KR20200039642A - The Centrifugal Tube Filtration Apparatus And The Particle Separation Method - Google Patents

Abstract

According to one embodiment of the present invention, a centrifugal tube filtration apparatus comprises: a centrifugal tube receiving a liquid sample and including a lid disposed at one end; at least one porous membrane disposed in a section which crosses an extending direction of the centrifugal tube from the inside of the centrifugal tube; and a restoring force applying unit disposed at the other end of the centrifugal tube for providing restoring force to the liquid sample. The liquid sample comprises a first particle larger than a pore size of the porous membrane and a second particle smaller than the pore size of the porous membrane. The second particle moves from one side of the porous membrane to the other side thereof by centrifugal force. If the centrifugal force is reduced, the restoring force applying unit removes the first particle which blocks the pore of the porous membrane. Accordingly, the present invention provides a particle separation apparatus for separating particles by controlling a flow of a biological liquid sample with centrifugal force and restoring force.

Description

Centrifugal Tube Filtration Apparatus And The Particle Separation Method

The present invention relates to a centrifuge tube filtration device including a porous membrane, and more particularly, to a biomolecule, particle, or cell separation device using a centrifugal tube, porous membrane, centrifugal force, and resilience.

Centrifuges are used in bioseparation processes. The centrifuge uses the density difference between the solid and the liquid surrounding the solid. However, when a centrifugal separator is used, particles having similar densities are precipitated at the same time and are difficult to separate from each other. Particles of similar density but different sizes are required to be spatially separated from each other.

 Most conventional methods of separating porous membranes are methods in which pressure or external force is applied in only one direction. Most of the systems using porous membranes have different sizes and concentrations of the original sample to be separated and concentrated. In particular, when a biological sample is used, in addition to the desired sample to be separated, impurities such as fine protein molecules and cell fragments having similar density or mass are contained. The original sample containing these impurities at a high concentration closes the pores of the porous membrane in the filtration process using the porous membrane. Therefore, samples smaller than the size of the pores do not pass through the porous membrane. Accordingly, the processing efficiency per unit pore is greatly reduced, and inefficient separation and concentration are performed.

In addition, pores clogged by high concentrations of impurities that did not pass through the pores increase the pressure. Accordingly, an increase in pressure causes loss or deformation of the porous membrane.

The present invention proposes a method for resolving the pore clogging phenomenon occurring in the conventional unidirectional porous membrane-based separation and concentration system.

One technical problem to be solved of the present invention is to provide a particle separation device for separating particles through a bidirectional flow rate control of a biological liquid sample using centrifugal force and restoring force.

One technical problem to be solved of the present invention is to provide a particle separation device for separating and concentrating particles by controlling the flow rate of a biological liquid sample and moving a porous membrane using centrifugal force and restoring force.

Centrifuge tube filtration apparatus according to an embodiment of the present invention, a centrifuge tube (centrifuge tube) for receiving a liquid sample and including a lid disposed on one end; At least one porous membrane disposed in a cross section in the centrifugation tube in the direction of extension of the centrifugation tube; And a restoring force applying unit disposed at the other end of the centrifuge tube and providing a restoring force to the liquid sample. The liquid sample includes first particles larger than the pore size of the porous membrane and second particles smaller than the pore size of the porous membrane, and the second particles move from one side of the porous membrane to the other side by centrifugal force. When the centrifugal force is reduced, the restoring force applying unit removes the first particles blocking the pores of the porous membrane.

In one embodiment of the present invention, the cap of the centrifuge tube includes a first pinhole through which the atmosphere can flow, the other end of the centrifuge tube is closed, and the restoring force applying unit is the other end of the centrifuge tube. It may include a balloon disposed therein and a balloon detachment preventing unit for restraining the balloon to a specific area.

In one embodiment of the present invention, the balloon departure prevention portion may be a mesh.

In one embodiment of the present invention, the balloon detachment prevention unit may be a connection between the balloon and the centrifugal tube or a connection between the balloon and the line.

In one embodiment of the present invention, the cap of the centrifuge tube includes a first pinhole through which the atmosphere can flow, and the other end of the centrifuge tube is closed and includes a second pinhole through which the atmosphere can flow. You can. The restoring force applying unit may include a plunger disposed inside the other end of the centrifuge tube and a spring disposed between the plunger and the other end of the centrifuge tube.

In one embodiment of the present invention, the cap of the centrifuge tube includes a first pinhole through which the atmosphere can flow, and the other end of the centrifuge tube is closed and includes a second pinhole through which the atmosphere can flow. You can. The restoring force applying unit may include a plunger disposed inside the other end of the centrifuge tube and a pair of magnets disposed between the plunger and the other end of the centrifuge tube.

In one embodiment of the present invention, the restoring force applying unit may be an air pocket formed by a partition wall.

In one embodiment of the present invention, the centrifugal tube is decomposed into a plurality of cylindrical shaped tubes, the tubes are fixed to each other, and the porous membrane can be disposed at the bonding site of the tube.

In one embodiment of the present invention, it may further include a porous membrane moving portion for moving the porous membrane.

In one embodiment of the present invention, the porous membrane moving portion, a porous membrane support portion in the form of a ring supporting the porous membrane; A first permanent magnet coupled to the porous membrane support; And a second permanent magnet disposed to surround the outer circumferential surface of the centrifuge tube and coupled with the first permanent magnet through magnetic force.

In one embodiment of the present invention, a first thread is formed on the outer circumferential surface of the porous membrane support, and a second thread is formed on the inner surface of the centrifuge tube facing the first thread. Can be.

In one embodiment of the present invention, the second permanent magnet supporting portion for supporting the second permanent magnet may be further included. The second permanent magnet support portion includes a third screw thread formed on a surface facing the outer circumferential surface of the centrifuge tube, and a fourth screw thread is formed on the outer circumferential surface of the centrifugal tube facing the third screw thread, and the second The position of the porous membrane may be changed as the permanent magnet rotates.

In one embodiment of the present invention, the centrifuge tube may include a cap, a first tube, a second tube, and a third tube in the form of a truncated cone, which are continuously connected. The lid, the first tube, the second tube, and the third tube may be fixed by being coupled to each other. The first tube includes a first ring portion protruding from the inner circumferential surface of one end, the second tube includes a second ring portion protruding from the outer circumferential surface of the one end, and the first ring portion comprises the second ring It is configured in a complementary form so as to be fastened to the site, and the first ring site of the first tube may include the porous membrane insert in the form of a recessed ring.

In one embodiment of the present invention, the first projection projecting locally from the outer peripheral surface of the porous membrane support; A second permanent magnet support unit supporting the second permanent magnet; And a second protrusion locally protruding from the inner peripheral surface of the second permanent magnet support. The first protrusion is inserted into a first groove formed along the inner circumferential surface of the centrifuge tube, and the second protrusion is inserted into a second groove formed along the outer circumferential surface of the centrifuge tube, and the first groove and the second Each of the grooves includes at least one of a vertical portion extending in the center axis direction of the cylinder, an azimuth portion extending in the azimuth direction, and a diagonal portion including both the vertical component and the azimuth component, as the second permanent magnet rotates. The position of the porous membrane can be changed.

In one embodiment of the present invention, the centrifugal tube may include a cap, a first tube, a second tube, a third tube, and a fourth tube in the form of a truncated cone that are continuously connected. The porous membrane includes a first porous membrane disposed between the first tube and the second tube and a second porous membrane disposed between the second tube and the third tube, and the pore size of the first porous membrane is the It may be larger than the pore size of the second porous membrane.

Particle separation apparatus according to an embodiment of the present invention, a centrifuge; And a centrifuge tube filtration device mounted on the centrifuge. The centrifugal tube filtration device includes a centrifuge tube containing a liquid sample and a lid disposed at one end; At least one porous membrane disposed in a cross section in the centrifugation tube in the direction of extension of the centrifugation tube; And a restoring force applying unit disposed at the other end of the centrifuge tube and providing a restoring force to the liquid sample. The liquid sample includes first particles larger than the pore size of the porous membrane and second particles smaller than the pore size of the first porous membrane, and the second particles move from one side of the porous membrane to the other side by centrifugal force. , When the centrifugal force is reduced, the restoration force applying unit removes the first particle blocking the pores of the porous membrane.

In one embodiment of the present invention, the first particle is an exosome, and the second particle may include at least one of liquid protein and cell-free DNA (cfDNA).

In one embodiment of the present invention, the first particles are stem cells, and the second particles may include at least one of red blood cells, white blood cells, and platelets.

In one embodiment of the present invention, the first particle is a circulating tumor cell (CTC), and the second particle may include at least one of red blood cells, white blood cells, and platelets.

Particle separation apparatus according to an embodiment of the present invention, a centrifuge; And a centrifuge tube filtration device mounted on the centrifuge. The centrifuge tube filtration apparatus includes a centrifuge tube containing a liquid sample and a lid disposed at one end; It includes at least one porous membrane disposed in a cross section in the centrifugation tube in the cross-section extending direction of the centrifugation tube; And it is disposed on the other end of the centrifuge tube and includes a restoring force applying unit for providing a resilience to the liquid sample. The particle separation method of the particle separation device comprises: opening the lid of the centrifugal tube filtration device, supplying a liquid sample containing first particles and second particles to the centrifugal tube filtration device from outside and covering the lid. ; Attaching the centrifugal tube filtration device to the centrifuge and accelerating to a first state to separate second particles smaller than the pore size of the porous membrane by centrifugal force; And decelerating the centrifuge to a second state to remove the first particles blocking the pores of the porous membrane.

In one embodiment of the present invention, the step of separating the second particles by centrifugal force and the step of removing the first particles blocking the pores of the porous membrane may be alternately repeated.

In one embodiment of the present invention, the step of separating the second particles by centrifugal force and the step of removing the first particles clogging the pores of the porous membrane may be performed by repeatedly increasing and decreasing the centrifugal force. .

In one embodiment of the present invention, it may further include the step of moving the porous membrane.

In one embodiment of the present invention, the step of separating the second particles by centrifugal force and the step of removing the first particles clogging the pores of the porous membrane are alternately repeated, and the step of moving the porous membrane is the The first particles may be concentrated by moving the porous membrane in a specific direction while reciprocating.

In one embodiment of the present invention, the restoring force providing unit is a balloon, and the restoring force of the restoring force providing unit may be pressure to restore the original volume of the balloon.

In one embodiment of the present invention, the restoring force providing unit is a plunger and a spring, and the restoring force of the restoring force providing unit may be an elastic force to restore the original position of the spring.

In one embodiment of the present invention, the restoring force providing unit is an air pocket formed by a partition wall, and the restoring force of the restoring force providing unit may be pressure to restore the original volume of the air pocket.

In one embodiment of the present invention, the restoring force applying unit includes a plunger disposed inside the other end of the centrifuge tube and a pair of magnets disposed between the plunger and the other end of the centrifuge tube, and the restoring force providing unit The restoring force may be the repulsive force of the pair of magnets.

The particle separation device according to an embodiment of the present invention can easily perform particle separation using centrifugal force and restoring force. The particle separation device provides centrifugal force to the particles to provide a forward flow or a first direction of movement through the porous membrane, and a reverse flow in a state in which the centrifugal force is reduced by using a restoring force formed by the centrifugal force. Accordingly, particles smaller than the pores of the porous membrane can be filtered by centrifugal force. Congestion of the porous membrane due to pore clogging due to particles or impurities larger than the pores can be resolved by resilience. Therefore, the particle separation efficiency can be increased.

The particle separation device according to an embodiment of the present invention can separate a specific biomolecule from a biological liquid sample containing various biomolecules.

The particle separation method according to an embodiment of the present invention can perform continuous separation or concentration without dilution of a high-concentration sample without depending on the concentration of the original sample, and selectively separate only biomolecules of a desired size in a short time with high efficiency. can do.

1A and 1B are conceptual views illustrating a particle separation device using a centrifuge tube device.
2A and 2B are cross-sectional views illustrating the centrifuge tube device of FIG. 1B.
3 is a view for explaining a particle separation method according to another embodiment of the present invention.
4 is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.
5 is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.
6 is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.
7A is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.
7B is a view for explaining the porous membrane moving part of the centrifugal tube filtration device of FIG. 7A.
8A is a cut perspective view illustrating a porous membrane moving part according to an embodiment of the present invention.
8B is a cross-sectional view taken along line BB 'of FIG. 8A.
8C is a cross-sectional view taken along line CC ′ of FIG. 8A.
9 is a view for explaining a particle separation method according to another embodiment of the present invention.
10 is a cross-sectional view illustrating a perspective view of a porous membrane and a moving portion of the porous membrane according to another embodiment of the present invention.
11 is a view for explaining a particle separation method according to another embodiment of the present invention.
12 is a view for explaining the operation of the particle separation method of FIG.
13 is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.

The particle separation device according to an embodiment of the present invention provides centrifugal force to particles to provide a forward flow through the porous membrane, and reverse flow by resilience to remove particles blocking the porous membrane in a state where the centrifugal force is reduced or removed. Gives By alternately applying a forward flow and a reverse flow to the porous membrane, the membrane permeation flow control in both directions of the porous membrane is repeatedly performed. Accordingly, the pore clogging phenomenon caused by particles and biomolecules accumulated locally on the porous membrane is removed using a reverse pressure, so that efficient separation and concentration can be continuously performed.

The particle separation device according to an embodiment of the present invention may use a centrifugal force for forward flow, and may use a plunger and a spring to provide reverse pressure (or flow rate).

The particle separation device according to an embodiment of the present invention may use a centrifugal force for forward flow and a balloon to provide reverse pressure (or flow rate).

The particle separation device according to an embodiment of the present invention may include a porous membrane moving unit that moves the position of the porous membrane for separation and concentration of particles. The porous membrane moving part may use a magnetic force by a pair of magnets. In addition, the movement of the porous membrane may be screwed to the centrifuge tube to prevent movement of the movement of the porous membrane by forward pressure or reverse pressure.

The particle separation device according to an embodiment of the present invention may selectively separate and concentrate nanometer-scale particles or biomolecules in a liquid sample including a micrometer-level particle or a complex biological sample.

The particle separation device according to an embodiment of the present invention can separate and concentrate exosomes. Exosomes are vesicles of 50 nm to 120 nm in size found in body fluids. In order to separate exosomes present in body fluids, a nano-sized nanoporous membrane is essential. However, it contains a lot of impurities such as fine protein molecules and cell fragments that are similar in density or mass to exosomes. In the separation and concentration of such a complex biological sample, since the volume of the exosome space must be reduced and concentrated among a number of factors, if control and exchange of bi-directional flow through the porous membrane is used, only biomolecules of a desired size are selected in a short time, High efficiency and high purity can be concentrated.

The particle separation device according to an embodiment of the present invention can separate and concentrate particles in a liquid sample using a simple mechanism without using an expensive device.

The particle separation device according to an embodiment of the present invention uses a porous membrane as a filtration filter in a storage space of a liquid sample in the form of a centrifuge tube. In addition, the filtration filter may transmit particles smaller than pores in a forward direction when centrifugal force is applied. On the other hand, in order to remove the pore clogging phenomenon due to the forward flow, the reverse pressure of the porous membrane may be provided by the restoring force applying unit.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the present invention is not limited or limited by experimental conditions, material types, and the like. The present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed contents are thorough and complete and that the spirit of the present invention is sufficiently conveyed to those skilled in the art. In the drawings, the components are exaggerated for clarity. Portions denoted by the same reference numerals throughout the specification denote the same components.

1A and 1B are conceptual views illustrating a particle separation device using a centrifuge tube device.

2A and 2B are cross-sectional views illustrating the centrifuge tube device of FIG. 1B.

1A, 1B, and 2A. 2B, the particle separation device 100 includes a centrifuge 20; And a centrifuge tube filtration device 101 mounted on the centrifuge 20. The centrifugal tube filtration device 101 includes a centrifuge tube (120) for storing a liquid sample (10) and including a lid disposed at one end; At least one porous membrane 130 disposed in a cross section in the centrifugation tube 120 in a direction transverse to the extending direction of the centrifugation tube; And a restoring force applying unit 140 disposed at the other end of the centrifuge tube 120 and providing a restoring force to the liquid sample 10. The liquid sample 10 includes first particles 12 larger than the size of the pores 132 of the porous membrane 130 and second particles 14 smaller than the size of the pores 132 of the porous membrane. The second particles 14 move from one side of the porous membrane 130 to the other side by centrifugal force. When the centrifugal force is reduced, the restoring force applying unit 140 removes the first particles 12 blocking the pores of the porous membrane 130.

The centrifugal separator 20 may be a swing-out rotor, or a fixed-angle rotor. The swing-neighbor rotor may be arranged to hook the centrifuge tube filtration device 101 with a ring 21.

The centrifugal tube filtration device 101 may filter the particles of the liquid sample received by the centrifugal force of the centrifugal separator 20 and the restoring force of the restoring force applying unit 140 into the porous membrane 130 according to the size. The centrifugal force may provide a forward flow in the direction of the centrifugal force, and the restoring force may provide a reverse flow in the direction of the restoring force. As the centrifugal separator 20 accelerates, the centrifugal force may be greater than the restoring force. As the centrifugal separator 20 decelerates, the restoring force may be greater than the centrifugal force. As the centrifuge 20 alternates the acceleration and deceleration alternately, the forward flow and the reverse flow may be repeated. Accordingly, the second particles 14 may be filtered through the porous membrane 130.

The centrifuge tube 120 has a cylindrical shape, and may be made of metal or plastic. The centrifugation tube 120 may include a lid 122 that accommodates a liquid sample and is disposed at one end. After the lid 122 is opened, the liquid sample may be stored in the centrifuge tube 120 and the lid 122 may be closed. The other end of the centrifuge tube 120 may be conical. The cap of the centrifuge tube 122 may include a first pinhole 122a through which air can flow, and the other end of the centrifuge tube 120 may be closed.

The centrifuge tube 120 may include a lid 122 that is continuously connected, a first tube 124, a second tube 126, and a third tube 128 having a truncated cone shape. The lid 122, the first tube 124, the second tube 126, and the third tube 128 may be disassembled from each other by being coupled to each other by screwing. A porous membrane 130 may be disposed at a bonding site between the first tube 124 and the second tube 126. The first tube 124 includes a first ring portion 124b protruding from an inner circumferential surface of one end thereof, and the second tube 126 includes a second ring portion 126a protruding from an outer circumferential surface of one end thereof can do.

The first ring portion 124b is configured in a complementary form to be fastened to the second ring portion 126a, and the first ring portion 124b of the first tube is the porous membrane in the form of a recessed ring It may include an insertion portion (124a). The first ring portion 124b and the second ring portion 126a may be screwed together to be sealed. The porous membrane 130 may be fixed by being inserted into the porous membrane insertion portion 124a.

The cap of the centrifuge tube includes a first pinhole through which air can flow, and the other end of the centrifuge tube can be closed. The restoring force applying unit 140 may include a balloon disposed inside the other end of the centrifugal tube and a balloon detachment prevention unit 141 for constraining the balloon to a specific area. The balloon departure prevention portion 141 may be a mesh. The balloon departure prevention unit 141 may be a connection between the balloon and the centrifugal tube or a connection between the balloon and the line.

The liquid sample 10 may fill a space excluding the balloon. When the balloon is compressed by the centrifugal force applied to the liquid sample, the balloon may cause a volume reduction to provide a forward flow. When the centrifugal force applied to the liquid sample decreases, the balloon may cause a volume expansion to provide a reverse flow.

The porous membrane 130 is the centrifugation tube 120 into the first space portion 121a formed on one side of the porous membrane 130 and the second space portion 121b formed on the other side of the porous membrane 130 Can be separated. The porous membrane 130 may include pores of a certain diameter. The pores 132 may have a constant diameter. The porous membrane 130 may be a porous membrane. The pores may have a diameter of several nanometers to several tens of micrometers. The porous membrane 130 may be polycarbonate track etched (PCTE). The porous film 130 may be formed through a photo-lithography process or a laser drilling process. The porous membrane 130 may be fixed in the centrifuge tube.

The liquid sample 10 may be a biological sample including biomolecules. The liquid sample may include a first particle 12, a second particle 14, and a fluid medium. The liquid sample 10 may be a bodily fluid that contains high concentrations of biomolecules and has not undergone a pretreatment process. The centrifugal tube 120 may be filled with a liquid sample containing particles.

The first particle may be an exosome (Exosome). The second particle may be liquid protein, cfDNA (cell-free DNA). The exosomes are vesicles of 50 nm to 120 nm in size found in body fluids. The state of the cell from which the exosome is derived can be diagnosed through analysis of the substance inside the exosome. In addition, exosomes can be used as therapeutic agents.

When exosomes are separated by a conventional exo point separation method, chemicals or antibodies are used. Accordingly, it is impossible to re-inject into body fluids again. The particle separation device according to an embodiment of the present invention can be separated by diluting proteins in the body as much as possible and concentrating pure exosomes at the same time. The isolated exosomes can be re-injected into the canine.

The second particles are adipose tissue-derived stem cells, and the first particles may include red blood cells, white blood cells, platelets, and the like smaller than the stem cells.

Due to the recent development of stem cell therapy, in order to increase the engraftment rate of fat transplantation, stromal vascular fraction (SVF) cells are put together during fat transplantation. This is called cell assisted lipotransfer (CAL) or cell assisted fat injection.

Stem cells to increase the survival rate of fat transplantation in fat transplantation SVF cells used in fat transplantation actually contain only 10% of adipose tissue derived stem cells (ADSC), and the rest are fibroblasts. ), Vascular endothelial cells (endothelial cells), pericyte, preadipocyte, and other various cells are present in about 90%. The injected SVF cells must survive by receiving oxygen and nutrients from surrounding tissues. However, in stem cell fat transplantation, if too many cells (or solvents) other than adipose tissue-derived stem cells are injected, adipose tissue-derived stem cells cannot survive because they do not receive oxygen and nutrients. The efficiency of cell therapy is reduced. Therefore, high-purity adipose-derived stem cells must be injected into a small solvent. Therefore, high-purity separation and concentration of adipose tissue-derived stem cells present only in 10% farmland in SVF cells is required.

The first particle is a circulating tumor cell (CTC), and the second particle may include at least one of red blood cells, white blood cells, and platelets.

3 is a view for explaining a particle separation method according to another embodiment of the present invention.

1A, 2A, and 3, the particle separation device 100 includes a centrifuge 20; And a centrifuge tube filtration device 101 mounted on the centrifuge. The centrifuge tube filtration device 101 includes a centrifuge tube (120) for storing a liquid sample and including a lid disposed at one end; At least one porous membrane 130 disposed in a cross section in the centrifugation tube 120 in a direction transverse to the extending direction of the centrifugation tube; And a restoring force applying unit 140 disposed at the other end of the centrifuge tube and providing a restoring force to the liquid sample.

In the particle separation method of the particle separation device, the lid 122 of the centrifugal tube filtration device 101 is opened, and the liquid sample 10 including the first particle and the second particle is centrifuged tube filtration device from the outside. After supplying to (101), covering the lid 122 (S110); After the centrifugation tube filtration device 101 is mounted on the centrifugal separator 20, it accelerates to a first state to separate second particles smaller than the pore size of the porous membrane by centrifugal force (S112); And removing the first particles blocking the pores of the porous membrane by decelerating the centrifuge to a second state (S114).

The step of separating the second particles by centrifugal force and the step of removing the first particles clogging the pores of the porous membrane are repeated. The alternating process of forward flow by centrifugal force and reverse flow by restoring force can filter the second particles. In the first state, the centrifugal force may be greater than the restoring force, and in the second state, the centrifugal force may be smaller than the restoring force.

4 is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.

Referring to Figure 4, the centrifugal tube filtration device 201, a centrifuge tube (centrifuge tube, 120) containing a lid 122 disposed at one end and receiving the liquid sample 10; At least one porous membrane 130 disposed in the centrifuge tube 120; And a restoring force applying unit 240 disposed at the other end of the centrifuge tube 120 and providing a restoring force to the liquid sample. The liquid sample includes first particles 12 larger than the pore size of the porous membrane 130 and second particles 14 smaller than the pore size of the porous membrane. The second particles move from one side to the other side of the porous membrane by centrifugal force, and when the centrifugal force is reduced, the restoring force applying unit 240 removes the first particles blocking the pores of the porous membrane.

The cap 122 of the centrifuge tube includes a first pinhole 122a through which the atmosphere can flow, and the other end of the centrifuge tube is closed and includes a second pinhole 128a through which the atmosphere can flow. You can.

The centrifuge tube 122 may include a lid 122 that is continuously connected, a first tube 124, a second tube 126, and a third tube 128 having a truncated cone shape. The lid 122, the first tube 124, the second tube 126, and the third tube 128 may be screwed together and disassembled from each other. A porous membrane 130 may be disposed at a bonding site between the first tube 124 and the second tube 126.

The restoring force applying unit 240 may include a plunger 242 disposed inside the other end of the centrifuge tube and a spring 244 disposed between the plunger 242 and the other end of the centrifuge tube 120. have. The spring may be compressed by centrifugal force to move the plunger in the direction of centrifugal force to provide forward flow. When the centrifugal force is reduced, the spring can expand to move the plunger in the direction of restoring force to provide reverse flow.

5 is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.

Referring to FIG. 5, the centrifugal tube filtration device 301 includes a centrifuge tube 120 that stores a liquid sample 10 and includes a lid 122 disposed at one end; At least one porous membrane 130 disposed in a cross section in the centrifugation tube 120 in a direction transverse to the extending direction of the centrifugation tube; And a restoring force applying unit 340 disposed at the other end of the centrifuge tube 120 and providing a restoring force to the liquid sample. The liquid sample includes first particles 12 larger than the pore size of the porous membrane 130 and second particles 14 smaller than the pore size of the porous membrane. The second particles move from one side to the other side of the porous membrane by centrifugal force, and when the centrifugal force is reduced, the restoring force applying unit 340 removes the first particles blocking the pores of the porous membrane.

The cap 122 of the centrifuge tube may include a first pinhole 122a through which air can flow. The other end of the centrifuge tube may be closed.

The centrifuge tube 122 may include a lid 122 that is continuously connected, a first tube 124, a second tube 126, and a third tube 128 having a truncated cone shape. The lid 122, the first tube 124, the second tube 126, and the third tube 128 may be screwed together and disassembled from each other. A porous membrane 130 may be disposed at a bonding site between the first tube 124 and the second tube 126.

The restoring force applying unit 340 may be a partition wall providing an air pocket. The partition wall may include a pipe region to form an air pocket when the liquid sample 10 is introduced. The air in the air pocket is compressed by centrifugal force to provide a forward flow in the direction of centrifugal force. When the centrifugal force is reduced, the air pocket can expand to provide a reverse flow in the direction of the restoring force.

6 is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.

Referring to FIG. 6, the centrifugal tube filtration device 401 includes a centrifuge tube 120 containing a liquid sample 10 and a lid 122 disposed at one end; At least one porous membrane 130 disposed in a cross section in the centrifugation tube 120 in a direction transverse to the extending direction of the centrifugation tube; And a restoring force applying unit 440 disposed at the other end of the centrifuge tube 120 and providing a restoring force to the liquid sample. The liquid sample includes first particles 12 larger than the pore size of the porous membrane 130 and second particles 14 smaller than the pore size of the porous membrane. The second particles move from one side to the other side of the porous membrane by centrifugal force, and when the centrifugal force is reduced, the restoring force applying unit 440 removes the first particles blocking the pores of the porous membrane.

The centrifuge tube 122 may include a lid 122 that is continuously connected, a first tube 124, a second tube 126, and a third tube 128 having a truncated cone shape. The lid 122, the first tube 124, the second tube 126, and the third tube 128 may be screwed together and disassembled from each other. A porous membrane 130 may be disposed at a bonding site between the first tube 124 and the second tube 126.

The cap 122 of the centrifuge tube includes a first pinhole 122a through which the atmosphere can flow, and the other end of the centrifuge tube is closed and includes a second pinhole 128a through which the atmosphere can flow. You can.

The restoring force applying unit 440 includes a plunger 242 disposed inside the other end of the centrifuge tube 120 and a pair of magnets 444a and 444b disposed between the plunger and the other end of the centrifuge tube. can do. The pair of magnets 444a and 444b are disk-shaped or washer-shaped, and may have magnetization directions opposite to each other to provide a repulsive force to each other.

The pair of magnets may be compressed with each other by centrifugal force, thereby providing forward flow in the direction of centrifugal force. When the centrifugal force is reduced, the pair of magnets may repel each other to provide a reverse flow in the direction of the restoring force.

7A is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.

7B is a view for explaining the porous membrane moving part of the centrifugal tube filtration device of FIG. 7A.

7A and 7B, a centrifuge tube filtration device 101a includes a centrifuge tube 120 containing a liquid sample 10 and a lid 122 disposed at one end; At least one porous membrane 130 disposed in a cross section in the centrifugation tube 120 in a direction transverse to the extending direction of the centrifugation tube; And a restoring force applying unit 140 disposed at the other end of the centrifuge tube 120 and providing a restoring force to the liquid sample. The liquid sample includes first particles 12 larger than the pore size of the porous membrane 130 and second particles 14 smaller than the pore size of the porous membrane. The second particles move from one side to the other side of the porous membrane by centrifugal force, and when the centrifugal force is reduced, the restoring force applying unit 140 removes the first particles blocking the pores of the porous membrane.

The cap 122 of the centrifuge tube includes a first pinhole 122a through which the atmosphere can flow, and the other end of the centrifuge tube is closed and includes a second pinhole 128a through which the atmosphere can flow. You can.

The centrifuge tube 120 may include a lid 122 that is continuously connected, a first tube 124, a second tube 126, and a third tube 128 having a truncated cone shape. The lid 122, the first tube 124, the second tube 126, and the third tube 128 may be screwed together and disassembled from each other. A porous membrane 130 may be disposed at a bonding site between the first tube 124 and the second tube 126.

The restoring force applying unit 140 may be a balloon disposed inside the other end of the centrifuge tube 120. The mesh 141 is fixed inside the other end of the centrifugation tube 120, and can prevent the balloon from escaping. The liquid sample 10 may fill a space excluding the balloon. When the balloon is compressed by the centrifugal force applied to the liquid sample, the balloon may cause a volume reduction to provide a forward flow. When the centrifugal force applied to the liquid sample decreases, the balloon may cause a volume expansion to provide a reverse flow.

The porous membrane moving unit 150 may concentrate the filtered particles by moving the porous membrane 130. The porous membrane moving part 150 includes: a porous membrane support portion 155 in the form of a ring supporting the porous membrane; A first permanent magnet 156 in the form of a ring coupled to the porous membrane support 155; And a second permanent magnet 154 in the form of a ring that is disposed to surround the outer circumferential surface of the centrifuge tube and is coupled to the first permanent magnet 156 through magnetic force.

A first screw thread may be formed on an outer circumferential surface of the porous membrane support part 155, and a second screw thread may be formed on an inner surface of the centrifugal tube 120 facing the first screw thread. .

The second permanent magnet support unit 152 may support the second permanent magnet 154. The second permanent magnet support 152 may include a third screw thread formed on a surface facing the outer circumferential surface of the centrifuge tube. A fourth screw thread may be formed on the outer circumferential surface of the centrifugal tube 120 facing the third screw thread. The position of the porous membrane may be changed as the second permanent magnet rotates.

8A is a cut perspective view illustrating a porous membrane moving part according to an embodiment of the present invention.

8B is a cross-sectional view taken along line B-B 'of FIG. 8A.

8C is a cross-sectional view taken along line C-C 'of FIG. 8A.

8A to 8C, the porous membrane moving part 550 may concentrate the filtered particles by moving the porous membrane 130. The porous membrane moving part 550 includes a ring-shaped porous film support part 155 for supporting the porous film; A first permanent magnet 156 in the form of a ring coupled to the porous membrane support 155; And a second permanent magnet 154 in the form of a ring that is disposed to surround the outer circumferential surface of the centrifuge tube and is coupled to the first permanent magnet 156 through magnetic force.

The first protrusion 157 may be locally protruded from the outer circumferential surface of the porous membrane support 155. The second permanent magnet support unit 152 may support the second permanent magnet 154. The second permanent magnet support 152 may have a cylindrical shape. The second protrusion 153 may protrude locally from the inner circumferential surface of the second permanent magnet support 152. The first protrusion 157 may be inserted into the first grooves 129a and 129b formed along the inner circumferential surface of the centrifuge tube 120. The second protrusion 153 may be inserted into the second groove 129c formed along the outer circumferential surface of the centrifuge tube 120. Each of the first groove and the second groove is at least among a vertical portion 129a extending in a central axis direction of the cylinder, an azimuth portion 129b extending in an azimuth direction, and a diagonal portion including both a vertical component and an azimuth component It can contain one. As the second permanent magnet 154 rotates, the position of the porous membrane 130 may be changed. When the position of the porous membrane 130 is changed, particles may be concentrated.

9 is a view for explaining a particle separation method according to another embodiment of the present invention.

Referring to Figure 9, the particle separation device 100 is a centrifuge 20; And a centrifuge tube filtration device 101a mounted on the centrifuge. The centrifugal tube filtration device 101a includes a centrifuge tube (120) for storing a liquid sample and including a lid disposed at one end; At least one porous membrane 130 disposed in a cross section in the centrifugation tube 120 in a direction transverse to the extending direction of the centrifugation tube; And a restoring force applying unit 140 disposed at the other end of the centrifuge tube and providing a restoring force to the liquid sample.

The particle separation method of the particle separation device comprises: opening the lid of the centrifugal tube filtration device, supplying a liquid sample containing first particles and second particles to the centrifugal tube filtration device from outside and covering the lid. (S110); Mounting the centrifugal tube filtration device to the centrifuge and accelerating to a first state to separate second particles smaller than the pore size of the porous membrane by centrifugal force (S112); And removing the first particles blocking the pores of the porous membrane 130 by decelerating the centrifuge to a second state (S114).

The step of separating the second particles by centrifugal force and the step of removing the first particles clogging the pores of the porous membrane are repeated. The alternating process of forward flow by centrifugal force and reverse flow by restoring force can filter the second particles. Separating the second particles by centrifugal force and removing the first particles clogging the pores of the porous membrane may be performed by repeatedly increasing and decreasing the centrifugal force.

Subsequently, a step of moving and concentrating the porous membrane 130 may be performed (S119). The porous membrane 130 may be moved by the porous membrane moving unit 150.

The centrifuge tube 120 may include a lid 122 that is continuously connected, a first tube 124, a second tube 126, and a third tube 128 having a truncated cone shape. The movement of the porous membrane 130 may be performed in the third tube direction within the second tube 126. Accordingly, the second particles can be concentrated.

10 is a cross-sectional view illustrating a perspective view of a porous membrane and a moving portion of the porous membrane according to another embodiment of the present invention.

11 is a view for explaining a particle separation method according to another embodiment of the present invention.

12 is a view for explaining the operation of the particle separation method of FIG.

10, 11 and 12, the particle separation device 100 includes a centrifuge 20; And a centrifuge tube filtration device 101a mounted on the centrifuge. The centrifugal tube filtration device 101a includes a centrifuge tube (120) for storing a liquid sample and including a lid disposed at one end; At least one porous membrane 130 disposed in a cross section in the centrifugation tube 120 in a direction transverse to the extending direction of the centrifugation tube; And a restoring force applying unit 140 disposed at the other end of the centrifuge tube and providing a restoring force to the liquid sample.

The particle separation method of the particle separation device comprises: opening the lid of the centrifugal tube filtration device, supplying a liquid sample containing first particles and second particles to the centrifugal tube filtration device from outside and covering the lid. (S110); Mounting the centrifugal tube filtration device to the centrifuge and accelerating to a first state to separate second particles smaller than the pore size of the porous membrane by centrifugal force (S112); And removing the first particles blocking the pores of the porous membrane 130 by decelerating the centrifuge to a second state (S114).

The step of separating the second particles by centrifugal force and the step of removing the first particles clogging the pores of the porous membrane are repeated. The alternating process of forward flow by centrifugal force and reverse flow by restoring force can filter the second particles. Separating the second particles by centrifugal force and removing the first particles clogging the pores of the porous membrane may be performed by repeatedly increasing and decreasing the centrifugal force. During the first time interval T1, the centrifugal force is set to be greater than the restoring force, so that the second particle can penetrate the porous membrane 130 (S112). During the second time interval T2, the restoring force is set to be greater than the centrifugal force, so that the second particles blocking the pores of the porous membrane can be removed (S114). If S112 and S114 are repeated multiple times, the second particles may pass through the porous membrane 130. The porous membrane 130 includes a first space portion 121a formed on one side of the porous membrane 130 and a second space portion 121b formed on the other side of the porous membrane 130. Can be separated by. When the volume V1 of the first space portion is repeated S112 and S114, the decrease and increase may be repeated. Specifically, as the steps of separating the second particles by centrifugal force and removing the first particles clogging the pores of the porous membrane are alternately repeated, the first particles and the second particles are the porous membrane 130 ).

Subsequently, a step of moving and concentrating the porous membrane 130 may be performed (S119). The porous membrane 130 may be moved by the porous membrane moving unit 150. The movement of the porous membrane 130 can be simply moved in a specific direction.

The centrifuge tube 120 may include a lid 122 that is continuously connected, a first tube 124, a second tube 126, and a third tube 128 having a truncated cone shape. The movement of the porous membrane 130 may be performed in the direction of the lid 122 within the first tube 124. Accordingly, the first particles can be concentrated.

According to a modified embodiment of the present invention, the porous membrane may concentrate in the centrifugal tube 120 while reciprocating in the specific direction while moving in a specific direction. For example, the moving distance of the porous membrane moving in the positive traveling direction may be smaller than moving in the negative traveling direction.

According to a modified embodiment of the present invention, after one cycle of separating the second particles and removing the first particles blocking the pores by centrifugal force, one cycle of moving the porous membrane may be performed. The movement of the porous membrane can be variously modified as long as concentration is performed.

13 is a conceptual diagram illustrating a centrifuge tube filtration device according to another embodiment of the present invention.

Referring to FIG. 13, the centrifugal tube filtration device 601 includes a centrifuge tube (620) for storing a liquid sample (10) and including a lid (122) disposed at one end; At least one porous membrane 130 disposed in a cross section in the centrifugation tube 620 transverse to an extension direction of the centrifugation tube; And a restoring force applying unit 140 disposed at the other end of the centrifuge tube 620 and providing a restoring force to the liquid sample. The liquid sample includes first particles 12 larger than the pore size of the porous membrane 130a and second particles 14 smaller than the pore size of the porous membrane. The second particles move from one side to the other side of the porous membrane by centrifugal force, and when the centrifugal force is reduced, the restoring force applying unit 140 removes the first particles blocking the pores of the porous membrane 130a.

The centrifuge tube 620 includes a lid 622 that is continuously connected, a first tube 624, a second tube 626, a third tube 628, and a truncated cone-shaped fourth tube 629. It can contain.

The liquid sample may include a first particle 12, a second particle 14, and a third particle 16. The size of the first particle may be larger than that of the second particle, and the size of the second particle may be larger than that of the third particle.

The porous membrane 130 is disposed between the first porous membrane 130a and the second tube 626 and the third tube 628 disposed between the first tube 624 and the second tube 626. It may include a second porous film (130b) disposed on. The pore size of the first porous membrane 130a may be larger than the pore size of the second porous membrane 130b. The size of the first particle may be larger than the pore size of the first porous membrane 130a. The size of the second particle may be smaller than the pore size of the first porous membrane 130a. The size of the third particle may be smaller than the pore size of the second porous membrane. Accordingly, after repeated processes of the forward flow and the reverse flow, the first particles may be concentrated in the first tube, the second particles may be concentrated in the second tube, and the third particles may be concentrated in the third tube. .

In the above, the present invention has been illustrated and described with respect to specific preferred embodiments, but the present invention is not limited to these embodiments, and those skilled in the art to which the present invention pertains are claimed in the claims. It includes all of the various types of embodiments that can be carried out without departing from the technical spirit.

20: centrifuge
100: particle separation device
101: centrifuge tube filtration device
120: centrifuge tube
130: porous membrane
140: resilience authorization unit

Claims (13)

Centrifuge; And
A centrifuge tube filtration device mounted on the centrifuge,
The centrifuge tube filtration device:
A centrifuge tube containing a liquid sample and a lid disposed at one end;
At least one porous membrane disposed in a cross section in the centrifugation tube in the direction of extension of the centrifugation tube; And
It is disposed on the other end of the centrifuge tube and includes a restoring force applying unit for providing a restoring force to the liquid sample,
The liquid sample includes first particles larger than the pore size of the porous membrane and second particles smaller than the pore size of the first porous membrane,
The second particles move from one side of the porous membrane to the other side when a forward flow of the liquid sample occurs by centrifugal force,
When the centrifugal force is reduced, the restoring force applying unit removes the first particles blocking the pores of the porous membrane by generating a reverse flow in the liquid sample,
The porous membrane is a porous membrane including pores of a certain diameter,
The pores are formed through the porous membrane,
The cap of the centrifuge tube includes a first pinhole through which the atmosphere can flow Particle separation device characterized in that. According to claim 1,
The first particle is an exosome, and the second particle is a particle separation device comprising at least one of liquid protein and cell-free DNA (cfDNA). According to claim 1,
The first particles are stem cells,
The second particle is a particle separation device, characterized in that it comprises at least one of red blood cells, white blood cells, platelets. According to claim 1,
The first particle is CTC (circulating tumor cell),
The second particle is a particle separation device, characterized in that it comprises at least one of red blood cells, white blood cells, platelets. Centrifuge; In the particle separation method of the particle separation device comprising a centrifuge tube filtration device mounted on the centrifugal separator,
The centrifuge tube filtration apparatus includes a centrifuge tube containing a liquid sample and a lid disposed at one end; At least one porous membrane disposed in a cross section in the centrifugation tube in the direction of extension of the centrifugation tube; And a restoring force applying unit disposed at the other end of the centrifuge tube and providing restoring force to the liquid sample,
Opening the lid of the centrifugal tube filtration device and supplying a liquid sample containing first particles and second particles to the centrifugal tube filtration device from outside to cover the lid;
Attaching the centrifugal tube filtration device to the centrifuge and accelerating to a first state to separate second particles smaller than the pore size of the porous membrane by centrifugal force; And
Including the step of removing the first particles blocking the pores of the porous membrane by decelerating the centrifuge to a second state to provide the restoring force;
The porous membrane is a porous membrane including pores of a certain diameter,
The pores are formed through the porous membrane,
The cap of the centrifugation tube comprises a first pinhole through which the atmosphere can flow. The method of claim 5,
Separating the second particles by centrifugal force and removing the first particles blocking the pores of the porous membrane alternately repeating the particle separation method. The method of claim 5,
Separating the second particles by centrifugal force and removing the first particles clogging the pores of the porous membrane are characterized in that the centrifugal force is increased and decreased by repeating the particle separation method. The method of claim 5,
The particle separation method further comprising the step of moving the porous membrane. The method of claim 8,
Separating the second particles by centrifugal force and removing the first particles clogging the pores of the porous membrane are alternately repeated,
The step of moving the porous membrane is a particle separation method characterized in that the first particles are concentrated by moving in a specific direction. The method of claim 5,
The resilience providing unit is a balloon,
The method of separating particles, wherein the restoring force of the restoring force providing unit is a pressure to restore the original volume of the balloon. The method of claim 5,
The restoring force providing unit is a plunger and a spring,
The restoring force of the restoring force providing unit is an elastic force to restore the original position of the spring. The method of claim 5,
The restoring force providing unit is an air pocket formed by a partition wall,
The method of separating particles, wherein the restoring force of the restoring force providing unit is a pressure to restore the original volume of the air pocket. The method of claim 5,
The restoring force applying unit includes a plunger disposed inside the other end of the centrifuge tube and a pair of magnets disposed between the plunger and the other end of the centrifuge tube,
The restoring force of the restoring force providing unit is a particle separation method, characterized in that the repulsive force of the pair of magnets.

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