CN212532816U - Magnetic separation device - Google Patents

Abstract

The utility model relates to a magnetic separation device, the device includes the supporting part, and this supporting part is used for bearing many container dish be provided with the magnetic response part that a plurality of arrays were arranged on the supporting part perpendicularly, magnetic response part evenly distributed is in around each container of many container dish, just the top of magnetic response part exceeds within 2 centimetres of container bottom.

Description

Magnetic separation device

Technical Field

The utility model belongs to the purification field, concretely relates to utilize magnetic separation device.

Background

The field of biological substance purification, particularly the field of Protein purification, is the mainstream of the traditional column chromatography process at present, and Protein A and AKTA series on the market are the most common consumables and instruments. The most commonly used Protein A affinity chromatography medium at present is prepared by immobilizing Protein A on the surface of agarose gel by chemical coupling method to prepare affinity filler, and then packing into chromatography column. The procedures of obtaining supernatant through centrifugal filtration from a fermentation liquid submerged tank, purifying through a column and the like are time-consuming, and a plurality of samples can only be purified in a mode of one by one, so that the flux is limited.

Magnetic particles have advantages in improving purification efficiency. Magnetic particles are used in various chemical processes in solid phase, and chemical components can adhere to the surface of the magnetic particles and move with the aid of a magnetic field. The use of magnetic particles makes the available surface of the solid phase as large as possible.

The magnetic particles take superparamagnetic microspheres as a substrate, have super-strong paramagnetism, can be rapidly gathered in a magnetic field, and can be uniformly dispersed after leaving the magnetic field. The magnetic particles have proper and uniform particle size, ensure strong enough magnetic responsiveness and are not easy to settle. In addition, the magnetic particles have abundant surface active groups so as to be coupled with biological substances and realize the separation from a sample to be detected under the action of an external magnetic field. The magnetic particles are used for separating complex components of a biochemical sample, separation and enrichment can be carried out simultaneously, separation speed and enrichment efficiency are effectively improved, and meanwhile sensitivity of analysis and detection is greatly improved. However, most of the laboratory magnetic particle purification still only stays on the aspect of manual pure manual operation, the efficiency is low, the uniformity is not guaranteed, and different purification results can be obtained by different human operations.

The traditional magnetic separation method is to place the container with magnetic particles on a bulk magnet directly, and then the magnetic microspheres can be adsorbed to the bottom of a microporous plate or a PCR. Although the method is simple, after the magnetic microspheres are adsorbed to the bottoms of the holes, when subsequent washing and separation processes are carried out, part of the magnetic microspheres are taken out of the micropores or the PCR tubes, so that subsequent reactions are influenced. The existing magnetic particle automatic purification instruments on the market at present comprise Kingfisher of Thermo and Am MagSA of Kisry, and the equipment and the consumable price are higher. Furthermore, these devices use magnetic rod heads to transfer magnetic particles for purification, which can collect on the rod surface, especially at the tip, when the magnet is in the lower position. When the magnet is raised to a higher position, the particles may be released from the magnetic bar accordingly. However, the limited plates form a bottleneck in the process, only limited samples can be processed at one time, and additional equipment is required for adding the neutralization solution after acid elution and adding the buffer solution in the washing well plate, and manual addition is time-consuming, labor-consuming and high in error rate. Furthermore, both the sample and the liquid remain on the surface of the bar magnet, which may lead to a risk of contamination in subsequent steps.

Therefore, the present invention is directed to a magnetic particle purifying apparatus with high compatibility for various scale purification processes.

SUMMERY OF THE UTILITY MODEL

The utility model provides a wide, the automatic magnetic separation device who moves of application scope. The method for transferring liquid by fixing magnetic particles is different from a semi-automatic mode of adsorbing magnetic particles by a conventional magnetic rod, high-flux purification is realized, cross contamination is avoided, and the recovery rate is increased.

Particularly, the utility model discloses a first aspect provides a magnetic separation device, including the supporting part, this supporting part is used for bearing many container dish be provided with the magnetic response part that a plurality of arrays were arranged perpendicularly on the supporting part, magnetic response part evenly distributed is in around each container of many container dish, just the top of magnetic response part is higher than the container bottom within 2 centimetres.

In one or more embodiments, four magnetically responsive members are evenly distributed around each vessel.

In one or more embodiments, the magnetically responsive member is a cylindrical structure or a square cylindrical structure.

In one or more embodiments, the top of the magnetically-responsive member is within 1 centimeter of the bottom of the container.

In one or more embodiments, the four corners of the carrier are chamfered structures suitable for facilitating multiple container trays of different skirts.

In one or more embodiments, the outer perimeter dimension of the carrier is slightly less than the skirt dimension of the multi-well tray.

In one or more embodiments, the carrier portion and the multi-well plate are secured to one another such that the multi-well plate is secured to the carrier portion.

In one or more embodiments, the apparatus further comprises a positioning device disposed on the carrier for securing the multi-well plate.

In one or more embodiments, the positioning device includes a plurality of support columns fixedly connected to the multi-well plate, and the bottom ends of the support columns are placed in positioning holes formed in the carrying portion to position the positioning device on the carrying portion.

In one or more embodiments, the space between every four magnetically responsive members corresponds to a receptacle of the multi-receptacle tray.

The beneficial effects of the utility model reside in that:

(1) when the multi-container tray is placed on the magnetic separation device, the biological substances are fully combined with the magnetic particles, the magnetic particles are adsorbed on the periphery of the tube wall through the magnetic field distributed on the side wall of the container, buffer solution is removed and added without extra equipment, mechanical operation is carried out without manpower, and the error rate is reduced. All steps are finished in a single container, so that the pollution risk is reduced;

(2) the magnetic separation device and the magnetic response component are fixed into a whole, so that the cost is low, and the device can be suitable for various microporous plates and PCR plates and is widely suitable by arranging different magnetic response components;

(3) the multi-container disc has 4 magnetic response components around each container, so that the magnetic flux is increased, and the magnetic adsorption efficiency is improved.

(4) Utilize the recovery rate and the purity that magnetic separation device realized have the uniformity with traditional technology.

(5) Utilize magnetic separation device is high-efficient quick than traditional technology, makes statistics of 1258 appearance, compares traditional column chromatography technology, can practice thrift hundreds of working days every year. (1258 × 2 h/sample 104.8 days, 1258 × 2h/24 days)

(6) Utilize magnetic separation device is than traditional technology with low costs, makes statistics of 1258 appearance, compares traditional column chromatography technology, can save ten thousand yuan each year.

Drawings

Fig. 1 is a top view of a magnetic separation device according to an embodiment of the present invention.

Fig. 2 is a schematic perspective view of a magnetic separation device according to an embodiment of the present invention.

Fig. 3 is a side view of a magnetic separation device according to an embodiment of the present invention.

Fig. 4 is a side view of a receptacle and a magnetically responsive member when a multi-receptacle tray is placed on a side of a carrier having magnetically responsive members according to an embodiment of the present invention.

Fig. 5 is a side view of a receptacle and a magnetically-responsive member when a multi-receptacle tray is placed on a side of a carrier having magnetically-responsive members according to another embodiment of the present invention.

Fig. 6 is a side view of a receptacle and a magnetically-responsive member when a multi-receptacle tray is placed on a side of a carrier having magnetically-responsive members according to another embodiment of the present invention.

Fig. 7 is a top view of a receptacle and a magnetically responsive member when a multi-receptacle tray is placed on a surface of a carrier having magnetically responsive members according to an embodiment of the present invention.

FIG. 8 is a comparison of the purity of proteins purified by the method of one embodiment of the present invention and column chromatography, respectively.

FIG. 9 is a comparison of the recovery yields of proteins purified by the method of one embodiment of the present invention and column chromatography, respectively.

Fig. 10 is a workstation layout.

Detailed Description

It is understood that within the scope of the present invention, the above-mentioned features of the present invention and those specifically described below (e.g., in the examples) can be combined with each other to form a preferred embodiment. The embodiments described herein are only used for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereto. All according to the technical thought that the utility model provides, any change of doing on the basis of technical scheme all falls into the scope of protection of the utility model claims.

The utility model relates to an automatic purification technology of perforated plate magnetic particle based on workstation adopts the mode of fixed magnetic particle transfer medium to realize the high flux purification, is different from the semi-automatization mode that conventional bar magnet adsorbs magnetic particle. The process does not need a special magnetic particle purifying instrument or special consumables, is compatible with various purifying processes and buffer solution systems, and is convenient to transplant to most liquid workstations for completion.

The utility model discloses an easily move magnetic separation device of liquid, including the supporting part, this supporting part is used for bearing many container dish be provided with the magnetic response part that a plurality of arrays were arranged on the supporting part perpendicularly, magnetic response part evenly distributed is in around each container of many container dish, just the top of magnetic response part is higher than the container bottom within 2 centimetres. The space between every four magnetic response components corresponds to one container of the multi-container tray.

Fig. 1 to 3 show a diagram of an embodiment of the magnetic separation device herein, comprising a carrier part 1, on which carrier part 15 rows and 7 columns of magnetically responsive components 2 are arranged. Fig. 4 to 6 are partial views of various embodiments of the carrier 1 carrying a 24-well plate, wherein the containers 3 are containers 31, 32, 33, respectively, and fig. 7 is a top view of fig. 6. In use, the 24-well plate is placed on the apparatus and the relative positions of the receptacle 3 and the magnetically-responsive member 2 are shown schematically in figures 4-6. Four cylindrical magnetic response components 2 are uniformly distributed around each container 3 of the 24-hole plate, and the side wall of each magnetic response component 2 is adjacent to the outer surface of the adjacent container 3, so that the magnetic flux is increased, and the magnetic adsorption efficiency is improved. The top of the magnetically responsive member is within 1 cm above the bottom of the container. Since the position of the magnet is always located around the lower portion of the tube, magnetic particles in the tube are attracted by the magnet to the inner wall of the tube extending upward from the bottom of the tube, which is free of magnetic particles. Therefore, the liquid transfer device is convenient to use and stretches into the bottom of the pipe to completely take out liquid, and the device and the 24-hole plate can be tilted and turned over together to move out the liquid due to the fact that the magnet is adjacent to the pipe wall and the magnetic adsorption force is strong.

In the device herein, four magnetically responsive members may be evenly distributed around each vessel. The magnetically responsive member may be a cylindrical structure or a square cylindrical structure with a sidewall adjacent the outer surface of the adjacent container.

In the device, the top of the magnetic response component can be within 1 cm, within 0.8 cm, within 0.7 cm, within 0.6 cm, within 0.5 cm, within 0.4 cm and within 0.3 cm higher than the bottom of the container. When in use, the magnetic particles are adsorbed on the inner side wall slightly higher than the bottom of the container. Preferably, the magnetic particles are adsorbed on the inner side wall less than or equal to 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the full length of the container from the bottom of the container.

The magnetically responsive component described herein may be a ferromagnetic, permanent magnet, or electromagnet. Preferably, the magnetically-responsive component comprises a material selected from the group consisting of: iron, neodymium iron boron, samarium cobalt and aluminum cobalt nickel. The bearing part can be made of metal or PC material or other engineering plastics which do not respond to magnetism.

The multi-well plate described herein can be any array of well plates, including microwell plates or PCR plates. Common multi-well plates include 24-well plates, 48-well plates, 96-well plates, or 384-well plates.

The bearing part in the device can be made of metal or PC material or other engineering plastics which do not respond to magnetism. The four corners of the bearing part can be chamfer structures suitable for multiple container discs with different skirt edges. The four corners of the bearing part can be cut off, so that the four corners of the bearing part are provided with chamfering structures convenient for different skirt edge micro-porous plates to be suitable. The peripheral dimension of the carrying portion can be set slightly smaller than the dimension of the skirt of the multi-container tray, so that the multi-container tray with skirt edges of different shapes can be suitable.

The magnetic response component or the bearing part can be mutually clamped and fixed with the multi-container plate, so that the multi-container plate is fixed on the bearing part. And a positioning device which is arranged on the bearing part and is used for the multi-container tray is also arranged. The positioning device can be a plurality of supporting columns fixedly connected with the multiple container plates, and the bottom ends of the supporting columns are placed in positioning holes formed in the bearing part to position the positioning device on the bearing part.

The magnetic response component in the device can be mutually clamped and fixed with the containers of the multi-container plate, so that the multi-container plate is fixed on the bearing part. Alternatively or additionally, the carrying part and the multi-container tray are mutually clamped, so that the multi-container tray is fixed on the carrying part.

The device of the utility model can be used in the following method: a method for separating biological material from a medium in one or more vessels using magnetic particles, the vessels comprising a sidewall and a bottom, comprising:

(1) contacting a medium comprising the biological substance and magnetic particles treated to associate with the biological substance and incubating under conditions in which the biological substance associates with the magnetic particles,

(2) applying a magnetic field to the side wall of the container to concentrate the magnetic particles onto the inner side wall of the container, said magnetic field covering at least 10%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the circumference of the side wall, whereby the magnetic particles can be concentrated in a horizontal direction onto the inner side wall of the container in a plurality of directions, preferably the magnetic field covering the entire circumference of the side wall,

(3) the medium in the container is removed, preferably by suction,

optionally (4) removing the magnetic field, washing the magnetic particles with the captured biological substances with the equilibration fluid, repeating steps (2) - (3),

(5) removing the magnetic field, eluting the biological substance from the magnetic particles with an eluent, repeating steps (2) - (3),

optionally (6) adjusting the biomass to an appropriate pH using a neutralization solution.

In one or more embodiments, the magnetic field is preferably located in the lower portion of the vessel, less than or equal to 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the full length of the vessel from the bottom of the vessel.

The magnetic field may be generated by a magnetic separation device as described herein, and step (2) comprises placing the multi-well plate on a face of the carrier having magnetically responsive members such that the magnetically responsive members gather magnetic particles around the bottom of the well.

The suction in step (3) includes a step of removing the medium from the container by using negative pressure. Preferably, the pumping removes the solution simultaneously from a plurality of vessels using a multichannel tip.

The containers described herein include, but are not limited to, any container useful for purifying biological material, preferably multi-well plates, such as 24-well plates, 96-well plates, and the like. Several parallel purifications can be done on one plate.

The biological material described herein may be any material derived from an organism, including but not limited to cells, viruses, subcellular organelles, DNA, RNA, proteins, or polypeptides. Exemplary biological substances are DNA, RNA and proteins.

The magnetic particles are preferably paramagnetic. The size of the particles is generally less than 50 μm, preferably from 0.1 to 10 μm, most preferably from 1 to 5 μm. The concentration of the particles may be, for example, 0.01-5mg/ml, preferably 0.05-3mg/ml, most preferably 0.2-2 mg/ml. Typically, the magnetic particles described herein are coated with groups that can be associated, coupled, bound, attracted to a biological substance of interest, such as silicon-based, amino, carboxyl, lectin and/or other reactive functional groups, such as oligonucleotides, antibodies, antigens, streptavidin or biotin. The magnetic particles used herein are activated prior to use, including but not limited to lye treatment, equilibration fluid treatment and/or water treatment. The alkali liquor is used for cleaning the magnetic particles, so that the heat source removing and impurity removing effects are achieved, and the balance liquid can regenerate the magnetic particles. Specifically, the activation method of the magnetic particles is as follows: removing the solution for storing the magnetic particles through magnetic separation to obtain the magnetic particles; then washing the magnetic particles with alkaline solution; and cleaning the magnetic particles by using the balance liquid for 2-5 times to obtain activated magnetic particles.

After the biological substance is incubated with the magnetic particles coated with the groups capable of associating with the biological substance, the biological substance is fixed on the magnetic particles, and the biological substance and the magnetic particles can move together through the magnetic field. After the magnetic particles coupled with the biological substances are treated with the eluent, the biological substances can be separated from the magnetic particles, thereby achieving purification.

Illustratively, the magnetic field attracts the magnetic particles around the inside wall of the vessel and then removes the medium other than the particles from the bottom of the vessel. The removal of the medium from the vessel may be accomplished by any method known in the art, such as by using a thin tube extending into the medium for suction. Preferably the pumping removes media from a plurality of vessels simultaneously using a multichannel tip. The medium comprises the remaining medium after the biological substance has been immobilized by the magnetic particles, or an equilibration fluid for treating or washing the magnetic particles, or an elution fluid containing the biological substance of interest.

Thus, the magnetic field described herein is capable of focusing magnetic particles in the container to the inner side walls of the container, preferably the magnetic field covers at least 30%, 40%, 50%, 60%, 70%, 80%, 90% of the circumference of the side walls, such that the magnetic particles can be focused in a horizontal direction to the inner side walls of the container in multiple directions. Preferably, the magnetic field covers the entire circumference of the side wall, so that the magnetic particles can be collected on the inner side wall of the container in all directions in the horizontal direction. The magnetic field is preferably located in the lower portion of the vessel less than or equal to 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the full length of the vessel from the bottom of the vessel, such that the magnetic particles are concentrated on the inner side walls of the lower portion of the vessel by 50%, 40%, 30%, 20%, 10%, 5%, or 1%.

The magnetic field herein is achieved by the magnetic separation device described herein. The utility model discloses a magnetic separation device includes the bearing part, and this bearing part is used for bearing many containers dish be provided with the magnetic response part that a plurality of arrays were arranged on the bearing part perpendicularly, magnetic response part evenly distributed is in around each container of many containers dish, just the top of magnetic response part is higher than the container bottom within 2 centimetres. The space between every four magnetic response components corresponds to one container of the multi-container tray.

After the biological material is immobilized, the magnetic particles are washed several times to remove all reaction components, caused by reactions or other contaminants, or non-specifically bound impurities. Washing can be performed by releasing and collecting the particles in the equilibration buffer and transferring the particles to another well containing fresh equilibration buffer.

To separate the magnetic particles from the biological substances bound thereto, the magnetic particles may be incubated with an elution buffer. Those skilled in the art know the composition of the eluent for different biological substances. For example, for isolating mRNA, a low salt eluent, such as a Tween-containing EDTA solution; for isolating DNA, use can be made; for the separation of proteins, 0.05-0.5M glycine solution with a pH of 2.5-3.5 can be used.

To ensure protein stability, the eluted biological material may require the addition of a neutralizing solution to adjust the pH to the appropriate pH. Those skilled in the art are aware of the optimal pH for various biological substances and the reagents and methods to adjust the pH for various biological substances.

The manner of incubation, washing, elution, neutralization described herein may be any form of treatment. Generally, the shaking can bring the medium into sufficient contact with the magnetic particles, shortening the processing time. Thus, the shaking time may be from 1 minute to 24 hours, such as from 1 minute to 10 minutes, from 1 hour to 4 hours. The oscillation herein may be implemented by a workstation oscillation module. Specifically, the container is placed into a workstation oscillation module, a workstation program is called, and the workstation is started after workstation parameters, such as sample volume, equilibrium liquid volume, operation times, eluent volume, neutralization liquid volume and the like, are set.

Examples

Example 1 magnetic particle purification of proteins

1) Treating magnetic particles, sterilizing the magnetic particles with 0.1M NaOH for 30min, removing alkali solution, adding balancing solution (PBS), balancing the volume of 10 magnetic beads, removing the balancing solution, and adding the balancing solution in an amount of 1:1 for later use;

2) suspending magnetic particles, and adding 200uL of magnetic particle suspension (the volume ratio of the magnetic particles to the balance liquid is 1:1) into a 24-pore plate fermentation plate;

3) putting the sample into a workstation oscillation module, calling a Method specially programmed by the workstation, setting parameters of the workstation, such as sample volume (mL), Wash buffer volume (mL), Wash times 3, Elution buffer volume (mL), and Neutralization buffer volume (mL) to be used, clicking 'OK', and operating the Method (exemplary parameters are shown in figure 10);

4) and (3) incubation: the oscillation module starts to operate, oscillation is carried out at 300rpm/min for 1h, the fermentation liquor and the magnetic particles are fully incubated, after 1h, the clamp module transfers the 24-hole plate to a special magnetic frame plate position, the magnetic base gathers the magnetic particles to the four walls of the bottom of the hole plate, and the mechanical arm 4 channel gun head removes the fermentation liquor;

5) balancing: adding 1mL of balance liquid, moving the 24-hole plate to a vibration module by a clamp module, vibrating for 1min, then transferring the sample hole plate to a magnetic frame module, and repeating the process for 3 times;

6) and (3) elution: adding 1mL of eluent (0.1M Glycine-HCl, pH 2-3 or 50mM Na-Citrate, 150mM NaCl, pH 3.0), transferring the 24-hole plate to a shaking module by a clamp module, shaking for 1min, transferring the hole plate to a magnetic frame module, gathering magnetic particles to the four walls of the bottom of the hole plate by a magnetic base, and transferring the eluent dissolved with the target protein to a new hole plate at the position of a final product plate by a mechanical arm 4-channel gun head;

7) neutralizing: adding 0.1mL of neutralizing solution (1M Tris-HCl, pH 9.0) into the new pore plate, transferring the 24-pore plate to a shaking module by a fixture module, shaking for 1min, transferring the pore plate to the plate position of the final product, and finishing the whole purification process.

Example 2 column chromatography purification of proteins

1) Centrifuging the fermentation liquor by 10000rcf for 30min to remove precipitate, and filtering the supernatant with 0.22um filter;

2) AKTA sample loading is utilized, and the fermentation liquid is pumped into an affinity chromatography column through a sample pump, so that the target protein is captured;

3) balancing, namely pumping a balance liquid into the chromatographic column to balance 10 column volumes;

4) eluting, namely pumping the eluent into a chromatographic column, eluting 5 column volumes, collecting components according to UV change, and adding a neutralizing solution;

the column chromatography purified protein is purified one sample at a time, 6 samples are purified one night, parallel purification cannot be realized, and the flux is lower than that of the automatic purification of a 24-well plate.

It should be noted that the drawings of the present invention are merely for convenience of illustration and are exemplarily shown, wherein the shapes, proportions and sizes of the multi-well plate, the magnetic response component and each component are not limited in the drawings, and the shape thereof can be arbitrarily changed into a circular shape, a square shape or an arbitrary shape according to the requirement.

The above-described embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and to implement the same, and all the equivalent changes or modifications made according to the spirit of the present invention, which are not limited by the scope of the present invention, are also included in the scope of the present invention.

Claims (10)

1. A magnetic separation device comprises a bearing part, wherein the bearing part is used for bearing a multi-container tray, and is characterized in that a plurality of magnetic response components which are arranged in an array mode are vertically arranged on the bearing part, the magnetic response components are uniformly distributed around each container of the multi-container tray, and the top of each magnetic response component is higher than the bottom of each container by less than 2 centimeters.

2. A magnetic separation device according to claim 1 wherein four magnetically responsive members are evenly distributed around each vessel.

3. A magnetic separation device according to claim 1 wherein the magnetically responsive member is of cylindrical or square cylindrical configuration.

4. A magnetic separation device according to claim 1 wherein the top of the magnetically responsive member is within 1 cm of the bottom of the vessel.

5. A magnetic separation device according to claim 1 wherein the four corners of the carrier are chamfered to facilitate the use of multiple receptacle trays of different skirts.

6. A magnetic separation device according to claim 1 wherein the peripheral dimension of the carrier is slightly less than the skirt dimension of the multi-well tray.

7. A magnetic separation device according to claim 1 wherein the carrier portion is secured to the multi-well plate such that the multi-well plate is secured to the carrier portion.

8. A magnetic separation device according to claim 1 wherein the device further comprises locating means provided on the carrier for securing the multi-well tray.

9. The magnetic separation device of claim 8, wherein the positioning device comprises a plurality of support columns fixedly connected to the multi-well plate, and bottom ends of the support columns are placed in positioning holes formed in the carrying portion to position the positioning device on the carrying portion.

10. A magnetic separation device according to claim 1 wherein the space between every fourth magnetically responsive member corresponds to a receptacle of the multi-receptacle tray.

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