Nanometer magnetic bead and preparation method thereof
By coating the magnetic core with polyethylene glycol and using a specific preservation solution, the problem of aggregation of magnetic nanobeads during preservation was solved, the preservation time of magnetic beads and the efficiency of cell sorting were improved, and the binding ability with antibodies, especially the specificity of CD4 antibodies, was enhanced.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NAYI BIOTECHNOLOGY (BEIJING) CO LTD
- Filing Date
- 2025-08-28
- Publication Date
- 2026-05-12
Smart Images
Figure CN120992923B_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of cell sorting technology, specifically to a magnetic nanobead and its preparation method. Background Technology
[0002] Magnetic beads, due to their simplicity, efficiency, and high specificity, have gradually become an important method for cell sorting. Typically, magnetic sorting relies on modifying magnetic beads with antibodies, proteins, or other additives. Through the specific binding of these modifiers to cell surface molecules, and under the influence of an external magnetic field, the target cells are rapidly separated.
[0003] In existing technologies, the synthetic route for magnetic beads typically involves first synthesizing functionalized magnetic nuclei, then coating the magnetic nuclei into magnetic beads, and finally coupling them with biological ligands to achieve specific processing. However, as the particle size of magnetic beads decreases, their aggregation effect becomes more pronounced, affecting the efficiency of specific processing and consequently impacting the separation effect on target cells.
[0004] In view of the above, this application is hereby submitted. Summary of the Invention
[0005] The purpose of this application is to provide a magnetic nanobead and a method for preparing the same, in order to solve at least one of the technical problems mentioned in the background art.
[0006] Specifically, in the first aspect of this application, a nanomagnetic bead is provided.
[0007] The nanomagnetic beads include a magnetic core formed of Fe3O4 and γ-Fe2O3, and a dextran layer coating the outside of the magnetic core;
[0008] The dextran layer contains polyethylene glycol.
[0009] By using the above technical solution, by coating the magnetic core with polyethylene glycol, the magnetic beads are less likely to agglomerate or aggregate during storage, thereby increasing the storage time of the magnetic beads and making them more efficient in sorting cells.
[0010] Preferably, the polyethylene glycol is polyethylene glycol 600.
[0011] Preferably, the dextran is a carboxylated dextran.
[0012] Preferably, the magnetic nanobeads further include an antibody linked to carboxylated dextran, wherein the antibody is a CD4 antibody.
[0013] A second aspect of this application provides a method for preparing magnetic nanobeads, comprising the steps of:
[0014] 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;
[0015] 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.
[0016] Preferably, the method for preparing the nanomagnetic beads further includes the step of:
[0017] The magnetic beads were placed in a preservation solution to form a magnetic bead suspension of approximately 10 mg / ml.
[0018] Preferably, the components of the preservation solution are:
[0019] Trehalose 2-8%, polyethylene glycol 2000 0.5-2%, sodium ascorbate 0.05-0.1%, BSA 0.2-1%, poloxamer 0.02-0.1%;
[0020] EDTA 0.4-0.6mM, sodium citrate 4-6mM;
[0021] The solution pH is 7.5-9.
[0022] Preferably, the magnetic beads are placed in the preservation solution for no more than 20 days.
[0023] Preferably, the method for preparing the magnetic nanobeads further includes an antibody conjugation step:
[0024] Take the dextran-coated magnetic beads, wash them with buffer solution to form a magnetic bead suspension;
[0025] Add 10 mg / ml EDC and 10 mg / ml NHS, and activate the carboxyl groups at 36-38℃ for 0.5-2 h;
[0026] After removing the supernatant, resuspend the sample in buffer solution.
[0027] Add antibody and incubate at 36-38℃ in a rotary culture device for 6-12 hours.
[0028] Preferably, the antibody is a CD4 antibody.
[0029] In summary, this application has the following beneficial effects:
[0030] First, the magnetic nanobeads provided in this application, by coating the magnetic core with polyethylene glycol, make the magnetic beads less prone to aggregation or clustering during storage, thereby increasing the storage time of the magnetic beads and making the magnetic beads more efficient in sorting cells.
[0031] Secondly, the method for preparing magnetic nanobeads provided in this application can be better combined with the magnetic bead preservation solution provided in this application to improve the preservation efficiency of magnetic beads and reduce the occurrence of agglomeration.
[0032] Third, the method for preparing magnetic nanobeads provided in this application has a higher binding efficiency with antibodies, especially CD4 antibodies, which makes the magnetic beads more specific when sorting cells containing CD4 antigen. Attached Figure Description
[0033] Figure 1 This is a flow cytometry plot of cells after sorting in Example 10;
[0034] Figure 2 This is a flow cytometry plot of cells after sorting in Example 11;
[0035] Figure 3 This is a flow cytometry plot of cells after sorting in Example 12;
[0036] Figure 4 This is a flow cytometry plot of cells after sorting in Example 13;
[0037] Figure 5 This is a flow cytometry plot of cells after sorting in Example 14;
[0038] Figure 6 This is a flow cytometry plot of cells after sorting in Example 15;
[0039] Figure 7 This is a flow cytometry plot of cells after sorting in Example 16;
[0040] Figure 8 This is a flow cytometry plot of cells after sorting in Example 17;
[0041] Figure 9 This is a flow cytometry plot of cells after sorting in Example 18;
[0042] Figure 10 This is the flow cytometry plot of cells after sorting in Comparative Example 1;
[0043] Figure 11 This is the flow cytometry plot of cells after sorting in Comparative Example 2;
[0044] Figure 12 This is the flow cytometry plot of Comparative Example 3 after cell sorting;
[0045] Figure 13This is the flow cytometry plot of cells after sorting in Comparative Example 4;
[0046] Figure 14 This is the flow cytometry plot of Comparative Example 5 after cell sorting. Detailed Implementation
[0047] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims.
[0048] 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. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.
[0049] The present application will be described in detail below through examples.
[0050] In existing technologies, the synthetic route for magnetic beads typically involves first synthesizing functionalized magnetic nuclei, then coating the magnetic nuclei into magnetic beads, and finally coupling them with biological ligands to achieve specific processing. However, as the particle size of magnetic beads decreases, their aggregation effect becomes more pronounced, affecting the efficiency of specific processing and consequently impacting the separation effect on target cells.
[0051] In view of this, in order to solve the existing technical problems in the background art, the inventive concept of this application is to provide a nanomagnetic bead and a method for preparing the same, wherein the nanomagnetic bead includes a magnetic core formed by Fe3O4 and γ-Fe2O3, and a dextran layer covering the outside of the magnetic core; wherein the dextran layer contains polyethylene glycol.
[0052] According to this invention, by coating the magnetic core with polyethylene glycol, the magnetic beads are less likely to agglomerate or cluster during storage, thereby increasing the storage time of the magnetic beads and making them more efficient in sorting cells.
[0053] To better understand the above technical solutions, the following detailed descriptions will be provided in conjunction with specific implementation methods. Those skilled in the art should also understand that the reaction times and drug dosages involved in this application cannot be absolutely precise in actual production, but are all within the allowable error range. For example, if the desired sample heating time is 30 minutes, the actual operation may be 30 minutes plus or minus 1 second; if the desired sample weight is 30g, the actual weight may be 30.001g or 29.998g.
[0054] Unless otherwise specified, in the experimental examples and embodiments of this application, the cells used for sorting are human peripheral blood mononuclear cells (PBMCs), and the CD4 antibody is an anti-human CD4 antibody.
[0055] Experiment Example 1: Magnetic Bead Agglomeration Experiment
[0056] The magnetic bead aggregation experiment uses existing technology, specifically:
[0057] A nano-Courte particle size analyzer was used to distinguish the particle size of single particles from that of aggregates by measuring the change in resistance when magnetic beads pass through nanopores, and the ratio of the number of magnetic beads larger than 500 nm to the number of magnetic beads smaller than 500 nm was calculated.
[0058] Example 2 Antibody Conjugation Experiment
[0059] The antibody conjugation assay uses existing technology, specifically:
[0060] Fresh human blood containing an anticoagulant was diluted 2-fold with PBS, and then dispersed in the lymphocyte separation fluid at a blood:lymphocyte separation fluid volume ratio of 1:1. Peripheral blood mononuclear cells were obtained after centrifugation, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / mL. 7 cells / ml;
[0061] Take 100 μl of the cell suspension obtained in the previous step and 5 μl of CD4 antibody-conjugated magnetic nanobeads (provided in this application), mix well and let stand at 4°C for 30 min; centrifuge the above suspension at 1500 rpm for 5 min and discard the supernatant, then add 500 μl of PBS buffer to the cell pellet to resuspend.
[0062] The cells were washed three times with a sorting column, each time with 500 μl of PBS buffer. Then, 1 ml of PBS buffer was added to rinse the cells and collect them into EP tubes.
[0063] Centrifuge again at 1500 rpm for 5 min and discard the supernatant. Add 500 μl of PBS buffer to the cell pellet to resuspend the pellet, add 10 μl of PE-labeled CD4 antibody, and incubate at 4°C for 20 min.
[0064] After sorting, the cells were collected in serum-containing medium to maintain cell viability. First, they were centrifuged at 500g for 10 minutes, the supernatant was discarded, and the cells were resuspended in PBS. This centrifugation and washing process was repeated 1-2 times. Finally, the cell concentration was adjusted to 1*102. 6 -1*10 7 Cells / mL were counted, and the cell sorting and recovery rate was detected by flow cytometry.
[0065] Experiment Example 3: Preparation of Preservative Solution
[0066] This embodiment aims to study the formulation of a preservation solution suitable for the magnetic beads of this application.
[0067] Technical Solution (A)
[0068] Preparation of preservation solution:
[0069] 1% polyethylene glycol-2000, 0.1% sodium ascorbate, 0.5% BSA, 0.05% Pluronic F-68, 0.5mM EDTA, 5mM sodium citrate;
[0070] Adjust the pH of the solution to 8.0 using 10mM Tris-HCl, and then filter it through a 0.22μm membrane for sterilization.
[0071] Preparation of magnetic beads:
[0072] Weigh 15 parts FeCl3·6H2O and 5 parts FeCl2·4H2O, dissolve them in 1000 parts deionized water, purge with nitrogen to remove oxygen for 15 min, add 10 parts 40 kDa dextran, and stir at 70℃ for 15 rpm / min for 30 min.
[0073] Add 8 mol / L ammonia solution to adjust the pH to 10; add 2 parts of polyethylene glycol 600 and continue the reaction at 70℃ for 1.5 h; stop the reaction and cool to 20℃ within 3 min.
[0074] After the reaction was completed, the synthesized magnetic beads were adsorbed using a magnet. After removing the supernatant, the magnetic cores were cleaned twice with 1.0 mol / L HCl, and then cleaned twice with deionized water.
[0075] The magnetic beads were placed in the preservation solution prepared in this method to form a magnetic bead suspension of approximately 10 mg / ml.
[0076] Technical Solution (B)
[0077] Preparation of preservation solution:
[0078] 5% trehalose, 0.1% sodium ascorbate, 0.5% BSA, 0.05% Pluronic F-68, 0.5mM EDTA, 5mM sodium citrate;
[0079] Adjust the pH of the solution to 8.0 using 10mM Tris-HCl, and then filter it through a 0.22μm membrane for sterilization.
[0080] Preparation of magnetic beads:
[0081] The method and technical solution (A) are the same.
[0082] Technical Solution (C)
[0083] Preparation of preservation solution:
[0084] 5% Trehalose, 1% Polyethylene Glycol-2000, 0.1% Sodium Ascorbate, 0.5% BSA, 0.05% Pluronic F-68, 0.5mM EDTA, 5mM Sodium Citrate;
[0085] Adjust the pH of the solution to 8.0 using 10mM Tris-HCl, and then filter it through a 0.22μm membrane for sterilization.
[0086] Preparation of magnetic beads:
[0087] Weigh 15 parts FeCl3·6H2O and 5 parts FeCl2·4H2O, dissolve them in 1000 parts deionized water, purge with nitrogen to remove oxygen for 15 min, add 10 parts 40 kDa dextran, and stir at 70℃ for 15 rpm / min for 30 min.
[0088] Add 8 mol / L ammonia solution to adjust the pH to 10, and continue the reaction at 70℃ for 1.5 h. Stop the reaction and cool to 20℃ within 3 min.
[0089] After the reaction was completed, the synthesized magnetic beads were adsorbed using a magnet. After removing the supernatant, the magnetic cores were cleaned twice with 1.0 mol / L HCl, and then cleaned twice with deionized water.
[0090] The magnetic beads were placed in the preservation solution prepared according to this method to form a magnetic bead suspension of approximately 10 mg / ml.
[0091] Technical Solution (D)
[0092] Preparation of preservation solution:
[0093] The method and technical solution (C) are the same.
[0094] Preparation of magnetic beads:
[0095] Weigh 15 parts FeCl3·6H2O and 5 parts FeCl2·4H2O, dissolve them in 1000 parts deionized water, purge with nitrogen to remove oxygen for 15 min, add 10 parts 40 kDa dextran, and stir at 70℃ for 15 rpm / min for 30 min.
[0096] Add 8 mol / L ammonia solution to adjust the pH to 10; add 2 parts of polyethylene glycol 600 and continue the reaction at 70℃ for 1.5 h; stop the reaction and cool to 20℃ within 3 min.
[0097] After the reaction was completed, the synthesized magnetic beads were adsorbed using a magnet. After removing the supernatant, the magnetic cores were cleaned twice with 1.0 mol / L HCl, and then cleaned twice with deionized water.
[0098] The magnetic beads were placed in the preservation solution prepared according to this method to form a magnetic bead suspension of approximately 10 mg / ml.
[0099] Technical Solution (E)
[0100] Preparation of preservation solution:
[0101] The method and technical solution (C) are the same.
[0102] Preparation of carboxyglucan:
[0103] Two parts of 40kDa dextran and five parts of succinic anhydride were dissolved in six parts of DMSO. After purging with nitrogen for 20 minutes to remove oxygen, one part of catalyst was added to the solution, and the mixture was stirred at 70°C for 20 hours.
[0104] After the reaction was complete, the solution was placed in ice-cold ethanol, and the precipitate was collected by filtration to obtain crystals. The crystals were dissolved in deionized water and then added to a dialysis bag for dialyzing for 48 hours (with water changed every six hours). The solution was then freeze-dried to obtain carboxyglucan.
[0105] The catalyst is a DMSO solution containing 10 mg / ml of 4-dimethylaminopyridine (DMAP).
[0106] Preparation of magnetic beads:
[0107] Weigh 15 parts FeCl3·6H2O and 5 parts FeCl2·4H2O, dissolve them in 1000 parts deionized water, purge with nitrogen to remove oxygen for 15 min, add 10 parts of the carboxyglucan prepared in this application, and stir at 70℃ for 15 rpm / min for 30 min.
[0108] Add 8 mol / L ammonia solution to adjust the pH to 10; add 2 parts of polyethylene glycol 600 and continue the reaction at 70℃ for 1.5 h; stop the reaction and cool to 20℃ within 3 min.
[0109] After the reaction was completed, the synthesized magnetic beads were adsorbed using a magnet. After removing the supernatant, the magnetic cores were cleaned twice with 1.0 mol / L HCl, and then cleaned twice with deionized water.
[0110] The magnetic beads were placed in the preservation solution prepared according to this method to form a magnetic bead suspension of approximately 10 mg / ml.
[0111] Technical Solution (F)
[0112] Preparation of preservation solution:
[0113] The method and technical solution (C) are the same.
[0114] Preparation of carboxyglucan:
[0115] Two parts of 40kDa dextran and five parts of succinic anhydride were dissolved in six parts of DMSO. After purging with nitrogen for 20 minutes to remove oxygen, one part of catalyst was added to the solution, and the mixture was stirred at 70°C for 20 hours.
[0116] After the reaction was complete, the solution was placed in ice-cold ethanol, and the precipitate was collected by filtration to obtain crystals. The crystals were dissolved in deionized water and then added to a dialysis bag for dialyzing for 48 hours (with water changed every six hours). The solution was then freeze-dried to obtain carboxyglucan.
[0117] The catalyst is a DMSO solution containing 10 mg / ml of 4-dimethylaminopyridine (DMAP).
[0118] Preparation of magnetic beads:
[0119] Weigh 15 parts FeCl3·6H2O and 5 parts FeCl2·4H2O, dissolve them in 1000 parts deionized water, purge with nitrogen to remove oxygen for 15 min, add 10 parts of the carboxyglucan prepared in this application, and stir at 70℃ for 15 rpm / min for 30 min.
[0120] Add 8 mol / L ammonia solution to adjust the pH to 10, and continue the reaction at 70℃ for 1.5 h. Stop the reaction and cool to 20℃ within 3 min.
[0121] After the reaction was completed, the synthesized magnetic beads were adsorbed using a magnet. After removing the supernatant, the magnetic cores were cleaned twice with 1.0 mol / L HCl, and then cleaned twice with deionized water.
[0122] The magnetic beads were placed in the preservation solution prepared according to this method to form a magnetic bead suspension of approximately 10 mg / ml.
[0123] The magnetic bead suspensions prepared by technical solutions (A)-(F) were stored at 4°C for 5, 10, 15 and 20 days, and the degree of aggregation was detected according to the method of Experimental Example 1. The proportion of magnetic beads with a size greater than 500 nm was calculated. The detection results are shown in Table 1.
[0124] Table 1. Cell aggregation detection results of technical solutions (A)-(F)
[0125]
[0126] Based on the experimental results in Table 1, those skilled in the art will know that:
[0127] First, the magnetic beads in technical solution (E) produce the least agglomeration and have a good preservation effect. This means that, firstly, the magnetic beads prepared using the technical solution of this application have a relatively uniform particle size distribution and less agglomeration. In addition, the magnetic beads can also achieve a better preservation effect in the preservation solution provided by this application, effectively reducing the generation of agglomeration.
[0128] Second, comparing technical solutions (C)-(F), although the same preservation solution is used, the preservation effects differ significantly due to the different magnetic bead preparation methods. Specifically, the carboxyglucan-coated magnetic beads prepared using the method of this application exhibit better preservation performance in the preservation solution.
[0129] Third, compared with technical solutions (E) and (F), technical solution (F) did not add polyethylene glycol when preparing magnetic beads, which significantly reduced their preservation effect in the preservation solution.
[0130] Example 1: Preparation of magnetic bead preservation solution
[0131] The solvent for the magnetic bead preservation solution is deionized water, and it includes the following components:
[0132] 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;
[0133] Adjust the pH of the solution to 8.0 using 10mM Tris-HCl, and then filter it through a 0.22μm membrane for sterilization.
[0134] Example 2: Preparation of magnetic bead preservation solution
[0135] The solvent of the magnetic bead preservation solution is deionized water, and it includes the following components:
[0136] 5% Trehalose, 1% Polyethylene Glycol-2000, 0.1% Sodium Ascorbate, 0.5% BSA, 0.05% Pluronic F-68, 0.5mM EDTA, 5mM Sodium Citrate;
[0137] Adjust the pH of the solution to 8.0 using 10mM Tris-HCl, and then filter it through a 0.22μm membrane for sterilization.
[0138] Example 3: Preparation of magnetic bead preservation solution
[0139] The solvent of the magnetic bead preservation solution is deionized water, and it includes the following components:
[0140] 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;
[0141] The solution pH was adjusted to 9.0 using 10 mM Tris-HCl, and then sterilized by filtration through a 0.22 μm filter membrane.
[0142] Example 4: Preparation of Carboxyglucose
[0143] Two parts of 40kDa dextran and four parts of succinic anhydride were dissolved in five parts of DMSO. After purging with nitrogen for 20 minutes to remove oxygen, one part of catalyst was added to the solution, and the mixture was stirred at 60℃ for 30 hours.
[0144] After the reaction was complete, the solution was placed in ice-cold ethanol, and the precipitate was collected by filtration to obtain crystals. The crystals were dissolved in deionized water and then added to a dialysis bag for dialyzing for 48 hours (with water changed every six hours). The solution was then freeze-dried to obtain carboxyglucan.
[0145] The catalyst is a DMSO solution containing 10 mg / ml of 4-dimethylaminopyridine (DMAP).
[0146] Example 5: Preparation of Carboxyglucose
[0147] Two parts of 40kDa dextran and five parts of succinic anhydride were dissolved in six parts of DMSO. After purging with nitrogen for 20 minutes to remove oxygen, one part of catalyst was added to the solution, and the mixture was stirred at 70°C for 20 hours.
[0148] After the reaction was complete, the solution was placed in ice-cold ethanol, and the precipitate was collected by filtration to obtain crystals. The crystals were dissolved in deionized water and then added to a dialysis bag for dialyzing for 48 hours (with water changed every six hours). The solution was then freeze-dried to obtain carboxyglucan.
[0149] The catalyst is a DMSO solution containing 10 mg / ml of 4-dimethylaminopyridine (DMAP).
[0150] Example 6: Preparation of Carboxyglucose
[0151] Three parts of dextran with a molecular weight of 40 kDa and five parts of succinic anhydride were dissolved in eight parts of DMSO. After purging with nitrogen for 20 min to remove oxygen, one part of catalyst was added to the solution, and the mixture was stirred at 70 °C for 30 h.
[0152] After the reaction was complete, the solution was placed in ice-cold ethanol, and the precipitate was collected by filtration to obtain crystals. The crystals were dissolved in deionized water and then added to a dialysis bag for dialyzing for 48 hours (with water changed every six hours). The solution was then freeze-dried to obtain carboxyglucan.
[0153] The catalyst is a DMSO solution containing 10 mg / ml of 4-dimethylaminopyridine (DMAP).
[0154] Example 7: Preparation of dextran-coated magnetic beads
[0155] Weigh 10 parts FeCl3·6H2O and 10 parts FeCl2·4H2O, dissolve them in 1000 parts deionized water, purge with nitrogen to remove oxygen for 10 min, add 5 parts of dextran as described in Example 4, and stir at 1000 rpm / min at 60°C for 40 min.
[0156] Add 5 mol / L ammonia water to adjust the pH to 9; add 1 part of polyethylene glycol 600, and continue the reaction at 60℃ for 2 hours. Stop the reaction and cool to 20℃ within 2 minutes.
[0157] After the reaction was completed, the synthesized magnetic beads were adsorbed using a magnet. After removing the supernatant, the magnetic cores were cleaned twice with 1.0 mol / L HCl, and then cleaned twice with deionized water.
[0158] The magnetic beads were placed in the preservation solution as described in Example 1 to form a magnetic bead suspension of approximately 10 mg / ml, and stored at 4°C for 7 days.
[0159] Example 8: Preparation of dextran-coated magnetic beads
[0160] Weigh 15 parts FeCl3·6H2O and 5 parts FeCl2·4H2O, dissolve them in 1000 parts deionized water, purge with nitrogen to remove oxygen for 15 min, add 10 parts of dextran as described in Example 5, and stir at 70°C and 15 rpm / min for 30 min.
[0161] Add 8 mol / L ammonia solution to adjust the pH to 10; add 2 parts of polyethylene glycol 600 and continue the reaction at 70℃ for 1.5 h; stop the reaction and cool to 20℃ within 3 min.
[0162] After the reaction was completed, the synthesized magnetic beads were adsorbed using a magnet. After removing the supernatant, the magnetic cores were cleaned twice with 1.0 mol / L HCl, and then cleaned twice with deionized water.
[0163] The magnetic beads were placed in the preservation solution as described in Example 2 to form a magnetic bead suspension of approximately 10 mg / ml, and stored at 4°C for 7 days.
[0164] Example 9: Preparation of dextran-coated magnetic beads
[0165] Weigh 20 parts FeCl3·6H2O and 10 parts FeCl2·4H2O, dissolve them in 1000 parts deionized water, purge with nitrogen to remove oxygen for 20 min, add 10 parts of dextran as described in Example 6, and stir at 80°C and 2000 rpm / min for 40 min.
[0166] Add 10 mol / L ammonia solution to adjust the pH to 13; add 2 parts of polyethylene glycol 600 and continue the reaction at 90℃ for 1 hour, then stop the reaction and cool to 25℃ within 5 minutes.
[0167] After the reaction was completed, the synthesized magnetic beads were adsorbed using a magnet. After removing the supernatant, the magnetic cores were cleaned twice with 1.0 mol / L HCl, and then cleaned twice with deionized water.
[0168] The magnetic beads were placed in the preservation solution as described in Example 3 to form a magnetic bead suspension of approximately 10 mg / ml, and stored at 4°C for 7 days.
[0169] Example 10: Preparation of antibody-conjugated magnetic beads
[0170] Take the magnetic beads obtained in Example 7, wash them twice with 25mM MES buffer at pH 5.0, and resuspend them to form a magnetic bead suspension of about 10mg / ml.
[0171] Add 10 μl of 10 mg / ml EDC and 20 μl of 10 mg / ml NHS, and activate the carboxyl group at 37 °C for 0.5 h.
[0172] The supernatant was removed by magnetic separation, and the solution was resuspended to 10 mg / ml using 25 mM pH 5.0 MES buffer. 60 μg of CD4 antibody was added, and the mixture was incubated at 37°C in a rotary incubator for 6 h.
[0173] The magnetic beads were separated using a magnet, sealed with PBS solution containing BSA for 1 hour, and then washed and stored using the preservation solution as described in Example 1.
[0174] Example 11 Preparation of antibody-conjugated magnetic beads
[0175] Take the magnetic beads obtained in Example 7, wash them twice with 30mM MES buffer at pH 5.0, and resuspend them to form a magnetic bead suspension of about 10mg / ml.
[0176] Add 10 μl of 15 mg / ml EDC and 20 μl of 20 mg / ml NHS, and activate the carboxyl group at 37 °C for 1 h.
[0177] The supernatant was removed by magnetic separation, and the solution was resuspended to 10 mg / ml using 35 mM pH 5.0 MES buffer. 50 μg of CD4 antibody was added, and the mixture was incubated at 37°C in a rotary culture system for 8 h.
[0178] The magnetic beads were separated using a magnet, sealed with PBS solution containing BSA for 1 hour, and then washed and stored using the preservation solution as described in Example 1.
[0179] Example 12 Preparation of antibody-conjugated magnetic beads
[0180] Take the magnetic beads obtained in Example 7, wash them twice with 30mM MES buffer at pH 5.0, and resuspend them to form a magnetic bead suspension of about 10mg / ml.
[0181] Add 15 μl of 10 mg / ml EDC and 15 μl of 10 mg / ml NHS, and activate the carboxyl group at 37 °C for 1 h.
[0182] The supernatant was removed by magnetic separation, and the solution was resuspended to 10 mg / ml using 30 mM pH 5.0 MES buffer. 50 μg of CD4 antibody was added, and the mixture was incubated at 37°C in a rotary incubator for 6 h.
[0183] The magnetic beads were separated using a magnet, sealed with PBS solution containing BSA for 1 hour, and then washed and stored using the preservation solution as described in Example 1.
[0184] Example 13 Preparation of antibody-conjugated magnetic beads
[0185] Take the magnetic beads obtained in Example 8, wash them twice with 25mM MES buffer at pH 5.0, and resuspend them to form a magnetic bead suspension of about 10mg / ml.
[0186] Add 10 μl of 10 mg / ml EDC and 20 μl of 10 mg / ml NHS, and activate the carboxyl group at 37 °C for 0.5 h.
[0187] The supernatant was removed by magnetic separation, and the solution was resuspended to 10 mg / ml using 25 mM pH 5.0 MES buffer. 60 μg of CD4 antibody was added, and the mixture was incubated at 37°C in a rotary incubator for 6 h.
[0188] The magnetic beads were separated using a magnet, sealed with PBS solution containing BSA for 1 hour, and then washed and stored using the preservation solution as described in Example 2.
[0189] Example 14 Preparation of antibody-conjugated magnetic beads
[0190] Take the magnetic beads obtained in Example 8, wash them twice with 30mM MES buffer at pH 5.0, and resuspend them to form a magnetic bead suspension of about 10mg / ml.
[0191] Add 10 μl of 15 mg / ml EDC and 20 μl of 20 mg / ml NHS, and activate the carboxyl group at 37 °C for 1 h.
[0192] The supernatant was removed by magnetic separation, and the solution was resuspended to 10 mg / ml using 35 mM pH 5.0 MES buffer. 50 μg of CD4 antibody was added, and the mixture was incubated at 37°C in a rotary culture system for 8 h.
[0193] The magnetic beads were separated using a magnet, sealed with PBS solution containing BSA for 1 hour, and then washed and stored using the preservation solution as described in Example 2.
[0194] Example 15 Preparation of antibody-conjugated magnetic beads
[0195] Take the magnetic beads obtained in Example 8, wash them twice with 30mM MES buffer at pH 5.0, and resuspend them to form a magnetic bead suspension of about 10mg / ml.
[0196] Add 15 μl of 10 mg / ml EDC and 15 μl of 10 mg / ml NHS, and activate the carboxyl group at 37 °C for 1 h.
[0197] The supernatant was removed by magnetic separation, and the solution was resuspended to 10 mg / ml using 30 mM pH 5.0 MES buffer. 50 μg of CD4 antibody was added, and the mixture was incubated at 37°C in a rotary incubator for 6 h.
[0198] The magnetic beads were separated using a magnet, sealed with PBS solution containing BSA for 1 hour, and then washed and stored using the preservation solution as described in Example 2.
[0199] Example 16 Preparation of antibody-conjugated magnetic beads
[0200] Take the magnetic beads obtained in Example 9, wash them twice with 25mM MES buffer at pH 5.0, and resuspend them to form a magnetic bead suspension of about 10mg / ml.
[0201] Add 10 μl of 10 mg / ml EDC and 20 μl of 10 mg / ml NHS, and activate the carboxyl group at 37 °C for 0.5 h.
[0202] The supernatant was removed by magnetic separation, and the solution was resuspended to 10 mg / ml using 25 mM pH 5.0 MES buffer. 60 μg of CD4 antibody was added, and the mixture was incubated at 37°C in a rotary incubator for 6 h.
[0203] The magnetic beads were separated using a magnet, sealed with PBS solution containing BSA for 1 hour, and then washed and stored using the preservation solution as described in Example 3.
[0204] Example 17 Preparation of antibody-conjugated magnetic beads
[0205] Take the magnetic beads obtained in Example 9, wash them twice with 30mM MES buffer at pH 5.0, and resuspend them to form a magnetic bead suspension of about 10mg / ml.
[0206] Add 10 μl of 15 mg / ml EDC and 20 μl of 20 mg / ml NHS, and activate the carboxyl group at 37 °C for 1 h.
[0207] The supernatant was removed by magnetic separation, and the solution was resuspended to 10 mg / ml using 35 mM pH 5.0 MES buffer. 50 μg of CD4 antibody was added, and the mixture was incubated at 37°C in a rotary culture system for 8 h.
[0208] The magnetic beads were separated using a magnet, sealed with PBS solution containing BSA for 1 hour, and then washed and stored using the preservation solution as described in Example 3.
[0209] Example 18 Preparation of antibody-conjugated magnetic beads
[0210] Take the magnetic beads obtained in Example 9, wash them twice with 30mM MES buffer at pH 5.0, and resuspend them to form a magnetic bead suspension of about 10mg / ml.
[0211] Add 15 μl of 10 mg / ml EDC and 15 μl of 10 mg / ml NHS, and activate the carboxyl group at 37 °C for 1 h.
[0212] The supernatant was removed by magnetic separation, and the solution was resuspended to 10 mg / ml using 30 mM pH 5.0 MES buffer. 50 μg of CD4 antibody was added, and the mixture was incubated at 37°C in a rotary incubator for 6 h.
[0213] The magnetic beads were separated using a magnet, sealed with PBS solution containing BSA for 1 hour, and then washed and stored using the preservation solution as described in Example 3.
[0214] Comparative Example 1
[0215] This is essentially the same as Example 11, except that the preservation solution is replaced with a prior art preservation solution, the composition of which is:
[0216] BSA: 1% (w / v);
[0217] Trehalose: 2% (w / v);
[0218] NaCl: 0.15 M;
[0219] Tween-20: 0.1% (v / v);
[0220] Proclin-300: 0.05% (v / v);
[0221] Polyethylene glycol 20000: 2% (w / v);
[0222] Tris-HCl: 50 mM, adjust pH to 7.4.
[0223] Comparative Example 2
[0224] This is essentially the same as Example 11, except that the carboxyglucan is replaced with the existing 40Kd glucan.
[0225] Comparative Example 3
[0226] The procedure is basically the same as in Example 11, except that the magnetic beads are stored in a preservation solution for 30 days before conjugating the antibody.
[0227] Comparative Example 4
[0228] The procedure was basically the same as in Example 11, except that the magnetic beads were stored in a preservation solution for 30 days before conjugating the antibody, then resuspended in Tris buffer containing 0.1% Tween-20, and then sonicated in a water bath at 40 Hz for 3 min.
[0229] Comparative Example 5
[0230] The procedure is basically the same as in Example 11, except that before conjugating the antibody, the sample is resuspended in Tris buffer containing 0.1% Tween-20 and then sonicated in a water bath at 40 Hz for 3 min.
[0231] Example 19
[0232] The binding efficiency of cells to magnetic beads was tested according to the detection method in Example 2, and the results are shown in Table 2.
[0233] Table 2 Test results of Examples 10-18 and Comparative Examples 1-5
[0234]
[0235] According to the results in Table 2:
[0236] First, the various embodiments proposed in this application all demonstrate qualified binding ability with CD4+ cells, and the recovery rates are generally higher than 85%.
[0237] Second, compared with Comparative Examples 1-3, the preservation solution provided in this application has a significantly better protective effect on magnetic beads under shorter preservation time; however, under longer preservation time, the binding ability of magnetic beads decreases significantly. Therefore, it is recommended to control the preservation time of magnetic beads to 20 days or even 7 days for conjugated antibody experiments, which is already a significant improvement in preservation time compared with the preservation solution in the prior art.
[0238] Third, comparing Example 11 with Comparative Examples 3-5, ultrasonic treatment has a positive effect on the aggregation and recovery of magnetic beads after long-term storage, but the effect of ultrasonic treatment on magnetic beads is not significant when stored for a short period of time.
[0239] In summary, the nanomagnetic beads and their preparation method provided by the embodiments of the present invention can solve the technical problem of magnetic beads being difficult to preserve in the prior art by coating the magnetic core with polyethylene glycol. This makes the magnetic beads less prone to aggregation or agglomeration during the preservation process, improves the cell sorting efficiency of the magnetic beads, and has a promising application prospect.
[0240] It should be noted that, for those skilled in the art, the technical features in the above embodiments can be freely combined, and the resulting technical solutions also belong to the embodiments disclosed in this application.
[0241] Furthermore, without departing from the principles of this application, several improvements and modifications may be made to this application, and these improvements and modifications also fall within the protection scope of the claims of this application.
Claims
1. A method for preparing nanomagnetic beads, characterized in that, Including the following steps: A solution containing Fe3+ and Fe2+ was prepared, and an appropriate amount of dextran was added. The components were then thoroughly mixed at 50-80℃. The concentration of Fe3+ was 0.01-0.05 mol / L, and the concentration of Fe2+ was 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. The magnetic beads were placed in the preservation solution to form a magnetic bead suspension of approximately 10 mg / ml; 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; 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.
2. The method for preparing nanomagnetic beads according to claim 1, characterized in that: The magnetic beads should not be placed in the preservation solution for more than 20 days.
3. The method for preparing nanomagnetic beads according to claim 1, characterized in that: The antibody is a CD4 antibody.
4. The method for preparing nanomagnetic beads according to claim 1, characterized in that: The prepared magnetic nanobeads consist of a magnetic core formed by Fe3O4 and γ-Fe2O3, and a dextran layer coating the outside of the magnetic core. The dextran layer contains polyethylene glycol.
5. The method for preparing nanomagnetic beads according to claim 4, characterized in that: The polyethylene glycol is polyethylene glycol 600.
6. The method for preparing nanomagnetic beads according to claim 4, characterized in that: The dextran is a carboxylated dextran.