Nanometer magnetic bead and preparation method thereof

Abstract

The invention relates to the technical field of cell sorting, in particular to a nano magnetic bead and a preparation method thereof. Wherein the nano magnetic bead comprises a magnetic core formed by Fe3O4 and gamma-Fe2O3, and a glucan layer coated on the outer part of the magnetic core; and the glucan layer contains polyethylene glycol. The magnetic core is coated with polyethylene glycol, so that the magnetic beads are not easy to agglomerate or aggregate in the storage process, the storage time of the magnetic beads is prolonged, and the efficiency of the magnetic beads during cell sorting is higher.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell sorting, in particular to a nano magnetic bead and a preparation method thereof. BACKGROUND

[0002] Magnetic beads have gradually developed into an important means for sorting cells due to their characteristics such as simplicity, high efficiency and strong specificity. Generally, magnetic sorting is mainly based on the modification of magnetic beads with antibodies, proteins and other modifiers, and through the specific binding of the modifiers and cell surface molecules, the purpose cells can be quickly separated under the action of an external magnetic field.

[0003] In the prior art, the synthesis route of the magnetic bead is usually to first synthesize a functionalized magnetic core, then coat the magnetic core into a magnetic bead and couple it with a biological ligand to realize specific treatment of the magnetic bead. However, as the particle size of the magnetic bead decreases, the agglomeration effect becomes more and more obvious, which affects the specific treatment efficiency of the magnetic bead, and thus affects the separation effect of the purpose cells.

[0004] Therefore, the present application is proposed. SUMMARY

[0005] The present application aims to provide a nano magnetic bead and a preparation method thereof to solve at least one technical problem mentioned in the background.

[0006] Specifically, the first aspect of the present application provides a nano magnetic bead, The nano magnetic bead comprises a magnetic core formed of Fe3O4 and γ-Fe2O3, and a dextran layer coated outside the magnetic core. The dextran layer contains polyethylene glycol.

[0007] By coating polyethylene glycol outside the magnetic core, the above technical solution can prevent the magnetic bead from agglomerating or gathering during storage, thereby prolonging the storage time of the magnetic bead and improving the efficiency of the magnetic bead in sorting cells.

[0008] Preferably, the polyethylene glycol is polyethylene glycol 600.

[0009] Preferably, the dextran is carboxylated dextran.

[0010] Preferably, the nano magnetic bead further comprises an antibody connected to the carboxylated dextran, and the antibody is a CD4 antibody.

[0011] The second aspect of the present application provides a preparation method of a nano magnetic bead, comprising the steps of: Preparation of a solution containing Fe 3+ , Fe 2+ , and mixing the components at 50-80℃ after adding an appropriate amount of dextran. The present application has the advantages of the following aspects:3 + Fe3+ concentration is 0.01-0.05mol / l, and the concentration of Fe2+ is 0.03-0.06mol / l; 2+ Fe3+ concentration is 0.01-0.05mol / l, and the concentration of Fe2+ is 0.03-0.06mol / l; Adjust the pH of the solution to be alkaline, add an appropriate amount of polyethylene glycol 600, and continue the reaction at a temperature of 50-80℃. After the reaction is completed, the solution is rapidly cooled to obtain the dextran-coated magnetic beads.

[0012] Preferably, the preparation method of the nanometer magnetic beads further comprises the steps of: The magnetic beads are placed in the storage solution to form a magnetic bead suspension of about 10mg / ml.

[0013] Preferably, the components of the storage solution are: Trehalose 2-8%, polyethylene glycol 2000 0.5-2%, sodium ascorbate 0.05-0.1%, BSA 0.2-1%, poloxamer 0.02-0.1%; EDTA 0.4-0.6mM, sodium citrate 4-6mM; The pH of the solution is 7.5-9.

[0014] Preferably, the magnetic beads are placed in the storage solution for no more than 20 days.

[0015] Preferably, the preparation method of the nanometer magnetic beads further comprises the step of connecting an antibody: The dextran-coated magnetic beads are washed with a buffer solution to form a magnetic bead suspension; Add 10mg / ml EDC and 10mg / ml NHS, and activate the carboxyl group at 36-38℃ for 0.5-2h; Resuspend the magnetic beads in the buffer solution after removing the supernatant; Add the antibody and incubate in a rotating incubator at 36-38℃ for 6-12h.

[0016] Preferably, the antibody is a CD4 antibody.

[0017] In summary, the present application has the following beneficial effects: First, the nanometer magnetic beads provided by the present application can prevent aggregation or aggregation during storage by coating polyethylene glycol on the outside of the magnetic core, thereby prolonging the storage time of the magnetic beads and improving the efficiency of the magnetic beads in sorting cells.

[0018] Second, the preparation method of the nanometer magnetic beads provided by the present application can better cooperate with the magnetic bead storage solution provided by the present application, improve the storage efficiency of the magnetic beads, and reduce the occurrence of aggregation.

[0019] Thirdly, the nano magnetic bead preparation method provided by the application has higher binding efficiency with antibodies, especially CD4 antibodies, so that the magnetic beads have stronger specificity in sorting cells containing CD4 antigens. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Flow cytometry diagram corresponding to the sorting of cells in Example 10; Figure 2 Flow cytometry diagram corresponding to the sorting of cells in Example 11; Figure 3 Flow cytometry diagram corresponding to the sorting of cells in Example 12; Figure 4 Flow cytometry diagram corresponding to the sorting of cells in Example 13; Figure 5 Flow cytometry diagram corresponding to the sorting of cells in Example 14; Figure 6 Flow cytometry diagram corresponding to the sorting of cells in Example 15; Figure 7 Flow cytometry diagram corresponding to the sorting of cells in Example 16; Figure 8 Flow cytometry diagram corresponding to the sorting of cells in Example 17; Figure 9 Flow cytometry diagram corresponding to the sorting of cells in Example 18; Figure 10 Flow cytometry diagram corresponding to the sorting of cells in Comparative Example 1; Figure 11 Flow cytometry diagram corresponding to the sorting of cells in Comparative Example 2; Figure 12 Flow cytometry diagram corresponding to the sorting of cells in Comparative Example 3; Figure 13 Flow cytometry diagram corresponding to the sorting of cells in Comparative Example 4; Figure 14 Flow cytometry diagram corresponding to the sorting of cells in Comparative Example 5. DETAILED DESCRIPTION

[0021] The exemplary embodiments will be described in detail herein with reference to the attached drawings. In the following description, like reference numerals refer to like elements, unless the context clearly dictates otherwise. The following exemplary embodiments are described in the context of the accompanying drawings, which are not necessarily to scale. The following description of the exemplary embodiments is not meant to limit or restrict the application to the embodiments set forth rather, the scope of the application is to be determined entirely by the claims as issued by the patent office.

[0022] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will also be understood that the term "and / or," as used herein, refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0023] The application will be described in detail below through examples.

[0024] In the prior art, the synthesis route of magnetic beads is generally to first synthesize a functionalized magnetic core, then coat the magnetic core into magnetic beads, and then couple with a biological ligand to realize specific treatment of the magnetic beads. However, as the particle size of the magnetic beads decreases, the agglomeration effect becomes more and more obvious, which affects the specific treatment efficiency of the magnetic beads, and thus affects the separation effect of the target cells.

[0025] In view of this, to solve the technical problems existing in the background art, the inventive concept of the present application provides a kind of nanometer magnetic beads and its preparation method, the nanometer magnetic beads include the magnetic core formed by Fe3O4 And γ-Fe2O3, and the dextran layer coated outside the magnetic core;Wherein, the dextran layer contains polyethylene glycol.

[0026] According to the inventive concept, by coating polyethylene glycol outside the magnetic core, the magnetic beads are less likely to agglomerate or aggregate during storage, thereby increasing the storage time of the magnetic beads and improving the efficiency of the magnetic beads in sorting cells.

[0027] In order to better understand the above technical solutions, the above technical solutions will be described in detail below in combination with specific embodiments, and those skilled in the art should understand that the reaction time, drug dosage involved in the present application cannot be absolutely accurate in actual production process, but are within the allowable error range. For example, if a sample is expected to be heated for 30 minutes, the actual operation may be 1 second more or less than 30 minutes; if a sample weighing 30g is expected, the actual operation may weigh 30.001g or 29.998g.

[0028] Unless otherwise specified, in the experimental examples and embodiments of the present application, the cells used for sorting are human peripheral blood mononuclear cells (PBMCs), and the CD4 antibody is an anti-human CD4 antibody.

[0029] Experimental Example 1 Magnetic bead agglomeration experiment The magnetic bead agglomeration experiment uses the prior art, specifically: A Nanosight particle size analyzer is used to distinguish single particles and agglomerate particle sizes by measuring the change in resistance of the magnetic beads passing through the nanopore, and to calculate the ratio of the number of magnetic beads above 500 nm to the number of magnetic beads at or below 500 nm.

[0030] Example 2 Antibody conjugation experiment The antibody conjugation experiment employs prior art, specifically: Dilute the fresh blood with anticoagulant with PBS by 2 times, then disperse the blood to the lymph separation liquid according to the volume ratio of blood:lymph separation liquid = 1:1; obtain the peripheral blood mononuclear cells after centrifugation, and adjust the cell concentration to 5*10 7 cells / ml; Take 100 μl of the cell suspension obtained in the previous step and mix with 5 μl of the CD4 antibody conjugated nanometer magnetic beads (provided in the present application), and then place at 4°C for 30 min; centrifuge the above suspension at 1500 rpm for 5 min, and then discard the supernatant, and resuspend the cell precipitate with 500 μl of PBS buffer; Wash the cells with the sorting column for 3 times, each time with 500 μl of PBS buffer, and then add 1 ml of PBS buffer to wash the cells down and collect into an EP tube; Centrifuge again at 1500 rpm for 5 min to discard the supernatant, resuspend the cell precipitate with 500 μl of PBS buffer, add 10 μl of CD4 antibody labeled with PE, and incubate at 4°C for 20 min; Collect the sorted cells in a serum-containing culture medium to maintain cell activity, first centrifuge at 500g for 10 minutes, discard the supernatant, resuspend the cells with PBS, repeat the centrifugal washing for 1-2 times, and finally adjust the cell concentration to 1*10 6 -1*10 7 -1*10

[0031] Example 3 Preparation experiment of preservation solution The present embodiment aims to study the formula of the preservation solution suitable for the magnetic beads of the present application.

[0032] Technical solution (A) Prepare the preservation solution: 1% polyethylene glycol-2000, 0.1% sodium ascorbate, 0.5% BSA, 0.05% Pluronic F-68, 0.5 mM EDTA, 5 mM sodium citrate; Adjust the pH of the solution to 8.0 with 10 mM Tris-HCl, and filter sterilization with a 0.22 μm filter membrane.

[0033] Prepare the magnetic beads: Take 15 parts of FeCl3·6H2O and 5 parts of FeCl2·4H2O, dissolve in 1000 parts of deionized water, pass nitrogen to remove oxygen for 15 min, then add 10 parts of 40 kDa dextran, and stir at 70°C and 15 rpm / min for 30 min; Add 8 mol / L ammonia water to adjust pH to 10; add 2 parts of polyethylene glycol 600 and control temperature at 70°C to continue reaction for 1.5 h, stop reaction and cool to 20°C within 3 min; After the reaction, the obtained magnetic beads were adsorbed by a magnet, and the supernatant was removed. Then, the magnetic beads were washed twice with 1.0 mol / l HCl and twice with deionized water.

[0034] The magnetic beads were suspended in the storage solution prepared in the present application to form a magnetic bead suspension of about 10 mg / ml.

[0035] Technical solution (B) Preparation of storage solution: 5% trehalose, 0.1% sodium ascorbate, 0.5% BSA, 0.05% Pluronic F-68, 0.5 mM EDTA, 5 mM sodium citrate; Adjust the pH of the solution to 8.0 with 10 mM Tris-HCl, and filter sterilize with a 0.22 μm filter.

[0036] Preparation of magnetic beads: The method is the same as that of technical solution (A).

[0037] Technical solution (C) Preparation of storage solution: 5% trehalose, 1% polyethylene glycol-2000, 0.1% sodium ascorbate, 0.5% BSA, 0.05% Pluronic F-68, 0.5 mM EDTA, 5 mM sodium citrate; Adjust the pH of the solution to 8.0 with 10 mM Tris-HCl, and filter sterilize with a 0.22 μm filter.

[0038] Preparation of magnetic beads: Weigh 15 parts of FeCl3·6H2O and 5 parts of FeCl2·4H2O, dissolve in 1000 parts of deionized water, and then pass nitrogen to remove oxygen for 15 min. Then, add 10 parts of 40 kDa dextran and stir at 70°C and 15 rpm / min for 30 min; Adjust the pH to 10 with 8 mol / L ammonia water, and control the temperature at 70°C to continue the reaction for 1.5 h. Stop the reaction and cool to 20°C within 3 min. After the reaction, the obtained magnetic beads were adsorbed by a magnet, and the supernatant was removed. Then, the magnetic beads were washed twice with 1.0 mol / l HCl and twice with deionized water.

[0039] The magnetic beads were suspended in the storage solution prepared in the present application to form a magnetic bead suspension of about 10 mg / ml.

[0040] Technical solution (D) Preparation of the storage solution: The method is the same as that of technical solution (C).

[0041] Preparation of the magnetic beads: Take 15 parts of FeCl3·6H2O and 5 parts of FeCl2·4H2O, dissolve them in 1000 parts of deionized water, and then pass nitrogen gas to remove oxygen for 15 minutes. Then, add 10 parts of 40 kDa dextran, and stir at 70℃ and 15 rpm / min for 30 minutes. Adjust the pH to 10 by adding ammonia water with a concentration of 8 mol / L. Then, add 2 parts of polyethylene glycol 600, and control the temperature at 70℃ to continue the reaction for 1.5 hours. Stop the reaction and cool it to 20℃ within 3 minutes. After the reaction is completed, use a magnet to adsorb the magnetic beads obtained by the synthesis. After removing the supernatant, use 1.0 mol / l HCl to clean the magnetic core twice, and then use deionized water to clean the magnetic core twice.

[0042] Put the magnetic beads in the storage solution prepared in this solution to form a magnetic bead suspension of about 10 mg / ml.

[0043] Technical solution (E) Preparation of the storage solution: The method is the same as that of technical solution (C).

[0044] Preparation of carboxyl dextran: Dissolve 2 parts of 40 kDa dextran and 5 parts of succinic anhydride in 6 parts of DMSO. After passing nitrogen gas to remove oxygen for 20 minutes, add 1 part of a catalyst to the solution, control the temperature at 70℃, and stir for 20 hours.

[0045] After the reaction is completed, place the solution in ice ethanol, and then perform suction filtration to obtain crystals. Dissolve the crystals in deionized water, and then add them to a dialysis bag. Dialyze for 48 hours (change the water every six hours during the period), and then freeze-dry to obtain carboxyl dextran.

[0046] The catalyst is a DMSO solution containing 10 mg / ml of 4-dimethylaminopyridine (DMAP).

[0047] Preparation of the magnetic beads: Take 15 parts of FeCl3·6H2O and 5 parts of FeCl2·4H2O, dissolve them in 1000 parts of deionized water, and then pass nitrogen gas to remove oxygen for 15 minutes. Then, add 10 parts of the carboxyl dextran prepared in this application, and stir at 70℃ and 15 rpm / min for 30 minutes. Add 8 mol / L ammonia water to adjust pH to 10; add 2 parts of polyethylene glycol 600 and control the temperature at 70°C to continue the reaction for 1.5 h; stop the reaction and cool to 20°C within 3 min; After the reaction, the obtained magnetic beads are adsorbed by a magnet, the supernatant is removed, and the magnetic core is washed twice with 1.0 mol / l HCl and twice with deionized water.

[0048] The magnetic beads are suspended in the storage solution prepared in the present application to form a magnetic bead suspension of about 10 mg / ml.

[0049] Technical solution (F) Preparation of the storage solution: The method is the same as that of technical solution (C).

[0050] Preparation of carboxyl dextran: Dissolve 2 parts of dextran with a molecular weight of 40 kDa and 5 parts of succinic anhydride in 6 parts of DMSO, and after 20 min of oxygen removal by nitrogen gas, add 1 part of a catalyst to the solution, control the temperature at 70°C, and stir for 20 h.

[0051] After the reaction, the solution is placed in ice ethanol, and the precipitate is obtained by suction filtration. The crystal is dissolved in deionized water and added to a dialysis bag, dialyzed for 48 h (with water changed every six hours), and freeze-dried to obtain carboxyl dextran.

[0052] The catalyst is a DMSO solution containing 10 mg / ml of 4-dimethylaminopyridine (DMAP).

[0053] Preparation of magnetic beads: Weigh 15 parts of FeCl3·6H2O and 5 parts of FeCl2·4H2O, dissolve in 1000 parts of deionized water, and after 15 min of oxygen removal by nitrogen gas, add 10 parts of the carboxyl dextran prepared in the present application, and stir at 70°C and 15 rpm / min for 30 min; Add 8 mol / L ammonia water to adjust pH to 10, and control the temperature at 70°C to continue the reaction for 1.5 h; stop the reaction and cool to 20°C within 3 min; After the reaction, the obtained magnetic beads are adsorbed by a magnet, the supernatant is removed, and the magnetic core is washed twice with 1.0 mol / l HCl and twice with deionized water.

[0054] The magnetic beads are suspended in the storage solution prepared in the present application to form a magnetic bead suspension of about 10 mg / ml.

[0055] The magnetic bead suspensions prepared by technical solutions (A)-(F) were stored at 4°C for 5 days, 10 days, 15 days, 20 days, and the cell aggregation degree was detected according to the method of experimental example 1, and the proportion of the number of magnetic beads above 500 nm was calculated, and the detection results are shown in Table 1.

[0056] Table 1 Detection results of cell aggregation degree of technical solutions (A)-(F)

[0057] According to the experimental results in Table 1, those skilled in the art can know that: First, the magnetic beads in technical solution (E) have the least aggregation and good storage effect, which indicates that, first, the magnetic beads prepared by the technical solution of the present application have a relatively uniform particle size distribution and less aggregation, and in addition, the magnetic beads can also obtain better storage effect in the storage solution provided by the present application, effectively reducing the generation of aggregation phenomenon.

[0058] Second, comparing technical solutions (C)-(F), the selected storage solution is the same, but due to the different preparation methods of the magnetic beads, the storage effect also has a more significant difference. Specifically, the carboxyl dextran coated magnetic beads prepared by the method of the present application have better storage effect in the storage solution.

[0059] Third, comparing technical solutions (E)-(F), technical solution (F) does not add polyethylene glycol when preparing the magnetic beads, which makes the storage effect in the storage solution have a more obvious decrease.

[0060] Example 1 Preparation of magnetic bead storage solution The solvent of the magnetic bead storage solution is deionized water, and includes the following components: 4% trehalose, 0.8% polyethylene glycol-2000, 0.08% sodium ascorbate, 0.4% BSA, 0.04% Pluronic F-68, 0.4mM EDTA, 4mM sodium citrate; The solution pH is adjusted to 8.0 with 10mM Tris-HCl, and sterilized by 0.22μm filter membrane.

[0061] Example 2 Preparation of magnetic bead storage solution The solvent of the magnetic bead storage solution is deionized water, and includes the following components: 5% trehalose, 1% polyethylene glycol-2000, 0.1% sodium ascorbate, 0.5% BSA, 0.05% Pluronic F-68, 0.5mM EDTA, 5mM sodium citrate; The solution pH is adjusted to 8.0 with 10mM Tris-HCl, and sterilized by 0.22μm filter membrane.

[0062] Example 3 Preparation of magnetic bead storage solution The solvent of the magnetic bead preservative solution is deionized water and includes the following components: 8% trehalose, 1.5% polyethylene glycol-2000, 0.15% sodium ascorbate, 0.6% BSA, 0.06% Pluronic F-68, 0.6mM EDTA, 6mM sodium citrate; The solution is adjusted to pH 9.0 with 10mM Tris-HCl and sterilized by filtration through a 0.22μm filter.

[0063] Example 4 Preparation of carboxyl dextran Two parts of dextran with a molecular weight of 40kDa and four parts of succinic anhydride are dissolved in five parts of DMSO. After 20 minutes of oxygen removal by nitrogen bubbling, one part of catalyst is added to the solution, and the temperature is controlled at 60°C for 30 hours of stirring.

[0064] After the reaction is completed, the solution is placed in ice ethanol, and the crystals are obtained by suction filtration after precipitation. The crystals are dissolved in deionized water and added to a dialysis bag, and dialysis is performed for 48 hours (with water changed every six hours), and then freeze-dried to obtain carboxyl dextran.

[0065] The catalyst is a DMSO solution containing 10mg / ml of 4-dimethylaminopyridine (DMAP).

[0066] Example 5 Preparation of carboxyl dextran Two parts of dextran with a molecular weight of 40kDa and five parts of succinic anhydride are dissolved in six parts of DMSO. After 20 minutes of oxygen removal by nitrogen bubbling, one part of catalyst is added to the solution, and the temperature is controlled at 70°C for 20 hours of stirring.

[0067] After the reaction is completed, the solution is placed in ice ethanol, and the crystals are obtained by suction filtration after precipitation. The crystals are dissolved in deionized water and added to a dialysis bag, and dialysis is performed for 48 hours (with water changed every six hours), and then freeze-dried to obtain carboxyl dextran.

[0068] The catalyst is a DMSO solution containing 10mg / ml of 4-dimethylaminopyridine (DMAP).

[0069] Example 6 Preparation of carboxyl dextran Three parts of dextran with a molecular weight of 40kDa and five parts of succinic anhydride are dissolved in eight parts of DMSO. After 20 minutes of oxygen removal by nitrogen bubbling, one part of catalyst is added to the solution, and the temperature is controlled at 70°C for 30 hours of stirring.

[0070] After the reaction is completed, the solution is placed in ice ethanol, and the crystals are obtained by suction filtration after precipitation. The crystals are dissolved in deionized water and added to a dialysis bag, and dialysis is performed for 48 hours (with water changed every six hours), and then freeze-dried to obtain carboxyl dextran.

[0071] The catalyst is a DMSO solution with 10 mg / ml 4-dimethylaminopyridine (DMAP) dissolved therein.

[0072] Example 7 Preparation of dextran-coated magnetic beads Take 10 parts of FeCl3-6H2O and 10 parts of FeCl2-4H2O, dissolve in 1000 parts of deionized water, pass nitrogen to remove oxygen for 10 min, then add 5 parts of dextran as described in Example 4, and stir at 60°C at 1000 rpm / min for 40 min; Add ammonia water with a concentration of 5 mol / L to adjust the pH to 9; add 1 part of polyethylene glycol 600, and control the temperature at 60°C to continue the reaction for 2 h, stop the reaction and cool to 20°C within 2 min; After the reaction is completed, the magnetic beads obtained by synthesis are adsorbed using a magnet, the supernatant is removed, and 1.0 mol / l HCl is used for two times of magnetic core cleaning, and deionized water is used for two times of cleaning of the magnetic core.

[0073] The magnetic beads are placed in the storage solution as in Example 1 to form a magnetic bead suspension of about 10 mg / ml, and stored at 4°C for 7 days.

[0074] Example 8 Preparation of dextran-coated magnetic beads Take 15 parts of FeCl3-6H2O and 5 parts of FeCl2-4H2O, dissolve in 1000 parts of deionized water, pass nitrogen to remove oxygen for 15 min, then add 10 parts of dextran as described in Example 5, and stir at 70°C at 15 rpm / min for 30 min; Add ammonia water with a concentration of 8 mol / L to adjust the pH to 10; add 2 parts of polyethylene glycol 600, and control the temperature at 70°C to continue the reaction for 1.5 h, stop the reaction and cool to 20°C within 3 min; After the reaction is completed, the magnetic beads obtained by synthesis are adsorbed using a magnet, the supernatant is removed, and 1.0 mol / l HCl is used for two times of magnetic core cleaning, and deionized water is used for two times of cleaning of the magnetic core.

[0075] The magnetic beads are placed in the storage solution as in Example 2 to form a magnetic bead suspension of about 10 mg / ml, and stored at 4°C for 7 days.

[0076] Example 9 Preparation of dextran-coated magnetic beads Take 20 parts of FeCl3-6H2O and 10 parts of FeCl2-4H2O, dissolve in 1000 parts of deionized water, pass nitrogen to remove oxygen for 20 min, then add 10 parts of dextran as described in Example 6, and stir at 80°C at 2000 rpm / min for 40 min; Add 10 mol / L ammonia water to adjust pH to 13; add 2 parts of polyethylene glycol 600 and control temperature at 90°C to continue reaction for 1 h; stop reaction and cool to 25°C within 5 min; After the reaction, the magnetic beads were separated by a magnet and the supernatant was removed. The magnetic beads were washed twice with 1.0 mol / L HCl and twice with deionized water.

[0077] The magnetic beads were suspended in the storage solution of Example 3 to form a magnetic bead suspension of about 10 mg / ml and stored at 4°C for 7 days.

[0078] Example 10 Preparation of antibody conjugated magnetic beads The magnetic beads obtained in Example 7 were washed twice with 25 mM MES buffer at pH 5.0 and resuspended to form a magnetic bead suspension of about 10 mg / ml.

[0079] Add 10 mg / ml of EDC 10 μl and 10 mg / ml of NHS 20 μl to activate the carboxyl group at 37°C for 0.5 h.

[0080] The supernatant was removed by magnetic separation and the magnetic beads were resuspended to 10 mg / ml using 25 mM MES buffer at pH 5.0. Add 60 μg of CD4 antibody and incubate in a rotary incubator at 37°C for 6 h.

[0081] The magnetic beads were separated using a magnet, blocked with a PBS solution containing BSA for 1 h, and then washed and stored using the storage solution of Example 1.

[0082] Example 11 Preparation of antibody conjugated magnetic beads The magnetic beads obtained in Example 7 were washed twice with 30 mM MES buffer at pH 5.0 and resuspended to form a magnetic bead suspension of about 10 mg / ml.

[0083] Add 15 mg / ml of EDC 10 μl and 20 mg / ml of NHS 20 μl to activate the carboxyl group at 37°C for 1 h.

[0084] The supernatant was removed by magnetic separation and the magnetic beads were resuspended to 10 mg / ml using 35 mM MES buffer at pH 5.0. Add 50 μg of CD4 antibody and incubate in a rotary incubator at 37°C for 8 h.

[0085] The magnetic beads were separated using a magnet, blocked with a PBS solution containing BSA for 1 h, and then washed and stored using the storage solution of Example 1.

[0086] Example 12 Preparation of antibody conjugated magnetic beads The magnetic beads obtained in Example 7 were washed twice with 30 mM MES buffer, pH 5.0, and resuspended to form a magnetic bead suspension of about 10 mg / ml.

[0087] EDC 10 μl, 10 mg / ml of NHS 20 μl were added, and the carboxyl groups were activated at 37°C for 0.5 h.

[0088] The supernatant was removed by magnetic separation, and the magnetic beads were resuspended to 10 mg / ml using 25 mM MES buffer, pH 5.0. 60 μg of CD4 antibody was added, and the mixture was incubated in a rotary incubator at 37°C for 6 h.

[0089] The magnetic beads were separated using a magnet, and were blocked by adding a PBS solution containing BSA for 1 h, and then were washed and stored using the storage solution as in Example 2.

[0090] Example 13 Preparation of antibody-conjugated magnetic beads The magnetic beads obtained in Example 8 were washed twice with 30 mM MES buffer, pH 5.0, and resuspended to form a magnetic bead suspension of about 10 mg / ml.

[0091] EDC 10 μl, 10 mg / ml of NHS 20 μl were added, and the carboxyl groups were activated at 37°C for 0.5 h.

[0092] The supernatant was removed by magnetic separation, and the magnetic beads were resuspended to 10 mg / ml using 25 mM MES buffer, pH 5.0. 60 μg of CD4 antibody was added, and the mixture was incubated in a rotary incubator at 37°C for 6 h.

[0093] The magnetic beads were separated using a magnet, and were blocked by adding a PBS solution containing BSA for 1 h, and then were washed and stored using the storage solution as in Example 2.

[0094] Example 14 Preparation of antibody-conjugated magnetic beads The magnetic beads obtained in Example 8 were washed twice with 30 mM MES buffer, pH 5.0, and resuspended to form a magnetic bead suspension of about 10 mg / ml.

[0095] EDC 10 μl, 10 mg / ml of NHS 20 μl were added, and the carboxyl groups were activated at 37°C for 0.5 h.

[0096] The supernatant was removed by magnetic separation, and the magnetic beads were resuspended to 10 mg / ml using 25 mM MES buffer, pH 5.0. 60 μg of CD4 antibody was added, and the mixture was incubated in a rotary incubator at 37°C for 6 h.

[0097] The magnetic beads were separated using a magnet, and were blocked by adding a PBS solution containing BSA for 1 h, and then were washed and stored using the storage solution as in Example 2.

[0098] Example 15 Preparation of antibody conjugated magnetic beads The magnetic beads obtained in Example 8 were washed twice with 30 mM MES buffer, pH 5.0 and resuspended to form a magnetic bead suspension of about 10 mg / ml.

[0099] EDC 15 μl of 10 mg / ml and NHS 15 μl of 10 mg / ml were added and the carboxyl groups were activated at 37°C for 1 h.

[0100] The supernatant was removed by magnetic separation and the beads were resuspended to 10 mg / ml using 30 mM MES buffer, pH 5.0. 50 μg of CD4 antibody was added and the beads were incubated in a rotating incubator at 37°C for 6 h.

[0101] The beads were separated using a magnet and blocked with a PBS solution containing BSA for 1 h. They were then washed and stored as in Example 2.

[0102] Example 16 Preparation of antibody conjugated magnetic beads The magnetic beads obtained in Example 9 were washed twice with 25 mM MES buffer, pH 5.0 and resuspended to form a magnetic bead suspension of about 10 mg / ml.

[0103] EDC 10 μl of 10 mg / ml and NHS 20 μl of 20 mg / ml were added and the carboxyl groups were activated at 37°C for 0.5 h.

[0104] The supernatant was removed by magnetic separation and the beads were resuspended to 10 mg / ml using 25 mM MES buffer, pH 5.0. 60 μg of CD4 antibody was added and the beads were incubated in a rotating incubator at 37°C for 6 h.

[0105] The beads were separated using a magnet and blocked with a PBS solution containing BSA for 1 h. They were then washed and stored as in Example 3.

[0106] Example 17 Preparation of antibody conjugated magnetic beads The magnetic beads obtained in Example 9 were washed twice with 30 mM MES buffer, pH 5.0 and resuspended to form a magnetic bead suspension of about 10 mg / ml.

[0107] EDC 10 μl of 15 mg / ml and NHS 20 μl of 20 mg / ml were added and the carboxyl groups were activated at 37°C for 1 h.

[0108] The supernatant was removed by magnetic separation and the beads were resuspended to 10 mg / ml using 35 mM MES buffer, pH 5.0. 50 μg of CD4 antibody was added and the beads were incubated in a rotating incubator at 37°C for 8 h.

[0109] The magnetic beads were separated using a magnet, blocked with a PBS solution containing BSA for 1 hour, and then washed and stored using the storage solution as in Example 3.

[0110] Example 18 Preparation of antibody conjugated magnetic beads The magnetic beads obtained in Example 9 were washed twice with 30 mM MES buffer, pH 5.0, and resuspended to form a suspension of magnetic beads at approximately 10 mg / ml.

[0111] Carboxyl groups were activated by adding 15 μl of 10 mg / ml EDC and 15 μl of 10 mg / ml NHS and incubating at 37°C for 1 hour.

[0112] The supernatant was removed by magnetic separation and the magnetic beads were resuspended to 10 mg / ml using 30 mM MES buffer, pH 5.0. 50 μg of CD4 antibody was added and the mixture was incubated at 37°C in a rotating incubator for 6 hours.

[0113] The magnetic beads were separated using a magnet, blocked with a PBS solution containing BSA for 1 hour, and then washed and stored using the storage solution as in Example 3.

[0114] Comparative Example 1 This example is essentially the same as Example 11, except that the storage solution was replaced with a prior art storage solution having the following composition: BSA: 1% (w / v); Trehalose: 2% (w / v); NaCl: 0.15 M; Tween-20: 0.1% (v / v); Proclin-300: 0.05% (v / v); Polyethylene glycol 20000: 2% (w / v); Tris-HCl: 50 mM, adjusted to pH 7.4. Comparative Example 2 This example is essentially the same as Example 11, except that the carboxydextran was replaced with a prior art 40Kd dextran.

[0115] Comparative Example 3 This example is essentially the same as Example 11, except that the magnetic beads were stored in the storage solution for 30 days prior to conjugation of the antibody.

[0116] Comparative Example 4 This example is essentially the same as Example 11, except that the magnetic beads were stored in the storage solution for 30 days prior to conjugation of the antibody, and then resuspended in Tris buffer containing 0.1% Tween-20 and sonicated for 3 minutes in a 40 Hz water bath.

[0117] Comparative Example 5 The same as example 11, the difference is that before coupling the antibody, resuspend with Tris buffer containing 0.1% Tween-20, and then ultrasonic treatment for 3 minutes in 40hz water bath.

[0118] Example 19 The cell and magnetic bead binding efficiency was tested according to the detection method of example 2, and the test results are shown in table 2.

[0119] Table 2 Test results of examples 10-18 and comparative examples 1-5

[0120] According to the results in table 2, it can be seen that: First, the binding capacity of each embodiment of the present application to CD4+ cells is qualified, and the recovery rate is basically higher than 85%.

[0121] Second, compared with comparative example 11 and comparative examples 1-3, the preservation solution provided by the present application has a better protection effect on magnetic beads in a shorter preservation time; but the binding capacity of the magnetic beads decreases significantly in a longer preservation time, so it is recommended to control the magnetic bead preservation time within 20 days, or even within 7 days for antibody coupling experiment, but this has significantly improved the preservation time compared with the preservation solution in the prior art. Third, compared with comparative example 11 and comparative examples 3-5, ultrasonic treatment has a positive effect on the recovery of magnetic bead aggregation after a long preservation time, but the effect of ultrasonic treatment on magnetic beads is not significant in a shorter preservation time.

[0122] In summary, the nanometer magnetic beads and the preparation method thereof provided by the embodiments of the present application can solve the technical problem of the difficulty of preservation of magnetic beads in the prior art, so that the magnetic beads are less likely to aggregate or gather during the preservation process, the cell sorting efficiency of the magnetic beads is improved, and the magnetic beads have a good application prospect.

[0123] It should be pointed out that the technical features in the above embodiments can be freely combined by those skilled in the art, and the formed technical solutions also belong to the embodiments disclosed by the present application.

[0124] Further, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A type of nanomagnetic bead, characterized in that: The nanomagnetic beads include a magnetic core formed of Fe3O4 and γ-Fe2O3, and a dextran layer coating the outside of the magnetic core; The dextran layer contains polyethylene glycol.

2. The nanomagnetic beads according to claim 1, characterized in that: The polyethylene glycol is polyethylene glycol 600.

3. The nanomagnetic beads according to claim 1, characterized in that: The dextran is a carboxylated dextran.

4. The nanomagnetic beads according to claim 1, characterized in that: The magnetic nanobeads also include an antibody linked to carboxylated dextran, wherein the antibody is a CD4 antibody.

5. A method for preparing nanomagnetic beads according to any one of claims 1-4, characterized in that, Including the following steps: Preparation of Fe 3+ Fe 2+ The solution was mixed thoroughly with an appropriate amount of dextran at 50-80℃; the Fe... 3+ The concentration is 0.01-0.05 mol / L, and the Fe... 2+ The concentration is 0.03-0.06 mol / L; Adjust the pH of the solution to alkaline, add an appropriate amount of polyethylene glycol 600, and continue the reaction at a controlled temperature of 50-80℃. After the reaction is complete, the solution is rapidly cooled to obtain dextran-coated magnetic beads.

6. The method for preparing nanomagnetic beads according to claim 5, characterized in that: The method for preparing the nanomagnetic beads further includes the following steps: The magnetic beads were placed in a preservation solution to form a magnetic bead suspension of approximately 10 mg / ml.

7. The nanomagnetic beads according to claim 6, characterized in that: The components of the preservation solution are: Trehalose 2-8%, polyethylene glycol 2000 0.5-2%, sodium ascorbate 0.05-0.1%, BSA 0.2-1%, poloxamer 0.02-0.1%; EDTA 0.4-0.6mM, sodium citrate 4-6mM; The solution pH is 7.5-9.

8. The method for preparing nanomagnetic beads according to claim 7, characterized in that: The magnetic beads should not be placed in the preservation solution for more than 20 days.

9. The nanomagnetic beads according to claim 5, characterized in that: The method for preparing the magnetic nanobeads further includes an antibody conjugation step: Take the dextran-coated magnetic beads, wash them with buffer solution to form a magnetic bead suspension; Add 10 mg / ml EDC and 10 mg / ml NHS, and activate the carboxyl groups at 36-38℃ for 0.5-2 h; After removing the supernatant, resuspend in buffer solution; Add antibody and incubate at 36-38℃ in a rotary culture device for 6-12 hours.

10. The method for preparing nanomagnetic beads according to claim 9, characterized in that: The antibody is a CD4 antibody.

Images