Preparation method of nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores
The FE3O4 magnetic nanoparticles are prepared through the improved co-sedimentation method and sodium citrate, and the ing was formed with agarose to form IDA-ni agarose magnetic beads, which solved the problem of uneven dispersion of magnetic nanoparticles and poor suspension. Protein purification.
Patent Information
- Application Number
- CN202411584206.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2025-01-24
AI Technical Summary
In the existing preparation methods for magnetic agarose microspheres, the magnetic nanoparticles are unevenly dispersed, resulting in some microspheres not being magnetized, making it difficult to completely recover during application; excessive coating of magnetic particles leads to damage to the microspheres and non-specific adsorption of hybrid proteins; The sphere contains more magnetic particles, resulting in poor suspension and low protein purification efficiency.
The improved co -precipitation method is used to prepare the FE3O4 magnetic nanoparticles, and a stable suspended magnetic flow is obtained by the sodium citrate solution. Mix the magnetic flow with the agarose solution evenly, and processing magnetic agarose particles through emulsification and cooling. Then under alkaline conditions, the cross-linked reaction is carried out, the branches of the IDA interval arm and chelated nickel ions to form IDA-ni agar glycogen magnet beads.
The prepared nickel chelated agar glycogen beads have good stability and distribution of uniform magnetic substances, solving the problem of incomplete magnetic separation and poor suspension, improved protein purification efficiency, and suitable Separation and purification of protein.
Smart Images
Figure CN119361309A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of magnetic nanomaterials, and in particular to a method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores. Background Art
[0002] Immobilized metal ion chelate affinity chromatography (IMAC) has become one of the most effective methods for separation and purification of proteins, especially recombinant proteins and peptides. It is a method based on the ability of some amino acids (such as His, Trp, Lys, etc.) on the surface of proteins to bind to transition metal ions (such as Cu 2+ 、Ni 2+ , Fe 2+ 、Zn 2+ 、Co 2+ The technology of separating and purifying specific proteins can be used to form complexes with proteins (such as proteins, proteins, etc.) to separate and purify specific proteins.
[0003] At present, the commonly used separation matrices for IMAC technology include agarose microspheres, polymer microspheres, silica microspheres, etc., among which agarose microspheres are the most widely used. Agarose microspheres have good biocompatibility, are rich in a large number of hydroxyl groups on the surface, and have low nonspecific adsorption to proteins. After the microspheres are modified and connected to spacer arms (such as IDA, TED, and NTA), they can chelate metal ions to achieve the separation and purification of specific tagged proteins. However, the IMAC technology based on agarose microspheres generally requires the separation medium to be loaded into the column, and different specifications of separation columns are selected for operation according to the volume of the separated material. The entire process is difficult to automate and takes a long time. In addition, problems such as column clogging, column poisoning, and large column dead volume are prone to occur during operation. In recent years, with the continuous deepening of research on magnetic nanomaterials, reports on magnetic agarose composite microspheres have increased day by day. After magnetic nanoparticles are added to agarose microspheres, superparamagnetism can be given to the microspheres. Directed enrichment of microspheres can be achieved under an external magnetic field, and uniform dispersion of microspheres can be achieved after the magnetic field is removed, thereby easily realizing the full automation of the separation and purification process. However, most of the currently reported methods for preparing magnetic agarose microspheres have the following problems: (1) After coating, the magnetic nanoparticles inside the microspheres are unevenly dispersed, resulting in some microspheres not being magnetized and difficult to be completely recovered during use; (2) The excessive coating of magnetic particles leads to increased risk of microsphere breakage and exposure, resulting in the problem of nonspecific adsorption of foreign proteins; (3) In order to increase the magnetic separation speed of the microspheres, the microspheres contain more magnetic particles, resulting in poor suspension of the microspheres and low protein purification efficiency. Summary of the invention
[0004] In view of the defects of the prior art, the present invention provides a method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores. The prepared nickel-chelated agarose magnetic beads have good stability and can separate and purify histidine-tagged proteins in fermentation broth.
[0005] In order to achieve the above purpose, the present invention solves the technical problem by adopting the following technical solutions: The present invention provides a method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores, the preparation method comprising the following steps: S1. Under alkaline conditions, Fe3O4 magnetic nanoparticles were prepared by hydrolyzing divalent iron ions and trivalent iron ions, and then treated with 1M sodium citrate solution to obtain a magnetic fluid; S2. adding the magnetic fluid to the agarose solution and mixing evenly to obtain a mixed system, adding the mixed system to the oil phase and stirring and emulsifying the mixed system, and cooling the mixed system to obtain uncross-linked magnetic agarose particles; S3. After magnetic separation of the magnetic agarose microparticles in step S2, an activator and an alkali solution are added and reacted at 30-40° C. for 5-7 hours; S4. After the reaction is completed, the magnetic agarose particles are washed and added to a sodium carbonate-sodium bicarbonate buffer solution and stirred at 200 rpm in a 30-40°C water bath, and then the IDA solution is added and reacted at 30-40°C overnight.
[0006] S5. Wash and magnetically separate the magnetic agarose microparticles after the reaction in step S4, then add them to a nickel sulfate hexahydrate solution, stir and react overnight at room temperature, wash them after the reaction, and then store them in 20% ethanol to obtain IDA-Ni agarose magnetic beads.
[0007] The present invention adopts sodium citrate to modify Fe3O4 magnetic nanoparticles according to the hydrophilic and hydrophobic properties of the system, thereby solving the problem of coexistence of bilayer and monolayer surface activity in the magnetic fluid modified with oleic acid in the traditional method, and avoiding the inability of the magnetic fluid to be stably suspended in the system and the phase separation of the magnetic core.
[0008] In some of the embodiments, in step S1, the Fe3O4 magnetic nanoparticles are prepared by reacting in a water bath at 75-85°C for 25-35 minutes.
[0009] In some embodiments, in step S2, the concentration of the agarose solution is 4-6 wt %.
[0010] In some embodiments, in step S2, the oil phase is prepared by stirring paraffin wax and Span 80 at 75-85°C and 650-750 rpm for 15-25 min; the mass ratio of the paraffin wax to Span 80 is 20:3.
[0011] In some of the embodiments, the stirring speed during the stirring emulsification is 650-750 rpm, and the stirring time is 55-65 min.
[0012] In some embodiments, in step S3, the activating agent is epichlorohydrin.
[0013] In some embodiments, the concentration of the IDA solution is 10 mg / mL.
[0014] In some embodiments, the concentration of the nickel sulfate hexahydrate solution is 0.1M.
[0015] On the one hand, the present invention provides nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores, which are prepared by the preparation method of nickel-chelated agarose magnetic beads.
[0016] On the other hand, the present invention also provides the use of the nickel-chelated agarose magnetic beads in selectively capturing, releasing and purifying His-tag tagged proteins.
[0017] Compared with the prior art, the present invention has the following beneficial effects: The preparation method of the invention can prepare superparamagnetic agarose composite microspheres with uniform magnetic material distribution and good stability, and solves the problems of incomplete magnetic separation and rapid precipitation of traditional agarose composite microspheres; at the same time, the magnetic agarose composite microspheres are used for separation and purification of histidine tag proteins in fermentation broth, and can obtain tag proteins with higher purity, which is beneficial to the research in the fields of subsequent pharmaceutical development, clinical diagnosis, personalized medicine, etc., and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the implementation mode will be briefly introduced below. Obviously, the drawings described below are only some implementation modes of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0019] Figure 1 The figure is a schematic diagram of the preparation process of nickel-chelated agarose magnetic beads provided in an embodiment of the present invention.
[0020] Figure 2 This is a real picture of the appearance of the steady-state magnetic fluid provided by an embodiment of the present invention.
[0021] Figure 3 It is a DLS graph of Fe3O4 nanoparticles provided in an embodiment of the present invention.
[0022] Figure 4 It is an optical microscope picture of agarose magnetic microspheres provided in an embodiment of the present invention.
[0023] Figure 5 It is an infrared spectrum of agarose magnetic microspheres provided in an embodiment of the present invention.
[0024] Figure 6 1 is a graph showing the test results of the carboxyl content of the IDA agarose magnetic microspheres provided in an embodiment of the present invention.
[0025] Figure 7 It is an optical microscope image of the IDA-Ni agarose beads provided in the embodiment of the present invention.
[0026] Figure 8 This is a graph showing the protein purification test results of the IDA-Ni agarose magnetic beads provided in an embodiment of the present invention.
[0027] Fig. 9 It is a graph showing the stability test results of agarose magnetic beads provided in an embodiment of the present invention.
[0028] Fig.10 It is a graph showing the test results of the magnetic separation speed of agarose magnetic beads provided in an embodiment of the present invention.
[0029] Fig.11 These are optical microscope images of different agarose magnetic beads, in which: A is a market-competing agarose magnetic bead; and B is the agarose magnetic bead of the present invention. DETAILED DESCRIPTION
[0030] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.
[0032] like Figure 1 As shown, the present invention firstly adopts a modified coprecipitation method to prepare Fe3O4 magnetic nanoparticles, and the particles can be stably suspended in an aqueous solution to form an ultra-stable magnetic fluid (no precipitation is observed after 3 months of suspension) after being treated with a sodium citrate solution; then the stable magnetic fluid is introduced into a high-temperature agarose solution to prepare a magnetic agarose solution with ultra-uniform distribution of magnetic particles; then the magnetic agarose solution is dripped into an oil phase containing a surfactant, and after high-temperature shear emulsification, cooling can be performed to prepare superparamagnetic agarose composite microspheres with uniform distribution of magnetic substances. After the surface of the microspheres is grafted with an IDA spacer arm to chelate nickel ions, specific separation and purification of histidine-tagged proteins in the fermentation broth is achieved.
[0033] Example 1 The coprecipitation method is used to prepare stably suspended Fe3O4 magnetic nanoparticles, and the method comprises the following steps: 1. Take a clean 2L glass beaker, add 80g ferric chloride hexahydrate, 30g ferrous sulfate heptahydrate, and 1237.5g deionized water, then place it in an ultrasonic machine, stir and ultrasonicate to dissolve it into a uniform solution.
[0034] 2. Take a clean 2L glass flask, use a glass rod to drain all the iron salt solution into the flask, place the flask in an 80℃ water bath, then set up the flask, constant pressure dropping funnel (containing 400mL concentrated ammonia water), condensation device and stirring motor, then seal the reaction system and set the stirring speed to 300rpm.
[0035] 3. Open the drain valve of the constant pressure dropping funnel and slowly add concentrated ammonia water into the flask. After all the concentrated ammonia water is added, react in a water bath at 80°C for 30 minutes.
[0036] 4. After the reaction is completed, pour the product in the flask into a clean 2L glass beaker, and discard the clear liquid after magnetic separation.
[0037] 5. Add deionized water to the beaker, stir evenly with ultrasound, and discard the supernatant after magnetic separation.
[0038] 6. Add 200 mL of sodium citrate solution (1 M sodium citrate solution) to the beaker and perform ultrasonic stirring and dispersion for 20 minutes. Magnetic separation is performed on the obtained uniform magnetic fluid to remove the supernatant.
[0039] 7. Add deionized water to the beaker and perform ultrasonic washing followed by magnetic separation until the supernatant is neutral.
[0040] 8. Add deionized water to the beaker to the 1000mL mark and disperse evenly by ultrasonic to prepare a stably dispersed magnetic fluid.
[0041] Depend on Figure 3 It can be seen that the hydrated particle size of the magnetic core prepared by the present invention is about 27nm, and the magnetic core is in a polydisperse state. Figure 2 As shown, after the magnetic fluid was left to stand for 3 months, no precipitated particles were found in the system, indicating that the magnetic fluid itself has good stability.
[0042] Example 2 The preparation of agarose magnetic microspheres comprises the following steps: 1. Weigh 200g of liquid paraffin and 30g of Span 80 in a 500mL three-necked flask, transfer it to an 80℃ water bath, and preheat at 700rpm for 20min.
[0043] 2. Weigh 1.6 g agarose powder and 30 g deionized water in a 100 mL beaker, heat in a microwave to dissolve them to obtain an agarose solution, and keep it warm in a 90 ° C water bath for later use to prevent the agarose from solidifying.
[0044] 3. Add 16 mL of treated magnetic fluid preheated to 90°C to the agarose solution in step 2 and stir evenly.
[0045] 4. Pour the uniform agarose solution containing magnetic fluid into the three-necked flask in step 1 quickly, and stir and emulsify at 700 rpm for 60 minutes to make the system into a uniform emulsion.
[0046] 5. Stop heating the three-necked flask, quickly replace the hot water in the water bath with ice water, and solidify the emulsion droplets into balls while stirring at 700 rpm.
[0047] 6. The product was washed three times with 300 mL of petroleum ether, 300 mL of ethanol and 300 mL of water respectively, and finally stored in 20% ethanol for later use.
[0048] The morphology of agarose magnetic microspheres was observed under an optical microscope and the -1 The transmittance of infrared light absorbed by agarose magnetic microspheres was measured within a wave number range.
[0049] Depend on Figure 4 and Figure 5 It can be seen that the yellow magnetic cores were successfully encapsulated in the agarose microspheres, and the magnetic cores were evenly distributed.
[0050] Example 3 The preparation of IDA-Ni agarose beads includes the following steps: 1. Set up the stirring device, set the stirring speed to 200 rpm, and heat in a 37°C water bath.
[0051] 2. Take 200 mL of uncross-linked agarose magnetic fluid (solid-liquid ratio 20%) in a 250 mL beaker.
[0052] 3. Remove the supernatant by magnetic separation, dilute to 200 mL with deionized water, stir well and transfer all to a 500 mL flask; rinse the beaker wall with 50 mL deionized water and transfer all the liquid to the flask; 5. Add 30 mL of sodium hydroxide solution (2M) to the flask.
[0053] 6. Add 10 mL of epichlorohydrin and continue stirring the reaction in a 37°C water bath for 6 h.
[0054] 7. The product was repeatedly washed with deionized water until no epoxy groups were detected in the washing solution.
[0055] 8. Wash the magnetic beads in step 7 and disperse them in 350 mL of sodium carbonate-sodium bicarbonate buffer (200 mM). Stir well and transfer to a flask. Stir at 200 rpm in a 37°C water bath. Rinse the beaker wall with 50 mL of sodium carbonate-sodium bicarbonate buffer (200 mM) and transfer all the washing liquid to the flask.
[0056] 9. Add 100 mL of IDA solution (10 mg / mL iminodiacetic acid, adjust the pH to 8.0 with sodium hydroxide) to the flask, stir and react at 37°C and 200 rpm overnight, wash the reaction product with deionized water several times until the pH of the supernatant is neutral, and obtain IDA agarose magnetic microspheres.
[0057] 10. Remove the supernatant by magnetic separation, disperse the magnetic beads in 300 mL of nickel sulfate hexahydrate solution (concentration of 0.1 M), stir and react at 200 rpm at room temperature overnight, wash the reaction product with deionized water several times and then disperse it in 250 mL of 20% ethanol for storage (the volume of magnetic fluid accounts for about 10%) to obtain IDA-Ni agarose magnetic beads.
[0058] The carboxyl content of IDA-Ni agarose beads was detected by conductivity titration, and the morphology of IDA-Ni agarose beads was observed under an optical microscope.
[0059] like Figure 6 and Figure 7 As shown in the figure, IDA was successfully grafted onto the surface of IDA-Ni agarose magnetic beads, and its carboxyl content was about 235 μmol. At the same time, microscopic observation showed that the appearance of the microspheres chelated with nickel ions did not change significantly, and the sphericity was good.
[0060] Test Example 1 The IDA-Ni agarose beads prepared above and commercially available IDA-Ni agarose beads were used for His-tag protein purification: 1. The buffer solution formula is as follows: Equilibration buffer: 20 mM phosphate buffer, 500 mM sodium chloride, 25 mM imidazole, pH 7.4.
[0061] Wash buffer: 20 mM phosphate buffer, 500 mM sodium chloride, 75 mM imidazole, pH 7.4.
[0062] Elution buffer: 20 mM phosphate buffer, 500 mM sodium chloride, 500 mM imidazole, pH 7.4.
[0063] 2. The steps for purification of His-tag protein are as follows: 1. Take 2 mg of IDA-Ni agarose beads into 1.5 mL centrifuge tubes.
[0064] 2. Dilute the bacterial lysate to 1 mg / mL with equilibrium buffer for use.
[0065] 3. Add 0.5 mL of equilibration buffer to the magnetic beads, vortex and separate by magnetic separation, and discard the supernatant. Repeat three times.
[0066] 4. Add 500uL of diluted protein solution in step 2 and invert at room temperature for 1 hour.
[0067] 5. Magnetic separation, retain the magnetic beads and collect the supernatant.
[0068] 6. Add 0.5 mL of washing buffer to the magnetic beads, vortex to mix, collect the supernatant by magnetic separation, and retain the magnetic beads. Repeat the step once.
[0069] 7. Add 0.25 mL of elution buffer to the magnetic beads, vortex and mix, invert and mix at room temperature for 10 minutes, collect the supernatant by magnetic separation, and retain the magnetic beads. Repeat the step once.
[0070] 8. Collect the two eluates into the same tube and test the protein concentration.
[0071] Table 1
[0072] From Table 1 and Figure 8 It can be seen that compared with the commercially available IDA-Ni agarose magnetic beads (competitor IDA-Ni magnetic beads), the IDA-Ni agarose magnetic beads prepared by the present invention have a higher His-tag tagged protein recovery efficiency, and their nonspecific adsorption is significantly lower than that of the competitor magnetic beads; the SDS-PAGE electrophoresis results also confirm that the tagged protein separated and purified by the magnetic beads prepared by the present invention has a high purity.
[0073] Test Example 2 1. Stability test At room temperature, the IDA-Ni agarose beads prepared in Example 3 and the commercially available IDA-Ni agarose beads were allowed to stand, respectively, and the changes in the magnetic bead suspension were observed.
[0074] 2. Magnetic separation speed test A magnet was placed on one side of the suspension containing IDA-Ni agarose beads in Example 3 or commercially available IDA-Ni agarose beads, and the time taken for the IDA-Ni agarose beads to be completely separated from the suspension was recorded.
[0075] 3. Observe the morphological characteristics of IDA-Ni agarose beads and commercially available IDA-Ni agarose beads under an optical microscope.
[0076] Depend on Figure 9-11 It can be seen that compared with the commercially available IDA-Ni agarose magnetic microspheres (competitive magnetic beads in the figure), the IDA-Ni agarose magnetic microspheres prepared by the present invention have better suspension stability, and the magnetic separation speed of the magnetic beads of the present invention is basically the same as that of the competitive magnetic beads on the market; at the same time, under a microscope, the magnetic cores (yellow substance) inside the magnetic beads of the present invention are evenly distributed, while the competitive magnetic beads obviously have the problem of uneven distribution of the magnetic cores (black substance).
[0077] In summary, the IDA-Ni agarose magnetic beads prepared by the preparation method of the present invention have good suspension stability, and the internal magnetic material is evenly dispersed, which ensures the sphericity of the product and avoids the problem that the high reaction temperature during the emulsification process makes the inner core of the aqueous phase unstable, the magnetic core leaks into the oil phase, and the product has poor magnetism and sphericity. At the same time, the magnetic separation speed of the magnetic beads is not reduced, and when separating and purifying the labeled protein, it can fully react with the substance to be separated, thereby obtaining a higher protein recovery efficiency.
[0078] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.
Claims
1. A method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores, characterized in that: The preparation method comprises the following steps: S1. Under alkaline conditions, Fe3O4 magnetic nanoparticles were prepared by hydrolyzing divalent iron ions and trivalent iron ions, and then treated with 1M sodium citrate solution to obtain a magnetic fluid; S2. adding the magnetic fluid to the agarose solution and mixing evenly to obtain a mixed system, adding the mixed system to the oil phase and stirring and emulsifying the mixed system, and cooling the mixed system to obtain uncross-linked magnetic agarose particles; S3. After magnetic separation of the magnetic agarose microparticles in step S2, an activator and an alkali solution are added and reacted at 30-40° C. for 5-7 hours; S4. After the reaction is completed, the magnetic agarose particles are washed and added to a sodium carbonate-sodium bicarbonate buffer solution and stirred at 200 rpm in a 30-40°C water bath, and then the IDA solution is added and reacted at 30-40°C overnight; S5. Wash and magnetically separate the magnetic agarose microparticles after the reaction in step S4, then add them to a nickel sulfate hexahydrate solution, stir and react overnight at room temperature, wash them after the reaction, and then store them in 20% ethanol to obtain IDA-Ni agarose magnetic beads.
2. The method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores according to claim 1, characterized in that: In step S1, the Fe3O4 magnetic nanoparticles are prepared by reacting in a water bath at 75-85°C for 25-35 minutes.
3. The method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores according to claim 1, characterized in that: In step S2, the concentration of the agarose solution is 4-6wt%.
4. The method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores according to claim 1, characterized in that: In step S2, the oil phase is prepared by stirring paraffin wax and Span 80 at 75-85°C and 650-750 rpm for 15-25 min; the mass ratio of the paraffin wax to Span 80 is 20:
3.
5. The method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores according to claim 1, characterized in that: The stirring speed in the stirring emulsification is 650-750 rpm, and the stirring time is 55-65 min.
6. The method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores according to claim 1, characterized in that: In step S3, the activating agent is epichlorohydrin.
7. The method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores according to claim 1, characterized in that: The concentration of the IDA solution is 10 mg / mL.
8. The method for preparing nickel-chelated agarose magnetic beads with uniformly dispersed magnetic cores according to claim 1, characterized in that: The concentration of the nickel sulfate hexahydrate solution is 0.1M.
9. A nickel-chelated agarose magnetic bead with uniformly dispersed magnetic cores, characterized in that: The nickel-chelated agarose magnetic beads are prepared by the preparation method of any one of claims 1 to 8.
10. Use of the nickel-chelated agarose magnetic beads according to claim 9 in selectively capturing, releasing and purifying His-tag tagged proteins.