, azobisisobutyronitrile, and benzoyl peroxide, and the content of the initiator is 1 wt% to 5 wt% of the styrene, preferably 1 wt% to 3 wt%;

[0020] Furthermore, the dispersant in step S11 can be one or more of polyvinyl pyrrolidone, alkylphenol polyoxyethylene ether, sodium phosphate, polyethylene glycol, polyacrylic acid, polyvinyl alcohol, and hydroxypropyl cellulose, and the content of the dispersant is 5wt% to 25wt% of the styrene, preferably 15wt% to 20wt%.

[0021] Preferably, the preparation method of carboxyl magnetic beads in step S2 includes:

[0022] Step S21: resuspending and dispersing the PS / SiO2 composite microspheres prepared in step S1, and synthesizing magnetic composite microspheres using a hydrophilic ferrosilicate dispersion in the presence of ethyl orthosilicate and ammonia water;

[0023] Step S22: Add an appropriate amount of aqueous ammonia and react with glyphosate at a certain pH at a first reaction temperature to modify the carboxyl groups to obtain carboxyl magnetic beads. Preferably, the first reaction temperature is 40-70°C, including but not limited to 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C, for 4-24 hours.

[0024] Preferably, the activation method of the carboxyl magnetic beads in step S3 includes:

[0025] Step S31: taking a carboxyl magnetic bead stock solution into a container, placing the container on a magnetic stand for magnetic separation, and removing the preservation solution; preferably, the container is an EP tube; preferably, the magnetic separation in step S31 is performed for two minutes;

[0026] Step S32: adding PBS buffer to wash the magnetic beads several times, and magnetically separating the magnetic beads to remove the supernatant; preferably, the concentration of the PBS buffer in step S32 is 0.01 moles per liter (0.01M); the several times in step S32 is two to three times;

[0027] Step S33: adding freshly prepared NHS and MES solutions and incubating in a constant temperature shaker at a first incubation temperature for a first incubation time to activate the carboxyl groups on the surface of the magnetic beads; wherein the NHS and MES solutions are 10 mg / mL NHS and 10 mg / mL MES; the first incubation temperature is 37° C.; and the first incubation time is 1 hour;

[0028] Step S34: After activation, the magnetic beads are magnetically separated and washed several times with PBS buffer to remove the activator. Preferably, the magnetic separation in step S34 is performed for two minutes, and the concentration of the PBS buffer in step S34 is 0.01 molar per liter (0.01M);

[0029] Preferably, the method for covalently coupling carboxyl magnetic beads and streptavidin in step S4 includes:

[0030] Step S41: resuspending the carboxyl magnetic beads activated in step S3 in a coupling buffer, adding streptavidin, and incubating on a constant temperature shaker for at least the second incubation time, separating the magnetic beads and removing the supernatant; in step S41, incubating on a constant temperature shaker at room temperature for at least 3 hours;

[0031] Step S42: Add PBS buffer (or phosphate buffer) to wash the magnetic beads several times, and add phosphate buffer to resuspend the magnetic beads. Preferably, the PBS buffer in step S42 is 0.01M PBS buffer; the phosphate buffer is 1000 μL of 0.01M phosphate buffer; the several times in step S42 can be three times;

[0032] Preferably, the method for stripping the physically bound streptavidin in step S5 comprises:

[0033] The streptavidin-coupled magnetic beads obtained in step S4 are placed in a dialysis bag, the dialysate is phosphate buffer, the dialysate is replaced during the period, and the dialysis is performed with magnetic stirring for a first period; preferably, the first period is 24 hours; the dialysate is 0.01M phosphate buffer.

[0034] Preferably, the method for blocking and storing the streptavidin magnetic beads in step S6 includes:

[0035] Step S61: Mixing the magnetic beads obtained by coupling carboxyl magnetic beads with streptavidin (i.e., streptavidin magnetic beads) with a blocking buffer to perform a blocking reaction; wherein the blocking buffer is prepared by phosphate buffer and BSA (the blocking buffer is prepared by 10 mL of phosphate buffer and 1% BSA);

[0036] Step S62: Add a preservation solution to the mixture obtained in step S61 for preservation. Further, the preservation solution (preservation buffer) contains 0.1% to 0.5% Proclin-300 and 0.1% to 1% BSA.

[0037] Preferably, the coupling buffer is prepared as follows: MES reagent is added to deionized water, and then an appropriate amount of potassium hydroxide solution is added to adjust the pH to a final pH of 5.0. After sterilization, it is stored at 4°C for future use.

[0038] Preferably, during the batch preparation of streptavidin magnetic beads, in step S2, the PS / SiO2 composite microspheres prepared in step S1 are resuspended and dispersed: the PS / SiO2 composite microspheres prepared in step S1 are added to anhydrous ethanol, and then stirred and dispersed using an ultrasonic stirring and dispersion device, wherein the ultrasonic stirring and dispersion device operates at corresponding target control parameters during the stirring and dispersion process;

[0039] The current method for obtaining target control parameters during the batch preparation of streptavidin magnetic beads includes:

[0040] Step S101: According to the specific process parameter requirements for the batch preparation of the current batch of streptavidin magnetic beads, PS / SiO2 composite microspheres obtained by a single execution of step S1 are obtained several times, and the PS / SiO2 composite microspheres obtained by each execution of step S1 are classified according to particle size to obtain parameters of each type of PS / SiO2 composite microspheres corresponding to each execution of step S1. The parameters of the PS / SiO2 composite microspheres include: the total weight of the corresponding type of PS / SiO2 composite microspheres and the equivalent diameter of the corresponding type of PS / SiO2 composite microspheres;

[0041] Step S102: determining the actual aggregation state coefficient W of the current batch of streptavidin magnetic beads based on the parameters of each type of PS / SiO2 composite microspheres obtained in step S101;

[0042] Step S103: determining a maximum equivalent diameter H based on the equivalent diameter of each type of PS / SiO2 composite microspheres obtained in step S101, and determining a target value of a dispersion intensity-related control parameter among target control parameters in the process of batch preparation of the current batch of streptavidin magnetic beads based on the maximum equivalent diameter and the actual aggregation state coefficient W;

[0043] Step S104: Determine the maximum equivalent diameter distribution ratio K based on the parameters of each type of PS / SiO2 composite microspheres obtained in step S101, and determine the target value of the working time of the ultrasonic stirring and dispersion device in the target control parameters during the batch preparation of the current batch of streptavidin magnetic beads based on the maximum equivalent diameter distribution ratio K and the actual agglomeration state coefficient W.

[0044] Preferably, the processes of step S11 and step S12 are both performed based on the reaction container, and the reaction container is passed through a nitrogen device to realize the deoxygenation process in the reaction container; the nitrogen source in the nitrogen device is a nitrogen balloon;

[0045] The nitrogen deoxygenation process includes:

[0046] Step S111: determining a target total deoxygenation amount for the current nitrogen deoxygenation based on the detection, and determining a target deoxygenation flow rate for the current nitrogen deoxygenation based on a target deoxygenation time for the current nitrogen deoxygenation process; and determining a target total nitrogen demand for the current nitrogen deoxygenation based on the target total deoxygenation amount for the current nitrogen deoxygenation process;

[0047] Step S112: determining the first nitrogen flow rate of the current nitrogen deoxygenation process based on the target deoxygenation flow rate-first nitrogen flow rate mapping table;

[0048] Based on the initial nitrogen amount of the nitrogen balloon before the current nitrogen and oxygenation, the target total nitrogen amount required for the current nitrogen and oxygenation, and the initial nitrogen amount of the nitrogen balloon before nitrogen and oxygenation - the target total nitrogen amount required for nitrogen and oxygenation - the average nitrogen pressure decay rate mapping table, determine the average nitrogen pressure decay rate corresponding to the current nitrogen and oxygenation, and calculate the time interval corresponding to the current nitrogen and oxygenation based on the average nitrogen pressure decay rate corresponding to the current nitrogen and oxygenation;

[0049] Divide the target deoxygenation time of the current nitrogen and deoxygenation process into several sub-time ranges according to the time interval corresponding to the current nitrogen and deoxygenation, and number the sub-time ranges;

[0050] Step S113: Dynamically control the nitrogen flow rate of the nitrogen supply device to the reaction container, wherein each sub-time range is nitrogen-deoxygenated at the target nitrogen flow rate of the corresponding sub-time range, and the target nitrogen flow rate of the next sub-time range is obtained by correcting the target nitrogen flow rate of the previous sub-time range by the total oxygen removal rate difference coefficient, the dissolved oxygen removal rate difference coefficient, and the pressure difference fluctuation coefficient of the previous sub-time range; the target nitrogen flow rate of the first sub-time range is the first nitrogen flow rate.

[0051] Preferably, step S113 includes:

[0052] Step S1131: In the current sub-time range, the nitrogen flow rate of the nitrogen supply device to the reaction vessel is controlled to be the target nitrogen flow rate corresponding to the current sub-time range, and in the current sub-time range: the oxygen flow rate of the oxygen outlet of the reaction vessel is detected multiple times, the dissolved oxygen concentration in the reaction vessel is detected multiple times, the nitrogen pressure of the nitrogen balloon is detected multiple times, and the ambient pressure in the reaction vessel is detected multiple times;

[0053] Step S1132: Calculating the dissolved oxygen exhaust rate difference coefficient for the current sub-time range based on the dissolved oxygen concentration in the reaction vessel detected in step S1131, calculating the total oxygen exhaust rate difference coefficient for the current sub-time range based on the oxygen flow rate at the oxygen outlet of the reaction vessel detected in step S1131, and calculating the pressure difference fluctuation coefficient for the current sub-time range based on the nitrogen pressure and ambient pressure detected in step S1131;

[0054] Step S1133: The target nitrogen flow rate for the next sub-time range is obtained by correcting the target nitrogen flow rate for the previous sub-time range by the total oxygen exhaust rate difference coefficient, dissolved oxygen exhaust rate difference coefficient, and pressure difference fluctuation coefficient for the previous sub-time range.

[0055] In a second aspect, the present invention provides streptavidin magnetic beads, which are obtained by the preparation scheme described in the first aspect.

[0056] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] The magnetic composite microspheres (streptavidin magnetic beads) prepared by the method of the present invention have controllable particle size, superparamagnetism, good stability, and strong activity. Compared with traditional methods, no additional cross-linking agent is required, which effectively simplifies the process and reduces costs. They have excellent performance during use on a chemiluminescence platform, and the process is easy to scale up and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] 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:

[0060] Figure 1 is a flow chart of the method of the present invention;

[0061] Figure 2 is a particle size distribution diagram of the streptavidin magnetic beads in Example 1 of the present invention;

[0062] Figure 3 is a SEM image of the streptavidin magnetic beads in Example 1 of the present invention;

[0063] Figure 4 is a VSM diagram of the streptavidin magnetic beads in Example 1 of the present invention;

[0064] Figure 5 This is a graph showing the measurement results of the streptavidin coupling efficiency of the invented streptavidin magnetic beads.

[0065] Figure 6 Schematic diagram of the determination of carboxyl content by conductivity titration of carboxyl magnetic composite microspheres in Example 1 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] The present invention provides streptavidin magnetic beads and a preparation method thereof. The present invention adopts prepared carboxyl magnetic beads to couple with streptavidin to prepare the streptavidin magnetic beads. Simultaneously, in combination with a specific process, the prepared streptavidin magnetic beads have high coupling efficiency, strong activity, high stability, and are conducive to process control.

[0069] The present invention provides the following embodiments:

[0070] Example 1:

[0071] This embodiment provides a method for preparing streptavidin magnetic beads, comprising the following steps:

[0072] 1. Preparation of PS / SiO2 composite microspheres:

[0073] (1) Weigh 2g of PVP (polyvinyl pyrrolidone) and dissolve it in 53g of anhydrous ethanol and 12g of deionized water. Transfer the mixture to a three-necked flask equipped with a condenser, nitrogen balloon, and mechanical stirrer. Stir at 400rpm for uniform dispersion and deoxygenate with nitrogen for 15min. Mix 0.15g of AIBN (azobisisobutyronitrile) and 10g of styrene and add the mixture. Then, heat the mixture to 70°C and allow the reaction to continue at this temperature for 11h.

[0074] (2) Take 0.3g of polystyrene and wash it by centrifugation. Then, ultrasonically disperse it with 63g of anhydrous ethanol and 7g of deionized water. Transfer it into a three-necked flask equipped with a condenser, a nitrogen balloon and a mechanical stirrer. Adjust the mechanical stirring rate to 500rpm to make it disperse evenly. When the temperature is raised to 70℃, add 0.3mL of methacryloyloxyethyl trimethylammonium chloride to modify the surface of the polystyrene microspheres. Deoxygenate with nitrogen for 15 minutes. After reacting for 6 hours, adjust the temperature to 50℃. After the temperature drops to 50℃, quickly add 0.4mL of ammonia water and 1.0mL of TEOS (tetraethyl orthosilicate) and continue the reaction for 2 hours.

[0075] 2. Preparation of carboxyl magnetic microspheres:

[0076] (1) The silicon-coated polystyrene microspheres obtained above were centrifuged and washed, and then added with 100 mL of anhydrous ethanol and dispersed with ultrasonic stirring for 30 minutes. 1 mL of a hydrophilic ferrosoferric oxide dispersion prepared by dispersing 0.2 mL of ammonia water, 0.4 mL of TEOS, and 20 mL of anhydrous ethanol was then added, and the mixture was reacted at 75°C for 7 hours. The obtained magnetic composite microspheres were washed three times with deionized water and three times with anhydrous ethanol magnetic separation.

[0077] (2) Add 50 mL of deionized water to disperse the washed magnetic composite microspheres by ultrasonic stirring for 30 minutes, add 1 g of glyphosate to the solution, adjust the pH to about 6 with ammonia water, and react at 40°C and 300 r / min for 2 hours. After the reaction is completed, use an external magnetic field to separate the resulting precipitate from the reaction system, and then wash it three times with ethanol and deionized water to obtain magnetic polystyrene microspheres modified with carboxyl groups on the surface.

[0078] 3. Activation of carboxyl magnetic composite microspheres:

[0079] (1) Take the magnetic bead stock solution and gently shake to mix, aspirate 5 mg of carboxyl magnetic bead stock solution into a 4 mL EP tube, add 2 mL of 0.01 M phosphate buffer and gently shake to wash, place on a magnetic stand for recovery, repeat this step three times, separate the magnetic beads and remove the supernatant.

[0080] (2) Add 100 μL of newly prepared 10 mg / mL NHS (abbreviation for N-hydroxysuccinimide; NHS is often used as a carboxyl activator) and 100 μL of newly prepared 10 mg / mL EDC solution (EDC solution refers to a solution containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl); EDC is a commonly used carboxyl activator) and incubate on a 37°C constant temperature shaker for 2 hours. After activation, the magnetic beads are separated for 2 minutes and 1 mL of 0.01 M phosphate buffer is added to wash the magnetic beads three times to remove the activator.

[0081] 4. Covalent coupling of carboxyl magnetic beads and streptavidin:

[0082] (1) Place carboxyl magnetic beads in 2 mL of 600 μg SA coupling buffer (usually refers to the buffer system used when coupling streptavidin (SA) to a carrier (such as magnetic beads or microspheres)). Add streptavidin and incubate on a constant temperature shaker for at least 3 hours. Separate the beads and remove the supernatant. The coupling buffer is prepared as follows: weigh 1.067 g MES (full name: 2-(N-morpholino)ethanesulfonic acid, a biological buffer) and dissolve it in 100 mL of deionized water. Then adjust the pH to 5.5 with 10% potassium hydroxide solution.

[0083] (2) Wash the magnetic beads three times with 2 mL of 0.01 M phosphate buffer and resuspend the magnetic beads with 500 μL of 0.01 M phosphate buffer.

[0084] 5. Stripping of physically bound streptavidin:

[0085] (1) Place the streptavidin-coupled magnetic beads into a dialysis membrane for dialysis. The dialysis solution is 500 mL of 0.01 M phosphate buffer. The dialysis solution is changed every 4 hours and magnetic stirring is applied for 24 hours.

[0086] (2) The stripped magnetic beads were incubated again with 1 mg / mL streptavidin coupling buffer, and then the magnetic beads were separated and washed three times with 1 mL of 0.01 M phosphate buffer.

[0087] 6. Blocking and storage of streptavidin magnetic beads:

[0088] (1) After magnetic bead separation and washing, add 500 μL of blocking buffer to the magnetic beads, mix gently, and incubate on a thermostatic shaker for 45 minutes. The blocking buffer is prepared by mixing BSA (BSA (Bovine Serum Albumin) is a globulin extracted from bovine serum) and phosphate buffer.

[0089] (2) Collect the magnetic beads using a magnetic rack and remove the supernatant.

[0090] (3) Add 500 μL of preservation solution to the magnetic beads for preservation. The preservation solution is prepared by 1% BSA, 0.05% proclin-300, and 0.01 M phosphate buffer.

[0091] Example 2:

[0092] This embodiment provides a method for preparing streptavidin magnetic beads, comprising the following steps:

[0093] 1. Preparation of PS / SiO2 composite microspheres:

[0094] (1) Weigh 2g of PVP and dissolve it in 53g of anhydrous ethanol and 12g of deionized water. Transfer the mixture to a three-necked flask equipped with a condenser, nitrogen balloon, and mechanical stirrer. Stir and disperse the mixture evenly at 400rpm. Deoxygenate with nitrogen for 15min. Mix 0.15g of AIBN and 10g of styrene and add the mixture. Then heat the mixture to 70°C and keep the mixture in the same temperature for 11h.

[0095] (2) Take 0.3g of polystyrene and wash it by centrifugation. Then, ultrasonically disperse it with 63g of anhydrous ethanol and 7g of deionized water. Transfer it into a three-necked flask equipped with a condenser, a nitrogen balloon and a mechanical stirrer. Adjust the mechanical stirring rate to 500rpm to make it disperse evenly. When the temperature is raised to 70℃, add 0.3mL of methacryloyloxyethyl trimethylammonium chloride to modify the surface of the polystyrene microspheres. Deoxygenate with nitrogen for 15min. After reacting for 6h, adjust the temperature to 50℃. After the temperature drops to 50℃, quickly add 0.4mL of ammonia water and 1.0mL of TEOS and continue the reaction for 2h.

[0096] 2. Preparation of carboxyl magnetic microspheres:

[0097] (1) The silicon-coated polystyrene microspheres obtained above were centrifuged and washed, and then added with 100 mL of anhydrous ethanol and dispersed with ultrasonic stirring for 30 minutes. 2 mL of a hydrophilic ferrosoferric oxide dispersion prepared by dispersing 0.2 mL of ammonia water, 0.4 mL of TEOS, and 20 mL of anhydrous ethanol was then added and reacted at 75°C for 7 hours. The obtained magnetic composite microspheres were washed three times with deionized water and three times with anhydrous ethanol magnetic separation.

[0098] (2) Add 50 mL of deionized water to disperse the washed magnetic composite microspheres by ultrasonic stirring for 30 minutes, add 1 g of glyphosate to the solution, adjust the pH to about 6 with ammonia water, and react at 300 r / min at 40°C for 2 hours. After the reaction is completed, use an external magnetic field to separate the resulting precipitate from the reaction system, and then wash it three times with ethanol and deionized water to obtain magnetic polystyrene microspheres modified with carboxyl groups on the surface.

[0099] 3. Activation of carboxyl magnetic composite microspheres:

[0100] (1) Take the magnetic bead stock solution and gently shake to mix, aspirate 5 mg of carboxyl magnetic bead stock solution into a 4 mL EP tube, add 2 mL of 0.01 M phosphate buffer and shake gently to wash, place on a magnetic stand for recovery, repeat this step three times, separate the magnetic beads and remove the supernatant.

[0101] (2) Add 200 μL of freshly prepared 10 mg / mL NHS and 200 μL of freshly prepared 10 mg / mL EDC solution and incubate at 37°C on a constant temperature shaker for 2 hours. After activation, the magnetic beads are separated for 2 minutes and washed three times with 1 mL of 0.01 M phosphate buffer to remove the activator.

[0102] 4. Covalent coupling of carboxyl magnetic beads and streptavidin

[0103] (1) Place carboxyl magnetic beads in 2 mL of coupling buffer containing 600 μg of SA, add streptavidin, and incubate on a shaker at room temperature for at least 3 hours. Separate the beads by magnetic separation and discard the supernatant. The coupling buffer is prepared as follows: weigh 1.067 g of MES and dissolve it in 100 mL of deionized water. Then adjust the pH to 5.5 with 10% potassium hydroxide solution.

[0104] (2) Wash the magnetic beads three times with 2 mL of 0.01 M phosphate buffer and resuspend the magnetic beads with 500 μL of 0.01 M phosphate buffer.

[0105] 5. Stripping of physically bound streptavidin:

[0106] (1) Place the streptavidin-coupled magnetic beads into a dialysis bag for dialysis. The dialysis fluid is 500 mL of 0.01 M phosphate buffer. The dialysis fluid is changed every 4 hours and magnetic stirring is performed for 24 hours.

[0107] (2) The stripped magnetic beads were incubated again with 500 μL of 1 mg / mL streptavidin coupling buffer, and then the magnetic beads were separated and washed three times with 1 mL of 0.01 M phosphate buffer.

[0108] 6. Blocking and storage of streptavidin magnetic beads:

[0109] (1) After magnetic bead separation and washing, the magnetic beads were added to 500 μL of blocking buffer, gently mixed, and incubated on a thermostatic shaker for 45 minutes. The blocking buffer was prepared by mixing BSA and phosphate buffer.

[0110] (2) Collect the magnetic beads using a magnetic rack and remove the supernatant.

[0111] (3) Add 500 μL of preservation solution to the magnetic beads for preservation. The preservation solution is prepared by 1% BSA, 0.05% proclin-300, and 0.01 M phosphate buffer.

[0112] Example 3:

[0113] This embodiment provides a method for preparing streptavidin magnetic beads, comprising the following steps:

[0114] 1. Preparation of PS / SiO2 composite microspheres:

[0115] (1) Weigh 2g of PVP and dissolve it in 53g of anhydrous ethanol and 12g of deionized water. Transfer the mixture to a three-necked flask equipped with a condenser, nitrogen balloon, and mechanical stirrer. Stir and disperse the mixture evenly at 400rpm. Deoxygenate with nitrogen for 15min. Mix 0.15g of AIBN and 10g of styrene and add the mixture. Then heat the mixture to 70°C and keep the mixture in the same temperature for 11h.

[0116] (2) Take 0.3g of polystyrene and wash it by centrifugation. Then, ultrasonically disperse it with 63g of anhydrous ethanol and 7g of deionized water. Transfer it into a three-necked flask equipped with a condenser, a nitrogen balloon and a mechanical stirrer. Adjust the mechanical stirring rate to 500rpm to make it disperse evenly. When the temperature is raised to 70℃, add 0.1mL of methacryloyloxyethyltrimethylammonium chloride to modify the surface of the polystyrene microspheres. Deoxygenate with nitrogen for 15min. After reacting for 6h, adjust the temperature to 50℃. After the temperature drops to 50℃, quickly add 0.4mL of ammonia water and 1mL of TEOS and continue the reaction for 2h.

[0117] 2. Preparation of carboxyl magnetic microspheres:

[0118] (1) The silicon-coated polystyrene microspheres obtained above were centrifuged and washed, and then added with 100 mL of anhydrous ethanol and dispersed with ultrasonic stirring for 30 minutes. 0.2 mL of ammonia water, 0.4 mL of LTEOS, and 2 mL of a hydrophilic ferrosoferric oxide dispersion dispersed in 20 mL of anhydrous ethanol were then added and reacted at 75°C for 7 hours. The obtained magnetic composite microspheres were washed three times with deionized water and three times with anhydrous ethanol magnetic separation.

[0119] (2) Add 50 mL of deionized water to disperse the washed magnetic composite microspheres by ultrasonic stirring for 30 minutes, add 1 g of glyphosate to the solution, adjust the pH to about 6 with ammonia water, and react at 300 r / min at 40°C for 2 hours. After the reaction is completed, use an external magnetic field to separate the resulting precipitate from the reaction system, and then wash it three times with ethanol and deionized water to obtain magnetic polystyrene microspheres modified with carboxyl groups on the surface.

[0120] 3. Activation of carboxyl magnetic composite microspheres:

[0121] (1) Take the magnetic bead stock solution and gently shake to mix, aspirate 5 mg of carboxyl magnetic bead stock solution into a 4 mL EP tube, add 2 mL of 0.01 M phosphate buffer and gently shake to wash, place on a magnetic stand for recovery, repeat this step three times, separate the magnetic beads and remove the supernatant.

[0122] (2) Add 100 μL of freshly prepared 10 mg / mL NHS and 100 μL of freshly prepared 10 mg / mL EDC solution and incubate at 37°C on a constant temperature shaker for 2 hours. After activation, the magnetic beads are separated for 2 minutes and washed three times with 1 mL of 0.01 M phosphate buffer to remove the activator.

[0123] 4. Covalent coupling of carboxyl magnetic beads and streptavidin

[0124] (1) Place carboxyl magnetic beads in 2 mL of coupling buffer containing 500 μg of SA, add streptavidin, and incubate on a constant temperature shaker at room temperature for at least 3 hours. Separate the beads by magnetic separation and remove the supernatant. The coupling buffer is prepared as follows: weigh 1.067 g of MES (full name: 2-(N-morpholino)ethanesulfonic acid, a biological buffer) and dissolve it in 100 mL of deionized water. Then adjust the pH to 5.5 with 10% potassium hydroxide (KOH) solution.

[0125] (2) Wash the magnetic beads three times with 2 mL of 0.01 M phosphate buffer and resuspend the magnetic beads with 500 μL of 0.01 M phosphate buffer.

[0126] 5. Stripping of physically bound streptavidin:

[0127] (1) Place the streptavidin-coupled magnetic beads into a dialysis bag for dialysis. The dialysis fluid is 500 mL of 0.01 M phosphate buffer. The dialysis fluid is changed every 4 hours and magnetic stirring is performed for 24 hours.

[0128] (2) The stripped magnetic beads were incubated again with 500 μL of 1 mg / mL streptavidin coupling buffer, and then the magnetic beads were separated and washed three times with 1 mL of 0.01 M phosphate buffer.

[0129] 6. Blocking and storage of streptavidin magnetic beads:

[0130] (1) After magnetic bead separation and washing, the magnetic beads were added to 500 μL of blocking buffer, gently mixed, and incubated on a thermostatic shaker for 45 minutes. The blocking buffer was prepared by mixing BSA and phosphate buffer.

[0131] (2) Collect the magnetic beads using a magnetic rack and remove the supernatant.

[0132] (3) Add 500 μL of preservation solution to the magnetic beads for preservation. The preservation solution is prepared by 1% BSA, 0.05% proclin-300, and 0.01 M phosphate buffer.

[0133] Figure 1 The particle size distribution diagram of the streptavidin magnetic beads obtained in Example 1 can be seen from the figure. It can be seen from the figure that the average particle size of the streptavidin magnetic beads is 1503 nm.

[0134] Figure 2 This is an SEM image of the streptavidin magnetic beads obtained in Example 1. It can be seen from the figure that the streptavidin magnetic beads have a good shape, uniform size, and are evenly coated with ferrosoferric oxide on the outer layer.

[0135] Figure 3This is the VSM diagram of the streptavidin magnetic beads obtained in Example 1. The dried magnetic composite microsphere powder was tested using a vibrating sample magnetometer. No remanence or coercive force was detected in the magnetic composite microsphere powder, indicating superparamagnetism. The magnetic saturation intensity of the magnetic nanoparticles was 22.1 emu / g.

[0136] Figure 4 This is a graph showing the measurement results of the streptavidin coupling efficiency of the streptavidin magnetic beads obtained in the present invention.

[0137] Figure 5 Schematic diagram of the determination of the carboxyl content of the carboxyl magnetic composite microspheres obtained in Example 1 by conductometric titration. It can be seen from the figure that the surface carboxyl content of the magnetic composite microspheres can reach 1.601 mmol / g.

[0138] Example 4, based on any one of Examples 1-3, during the batch preparation of streptavidin magnetic beads, in step S2, during the resuspending and dispersing process of the PS / SiO2 composite microspheres prepared in step S1: the PS / SiO2 composite microspheres prepared in step S1 are added to anhydrous ethanol, and then stirred and dispersed using an ultrasonic stirring and dispersing device, and the ultrasonic stirring and dispersing device operates according to the corresponding target control parameters during the stirring and dispersing process;

[0139] The current method for obtaining target control parameters during the batch preparation of streptavidin magnetic beads includes:

[0140] Step S101: According to the specific process parameter requirements for the batch preparation of the current batch of streptavidin magnetic beads, the PS / SiO2 composite microspheres obtained by a single execution of step S1 are obtained several times, and the PS / SiO2 composite microspheres obtained each time step S1 is executed are classified according to the particle size, and the parameters of each type of PS / SiO2 composite microspheres corresponding to each execution of step S1 are obtained. The parameters of the PS / SiO2 composite microspheres include: the total weight of the corresponding type of PS / SiO2 composite microspheres, and the equivalent diameter of the corresponding type of PS / SiO2 composite microspheres (each type of PS / SiO2 composite microspheres corresponds to a particle size range, and the median of the particle size range of the corresponding type of PS / SiO2 composite microspheres is the equivalent diameter);

[0141] Step S102: determining the actual aggregation state coefficient W of the current batch of streptavidin magnetic beads based on the parameters of each type of PS / SiO2 composite microspheres obtained in step S101;

[0142] Step S103: determining a maximum equivalent diameter H based on the equivalent diameter of each type of PS / SiO2 composite microspheres obtained in step S101, and determining a target value of a dispersion intensity-related control parameter among target control parameters in the process of batch preparation of the current batch of streptavidin magnetic beads based on the maximum equivalent diameter and the actual aggregation state coefficient W;

[0143] Step S104: Determine the maximum equivalent diameter distribution ratio K based on the parameters of each type of PS / SiO2 composite microspheres obtained in step S101, and determine the target value of the working time of the ultrasonic stirring and dispersion device in the target control parameters during the batch preparation of the current batch of streptavidin magnetic beads based on the maximum equivalent diameter distribution ratio K and the actual agglomeration state coefficient W.

[0144] The actual aggregation coefficient W of the current batch of streptavidin magnetic beads is calculated based on the following formula:

[0145]

[0146] M is the total number of PS / SiO2 composite microspheres classified; d i G is the equivalent diameter of the i-th type of PS / SiO2 composite microspheres obtained in the current step S101; i is the average total weight of the i-th type of PS / SiO2 composite microspheres obtained in a single execution of step S1 obtained in the current step S101; G is the average total weight of all types of PS / SiO2 composite microspheres obtained in a single execution of step S1 obtained in the current step S101; W is the actual aggregation state coefficient of the current batch of streptavidin magnetic beads;

[0147] in,

[0148] K is the maximum equivalent diameter distribution ratio; G0 is the average value of the total weight of the PS / SiO2 composite microspheres corresponding to the maximum equivalent diameter obtained in a single execution of step S1 obtained in the current step S101;

[0149] The control parameter related to the dispersion intensity can be any one of the power of the ultrasonic wave (in one embodiment of the present invention, the value can be 150-350W) and the frequency (in one embodiment of the present invention, the value can be 30-60KHZ), preferably the ultrasonic frequency;

[0150]

[0151] U is the target value of the dispersion strength-related control parameter among the target control parameters in the batch preparation process of the current batch of streptavidin magnetic beads; U0 is the standard value of the dispersion strength-related control parameter corresponding to U; R0 is the standard equivalent diameter; W0 is the standard aggregation state coefficient;

[0152] U0 is obtained based on the ultrasonic stirring dispersion test. Under the U0 condition, the ultrasonic dispersion effect meets the requirements. The diameter of the PS / SiO2 composite microspheres in the ultrasonic stirring dispersion test is the standard diameter (PS / SiO2 composite microspheres with similar diameters can be selected for testing, and the average diameter of the PS / SiO2 composite microspheres selected during the test is the standard diameter). During the ultrasonic stirring dispersion test, the agglomeration state coefficient of the PS / SiO2 composite microspheres is the standard agglomeration state coefficient;

[0153] in,

[0154]

[0155] t is the target value of the working time of the ultrasonic stirring and dispersing device in the target control parameter in the process of batch preparation of the current batch of streptavidin magnetic beads (a single execution of the dispersion of step S2); t0 is the standard stirring time of the ultrasonic stirring and dispersing device (the time for the PS / SiO2 composite microspheres to be uniformly dispersed during a single ultrasonic stirring and dispersing test); G ’ It is the total weight of the dispersed PS / SiO2 composite microspheres in a single ultrasonic stirring dispersion test.

[0156] Under the target values of the dispersion intensity-related control parameters among the target control parameters in the process of batch preparation of the current batch of streptavidin magnetic beads and the target value of the working time of the ultrasonic stirring dispersion device among the target control parameters in the process of batch preparation of the current batch of streptavidin magnetic beads, the PS / SiO2 composite microspheres are uniformly dispersed;

[0157] In one embodiment, the absorbance of different parts of the microsphere resuspension is measured at a specific wavelength using a spectrophotometer. The absorbance fluctuation is small, and the absorbance standard deviation is less than or equal to 0.03.

[0158] The beneficial effects of the above technical solution are:

[0159] The specific process parameter requirements (including raw material parameters and process parameters) for the batch preparation of different batches of streptavidin magnetic beads may be different, resulting in different PS / SiO2 composite microspheres obtained by a single execution of step S1 (including different diameters and particle size distributions, thus corresponding to different actual aggregation state coefficients, maximum equivalent diameters, and maximum equivalent diameter distribution ratios).

[0160] According to the specific process parameter requirements for the batch preparation of the current batch of streptavidin magnetic beads, the PS / SiO2 composite microspheres obtained by a single execution of step S1 are obtained several times, and the actual agglomeration state coefficient, maximum equivalent diameter, and maximum equivalent diameter distribution ratio corresponding to the current batch of streptavidin magnetic beads are classified and determined. Then, the target values of the dispersion intensity-related control parameters in the target control parameters in the process of batch preparation of the current batch of streptavidin magnetic beads and the target value of the working time of the ultrasonic stirring dispersion device in the target control parameters in the process of batch preparation of the current batch of streptavidin magnetic beads are determined based on the actual agglomeration state coefficient, maximum equivalent diameter, and maximum equivalent diameter distribution ratio, to ensure that the appropriate working parameters of the ultrasonic stirring dispersion device are determined and the stability and reliability of the batch preparation of the current batch of streptavidin magnetic beads are guaranteed.

[0161] Example 5, based on any one of Examples 1-4,

[0162] The processes of step S11 and step S12 are both performed based on the reaction container, and the reaction container is nitrogen-deoxygenated by a nitrogen gas supply device; the nitrogen source in the nitrogen gas supply device is a nitrogen balloon; the nitrogen gas supply device includes a nitrogen balloon, which is connected to the nitrogen gas outlet of the reaction container through a pipeline, and the pipeline is connected to a control valve;

[0163] The nitrogen deoxygenation process includes:

[0164] Step S111: determining a target total deoxygenation amount for the current nitrogen deoxygenation based on the detection, and determining a target deoxygenation flow rate for the current nitrogen deoxygenation based on a target deoxygenation time for the current nitrogen deoxygenation process; and determining a target total nitrogen demand for the current nitrogen deoxygenation based on the target total deoxygenation amount for the current nitrogen deoxygenation process;

[0165] Step S112: Determine the first nitrogen flow rate for the current nitrogen deoxygenation process based on a target deoxygenation flow rate-first nitrogen flow rate mapping table (determined based on testing, in the current reaction vessel, under the same solution conditions as those for the current nitrogen deoxygenation process);

[0166] Based on the initial nitrogen amount of the nitrogen balloon before the current nitrogen deoxygenation, the target total nitrogen amount required for the current nitrogen deoxygenation, and the initial nitrogen amount of the nitrogen balloon before the nitrogen deoxygenation-the target total nitrogen amount required for the nitrogen deoxygenation-the average nitrogen pressure decay rate mapping table, the average nitrogen pressure decay rate corresponding to the current nitrogen deoxygenation is determined, and the time interval corresponding to the current nitrogen deoxygenation is calculated based on the average nitrogen pressure decay rate corresponding to the current nitrogen deoxygenation; in the present invention, the nitrogen balloon is filled with air and then the nitrogen is discharged to construct a curve of the total nitrogen remaining in the nitrogen balloon-the pressure change in the nitrogen balloon (the horizontal axis is the total nitrogen remaining in the nitrogen balloon, and the vertical axis is the pressure in the nitrogen balloon). pressure), based on the curve, a mapping table of the initial nitrogen amount of the nitrogen balloon before nitrogen and deoxygenation - the target total nitrogen amount required for nitrogen and deoxygenation - the average nitrogen pressure decay rate can be obtained (a curve segment of the curve of the initial nitrogen amount of the nitrogen balloon before the current nitrogen and deoxygenation - the target total nitrogen amount required for nitrogen and deoxygenation corresponds to the remaining total nitrogen amount in the nitrogen balloon - the pressure change curve in the nitrogen balloon (the initial ordinate of the curve segment is the initial nitrogen amount of the nitrogen balloon before the current nitrogen and deoxygenation, and the end ordinate of the curve segment is the initial nitrogen amount of the nitrogen balloon before the current nitrogen and deoxygenation minus the target total nitrogen amount required for the current nitrogen and deoxygenation), and the average nitrogen pressure decay rate of the curve segment can be calculated);

[0167] Divide the target deoxygenation time of the current nitrogen and deoxygenation process into several sub-time ranges according to the time interval corresponding to the current nitrogen and deoxygenation, and number the sub-time ranges;

[0168] Step S113: Dynamically control the nitrogen flow rate of the nitrogen supply device to the reaction container, wherein each sub-time range is nitrogen-deoxygenated at the target nitrogen flow rate of the corresponding sub-time range, and the target nitrogen flow rate of the next sub-time range is obtained by correcting the target nitrogen flow rate of the previous sub-time range by the total oxygen removal rate difference coefficient, the dissolved oxygen removal rate difference coefficient, and the pressure difference fluctuation coefficient of the previous sub-time range; the target nitrogen flow rate of the first sub-time range is the first nitrogen flow rate.

[0169] Among them, the target total oxygen removal amount of the current nitrogen deoxygenation is detected and determined as follows: the target total oxygen removal amount of the current nitrogen deoxygenation is obtained by detecting and determining the total amount of oxygen that needs to be removed in the environment of the reaction container and the amount of dissolved oxygen that needs to be removed in the solution; this is the existing technology and will not be repeated here.

[0170] in,

[0171] t is the time interval corresponding to the current nitrogen deoxygenation; ΔP is the maximum difference in the air pressure in the nitrogen balloon allowed in each sub-time range (the value can be 0.05 MPa); is the rounding down symbol; θ0 is the average nitrogen pressure decay rate corresponding to the current nitrogen deoxygenation;

[0172] The beneficial effects of the above technical solution are:

[0173] The nitrogen flow rate from the nitrogen supply device to the reaction vessel is dynamically controlled, wherein each sub-time range is nitrogen-deoxygenated at the target nitrogen flow rate of the corresponding sub-time range, and the target nitrogen flow rate of the next sub-time range is obtained by correcting the target nitrogen flow rate of the previous sub-time range by the total oxygen removal rate difference coefficient, the dissolved oxygen removal rate difference coefficient, and the pressure difference fluctuation coefficient of the previous sub-time range, thereby ensuring that the nitrogen supply effect and oxygen removal in each sub-time range meet the requirements, and ensuring the stability and reliability of nitrogen supply and deoxygenation.

[0174] Example 6, based on Example 5, step S113 includes:

[0175] Step S1131: In the current sub-time range, the nitrogen flow rate of the nitrogen supply device to the reaction vessel is controlled to be the target nitrogen flow rate corresponding to the current sub-time range, and in the current sub-time range: the oxygen flow rate of the oxygen outlet of the reaction vessel is detected multiple times, the dissolved oxygen concentration in the reaction vessel is detected multiple times, the nitrogen pressure of the nitrogen balloon is detected multiple times, and the ambient pressure in the reaction vessel is detected multiple times;

[0176] Step S1132: Calculating the dissolved oxygen exhaust rate difference coefficient for the current sub-time range based on the dissolved oxygen concentration in the reaction vessel detected in step S1131, calculating the total oxygen exhaust rate difference coefficient for the current sub-time range based on the oxygen flow rate at the oxygen outlet of the reaction vessel detected in step S1131, and calculating the pressure difference fluctuation coefficient for the current sub-time range based on the nitrogen pressure and ambient pressure detected in step S1131;

[0177] Step S1133: The target nitrogen flow rate for the next sub-time range is obtained by correcting the target nitrogen flow rate for the previous sub-time range by the total oxygen exhaust rate difference coefficient, dissolved oxygen exhaust rate difference coefficient, and pressure difference fluctuation coefficient for the previous sub-time range.

[0178]

[0179] Among them, W 1j is the coefficient of difference in dissolved oxygen removal rate in the jth sub-time range, W 2j is the total oxygen exhaust rate difference coefficient of the jth sub-time range, U 1j is the pressure difference fluctuation coefficient of the jth sub-time range, Q js is the detection value of the dissolved oxygen concentration in the reaction container for the sth time in the jth sub-time range (N is greater than or equal to 2); Q j(s-1) is the detected value of the dissolved oxygen concentration in the reaction container during the s-1th detection in the jth sub-time range (N is greater than or equal to 2); j0is the time difference between the two detections of dissolved oxygen concentration in the reaction container within the jth sub-time range; N is the number of dissolved oxygen concentration detections in each sub-time range; G0 is the target dissolved oxygen exhaust rate ( corresponding target value); H j is the average oxygen flow rate of the oxygen outlet of the reaction vessel detected in the jth sub-time range; H2 is the target deoxygenation flow rate of the current nitrogen deoxygenation; P j1 is the average detected value of the nitrogen pressure of the nitrogen balloon in the jth sub-time range; P j2 is the average detected value of the ambient pressure in the reaction vessel during the jth sub-time range; P3 is the target difference between the nitrogen pressure and the ambient pressure during nitrogen ventilation and oxygen removal (the value can be 0.1-0.3 MPa);

[0180] Q j+1 =ln(e+γ1W 1j +γ2W 2j +γ3U 1j )*Q j ;

[0181] Q j+1 is the target nitrogen flow rate in the j+1th sub-time range; Q j is the target nitrogen flow rate for the jth sub-time range; γ1, γ2, and γ3 are respectively the first weight (a value greater than 0 and less than 1, such as 0.38-0.62), the second weight (a value greater than 0 and less than 1, such as 0.3-0.6), and the third weight (a value greater than 0 and less than 1, such as 0.08-0.32). ln is the natural logarithm, and e is a natural constant;

[0182] The beneficial effects of the above technical solution are:

[0183] The formula comprehensively considers multiple key factors, including the dissolved oxygen removal rate variance coefficient, the total oxygen removal rate variance coefficient, and the pressure differential fluctuation coefficient. These factors reflect the state of the deoxygenation process from different perspectives. By incorporating them into the calculation, it can comprehensively and accurately reflect the nitrogen flow rate requirements of the current deoxygenation conditions, avoiding the deviation caused by adjusting based on only one factor, making the nitrogen flow rate adjustment more in line with actual conditions and improving the accuracy and effectiveness of deoxygenation.

[0184] The formula provides a specific quantitative calculation method for automated control of the nitrogen deoxygenation process. Relevant test data can be input into the control system, and the nitrogen flow rate can be automatically calculated and adjusted based on the formula, achieving automated and intelligent control of the deoxygenation process, reducing manual intervention errors and improving control accuracy and efficiency.

[0185] A reasonable nitrogen flow rate can maintain a stable gas environment and pressure conditions within the reactor, which is conducive to the deoxygenation reaction proceeding under appropriate conditions. Stable process conditions can reduce the adverse effects of environmental fluctuations on the deoxygenation effect, improve the stability and reliability of the deoxygenation process, and thus enhance the quality of the deoxygenated product or process.

[0186] 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 streptavidin magnetic beads, characterized in that: include: Step S1: Preparation of PS / SiO2 composite microspheres; Step S2: preparing carboxyl magnetic beads based on the PS / SiO2 composite microspheres prepared in step S1; Step S3: Activation of carboxyl magnetic beads; Step S4: covalent coupling of carboxyl magnetic beads and streptavidin; Step S5: stripping of physically bound streptavidin; Step S6: Blocking and storage of streptavidin magnetic beads.

2. The method for preparing streptavidin magnetic beads according to claim 1, wherein: The preparation method of PS / SiO2 composite microspheres in step S1 includes: Step S11: using styrene as a monomer and ethanol and deionized water as a mixed solvent, reacting under the action of an initiator to produce polystyrene microspheres; Step S12: The polystyrene microspheres obtained in step S11 are synthesized into core-shell PS / SiO2 microspheres using tetraethyl orthosilicate in the presence of ammonia water. The core-shell PS / SiO2 microspheres are the PS / SiO2 composite microspheres.

3. The method for preparing streptavidin magnetic beads according to claim 1, wherein: The preparation method of carboxyl magnetic beads in step S2 includes: Step S21: resuspending and dispersing the PS / SiO2 composite microspheres prepared in step S1, and synthesizing magnetic composite microspheres using a hydrophilic ferrosilicate dispersion in the presence of ethyl orthosilicate and ammonia water; Step S22: adding an appropriate amount of ammonia water, reacting with glyphosate at a certain pH and the first reaction temperature to modify the carboxyl group, thereby obtaining carboxyl magnetic beads.

4. The method for preparing streptavidin magnetic beads according to claim 1, wherein: The activation method of the carboxyl magnetic beads in step S3 includes: Step S31: taking a carboxyl magnetic bead stock solution into a container, placing the container on a magnetic stand for magnetic separation, and removing the preservation solution; Step S32: adding PBS buffer to wash the magnetic beads several times, and magnetically separating the magnetic beads to remove the supernatant; Step S33: adding freshly prepared NHS and MES solutions and incubating in a constant temperature shaker at a first incubation temperature for a first incubation time to activate the carboxyl groups on the surface of the magnetic beads; Step S34: After activation, the magnetic beads are magnetically separated and PBS buffer is added to wash the magnetic beads several times to remove the activator.

5. The method for preparing streptavidin magnetic beads according to claim 1, wherein: The method for covalently coupling carboxyl magnetic beads and streptavidin in step S4 includes: Step S41: resuspending the carboxyl magnetic beads activated in step S3 in a coupling buffer, adding streptavidin, incubating on a constant temperature shaker for at least the second incubation time, and separating the beads by magnetic separation and removing the supernatant; Step S42: adding PBS buffer to wash the magnetic beads several times, and adding phosphate buffer to resuspend the magnetic beads.

6. The method for preparing streptavidin magnetic beads according to claim 1, wherein: The method for stripping the physically bound streptavidin in step S5 includes: The streptavidin-coupled magnetic beads obtained in step S4 are placed in a dialysis bag, the dialysate is phosphate buffered saline, the dialysate is replaced during the dialysis, and the dialysis is performed for the first time under magnetic stirring; The method for blocking and storing the streptavidin magnetic beads in step S6 includes: Step S61: mixing the magnetic beads obtained by coupling carboxyl magnetic beads with streptavidin with a blocking buffer to perform a blocking reaction; Step S62: Add preservation solution to the mixture obtained in step S61 for preservation.

7. The method for preparing streptavidin magnetic beads according to claim 1, wherein: The coupling buffer was prepared as follows: MES reagent was added to deionized water, and then an appropriate amount of potassium hydroxide solution was added to adjust the pH to 5.

0. The final pH value was stored at 4° C. after sterilization.

8. The method for preparing streptavidin magnetic beads according to claim 1, wherein: During the batch preparation of streptavidin magnetic beads, in step S2, the PS / SiO2 composite microspheres prepared in step S1 are resuspended and dispersed: the PS / SiO2 composite microspheres prepared in step S1 are added to anhydrous ethanol, and then stirred and dispersed using an ultrasonic stirring and dispersion device, wherein the ultrasonic stirring and dispersion device operates according to corresponding target control parameters during the stirring and dispersion process; The current method for obtaining target control parameters during the batch preparation of streptavidin magnetic beads includes: Step S101: According to the specific process parameter requirements for the batch preparation of the current batch of streptavidin magnetic beads, PS / SiO2 composite microspheres obtained by a single execution of step S1 are obtained several times, and the PS / SiO2 composite microspheres obtained by each execution of step S1 are classified according to particle size to obtain parameters of each type of PS / SiO2 composite microspheres corresponding to each execution of step S1. The parameters of the PS / SiO2 composite microspheres include: the total weight of the corresponding type of PS / SiO2 composite microspheres and the equivalent diameter of the corresponding type of PS / SiO2 composite microspheres; Step S102: determining the actual aggregation state coefficient W of the current batch of streptavidin magnetic beads based on the parameters of each type of PS / SiO2 composite microspheres obtained in step S101; Step S103: determining a maximum equivalent diameter H based on the equivalent diameter of each type of PS / SiO2 composite microspheres obtained in step S101, and determining a target value of a dispersion intensity-related control parameter among target control parameters in the process of batch preparation of the current batch of streptavidin magnetic beads based on the maximum equivalent diameter and the actual aggregation state coefficient W; Step S104: Determine the maximum equivalent diameter distribution ratio K based on the parameters of each type of PS / SiO2 composite microspheres obtained in step S101, and determine the target value of the working time of the ultrasonic stirring and dispersion device in the target control parameters during the batch preparation of the current batch of streptavidin magnetic beads based on the maximum equivalent diameter distribution ratio K and the actual agglomeration state coefficient W.

9. The method for preparing streptavidin magnetic beads according to claim 1, wherein: The processes of step S11 and step S12 are both performed based on the reaction container, and the reaction container is subjected to a nitrogen-gas ... The nitrogen deoxygenation process includes: Step S111: determining a target total deoxygenation amount for the current nitrogen deoxygenation based on the detection, and determining a target deoxygenation flow rate for the current nitrogen deoxygenation based on a target deoxygenation time for the current nitrogen deoxygenation process; and determining a target total nitrogen demand for the current nitrogen deoxygenation based on the target total deoxygenation amount for the current nitrogen deoxygenation process; Step S112: determining the first nitrogen flow rate of the current nitrogen deoxygenation process based on the target deoxygenation flow rate-first nitrogen flow rate mapping table; Based on the initial nitrogen amount of the nitrogen balloon before the current nitrogen and oxygenation, the target total nitrogen amount required for the current nitrogen and oxygenation, and the initial nitrogen amount of the nitrogen balloon before nitrogen and oxygenation - the target total nitrogen amount required for nitrogen and oxygenation - the average nitrogen pressure decay rate mapping table, determine the average nitrogen pressure decay rate corresponding to the current nitrogen and oxygenation, and calculate the time interval corresponding to the current nitrogen and oxygenation based on the average nitrogen pressure decay rate corresponding to the current nitrogen and oxygenation; Divide the target deoxygenation time of the current nitrogen and deoxygenation process into several sub-time ranges according to the time interval corresponding to the current nitrogen and deoxygenation, and number the sub-time ranges; Step S113: Dynamically control the nitrogen flow rate of the nitrogen supply device to the reaction container, wherein each sub-time range is nitrogen-deoxygenated at the target nitrogen flow rate of the corresponding sub-time range, and the target nitrogen flow rate of the next sub-time range is obtained by correcting the target nitrogen flow rate of the previous sub-time range by the total oxygen removal rate difference coefficient, the dissolved oxygen removal rate difference coefficient, and the pressure difference fluctuation coefficient of the previous sub-time range; the target nitrogen flow rate of the first sub-time range is the first nitrogen flow rate.

10. The method for preparing streptavidin magnetic beads according to claim 9, wherein: Step S113 includes: Step S1131: In the current sub-time range, the nitrogen flow rate of the nitrogen supply device to the reaction vessel is controlled to be the target nitrogen flow rate corresponding to the current sub-time range, and in the current sub-time range: the oxygen flow rate of the oxygen outlet of the reaction vessel is detected multiple times, the dissolved oxygen concentration in the reaction vessel is detected multiple times, the nitrogen pressure of the nitrogen balloon is detected multiple times, and the ambient pressure in the reaction vessel is detected multiple times; Step S1132: Calculating the dissolved oxygen exhaust rate difference coefficient for the current sub-time range based on the dissolved oxygen concentration in the reaction vessel detected in step S1131, calculating the total oxygen exhaust rate difference coefficient for the current sub-time range based on the oxygen flow rate at the oxygen outlet of the reaction vessel detected in step S1131, and calculating the pressure difference fluctuation coefficient for the current sub-time range based on the nitrogen pressure and ambient pressure detected in step S1131; Step S1133: The target nitrogen flow rate for the next sub-time range is obtained by correcting the target nitrogen flow rate for the previous sub-time range by the total oxygen exhaust rate difference coefficient, dissolved oxygen exhaust rate difference coefficient, and pressure difference fluctuation coefficient for the previous sub-time range.

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