KR101409720B1 - Target material using a magnetic particle separation method and separation device - Google Patents

Abstract

The present invention relates to a target material separating method and a target material separating device using magnetic particles, wherein a target material is separated and purified by making the target material selectively combined in a mixture in the process of combining magnetic particles with a material having affinity with the target material. The present invention provides a target material separating method comprising the steps of: providing the inner main body of the separating device with magnetic particles having a functional group, which can be combined with the target material, formed on the surface thereof; removing foreign materials by inputting a uniform-decomposition washing buffer solution into the inner main body, uniformly mixing the uniform-decomposition washing buffer solution with the magnetic particles, and discharging the uniform-decomposition washing buffer solution out of the inner main body while constraining the magnetic particles by applying a magnetic field; forming a mixture of the magnetic particles and the target material combined by providing the inner main body with a sample including the target material to be separated and dispersing the sample together with the magnetic particles; constraining the magnetic particles combined with the target material by applying a magnetic field to the mixture, and removing foreign materials by discharging a remaining mixture out of the inner main body; separating the magnetic particles from the target material by intercepting the applied magnetic field and an extraction buffer solution; and constraining the magnetic particles and purifying the separated target material by applying a magnetic field again if the magnetic particles are separated from the target material. By realizing a separating device according to the present invention, a large amount of samples can be separated and purified by a small amount of magnetic particles, and time can be reduced and a reaction device can be miniaturized by simplifying steps.

Description

TECHNICAL FIELD The present invention relates to a method for separating a target material using magnetic particles,

The present invention relates to a method and apparatus for separating a target material using magnetic particles, and more particularly, to a method and apparatus for separating and separating a target material from a magnetic material by combining a target substance and a substance having affinity to magnetic particles, The target substance bound to the magnetic particles is removed, the impurities are removed, and only the magnetic particles and the target substance are left, the target substance is separated from the extraction buffer solution, and the magnetic field is applied again to restrain only the magnetic particles, And a separation device using the magnetic particles.

Chromatography, the most commonly used method for separating mixtures, is a separation method that can separate similar components that make up a complex mixture. The mixture is separated using the difference of attraction between the mobile phase and the stationary phase. There are various kinds of chromatography depending on the characteristics of the stationary phase and the mobile phase. The types thereof include column chromatography, planar chromatography, affinity chromatography, molecular excursion chromatography, ion exchange chromatography, gas chromatography, Gas chromatography, liquid chromatography and the like, and since each chromatography has different characteristics, it is necessary to select it appropriately according to the properties and efficiency of the mixture.

Among the chromatographic techniques, there is a protein separation technique using agarose beads as a purification technique which is often used in the biotechnology field. The technique is to fill the column with agarose beads and flow the sample with different buffer solutions. The mixture is collected at each time interval and obtained proteins. The sample obtained at this time needs a process of increasing purity through dialysis.

The length and volume of the column, the rate at which the solution flows, and the constant filling of the agarose beads are many factors affecting resolution. There are a number of considerations to be taken into account, and there are drawbacks that must be worked manually throughout the mixture separation. In addition, since the volumetric fractions eluted by the time difference are large and eluted together with the buffer solution, a dialysis process is necessarily required, and it takes a long time to separate the mixture. This method is proportional to the amount of the filler and the solution proportional to the amount of the mixture to be separated, so that the size of the reaction apparatus is large and it is not suitable for mass separation of the mixture.

In addition, there is a separation method using glass fiber, silica, magnetic particles, etc., and a method mainly using magnetic particles is widely used. In Publication No. 10-2012-0031724 and Registration No. 10-0762969, magnetic particles are coated with silica to be used for protein separation. In Registration No. 10-0738496, a method for separating antibody proteins using magnetic fine particles and an orientation-immobilized ligand has been introduced . The method of separating the target material using the magnetic particles is excellent in that the surface of the magnetic particles is modified to improve the separation ability, but they also have a long reaction time and a manual process step by step.

In order to solve the above-mentioned problems, the method and apparatus for separating a target material using magnetic particles according to the present invention simplify a dispersion operation by dispersing and dissolving a target substance at a time in a separation apparatus, So as to shorten the time required for the operation.

The object of the present invention is to separate and purify a large amount of sample with a small amount of magnetic particles.

Disclosure of the Invention The object of the present invention is to reduce the size of the reaction body in which the reaction of the mixture takes place.

The object of the present invention is to provide a method and apparatus for separating a target material using magnetic particles so that all processes can be automatically performed even after the injection of the mixture is not involved.

According to an aspect of the present invention, there is provided a method for separating a target material using magnetic particles comprising the steps of: providing a magnetic particle having a reactor capable of binding to a target material on a surface thereof, Uniformly washing buffer solution into the inner body to uniformly mix the magnetic particles with the magnetic particles, and flowing the decomposed equivalent washing buffer solution to the outside of the inner body in a state where the magnetic particles are confined by applying a magnetic field, thereby removing impurities; Providing a sample containing a target substance to be separated in the inner body and dispersing the sample together with the magnetic particles to form a mixture by binding the magnetic particles and the target substance; Applying a magnetic field to the mixture to confine the magnetic particles bound to the target material, and flowing the remaining mixture to the outside of the inner body to remove impurities; Blocking the applied magnetic field and providing an extraction buffer solution to separate the magnetic particles and the target material; Separating the magnetic particles and the target material, applying a magnetic field again to confine the magnetic particles, and purifying the separated target material; The present invention can solve the technical problem of the present invention.

In order to separate and purify a target material using magnetic particles in a sample containing a target material, an apparatus for separating a target material using magnetic particles according to the present invention comprises: an inner body provided inside the outer body; A temperature holding device provided between the inner body and the outer body or inside the inner body; At least one electromagnet extending downward from an outer circumference of the inner body or an upper portion of the inner body; A solution injector connected to an upper portion of the inner body to inject the solution into the inner body; A sample injecting unit connected to an upper portion of the inner body and injecting the sample into the inner body; A filtrate discharge unit provided at a lower portion of the inner body to discharge the filtrate to the outside; The present invention can solve the technical problem of the present invention.

INDUSTRIAL APPLICABILITY The target substance separation method and separation apparatus using the magnetic particles of the present invention have the effect of purifying a large amount of sample with a small amount of magnetic particles.

INDUSTRIAL APPLICABILITY The target substance separation method and separation apparatus using the magnetic particles of the present invention have the effect of simplifying the steps and shortening the separation operation time.

In the method and apparatus for separating a target material using magnetic particles according to the present invention, by separating a target material using magnetic particles, the amount of the buffer solution used in the conventional method is remarkably reduced, The effect of reducing the size of the reaction body can be obtained.

The method and apparatus for separating a target material using magnetic particles according to the present invention are capable of improving the efficiency of the operator and efficiently separating the target material since the whole process is automatically performed even after the injection of the mixture is not involved, It has the effect of keeping the material at a high quality.

1 is a flowchart showing a method of separating a target material using magnetic particles according to the present invention.
2 is a schematic view showing a method of separating a target material using magnetic particles according to the present invention.
3 is a view showing an electromagnet type separator of a flask spin type according to the present invention.
4 is a view showing an electromagnet type external separator of a flask spin type according to the present invention.
5 is a view showing an electromagnet type separator of a screw impeller type according to the present invention.
FIG. 6 is a view showing a magnet type external separator of a screw impeller type according to the present invention. FIG.
FIG. 7 is a view showing an internal type separator of an aeration type electro-magnet according to the present invention; FIG.
FIG. 8 is a view showing an electromagnet type external separation device of an aeration type according to the present invention. FIG.
9 shows an inclined structure located underneath an internal body A according to the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a method and apparatus for separating a target material using magnetic particles according to the present invention will be described in detail with reference to the accompanying drawings. In the following description of the present invention, the following terms are defined in consideration of the function of the present invention, and this may be changed according to the intention of the user and the operator. Accordingly, Should be interpreted on the basis of. It should also be noted that the appended drawings do not limit the separating apparatus according to the invention, and that the respective apparatuses can be combined and applied to one another.

A method for separating a target material using magnetic particles according to the present invention comprises the steps of: providing a magnetic particle having a reactor capable of binding to a target material on its surface to an inner body of a separating device; Equilibration wash buffer), mixing the magnetic particles uniformly, applying a magnetic field to confine the magnetic particles, and flowing the decomposed equivalent washing buffer solution to the outside of the inner body to remove impurities; Providing a sample containing the target substance to be separated in the inner body and dispersing the sample together with the magnetic particles to form a mixture with the magnetic particles to form a mixture; and applying a magnetic field to the mixture to form a target substance The bound magnetic particles are confined and the remaining mixture is flowed out of the inner body to remove the foreign matter System; and the step of blocking the magnetic field was applied and the service extraction buffer (Elution buffer) to remove the magnetic particles and the target substance; And separating the magnetic particles and the target material, applying a magnetic field again to confine the magnetic particles and purifying the separated target material; Lt; / RTI >

The magnetic particles are bonded to a substance having an affinity according to a target substance to be separated.

The surface of the magnetic particles is modified and coated with a ligand, or the reactor is attached to the surface of magnetic particles to increase the reactivity with the target material.

First, a magnetic particle having a reactor capable of binding to a target substance is provided on the inner surface of the separating device, and a decomposition equivalent washing buffer solution is added to the inner surface of the separating device to mix the same with the magnetic particles .

A magnetic field is applied to the inner body to confine the magnetic particles with the magnetic field, and the decomposed equalization wash buffer is flowed to the outside of the inner body to remove impurities.

A sample containing the target substance to be separated is provided in the inner body and dispersed together with the magnetic particles to form a mixture by binding the magnetic particles and the target substance.

A magnetic field is applied to the inner body to confine the magnetic particles bound to the target material in the mixture, and the remaining mixture is flowed to the outside of the inner body to remove the foreign substances.

The magnetic field applied to the inner body is blocked and an extraction buffer is provided to separate the magnetic particles and the target material.

When the magnetic particles and the target material are separated from each other, a magnetic field is applied to the inner body again to confine the magnetic particles, and the separated target material is purified, whereby the target substance as a technical problem of the present invention can be separated using magnetic particles .

It goes without saying that the reactor to be formed on the surface of the magnetic particles must be changed according to the target material to be separated.

Hereinafter, an embodiment according to a method for separating a target material using the magnetic particles of the present invention will be described.

[Example 1]

 To express the protein labeled with 6X His in the animal cell and recover the secreted protein into the media, proceed as follows.

1. Concentrate the total protein in the media and replace the buffer by dialysis with 50 mM NaH ₂ PO ₄ , 300 mM NaCl, pH 8.0 Lysis Equilibration Wash buffer.

2. Transfer the sample to the instrument.

3. Add 20 mg of SPM-Ni (magnetic particles with Ni) to the sample.

4. Mix by rotating and incubate for 3 hours.

5. Apply a magnetic field to pull the magnetic particles.

6. Release the buffer solution from the inner body to the outside.

7. Add lysis equilibration wash buffer containing 0.2 mM imidazole.

8. Remove the magnetic field and disperse the magnetic particles through rotation and sonication.

9. Apply a magnetic field to pull the magnetic particles.

10. Release the buffer from the inner body to the outside.

11. Add 50 mM NaH ₂ PO ₄ , 300 mM NaCl, elution buffer containing 250 mM imidazole.

12. Remove the magnetic field and disperse the magnetic particles through rotation and sonication.

13. Apply a magnetic field to pull the magnetic particles.

14. Collect only the solution and recover the protein.

[Example 2]

 To recover the antibody in the serum, perform as follows.

1. Put serum into the instrument.

2. Add 20 mg of SPM-protein A / G (magnetic particle with protein A / G) to the sample.

3. Mix by rotating and incubate for 10 minutes.

4. Apply a magnetic field to pull the magnetic particles.

5. Release the filtrate from the inner body to the outside.

6. Add phosphate-buffered saline (PBS).

7. Remove the magnetic field and disperse the magnetic particles through rotation and sonication.

8. Apply a magnetic field to pull the magnetic particles.

9. Discharge the buffer from the inner body to the outside.

10. Repeat step 9 three times.

11. Add 0.1 M glycine-HCl, pH 2.8 acidic elution buffer.

12. Remove the magnetic field and disperse the magnetic particles through rotation and sonication.

13. Apply a magnetic field to pull the magnetic particles.

14. Collect only the solution and recover the protein.

15. Add 1/10 of the total volume of 1M Tris-HCl at pH 8.5.

Hereinafter, the target material separating apparatus using the magnetic particles of the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 is a flow chart showing a method for separating a target material using magnetic particles according to the present invention, and FIG. 2 is a schematic view showing a method for separating a target material using magnetic particles according to the present invention. FIG. 3 is a view showing an electromagnet separator of a flask spin type according to the present invention, FIG. 4 is a view showing an electromagnet external separator of a flask spin type according to the present invention, and FIG. FIG. 6 is a view showing an electromagnet type external separator of a screw impeller type according to the present invention, FIG. 7 is a view showing an electromagnet type external separator of a screw impeller type according to the present invention, FIG. 8 is an aeration type electromagnet external separator of an aeration type according to the present invention, FIG. 9 is a sectional view of an electromagnet type external separator of an aeration type internal body A according to the present invention, 0.0 > a < / RTI >

In order to separate and purify a target material using magnetic particles in a sample containing a target material, an apparatus for separating a target material using magnetic particles according to the present invention comprises an inner body A provided inside the outer body B; At least one electromagnet (3) extending downward from an upper portion of the inner body (A); A solution injector 1 connected to an upper portion of the inner body A to inject the solution into the inner body A; A sample injection unit 2 connected to an upper part of the inner body A and injecting the sample into the inner body A; A filtrate outlet 4 provided at the lower portion of the inner body A to discharge the filtrate to the outside; .

The inner main body A is rotated and mixed with the material introduced into the inner main body A. The inner main body A may be reciprocated up and down to rotate and mix, It is of course also possible to swing and rotate and mix.

The electromagnet 3 is rotated and mixed with the substance introduced into the inner body A. The electromagnet 3 may be reciprocated up and down and rotated and mixed. And may be rotated and mixed.

And a screw type impeller 5 having the electromagnet 3 fixed inside the screw type impeller 5 and having a screw type impeller on the outer circumference.

One or more sound wave devices (a, a ') for generating sound waves may be provided on the outer periphery of the inner body A or on the inner lower side of the inner body A.

One or more aeration devices (b, b ') for generating aeration may be further provided on the outer periphery of the inner body A or on the inner lower side of the inner main body A.

At least one structure (d) having an inclination angle at the lower portion of the inner body (A) may be further provided.

A temperature holding device (c) may be provided between the inner body (A) and the outer body (B) or within the inner body (A).

In order to separate and purify the target material using the magnetic particles in the sample containing the target material, the apparatus for separating the target material using the magnetic particles according to the present invention comprises an inner body A provided inside the outer body B, ; At least one electromagnet (3) provided on the outer periphery of the inner body (A); A solution injection unit 1 connected to an upper part of the inner body A and injecting a solution into the inner body A; A sample injection unit 2 connected to an upper part of the inner body A and injecting the sample into the inner body A; A filtrate outlet 4 provided at the lower portion of the inner body A to discharge the filtrate to the outside; . ≪ / RTI >

The inner body A may be rotated and mixed with the material introduced into the inner body A, and the inner body A may reciprocate up and down to rotate and mix. Alternatively, the inner body A may swing vertically and horizontally, And mixed.

And a screw type impeller 5 extending downward from the upper side of the inner body A and rotating and having a screw-shaped impeller at its outer periphery.

The screw-type impeller 5 may be rotated up and down and rotated, and the screw-type impeller 5 may be swung up and down and left and right.

At least one sound wave device (a, a ') for generating a sound wave may be further provided inside the inner body (A).

One or more aeration devices (b, b ') for generating aeration may be further provided inside the inner body (A).

At least one structure (d) having an inclination angle at the lower portion of the inner body (A) may be further provided.

A temperature holding device (c) may be provided between the inner body (A) and the outer body (B) or within the inner body (A).

Hereinafter, the types described with reference to the accompanying drawings are used to describe the present invention. The terms used herein may be varied depending on the intention of the user or the operator, and the meanings of the terms should be interpreted based on the contents throughout the specification. Of course.

The flask spin type refers to the type in which the inner body A rotates.

A screw impeller type refers to a type in which a threaded screw-type impeller 5 rotates.

The aeration type refers to the type distributed by the aerator (b, b ').

FIGS. 3 and 4 are flask spin type, in which the inner body A is rotated to disperse and mix the magnetic particles and the mixture, so that the magnetic particles and the target material can be bonded.

At this time, the electromagnet 3 can be located inside or outside, and the structure of the electromagnet 3 varies according to its position.

3, which is an internal form of the electromagnet, the electromagnet 3 is located inside and the sonic device a, a 'can be located on the bottom and side of the inner main body A, The sound wave device (a) can be positioned under the inner main body A because the inner wall (3) surrounds the side surface of the inner body (A).

In the target material separating apparatus using the magnetic particles as described above, first, the filtrate discharging portion 4 provided at the lower portion of the inner body A is closed, and a reactor capable of binding to the target substance is formed, Is supplied to the inner main body A through the injection part 1 and the decomposed equivalent washing buffer is put into the inner main body A through the solution injecting part 1 to mix the same with the magnetic particles give.

As described above, the inner main body A is rotated and mixed while the inner main body A is reciprocating up and down to mix and rotate. In addition, the inner main body A swings vertically, And may be rotated and mixed.

The electromagnet 3 is rotated and mixed with the substance introduced into the inner body A. The electromagnet 3 may be reciprocated up and down and rotated and mixed with the electromagnet 3. The electromagnet 3 may be vertically, It is of course also possible to mix and rotate by rocking.

A magnetic field is applied through the electromagnet 3 provided in the inner body A to open the filtrate discharging part 4 in a state where magnetic particles are confined by the magnetic field and to remove the decomposed equivalent washing buffer, Is flowed to the outside of the inner main body (A) to remove impurities.

The magnetic field applied through the electromagnet 3 is blocked and the sample containing the target substance to be separated into the inner main body A through the sample injecting section 2 Is dispersed together with the magnetic particles to form a mixture of the magnetic particles and the target material.

A magnetic field is applied through the electromagnet 3 provided in the inner body A to confine the magnetic particles combined with the target material in the mixture and to open the filtrate discharge portion 4 to remove the remaining mixture from the inner body A Remove foreign matter by flowing to the outside.

The filtrate discharging unit 4 is closed and the magnetic field applied through the electromagnet 3 is blocked and an extraction buffer is provided to separate the magnetic particles and the target material.

When the magnetic particles and the target material are separated from each other, a magnetic field is applied through the electromagnet 3 provided in the inner body A to confine the magnetic particles, and the separated target material is purified, To perform the target material separation.

5 and 6 show a screw impeller type spiral type impeller in which a screw type impeller 5 having a screw-shaped wing is disposed on the upper part of the inner body A and disperses the magnetic particles and the mixture, To be combined.

At this time, depending on the position of the electromagnet 3, the structure is divided into an inner type or an outer type. 5, which is an internal form of the electromagnet, fixes the electromagnet 3 inside the threaded impeller 5, and the threaded impeller 5 rotates separately from the electromagnet 3. The sound wave devices (a, a ') may be located on the lower and the side surfaces of the inner body A, respectively. FIG. 6 shows an external structure of the electromagnet, in which the electromagnet 3 surrounds the side surface of the inner body A and the sound wave device a can be positioned below the inner body A, thereby showing a difference in structure.

FIGS. 7 and 8 are views showing an aeration type an aeration tank used for purifying water quality is mounted on the inner body A and the magnetic particles and the target material are dispersed and combined with air bubbles. At this time, the aeration tank type can also be divided into an electromagnet type or an external type according to the engagement position of the electromagnet 3.

7 which is an internal form of the electromagnet is that the electromagnet 3 is located on the upper part of the inner body A and the aerator b and b 'are located on the lower or side of the inner body A, The electromagnet (3) surrounds the side surface of the inner body (A) and the aerator (b) is located at the bottom.

3, FIG. 5, FIG. 6 and FIG. 7 can be applied as a lift type. The lift type means that the electromagnet 3 or the threaded impeller 5 on the upper part of the inner main body A And the inner body (A) moves in a vertical reciprocating motion, thereby helping dispersion and mixing.

The electromagnet 3 or the screw type impeller 5 or the inner body A may be pivoted and rotated to be vertically and laterally and laterally rotated to assist dispersion and mixing.

It should be understood that the above-described four types of devices may be used in various combinations, and this is within the scope of the present invention.

That is, when the inner body A is rotated in the screw-type impeller type and the aeration tank type, the apparatus is applied to the flask rotation type, and the electromagnet 3 or the screw-type impeller 5 provided at the upper part is reciprocated upward and downward The lift type can be applied.

It is needless to say that the aeration devices b, b 'may also be provided at the portion where the sonic device a, a' can be positioned in the flask rotation type and the screw type impeller type.

The above listed devices may be equipped with a temperature holding device (c), which may be used when the target material to be separated is sensitive to temperature. As the temperature holding device (c), a double jacket c 'may be provided between the inner body A and the outer body B, or a movable cooler c' 'may be used.

A structure (d) having an inclination angle may be provided below the inner body (A). (D) having an inclination angle at the lower part of the inner body (A) in addition to the dispersion of the electromagnet, the impeller, and the inner body by the rotation, the vertical movement and the vertical movement, . And has an effect of facilitating the discharge of the filtrate by using the inclination.

1 - Solution injection part 2 - Sample injection part
3 - electromagnet 4 -
5 - Screw type impeller
A - Internal body B - External body
a, a '- sonic device b, b' - aeration device
c-Temperature holding device (c'-double jacket, c "-mobile cooler)
d inclination structure

Claims (27)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete An inner body (A) provided inside the outer body (B) for separating and purifying the target material using magnetic particles in a sample containing the target material;
At least one electromagnet (3) provided on an outer periphery of the inner body (A) and restraining the magnetic particles by applying a magnetic field;
A solution injection unit 1 connected to an upper part of the inner body A and injecting a solution into the inner body A;
A sample injection unit 2 connected to an upper part of the inner body A and injecting the sample into the inner body A;
A filtrate outlet 4 provided at the lower portion of the inner body A to discharge the filtrate to the outside; / RTI >
Wherein the inner body (A) rotates and mixes the substance introduced into the inner body (A). delete 18. The method of claim 17,
Wherein the inner body (A) rotates while reciprocating up and down. 18. The method of claim 17,
Wherein the inner body (A) swings vertically and horizontally and rotates. 18. The method of claim 17,
Further comprising a screw-type impeller (5) extending downward from an upper portion of the inner body (A) to rotate and having a screw-shaped impeller formed on an outer circumference thereof. . 22. The method of claim 21,
Wherein the screw-type impeller (5) rotates reciprocally up and down. 22. The method of claim 21,
Wherein the screw-type impeller (5) swings in the up, down, left, and right directions and rotates. 24. The method according to any one of claims 17 to 22,
Wherein at least one of the sound wave devices (a, a ') for generating sound waves is further provided inside the inner body (A). 24. The method according to any one of claims 17 to 22,
Wherein at least one of aeration devices (b, b ') generating aeration is further provided inside the inner body (A). 24. The method according to any one of claims 17 to 22,
Characterized in that one or more structures (d) having an inclination are provided on the inner lower side of the inner main body (A). Providing a magnetic particle having a surface on a reactor capable of binding to a target material to an inner body provided inside an outer body of the separating apparatus;
The decomposition equal washing buffer solution is put into the inner body, the inner body is rotated to uniformly mix with the magnetic particles, and a magnetic field is applied using the electromagnets provided on the outer circumference of the inner body to restrain the magnetic particles Removing the decomposed equivalent washing buffer solution to the outside of the inner body to remove impurities;
Providing a sample containing the target substance to be separated in the inner body, and then rotating the inner body to disperse the magnetic body together with the magnetic particles, thereby binding the magnetic particles and the target substance to form a mixture;
Applying a magnetic field to the mixture through the electromagnet to confine the magnetic particles bound to the target material, and flowing the remaining mixture to the outside of the inner body to remove the foreign substances;
Blocking the applied magnetic field and providing an extraction buffer solution to separate the magnetic particles and the target material;
When the magnetic particles and the target material are separated, applying a magnetic field through the electromagnet again to confine the magnetic particles and purifying the separated target material;
And separating the magnetic particles from the magnetic particles.

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