CN102586225B - Method for separating target molecules by controlling magnetic beads - Google Patents

Abstract

The invention discloses a method for manipulating magnetic beads to separate target molecules. The method comprises the following steps: (1) binding: the magnetic beads, the solution containing the target molecule and the binding buffer are placed in a container and mixed, the magnetic beads combine with the target molecule to form a complex; impurities are removed, and the complex is retained in the container Middle; (2) washing: add washing buffer to the container, make the washing buffer mix with the complex, remove impurities, and keep the complex in the container; (3) elution: add to the container Add an elution buffer to the mixture to mix the elution buffer with the complex; then remove the container from the magnetic field, incubate to separate the magnetic beads from the target molecule, and collect the target molecule. Experiments have proved that the yield of the method of the invention is higher than that of the traditional suction method, and the consistency between the centrifuge tubes is better than the traditional suction method. Therefore, the method of the invention will have broad application prospects in the field of separation and purification of biomolecules.

Description

A method of manipulating magnetic beads to separate target molecules

technical field

The invention relates to a method for manipulating magnetic beads to separate target molecules.

Background technique

A large number of biological experiments and clinical diagnosis are inseparable from sample preparation, and biomolecular extraction and purification techniques are widely used in sample preparation. With the development of related technologies, the number of samples processed in clinical diagnosis and biological experiments is also increasing.

Magnetic bead manipulation is a commonly used technique for sample preparation. At present, there are two main types of magnetic bead mixing and separation technologies: one is to realize the collection, release, and transfer of magnetic beads through the relative movement of the magnetic rod and the magnetic sleeve, and then complete the extraction of biomolecules. The mechanical structure adopted by this method is relatively complicated, and there is a problem of high automation cost, and the magnetic sleeve is used as a consumable, and the price is relatively high. At the same time, due to the full contact between the magnetic sleeve and the sample, there is a risk of contamination of the sample, and there is a problem of magnetic bead loss during the transfer of the magnetic beads. In addition, due to the violent movement of the magnetic sleeve, the integrity of the purified biomolecules cannot be guaranteed.

The other is the pipette suction method, that is, the pipette is blown and mixed evenly, and the magnet absorbs the magnetic beads to separate the magnetic beads. This method is expensive to automate, requires several robotic arms and pumps, and has a complex structure. In addition, there is also the problem that the number of samples processed at one time is limited by the robotic arm. In short, many factors such as efficiency and the number of samples processed at one time need to be improved to meet the requirements of low consumables and high efficiency of biomolecular extraction and purification.

Contents of the invention

The purpose of the present invention is to provide a method for manipulating magnetic beads to separate target molecules from a solution, specifically a magnetic bead separation method that enables more sufficient binding, washing and separation, and higher purity and yield of target molecules. method.

The method for separating the target molecule from the solution using magnetic beads of the present invention comprises the following steps:

(1) Binding: the magnetic beads, the solution containing the target molecule and the binding buffer are placed in a container and mixed, the container is placed in the magnetic field of the magnetic field and the direction of the magnetic field force on the magnetic beads in the container is And/or the size is in a state of continuous change, so that all the substances in the container are in full contact with each other, and the magnetic beads combine with the target molecule to form a complex; remove impurities and keep the complex in the container;

(2) Washing: add washing buffer to the container, mix the washing buffer with the complex, place the container in the magnetic range of the magnetic field and make the magnetic beads in the container subjected to the magnetic force The direction and/or size of are in a state of continuous change, so that all substances in the container are in full contact with each other; impurities are removed, and the complex is retained in the container;

(3) Elution: Add elution buffer to the container, mix the elution buffer with the complex, place the container in the magnetic range of the magnetic field and make the magnetic beads in the container The direction and/or magnitude of the magnetic force is in a state of continuous change, so that all the substances in the container are in full contact with each other; then the container is separated from the magnetic field and incubated to separate the magnetic beads from the target molecule , to collect target molecules.

In the above process, in the step (1), (2) or (3), the method of making the direction and/or size of the magnetic bead force in a continuously changing state can be specifically as follows I, II or III shown, but not excluding other methods;

1. Make the container move relative to the magnetic field, wherein the magnet generating the magnetic field is a permanent magnet;

II. Change the magnitude and/or direction of the magnetic field force, wherein the magnet that generates the magnetic field is an electromagnet; specifically, the magnitude of the magnetic force can be changed by changing the magnitude of the current of the electromagnet; changing the current direction of the electromagnet to change the N\S pole .

III. Changing the magnitude and/or direction of the magnetic field force and moving the container relative to the magnetic field, wherein the magnet generating the magnetic field is an electromagnet.

In the above process, the method for making the container move relative to the magnetic field may specifically be as shown in 1), 2) or 3) as follows:

1) The container moves between two opposite rows of magnets;

2) the container moves around the magnet;

3) The magnet moves around the container.

In the above process, in the above 1), 2) or 3), the motion may be circular motion, uniform motion or uniform circular motion.

In the above process, the speed of the uniform circular motion is 8rpm-75rpm, preferably 12rpm-30rpm or 15rpm-30rpm, specifically 15rpm, 19rpm, 23rpm or 30rpm.

If the movement speed is too fast, the magnetic beads will return to their original position before they can move in the tube, which will not only make them unable to move fully in the tube, but also may destroy the integrity of the original biomolecules; if the movement speed is too slow, The efficiency will be very low and the magnetic beads will not be sufficiently dispersed to contact the liquid.

In the above process, when the container is moving between two opposite rows of magnets, the container is a row of tubes connected by several individual tubes, and the row of tubes is in a straight line.

In the above process, in order to make the effects in each tube consistent, it is best to keep the magnetic force of each individual tube in the row of tubes at the same time to be consistent.

In the above-mentioned process, the method for making each individual tube in the row of tubes at the same time be subjected to the same magnetic field force can be specifically as follows, but is not limited to this method: each row of magnets is connected sequentially by several rectangular parallelepiped permanent magnets. As a result, the connection direction of the permanent magnet is straight and perpendicular to the direction of the N pole and S pole of the permanent magnet itself, and the magnetic pole directions of the two adjacent permanent magnets are opposite; the edge connection of the two opposite permanent magnets between the two rows of magnets It is rectangular or square, and the magnetic poles of the two opposite permanent magnets are the same; the row pipes are kept parallel to the two rows of magnets, and the width of the cut surface of each rectangular parallelepiped permanent magnet along the direction connecting its own N pole and S pole is equal to that of each The width of the widest point of the tangent plane of the single tube along the direction of the N pole and the S pole of the magnet; and the row tube moves in the non-edge effect area of the two rows of magnets, and the edge effect is the edge produced by the two ends of each row of magnets effect.

In the above process, the individual tubes are test tubes or centrifuge tubes; the row of tubes formed by connecting several individual tubes is eight consecutive rows of centrifuge tubes.

In the above process, the target molecule is a biomolecule; or the biomolecule is DNA, RNA, carbohydrate or protein.

In the method of the present invention, by changing the magnitude and direction of the magnetic field force on the magnetic beads, the magnetic beads are more fully combined or separated from the target molecules, and finally the target molecules are obtained. Specifically, the following four methods can be adopted to change the magnitude and direction of the magnetic field force on the magnetic beads: 1) the magnet is fixed, and the magnetic bead system moves; 2) the magnetic bead system is fixed, while the magnet moves; 3) the magnetic bead Both the system and the magnet move; 4) Electromagnets are used to change the magnitude and direction of the magnetic force.

The magnet can be an electromagnet or a permanent magnet.

The permanent magnet is mainly used to provide a magnetic field, and to control the movement of the magnetic beads by adjusting the distance and direction from the sample, thereby changing the magnitude and direction of the magnetic field force on the magnetic bead system.

The electromagnet mainly changes the magnitude and direction of the magnetic field force on the magnetic bead system by adjusting the magnitude and direction of its own current, combined with the adjustment of the distance and direction from the sample.

In the method of the present invention, the effect that makes magnetic bead be subjected to the size of magnetic field force and direction change is specifically as follows:

1. Combination: Keep the distance and/or position of the container with the magnetic bead system relative to the magnet in a state of continuous change, so that the magnetic beads can fully move in the system under the action of the changing magnetic field force, and then make the magnetic beads and biomolecules Thoroughly mix and contact to form complexes of magnetic beads and biomolecules.

2. Washing: The container is close to the magnet for a period of time. After removing excess liquid, add the washing liquid to the combined magnetic beads, so that the distance and/or position of the container with the magnetic bead system relative to the magnet is in a state of continuous change, so that the magnetic The beads move fully in the system under the action of the changing magnetic field force, so that the complex is fully mixed and fully contacted with the washing solution, so that it is fully washed.

3. Elution: The container is close to the magnet for a period of time. After removing excess liquid, add the eluent to the washed magnetic beads, so that the distance and/or position of the container with the magnetic bead system relative to the magnet is in a state of continuous change. Control the movement of the magnetic beads in the eluent, so that the magnetic beads can fully move in the system under the action of the changing magnetic field force, so that the complex can be fully mixed and contacted with the eluent, so as to facilitate the elution, so as to obtain high concentration, High purity target molecule.

The present invention has the following advantages due to the adoption of the above technical scheme:

1) Since the centrifuge tube adopts a slower moving speed, the target biomolecules are subjected to less shear force and are less likely to generate air bubbles, ensuring the integrity of the purified biomolecules.

2) There is no liquid splashing during operation, which reduces the possibility of cross-contamination.

3) No other experimental consumables are involved in the mixing process, which reduces possible pollution while reducing costs.

4) Multiple samples can be placed in the magnetic field for mixing, which improves the throughput of biomolecule extraction and purification.

5) It is only necessary to adjust the relative position of the magnet and the magnetic bead system, or to change the magnetic force of the electromagnet, and the movement is relatively simple, so it is easy to realize automation, and the cost is low.

6) No special container is required, only common consumables are required, and the practicability is very strong

7) There is no magnetic bead transfer in the whole process, which reduces the loss of magnetic beads.

Experiments prove that the biomolecule extraction yield of the method of the invention is higher than that of the traditional suction method, and the consistency among multiple tubes is better than that of the traditional suction method. Therefore, the method of the present invention will have broad application prospects in the field of sample preparation of biomolecules.

Description of drawings

FIG. 1 is a schematic diagram of Embodiment 1 of the present invention.

Fig. 2 is a schematic diagram of Embodiment 1 of the present invention.

Fig. 3 is a schematic diagram of Embodiment 2 of the present invention.

Fig. 4 is a schematic diagram of Embodiment 2 of the present invention.

Fig. 5 is a schematic diagram of Embodiment 3 of the present invention.

Fig. 6 is a schematic diagram of Embodiment 3 of the present invention.

Fig. 7 is a schematic diagram of Embodiment 3 of the present invention.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

In the drawings, the marks are as follows: 1. magnetic bead system; 2. magnet.

Buffer GD, washing solution PW, eluent TB, and magnetic bead suspension B in the following examples are all products in the magnetic bead method genomic DNA extraction kit. The magnetic bead method genomic DNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., the product catalog number is DP329.

The types and amounts of solutions that need to be added in each step of the experiment were operated according to the instructions of the magnetic bead method genomic DNA extraction kit.

The PCR products used in the following examples 1, 2, 3 and 4 were the same batch of PCR products, and what was used in Example 5 was another batch of PCR products. The PCR products all contain the DNA bound to the magnetic beads (ie, the DNA to be separated).

Example 1. Method for separating target molecules from solution using magnetic beads - the container moves around the magnet

A centrifuge tube and a magnet form a group, the magnet is fixed, and the centrifuge tube performs uniform circular motion around the magnet. The magnet is a single permanent magnet, and the single permanent magnet is a cuboid (Fig. 1 and 2). The centrifuge tubes are all within the magnetic force range of the magnetic field generated by the magnet during the movement.

1. Combination

1. Preparation system:

1) Add 40 μL of PCR product to each tube of the three groups of centrifuge tubes;

2) Add 10 μL buffer GD to each tube;

3) Add a mixture of 110 μL PW and 20 μL absolute ethanol to each tube, and vortex to mix;

4) Add 5 μL, 10 μL or 15 μL of magnetic bead suspension B to the three groups of centrifuge tubes respectively;

2. Combination operation

Move a single centrifuge tube around a permanent magnet at a speed of 15rpm in a uniform circular motion (as shown in Figures 1 and 2). During this process, the magnetic beads fully contact with the target molecule to form a complex. After 2 to 3 minutes of movement, close to the magnet Stop, and after staying for 30 seconds, the complex is adsorbed by the magnet to the tube wall of the container, and the liquid is removed with a pipette gun to retain the complex;

Two, washing

1. The first washing

Add 200 μL of buffer GD to the centrifuge tube containing the complex obtained in step 1, and move the centrifuge tube around the permanent magnet at a speed of 15 rpm to make a uniform circular motion. After 3 minutes, stop close to the magnet, and after staying for 30 seconds, the complex is adsorbed to the tube wall by the magnet, and the liquid is removed with a pipette gun to retain the complex;

2. The second washing

Add 200 μL of rinse solution PW to the centrifuge tube in step 1, and move the centrifuge tube around the permanent magnet at a constant speed of 15 rpm. After moving for 2 to 3 minutes, stop close to the magnet. After staying for 30 seconds, the complex is absorbed by the magnet. On the tube wall of the consumable, remove the liquid with a pipette and retain the complex; allow to dry at room temperature for 5 minutes.

3. Elution

Add 50-100 μL of eluent TB to the centrifuge tube in step 2, and move the centrifuge tube around the permanent magnet at a speed of 15 rpm for a uniform circular motion. After moving for 2 to 3 minutes, remove the centrifuge tube from the magnetic field (that is, leave the magnetic field Magnetic range), then place the centrifuge tube at 56°C and incubate for 10 minutes. Then stop the centrifuge tube close to the magnet. After staying for 30 seconds, the magnetic beads are adsorbed to the tube wall by the magnet, and the target molecule exists in the separation buffer (that is, the target molecule solution), and the target molecule solution is transferred to a new tube.

Fourth, the detection method of recovery

Method: Use a spectrophotometer to test the concentration of nucleic acid. Note that the nucleic acid needs to be mixed with a tip before the test.

Example 2, the method of using magnetic beads to separate the target molecule from the solution - the magnet moves around the container

The method is basically the same as in Example 1, except that in the steps of combining, washing and eluting, the relative motion of the centrifuge tube and the permanent magnet is different, specifically: the permanent magnet moves in a uniform circular motion around the centrifuge tube at a speed of 15 rpm (such as Figure 3,4 shown). Specifically, a centrifuge tube and a magnet form a group, the centrifuge tube is fixed, and the magnet performs uniform circular motion around the centrifuge tube. The magnet is a single permanent magnet, and the single permanent magnet is a cuboid (Fig. 3 and 4). During the movement of the magnet, it is necessary to ensure that the centrifuge tube is within the range of the magnetic force generated by the magnet.

Example 3. Method for separating target molecules from solution using magnetic beads - the container moves between two rows of magnets

The method is basically the same as in Example 1, except that in the steps of binding, washing and eluting, the relative movement of the centrifuge tube and the permanent magnet is different, as follows:

Two rows of opposite and parallel permanent magnets and a row of centrifuge tubes form a group. A row of centrifuge tubes is formed by connecting 8 individual centrifuge tubes in a straight line. The positions of the two rows of opposite magnets are fixed, and the eight centrifuge tubes as a whole move in a uniform circular motion at a speed of 15 rpm between the two rows of magnets (Fig. 5, 6, 7). The uniform circular motion can be realized by existing technology, such as placing eight centrifuge tubes in a corresponding rotor, and the rotor is driven by a motor to perform uniform circular motion, so that the eight centrifuge tubes also perform uniform circular motion. During the whole movement process, The relative positions of the eight centrifuge tubes and the rotor are constant. A row of centrifuge tubes are all within the range of the magnetic force generated by the two rows of magnets during the movement.

The magnets are permanent magnets, and the single magnets are in the shape of a cuboid; each row of magnets is formed by arranging the single permanent magnets of the same size next to each other in sequence.

In order to ensure that the force of each tube in the eight centrifuge tubes is consistent at the same time (that is, the magnitude and direction of the magnetic field force on each tube are consistent), the magnet structure is set as follows, and the eight centrifuge tubes are moved as follows:

Arrange the single magnets next to each other in a row, the arrangement direction of the single magnets is straight and perpendicular to the direction of the N pole and the S pole of the single magnet itself, and the magnetic pole directions of the two adjacent single magnets are opposite; The line connecting the edges of the two opposite single magnets between the row magnets is rectangular, and the magnetic poles of the two opposite single magnets are the same; no matter at the initial placement moment of the row tube or during the movement of the row tube, the row The tube is relatively parallel to the two rows of magnets; the width of the cut surface of each cuboid permanent magnet along the direction connecting its own N pole and S pole is equal to the widest width of the cut surface of each single tube along the direction connecting the N pole and S pole of the magnet It is also necessary to make the tubes move in the non-edge effect area between the two rows of magnets (to avoid the edge effects produced by the two ends of the two rows of magnets from causing uneven force on the tubes), and the edge effect refers to that each row The edge effect produced by the ends of the magnet.

The two rows of magnets arranged in this way are in the non-edge effect area, although the magnetic field strengths of each point along the direction parallel to the magnets are not exactly the same (generally the magnetic field strength of the connected part of the two magnets is stronger than that of the non-connected part of the magnet), But because the widest part of the longitudinal section of each tube is equal to the width of the longitudinal section of the magnet, no matter where the row of tubes moves, the stress on each tube at the same time is the same.

Avoiding the above-mentioned edge effects can be achieved by existing technologies, specifically, the number of single magnets in each row of magnets is much more than the number of single tubes in the row of tubes, and the row of tubes moves in the middle area of each row of magnets without approaching both ends of each row of magnets.

Such setting ensures that the magnitude and direction of the magnetic field force received by each of the eight centrifuge tubes at the same time is the same, thereby ensuring the consistency among the tubes.

In addition, experiments have shown that when the individual magnets in each row of magnets are separated by a certain distance, the dispersion of the magnetic beads in each centrifuge tube is obviously different, indicating that in this case, the strength of the magnetic field that each tube receives at the same time is different. Therefore, the tube-to-tube consistency is not as good as above next to each other.

The experiment also shows that when the magnetic pole directions of two adjacent single magnets in each row of magnets are the same, the dispersion degree of the magnetic beads in each centrifuge tube is obviously different, indicating that in this case the magnetic field strength of each tube at the same time Not the same, therefore, the consistency between the tubes is not as good as the above-mentioned adjacent magnets placed in opposite directions.

In the above-mentioned embodiments, three groups of experiments are set up, and in actual application, N (N≥1) groups of the same reactions can be produced according to the requirement, so as to improve the efficiency.

Embodiment 4, contrast (traditional mode)

1. Add 40 μL of PCR product (same as the PCR product described in Example 1-3, the same batch) to each tube of the three groups of centrifuge tubes.

2. Add 10 μL buffer GD to each tube;

3. Add a mixture of 110 μL PW and 20 μL absolute ethanol to each tube, and vortex to mix;

4. Add 5 μL, 10 μL and 15 μL of magnetic bead suspension B to the three groups of centrifuge tubes respectively;

5. Place the centrifuge tube on the magnetic stand and let it stand for 30 seconds, and carefully remove the liquid when the magnetic beads are completely absorbed;

6. Remove the centrifuge tube from the magnetic stand, add 200 μL of washing solution GD, and vortex to mix.

7. Place the centrifuge tube on the magnetic stand and let it stand for 30 seconds. When the magnetic beads are completely absorbed, carefully remove the liquid.

8. Add 200 μL of washing solution PW, and vortex to mix.

9. Place the centrifuge tube on the magnetic stand and let it stand for 30 seconds. When the magnetic beads are completely absorbed, carefully remove the liquid and dry at room temperature for 5 minutes.

10. Add 50-100 μL of eluent TB, mix according to step 5, and incubate at 56°C for 10 minutes.

11. Put the centrifuge tube close to the magnet for 30 seconds. When the magnetic beads are completely absorbed, carefully transfer the DNA solution to a new tube and measure.

The results are shown in Table 1.

Table 1, product recovery result

It can be seen from the table that regardless of the amount of magnetic beads added, the yield of the method of the present invention is higher than that of the traditional suction method, and the consistency between tubes is better than that of the traditional suction method.

Embodiment 5, different motion speeds

Method: the same as described in Example 3; the difference is that the speed of the uniform circular motion is 23rpm and 30rpm.

Results: as shown in Table 2.

Table 2, product recovery result

Claims (3)

1. a method of utilizing magnetic bead that target molecule is separated from solution, comprises the steps:

(1) combination: magnetic bead, the solution that contains target molecule and binding buffer liquid are placed in to container and mix, described container is placed in to the magnetic force scope in magnetic field and makes the magnetic field force induced direction of the magnetic bead of described container and/or size in continuous change state, so that the fully contact each other of all substances in described container, magnetic bead is combined with target molecule and is formed mixture; Remove impurity, retain mixture in described container;

(2) washing: add lavation buffer solution in described container, lavation buffer solution is mixed with described mixture, described container is placed in to the magnetic force scope in magnetic field and makes the magnetic field force induced direction of the magnetic bead of described container and/or size in continuous change state, so that the fully contact each other of all substances in described container; Remove impurity, retain mixture in described container;

(3) wash-out: add elution buffer in described container, elution buffer is mixed with described mixture, described container is placed in to the magnetic force scope in magnetic field and makes the magnetic field force induced direction of the magnetic bead of described container and/or size in continuous change state, so that the fully contact each other of all substances in described container; Again described container is departed to magnetic field, hatch, so that described magnetic bead separates with described target molecule, collect target molecule; In described step (1), (2) or (3), described in make the magnetic field force induced direction of magnetic bead and/or the method for size in continuous change state be

Make described container and the relative movement of described magnetic field, the magnet that wherein produces magnetic field is permanent magnet;

The described method that makes described container and the relative movement of described magnetic field is that described container is taken exercises between two relative row's magnet; The described container comb that several independent pipes are formed by connecting of serving as reasons, and described comb is straight line; In described comb, each independent pipe causes at the magnetic field force induced homogeneous of synchronization; Make each independent pipe in described comb synchronization the method that causes of magnetic field force induced homogeneous be: every row's magnet is connected in sequence by several rectangular-shaped permanent magnets, permanent magnet closure linearly and perpendicular to the N utmost point and the S utmost point line direction of permanent magnet self, the pole orientation of two adjacent permanent magnets is contrary; Two permanent magnet edge lines relative between two row's magnet are rectangular or square, and the magnetic pole of two relative permanent magnets is identical; Comb and two row's magnet equal keeping parallelisms, each rectangular-shaped permanent magnet equals the width of each single tube along the widest part of the N utmost point of magnet and the tangent plane of S utmost point line direction along the width of the tangent plane of himself N utmost point and S utmost point line direction; And comb is in the motion of the non-fringing effect region of two row's magnet, and described fringing effect is the fringing effect that the two ends of every row's magnet produce;

Described 1), 2) or 3) in, described motion is uniform circular motion, is that comb does uniform circular motion along the horizontal plane of two row's magnet lines; The speed of described uniform circular motion is 15rpm-30rpm;

Described target molecule is DNA, RNA, carbohydrate or protein.

2. method according to claim 1, is characterized in that: the speed of described uniform circular motion is 15rpm, 19rpm, 23rpm or 30rpm.

3. method according to claim 1 and 2, is characterized in that: the described comb being formed by connecting by several independent pipes is eight platoon centrifuge tubes.

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