KR102314901B1 - Crosslinked magnetic agarose bead having enhanced physicochemical stability and preparation method thereof - Google Patents

Abstract

The cross-linked magnetic agarose beads provided in one aspect of the present invention have improved chemical stability, mechanical strength and thermal stability compared to conventional magnetic agarose beads, so they can be used in harsh conditions such as high pressure, high temperature, and very high or low pH And, there is no leakage of magnetic nanoparticles from the magnetic agarose beads,
Since the manufacturing process is simple as an emulsion droplet gel forming method, when applied to protein separation by affinity tag, it can be used as an affinity chromatography medium that can improve protein separation efficiency and yield.

Description

Crosslinked magnetic agarose bead having enhanced physicochemical stability and preparation method thereof

The present invention prepares magnetic agarose beads in which magnetic nanoparticles are impregnated in agarose beads, and then reacts the magnetic agarose beads with two types of bifunctional crosslinking agents in turn to crosslinked magnetic agarose beads with improved physicochemical stability and preparation thereof it's about how

Recently, in the field of proteomics to understand the function of a protein, the importance of protein isolation and purification is increasing as a core technology for studying the function of a specific protein biosynthesized from 20 kinds of amino acids.

Materials using magnetic beads, which are currently attracting attention as materials for protein separation and purification, have higher separation ease compared to existing materials, and significantly improve the time required for separation and purification compared to conventional separation methods, making them efficient and ultra-fast. Since separation and purification are possible, it is currently evaluated as an essential purification material for protein function studies through high throughput system (HTS). Separation technology using silica-coated magnetic beads has been predominant around the world, but in recent years, agarose-based Technology has an advantage.

As a filler for chromatography columns, agarose has been widely used for separation and purification of various substances due to its high selectivity, excellent biocompatibility, and mild operating conditions. Agarose is obtained from agar, a marine hydrocolloid. Agar agarose is a disaccharide composed of 3-linked β-D-galactopyranosyl and 4-linked 3,6-anhydro-α-L-galactopyranosyl units. It has a common polymer backbone structure with repeating units. Unlike agarose, agar can cause non-specific adsorption with charged molecules because it has sulfate and carboxyl residues. By heating agar in an alkaline solution, the charged groups can be removed (Non-Patent Document 1, Chunling Ge et al., New agar microsphere for the separation and purification of natural products, Journal of Separation Science 2014, 37, 3253-3259).

Currently, magnetic agarose beads used for protein separation and purification are commercially available, but their chemical and mechanical stability is low, so that when used repeatedly several times, the separation efficiency of the target protein is lowered. In addition, due to the low chemical and mechanical stability of commercially available magnetic agarose beads, problems such as bead decomposition, ionization of beads, leakage of magnetic nanoparticles from the beads under low pH conditions or high pressure conditions, etc. There is a problem in that the separation efficiency is lowered.

To improve this problem of uncrosslinked magnetic agarose beads, magnetic agarose beads crosslinked with agarose have been developed and marketed, but there is no significant difference in stability compared to uncrosslinked magnetic agarose beads. There is a problem in that the separation efficiency of the protein is lowered.

There is a need for a technique for improving the thermal stability of agarose beads so as to overcome such a problem of low separation efficiency of proteins and to ensure stability in heat sterilization and separation under severe conditions.

New agar microsphere for the separation and purification of natural products, Journal of Separation Science 2014, 37, 3253-3259

An object of one aspect of the present invention is,

By sequentially crosslinking agarose beads containing magnetic nanoparticles with two types of crosslinking agents, chemical, mechanical and thermal stability is improved and swelling degree is improved compared to magnetic agarose beads that are not crosslinked or crosslinked with one type of crosslinking agent To provide a method for producing controlled cross-linked magnetic agarose beads.

The object of another aspect of the present invention is,

It is to provide a cross-linked magnetic agarose beads prepared by the above manufacturing method.

An object of another aspect of the present invention is,

It is to provide a method for separating and purifying a protein, comprising the step of contacting the protein with the cross-linked magnetic agarose beads.

In order to achieve the above object,

One aspect of the present invention is

preparing an aqueous solution containing agarose and magnetic nanoparticles;

preparing an oily solution containing a surfactant;

preparing magnetic agarose beads containing magnetic nanoparticles by dropping an aqueous solution to the oil phase solution, and forming a W/O emulsion;

preparing magnetic agarose beads containing primary crosslinked magnetic nanoparticles by mixing and stirring dihaloalkyl alcohol as a first crosslinking agent to the prepared magnetic nanoparticle-containing magnetic agarose beads;

Mixing and stirring the prepared first cross-linked magnetic nanoparticles-containing magnetic agarose beads with epihalohydrin, a second cross-linking agent, to prepare magnetic agarose beads containing secondary cross-linked magnetic nanoparticles; containing,

Provided is a method for preparing crosslinked magnetic agarose beads.

Another aspect of the present invention is

Provided is a cross-linked magnetic agarose bead prepared by the above manufacturing method.

Another aspect of the present invention is

It provides a method for separating and purifying a protein, comprising the step of contacting the protein with the cross-linked magnetic agarose beads.

The cross-linked magnetic agarose beads provided in one aspect of the present invention have improved chemical stability, mechanical strength and thermal stability compared to conventional magnetic agarose beads, so they can be used in harsh conditions such as high pressure, high temperature, and very high or low pH And, there is no leakage of magnetic nanoparticles from the magnetic agarose beads,

As the emulsion droplet gel-forming method has a simple manufacturing process, when applied to protein separation by affinity tag, it can be used as an affinity chromatography medium that can improve protein separation efficiency and yield.

1 is a diagram showing the morphology of MAB.
2 is a diagram showing the particle size distribution of MAB.
3 is a diagram showing the morphology of CL-MAB-D.
4 is a diagram showing the morphology of CL-MAB-DE.
5 is a view showing an X-ray diffraction pattern of iron oxide nanoparticles (IONP) and MAB.
6 shows FT-IR spectra of MAB, CL-MAB-D and CL-MAB-DE.
Figure 7a shows the compressive load and elongation of the MAB.
Figure 7b shows the compressive load and elongation of CL-MAB-D and CL-MAB-DE.
Figure 8 shows the swelling degree of MAB, CL-MAB-D and CL-MAB-DE.
9 shows thermogravimetric analysis curves of MAB, CL-MAB-D and CL-MAB-DE.
Figure 10 shows the DSC analysis curves of MAB, CL-MAB-D and CL-MAB-DE.

Hereinafter, the present invention will be described in detail.

On the other hand, the embodiment of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided in order to more completely explain the present invention to those of ordinary skill in the art.

Furthermore, "including" a certain component throughout the specification means that other components may be further included, rather than excluding other components, unless otherwise stated.

One aspect of the present invention is

preparing an aqueous solution containing agarose and magnetic nanoparticles;

preparing an oily solution containing a surfactant;

preparing magnetic agarose beads containing magnetic nanoparticles by dropping an aqueous solution to the oil phase solution, and forming a W/O emulsion;

preparing magnetic agarose beads containing primary crosslinked magnetic nanoparticles by mixing and stirring dihaloalkyl alcohol as a first crosslinking agent to the prepared magnetic nanoparticle-containing magnetic agarose beads;

Mixing and stirring the prepared first cross-linked magnetic nanoparticles-containing magnetic agarose beads with epihalohydrin, a second cross-linking agent, to prepare magnetic agarose beads containing secondary cross-linked magnetic nanoparticles; containing,

Provided is a method for preparing crosslinked magnetic agarose beads.

Hereinafter, the method for producing crosslinked magnetic agarose beads provided in one aspect of the present invention will be described in detail step by step.

<1> Preparing an aqueous solution containing agarose and magnetic nanoparticles

The temperature of the aqueous solution may be 70 to 95 ℃, 75 to 95 ℃, 80 to 95 ℃, 85 to 95 ℃, 70 to 90 ℃, 70 to 85 ℃ may be, may be 80 to 90 ℃, may be 84 to 86 ℃, most preferably may be a high temperature of 85 ℃.

The magnetic nanoparticles may be iron oxide nanoparticles having an average diameter of 50 to 500 nm, and the iron oxide nanoparticles are preferably paramagnetic nanoparticles made _{of Fe 3} O _{4 crystals.} In another aspect, the average diameter of the iron oxide nanoparticles may be 50 to 500 nm, may be 100 to 500 nm, may be 150 to 500 nm, may be 200 to 500 nm, may be 250 to 500 nm It can be 300-500 nm, 350-500 nm, 400-500 nm, 50-450 nm, 50-400 nm, 350-450 nm, It may be 380 to 420 nm, and most preferably 400 nm.

The magnetic nanoparticles may be those in which iron oxide nanoparticles are coated with a low-molecular material or a high-molecular material, or may not be coated with a low-molecular or high-molecular material. The coating material may be a low-molecular material such as oleic acid, linoleic acid, or citric acid, and a synthetic polymer or dextrose such as polyacrylic acid, polyvinylpyrrolidone, etc. It may be a natural polymer material such as dextran or the like.

The agarose concentration in the aqueous solution may be 0.1 to 10% by weight, 1 to 8% by weight, 3 to 7.5% by weight, 5 to 7% by weight, and 5.5 to 6.5% by weight. and most preferably 6% by weight.

The gel strength of the agarose is 750 to 1500 g/cm ² , preferably 1000 g/cm ² or more, and most preferably 1200 g/cm ² or more in a 1% by weight aqueous solution.

<2> Preparing an oily solution containing a surfactant

The organic solvent of the oily solution is preferably any one or two or more selected from cyclohexane and toluene.

In the oil phase solution containing the surfactant, the surfactant is preferably a Span series, which is a nonionic surfactant, and the concentration in the oil phase may be 0.1 to 15% by volume, 5 to 10% by volume, and 7 to It may be included in 9% by volume, most preferably 8% by volume.

<3> Dropping an aqueous solution to the oil phase solution to prepare magnetic nanoparticles-containing magnetic agarose beads through W/O emulsion formation

The magnetic agarose beads were prepared by mixing an aqueous phase (W, water phase) prepared by dissolving iron oxide nanoparticles and agarose in high-temperature deionized water, and an oil phase (O, oil phase) containing a surfactant. Then, the emulsion is slowly cooled to room temperature while stirring at a constant speed to form droplets of the emulsion gel.

At this time, the mixing volume ratio of the aqueous solution and the oil phase solution may be 1:1 to 40, may be 1:5 to 20, may be 1:8 to 15, may be 1:9 to 12, most preferably For example, it may be 1:10. The cooling rate for gelation of the droplets formed in the W/O emulsion is 0.1 to 2.0°C per minute, preferably 0.3 to 1.2°C, most preferably 0.6°C.

<4> A step of preparing magnetic agarose beads containing primary crosslinked magnetic nanoparticles by mixing and stirring dihaloalkyl alcohol as a first crosslinking agent to the prepared magnetic agarose beads containing magnetic nanoparticles (MAB → CL -MAB-D)

The first crosslinking agent, dihaloalkyl alcohol, is selected from 1,3-dichloro-2-propanol, 2,3-dichloropropanol, 1,3-dibromo-2-propanol and 2,3-dibromopropanol. Any one or two or more may be used. Most preferred is when 1,3-dichloro-2-propanol is used as the primary crosslinking agent.

The catalyst used for crosslinking MAB is preferably NaOH, KOH, or the like, and most preferably NaOH.

Whether CL-MAB-D is cross-linked in cross-linking of MAB can be confirmed from the fact that cross-linked magnetic agarose beads are not dissolved when CL-MAB-D is added to DMSO, a representative solvent of agarose.

<5> Mixing and stirring the prepared first cross-linked magnetic nanoparticles-containing magnetic agarose beads with epihalohydrin, which is a second cross-linking agent, to prepare magnetic agarose beads containing secondary cross-linked magnetic nanoparticles Step (CL-MAB-D → CL-MAB-DE)

The second crosslinking agent epihalohydrin is epifluorohydrin, epichlorohydrin, epibromohydrin, epiiodohydrin, methylepichlorohydrin, methylepibromohydrin, and methylepiiodohydrin. Any one or two or more selected from may be used. Most preferred is when epichlorohydrin is used.

The average diameter of the finally prepared crosslinked magnetic agarose beads may be 15 to 300 μm, may be 15 to 300 μm, may be 30 to 270 μm, may be 50 to 250 μm, and may be 70 to 200 μm. It may be 80 to 150 μm, may be 90 to 110 μm, and may be 100 μm.

As the type of reducing agent used for crosslinking CL-MAB-D, sodium borohydride, sodium aluminum hydride, lithium borohydride, sodium hydroxide, etc. are preferable, and the most Preferred is sodium borohydride.

Another aspect of the present invention is

Provided is a cross-linked magnetic agarose bead prepared by the above manufacturing method.

Another aspect of the present invention is

It provides a method for separating and purifying a protein, comprising the step of contacting the protein with the cross-linked magnetic agarose beads.

The cross-linked magnetic agarose beads provided in one aspect of the present invention have improved chemical stability, mechanical strength and thermal stability compared to conventional magnetic agarose beads, so that they can be used in harsh conditions such as high pressure, high temperature, and very high or low pH And, there is no leakage of magnetic nanoparticles from the magnetic agarose beads,

As the emulsion droplet gel-forming method has a simple manufacturing process, when applied to protein separation by affinity tag, it can be used as an affinity chromatography medium that can improve protein separation efficiency and yield.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples.

However, the Examples and Experimental Examples described below are merely illustrative of the present invention in detail in one aspect, and the present invention is not limited thereto.

<Example 1> Preparation of double cross-linked magnetic agarose beads (CL-MAB-DE)

[Production of MAB]: 1.2 g of agarose (Sigma-Aldrich), 0.3 g of iron oxide nanoparticles (average diameter: 400 nm) and 20 mL of deionized water were stirred at 85° C. for 30 minutes at 250 rpm using a magnetic stirrer. Thus, an agarose solution containing iron oxide nanoparticles was prepared to prepare a water phase solution. As an oil phase solution, 200 mL of cyclohexane (Daejeong Hwageum Co., Ltd.) solution containing 8 v/v% of Span 85 (Sigma-Aldrich) was prepared, and the oil phase was formed with a three-neck round bottom. After adding to the flask, the temperature was raised to 75°C. A W/O emulsion was prepared by slowly adding the aqueous phase with a micropipette while stirring the oil phase at 800 rpm using a mechanical stirrer. At this time, the mixing volume ratio of the aqueous solution: the oil phase solution was adjusted to 1:10. While maintaining the temperature of the prepared W/O emulsion at 60° C., the W/O emulsion was stirred at 900 rpm for 1 hour using a mechanical stirrer, and then the temperature of the solution was cooled to room temperature while maintaining the stirring. The produced magnetic agarose beads (MAB) containing iron oxide nanoparticles were separated using a magnet, and the separated beads were washed with 50 v/v% ethanol/water, redispersed in 20 v/v% ethanol/water, and used It was stored at 4°C until

[First crosslinking]: 1.5 g of the MAB prepared above, 0.1 mL of 1,3-dichloro-2-propanol (DCP) and 40 mL of 0.3 M NaOH solution were added to a round bottom flask, and then 500 rpm with a mechanical stirrer stirred in. After the temperature of the reaction mixture was set to 50° C., the reaction was allowed to proceed for 2 hours. After the reaction is complete, the magnetic agarose beads (CL-MAB-D) crosslinked with DCP are washed with distilled water until the pH of the solution becomes neutral, and then redispersed in 20 v/v% ethanol/water, until use. Stored at 4°C.

[Secondary crosslinking]: 2.0 g of the swollen CL-MAB-D was transferred to DMSO through a repeated washing process, and the solution was dispersed in DMSO to a volume of 50 mL. 0.17 mL of epichlorohydrin (ECH) was added, and the temperature was raised to 50 °C. After adding 7.04 mL of 0.3 M NaOH solution so that the molar ratio of ECH:NaOH becomes 1:1, the reaction mixture was stirred overnight. The temperature of the reactor was lowered to room temperature, and the product was repeatedly precipitated and washed with deionized water.

<Example 2> Preparation of CL-MAB-DE using DBP

Cross-linked magnetic agarose beads were prepared in the same manner as in Example 1, except that 1,3-dibromo-2-propanol (DBP) was used instead of DCP as a crosslinking agent for agarose. .

<Example 3> Preparation of CL-MAB-DE using 50 nm iron oxide nanoparticles

Instead of using iron oxide nanoparticles having an average diameter of 400 nm, CL-MAB-DE was prepared in the same manner as in Example 1, except that iron oxide nanoparticles having an average diameter of 50 nm were used.

<Example 4> Preparation of CL-MAB-DE using toluene

CL-MAB-DE was prepared in the same manner as in Example 1, except that toluene was used instead of cyclohexane as an oil phase.

<Example 5> Preparation of CL-MAB-DE using Span 60

Instead of using 8 v/v% of Span 85 as a surfactant, CL-MAB-DE was prepared in the same manner as in Example 1, except that 10 v/v% of Span 60 was used. .

<Comparative Example 1> Preparation of MAB

MAB was prepared in the same manner as in Example 1 without performing the step of crosslinking MAB with DCP and ECH.

<Comparative Example 2> Preparation of CL-MAB-D

CL-MAB-D was prepared in the same manner as in Example 1 without performing the step of secondary crosslinking of the agarose beads with ECH.

<Experimental Example 1> Morphology analysis of MAB

The MAB prepared in the intermediate process of Example 1 was observed using an optical microscope (Leica DMI3000 B, Leica Microsystems, Wetzlar, Germany). The results are shown in FIG. 1 .

1 is a diagram showing the morphology of MAB.

As shown in FIG. 1 , MAB had a spherical shape and iron oxide nanoparticles were evenly distributed in the beads. Scale bars here are 100 micrometers (μm).

<Experimental Example 2> Analysis of particle size distribution of MAB

A solution obtained by suspending the MABs prepared in the intermediate process of Example 1 in deionized water was added to deionized water containing an analytical probe at 25° C. using a particle size analyzer (Microtrac 3000, Microtrac Inc., Montgomeryville, PA, USA). The size and size distribution of the particles were measured in The results are shown in FIG. 2 .

2 is a diagram showing the particle size distribution of MAB.

As shown in FIG. 2 , the MAB prepared in the intermediate process of Example 1 was found to have a diameter range of about 15 to 300 micrometers, and an average diameter was found to be about 100 micrometers (㎛).

<Experimental Example 3> Morphology analysis of CL-MAB-D

CL-MAB-D first crosslinked with DCP in the intermediate process of Example 1 was observed using an optical microscope (Leica DMI3000 B, Leica Microsystems, Wetzlar, Germany). The results are shown in FIG. 3 .

3 is a diagram showing the morphology of CL-MAB-D.

As shown in FIG. 3, CL-MAB-D first crosslinked with DCP has a spherical shape without any difference from that of MAB, and the iron oxide nanoparticles are evenly distributed in the beads, so DCP crosslinking is the morphological characteristic of agarose beads It can be seen that there is no effect on Scale bars here are 50 micrometers (μm).

<Experimental Example 4> Morphology analysis of CL-MAB-DE

The CL-MAB-DE prepared in Example 1 was observed using an optical microscope (Leica DMI3000 B, Leica Microsystems, Wetzlar, Germany). The results are shown in FIG. 4 .

4 is a diagram showing the morphology of CL-MAB-DE.

As shown in Figure 4, CL-MAB-DE crosslinked with DCP and ECH,

It has a spherical shape similar to that of MAB or CL-MAB-D first crosslinked with DCP, and iron oxide nanoparticles are evenly distributed in the beads, so that double crosslinking using DCP and ECH affects the morphological properties of agarose beads It can be seen that the . Scale bars here are 100 micrometers (μm).

<Experimental Example 5> X-ray diffraction analysis of MAB

After drying the MAB prepared in the intermediate process of Example 1 under vacuum, X-ray powder diffractometer (D8 ADVANCE, Bruker AXS GmbH, Kalsruhe, Germany) using Cu Kα radiation was used to X- The line diffraction pattern was analyzed, and iron oxide nanoparticles (IONP) having an average diameter of 400 nm and 50 nm, respectively, were analyzed as a control sample. The results are shown in FIG. 5 .

5 is a diagram illustrating an X-ray diffraction pattern of MAB.

As shown in FIG. 5, the diffraction peak shown by the MAB prepared in the intermediate process of Example 1 is a unique diffraction peak shown by _{the Fe 3} O _{4 crystal of IONP having an average diameter of 400 nm or 50 nm as a control sample.} Therefore, it can be seen that the incorporation of IONP into agarose does not affect the crystallinity of IONP.

<Experimental Example 6> FT-IR analysis of MAB, CL-MAB-D and CL-MAB-DE

After drying the MAB, CL-MAB-D and CL-MAB-DE prepared in Example 1 under vacuum, KBr pellets were prepared and analyzed by FT-IR (Nicolet 5700, Nicolet Corporation). The results are shown in FIG. 6 .

6 shows FT-IR spectra of MAB, CL-MAB-D and CL-MAB-DE.

^{As shown in Fig. 6, the characteristic peak of 1037 cm -1} indicating the COC stretching vibration of agarose in CL-MAB-DE and CL-MAB-D is larger than that of MAB, so that the hydroxyl group of agarose is crosslinked. It can be confirmed that a new COC bond is generated.

<Experimental Example 7> Evaluation of compressive strength of MAB, CL-MAB-D and CL-MAB-DE

After drying MAB, CL-MAB-D and CL-MAB-DE in Example 1 under vacuum, the compressive strength of the sample was evaluated with a micro material tester (Instron 5848, Instron Co., Norwood, MA, USA). . The results are shown in FIG. 7 .

Figure 7a shows the compressive load and elongation of the MAB.

Figure 7b shows the compressive load and elongation of CL-MAB-D and CL-MAB-DE.

7A and 7B, MAB had a maximum compressive load of 1.1 N and a maximum compressive elongation of 1.2 mm. CL-MAB-D had a maximum compressive load of 3.2 N and a maximum compressive elongation of 1.1 mm. From these results, it was confirmed that the primary crosslinking of agarose by DCP could improve the compressive load by about 3 times without significantly affecting the compressive elongation of the MAB. On the other hand, CL-MAB-DE in which CL-MAB-D was secondarily crosslinked with ECH had a maximum compressive load of 2.3 N and a maximum compressive elongation of 0.9 mm.

<Experimental Example 8> Evaluation of swelling degree of MAB, CL-MAB-D and CL-MAB-DE

The CL-MAB-DE prepared in Example 1, the MAB prepared in Comparative Example 1, and the CL-MAB-D prepared in Comparative Example 2 were dried under vacuum, and then the dry weight was measured. Each bead was added to a vial containing a certain amount of deionized water, and samples were collected every predetermined time to measure the wet weight. The result of obtaining the swelling degree by the following formula is shown in FIG. 8: Swelling ratio (%) = [(wet sample weight - dry sample weight)/dry sample weight]×100.

Figure 8 shows the swelling degree of MAB, CL-MAB-D and CL-MAB-DE.

As shown in FIG. 8 , MAB showed a high degree of swelling with respect to deionized water, whereas CL-MAB-D and CL-MAB-DE showed a relatively low degree of swelling due to crosslinking, and CL-MAB-D and CL -MAB-DE showed similar swelling degree.

<Experimental Example 9> Thermogravimetric analysis (TGA) of agarose, MAB, CL-MAB-D and CL-MAB-DE

After drying the CL-MAB-DE prepared in Example 1, the MAB prepared in Comparative Example 1, and the CL-MAB-D prepared in Comparative Example 2 under vacuum, TGA results are shown in FIG. 9 .

9 shows thermogravimetric analysis curves of MAB, CL-MAB-D and CL-MAB-DE.

As shown in FIG. 9 , the decomposition initiation temperature of beads was higher in CL-MAB-D than MAB and CL-MAB-DE than CL-MAB-D. As for the decomposition behavior to heat, CL-MAB-D was more gentle than MAB and CL-MAB-DE was more gentle than CL-MAB-D, so it was confirmed that the resistance to thermal decomposition increased according to the degree of crosslinking.

<Experimental Example 10> Differential scanning calorimetry (DSC) analysis of MAB, CL-MAB-D and CL-MAB-DE

After drying the CL-MAB-DE prepared in Example 1, the MAB prepared in Comparative Example 1, and the CL-MAB-D prepared in Comparative Example 2 under vacuum, the results of DSC analysis are shown in FIG. 10 .

Figure 10 shows the DSC analysis curves of MAB, CL-MAB-D and CL-MAB-DE.

As shown in FIG. 10 , the melting point temperature (T _m ) of MAB is the same as CL-MAB-D and CL-MAB-DE, but the melting onset temperature (onset temperature) of CL-MAB-D and CL-MAB-DE ) increased significantly compared to the value of MAB.

Claims (12)

preparing an aqueous solution containing agarose and magnetic nanoparticles;
preparing an oily solution containing a surfactant;
preparing magnetic agarose beads containing magnetic nanoparticles by dropping an aqueous solution to the oil phase solution, and forming a W/O emulsion;
1,3-dichloro-2-propanol or 1,3-dibromo-2-propanol, which is a first crosslinking agent, is mixed with the prepared magnetic agarose beads containing magnetic nanoparticles and stirred, and the first crosslinked magnetic nano preparing particle-containing magnetic agarose beads;
In DMSO (dimethyl sulfoxide), the prepared magnetic agarose beads containing the primary cross-linked magnetic nanoparticles were mixed with epihalohydrin, a second cross-linking agent, and stirred, and the magnetic agarose containing the secondary cross-linked magnetic nanoparticles was stirred. Including; preparing a loss bead;
A method for producing cross-linked magnetic agarose beads,

The magnetic nanoparticles are iron oxide nanoparticles having an average diameter of 350 to 450 nm, and the iron oxide nanoparticles are paramagnetic nanoparticles composed _{of Fe 3} O _{4 crystals,}
In the step of preparing the oil phase solution containing the surfactant, the organic solvent of the oil phase solution is any one or two or more selected from cyclohexane and toluene,
The concentration of the surfactant in the oil phase solution containing the surfactant is included in 7 to 9% by volume,
In the step of preparing the magnetic nanoparticle-containing magnetic agarose beads by dropping the aqueous phase solution to the oil phase solution and forming a W / O emulsion, the mixing volume ratio of the aqueous phase solution and the oil phase solution is 1:9 to 12 to do,
Method for producing crosslinked magnetic agarose beads.
delete According to claim 1,
The epihalohydrin is any one selected from epifluorohydrin, epichlorohydrin, epibromohydrin, epiiodohydrin, methylepichlorohydrin, methylepibromohydrin and methylepiiodohydrin A method for producing crosslinked magnetic agarose beads, characterized in that one or two or more.
delete delete According to claim 1,
In the step of preparing an aqueous solution containing agarose and magnetic nanoparticles,
The method for producing a cross-linked magnetic agarose beads, characterized in that the temperature of the aqueous solution is 70 to 95 ℃.
delete delete delete According to claim 1,
The method for producing cross-linked magnetic agarose beads, characterized in that the average diameter of the prepared cross-linked magnetic agarose beads is 15 to 300 ㎛.
A cross-linked magnetic agarose bead produced by the method of claim 1.
A method for separating and purifying a protein, comprising the step of contacting the protein with the cross-linked magnetic agarose beads of claim 11 .

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