Porous membrane-based particle separation-concentration method and particle separation-concentration apparatus using bidirectional membrane permeation flow rate control and air bubbles

Abstract

The present invention relates to a porous membrane-based particle separation-concentration method and particle separation-concentration apparatus, using bidirectional membrane permeation movement control and air bubbles, in which, by preventing pore clogging of a porous membrane, which is used in separating and concentrating size-selective biomolecules, particles, or cells, by using bidirectional membrane permeation movement, especially reverse membrane permeation movement, and air bubbles, loss or deformation of the porous membrane can be prevented and a sample can be dispersed with high efficiency. According to the present invention, provided is a porous membrane-based particle separation-concentration method comprising: a forward flow forming step of applying a first force to form a forward flow that causes a liquid sample to flow from an upper space to a lower space; and a reverse flow forming step of applying, after the forward flow forming step, a second force to form a reverse flow that causes a pore unclogging means for unclogging pores of a porous membrane to flow from the lower space to the upper space.

Description

A porous membrane-based particle separation-concentration method and particle separation-concentration device using two-way membrane permeation flow control and air bubbles

[0001] The present invention relates to a particle separation-concentration method and a particle separation-concentration device based on a porous membrane.

[0002] More specifically, the present invention relates to a porous membrane-based particle separation-concentration method and particle separation-concentration device using bidirectional membrane permeation control and air bubbles, which are used for separation and concentration of size-selective biomolecules, particles, or cells, and which prevent loss or deformation of the porous membrane by preventing blockage of the pores of the porous membrane using bidirectional membrane permeation movement, especially reverse membrane permeation movement, and air bubbles, thereby enabling high-efficiency dispersion of the sample.

[0003] Microvesicles in vivo are small membrane-structured vesicles that exist in or are secreted from various types of cells.

[0004] Microvesicles secreted extracellularly include (i) exosomes: membrane vesicles with a diameter of 30 to 100 nm of phagocytic origin, (ii) exosomes (also called shedding microvesicles (SMVs)): large membrane vesicles with a diameter of 50 to 1000 nm that are shed directly from the plasma membrane, and (iii) apoptotic blebs: vesicles with a diameter of 50 to 5000 nm shed by dying cells.

[0005] The above-mentioned microvesicles in vivo, such as exosomes, are vesicles of several tens of nanometers in size secreted from cells, and are structures containing proteins and RNA produced in the cytoplasm or cells inside a lipid bilayer or lipid monolayer.

[0006] Exosomes are a means of intercellular communication through the exchange of proteins and RNA, and are also responsible for the function of excreting unnecessary substances within the cell. They contain microRNA (miRNA), so they can be used as useful markers in molecular diagnosis, such as the early diagnosis of diseases such as cancer.

[0007] Although the importance and value of microvesicles in vivo have been revealed as described above, obtaining the microvesicles is difficult.

[0008] For example, centrifuges are used in bioseparation processes. Centrifuges utilize the density difference between a solid and the liquid surrounding it.

[0009] However, when a centrifuge is used, particles with similar densities precipitate at the same time and are difficult to separate from each other.

[0010] Particles with similar densities but different sizes require a technique to spatially separate them.

[0011] Another conventional method for isolating microvesicles is to combine microvesicles with antibodies to immuno-capture and isolate the microvesicles.

[0012] These methods may cause bias depending on the separation or detection target due to masking of antibody recognition sites by changes in protein structure, microvesicle heterogeneity, protein interactions, etc.

[0013] Separation or detection may require complex processes or expensive equipment, and may result in high sample consumption.

[0014] In addition, although small-volume separation of in vivo microvesicles such as exosomes is possible with a microfluidic chip, the microfluidic chip has the disadvantage of being unsuitable for separating exosomes because it is difficult to separate large volumes and the separation membrane can be damaged due to pressure, etc.

[0015] As part of an effort to solve this problem, a separation method using a porous membrane has been proposed, but the conventional porous membrane separation method is a method in which pressure or external force is applied in only one direction.

[0016] Most systems that utilize these porous membranes have a mixture of raw samples to be separated and concentrated, each with a different size and concentration.

[0017] Especially in the case of biological samples, in addition to the sample to be separated, there are many impurities such as microscopic protein molecules and cell fragments with similar density or mass.

[0018] The raw sample containing these impurities in high concentrations blocks each pore of the porous membrane during the filtration process using the porous membrane.

[0019] Therefore, samples smaller than the pore size cannot pass through the porous membrane. Consequently, the processing efficiency per unit pore is significantly reduced, resulting in inefficient separation and concentration.

[0020] Additionally, the pores are clogged with high concentrations of impurities that cannot pass through, increasing the pressure.

[0021] Accordingly, the increase in pressure causes loss or deformation of the porous membrane.

[0022] Therefore, there is a need for research and development of methods and devices that can resolve the pore blockage and stagnation phenomenon that occurs in conventional unidirectional porous membrane-based separation and concentration methods and enable high-efficiency separation and concentration.

[0023] Accordingly, the present invention, which aims to solve the above-mentioned conventional problems, is to prevent loss or deformation of a porous membrane by preventing blockage of the pores of the porous membrane using air bubbles and by preventing bidirectional membrane permeation, especially reverse membrane permeation, of a porous membrane used for separation and concentration of size-selective biomolecules, particles, or cells.

[0024] The purpose is to provide a particle separation-concentration method and a particle separation-concentration device based on a porous membrane using air bubbles and a two-way membrane permeation flow control capable of highly efficiently dispersing a sample.

[0025] According to one aspect of the present invention for achieving the above objects and other features of the present invention, a porous membrane-based particle separation-concentration method is provided, which uses a separation-concentration chamber configured such that the interior is divided into an upper space and a lower space by a porous membrane to separate and concentrate first particles of a predetermined size contained in a liquid sample introduced into the separation-concentration chamber and second particles of a predetermined size smaller than the size of the first particles, the method comprising: a forward flow forming step of applying a first force to form a forward flow that causes the liquid sample to flow from the upper space to the lower space; and, after the forward flow forming step, a reverse flow forming step of applying a second force to form a reverse flow that causes a pore unclogging means for unclogging pores of the porous membrane to flow from the lower space to the upper space.

[0026] The method and device for particle separation and concentration based on a porous membrane using a two-way membrane permeation flow control and air bubbles according to the present invention provide the following effects.

[0027] First, unlike the existing one-way forward porous separation method, the present invention can continuously relieve congestion in a porous membrane clogged with large particles or impurities through repeated forward and reverse bidirectional flow through membrane permeation flow control, thereby having the effect of greatly increasing the processing efficiency per unit pore.

[0028] Second, the present invention has the effect of further increasing the separation and concentration efficiency by injecting air bubbles in the reverse direction in the two-way flow control to drop large particles accumulated at the pore inlet and mixing the sample by causing the air bubbles to rise through the one-side space (the space upstream of the porous membrane).

[0029] Third, unlike existing methods, the present invention can perform continuous separation or concentration of a high-concentration sample without dilution and without depending on the concentration of the original sample in separating a specific biomolecule from a biological sample containing various biomolecules, and has the effect of selectively and efficiently separating only biomolecules of a desired size within a relatively short period of time.

[0030] Fourth, the present invention has the effect of enabling highly efficient separation and concentration to be achieved through full automation even by non-trained experimenters by using a biomolecule separation and concentration device through continuous membrane permeation flow control.

[0031] Fifth, the present invention has the effect of contributing to the improvement of porous membrane-based technology capable of separating and concentrating not only macro- and micro-level particles and samples, but also nano- (nm)-scale particles or biomolecules in complex biological samples.

[0032] Figure 1 is a flow chart showing the processing process of a porous membrane-based particle separation-concentration method using two-way membrane permeation flow control and air bubbles according to the present invention.

[0033] Figure 2 is a schematic diagram illustrating a method for separating and concentrating particles based on a porous membrane using a two-way membrane permeation flow control and air bubbles according to the present invention.

[0034] Figure 3 is a schematic diagram schematically showing a concentration process through reverse injection of air bubbles in a porous membrane-based particle separation-concentration method using air bubbles and a two-way membrane permeation flow control according to the present invention.

[0035] FIG. 4 is a drawing schematically showing the configuration of a particle separation-concentration device based on a porous membrane using a two-way membrane permeation flow control and air bubbles (air) according to one embodiment of the present invention.

[0036] FIG. 5 is a drawing showing the configuration of a particle separation-concentration device unit included in a porous membrane-based particle separation-concentration device using a two-way membrane permeation flow control and air bubbles according to one embodiment of the present invention.

[0037] FIG. 6 is a drawing showing a porous membrane module included in a porous membrane-based particle separation-concentration device using a two-way membrane permeation flow control and air bubbles according to one embodiment of the present invention.

[0038] FIG. 7 is a schematic diagram illustrating the operation of a porous membrane-based particle separation-concentration device using a two-way membrane permeation flow control and air bubbles according to one embodiment of the present invention.

[0039] FIG. 8 is a schematic diagram showing the configuration of a porous membrane-based particle separation-concentration device using air bubbles and a two-way membrane permeation flow control according to another embodiment of the present invention.

[0040] FIG. 9 is a schematic diagram illustrating the operation of one embodiment of a porous membrane-based particle separation-concentration device using air bubbles (air) and a two-way membrane permeation flow control according to another embodiment of the present invention.

[0041] FIG. 10 is a schematic diagram illustrating the operation of another embodiment of a porous membrane-based particle separation-concentration device using air bubbles (air) and a two-way membrane permeation flow control according to another embodiment of the present invention.

[0042] Figure 11 is a graph showing the particle size distribution analyzed by NTA (Nano particle Tracking Analysis) as a result of separating 140 and 400 nm beads through a 200 nm porous membrane.

[0043] Figure 12 is a scanning electron microscope (SEM) photograph of a 200 nm porous membrane after a separation experiment (A), and a scanning electron microscope (SEM) photograph of a sample separated with a 200 nm porous membrane after concentrating it with a 50 nm porous membrane.

[0044] Figure 13 is a graph(s) showing the results of isolating exosomes from biological samples.

[0045] Figure 14 is a drawing showing the results confirmed by western blot after exosome separation using the present invention and a conventional separation method.

[0046] Figure 15 is a transmission electron microscope (TEM) photograph of exosomes separated using the present invention and a conventional separation method.

[0047] Hereinafter, a method for separating and concentrating particles based on a porous membrane and a particle separation and concentration device using a two-way membrane permeation flow control and air bubbles according to a preferred embodiment of the present invention will be described in detail with reference to the attached drawings.

[0048] First, the two-way membrane permeation flow control according to the present invention and the particle separation-concentration method based on a porous membrane using air bubbles will be described in detail with reference to FIGS. 1 to 3.

[0049] Figure 1 is a flow chart showing the processing process of a porous membrane-based particle separation-concentration method using two-way membrane permeation flow control and air bubbles according to the present invention.

[0050] Figure 2 is a schematic diagram illustrating a method for separating and concentrating particles based on a porous membrane using a two-way membrane permeation flow control and air bubbles according to the present invention.

[0051] FIG. 3 is a schematic diagram schematically showing a concentration process through reverse injection of air bubbles in a porous membrane-based particle separation-concentration method using air bubbles and a two-way membrane permeation flow control according to the present invention.

[0052]

[0053] In the description and patent claims of the present invention below, the upper space (or first space) is defined as the space on the upstream side based on the porous membrane, that is, the space on the upstream side of the porous membrane, and the lower space (or second space) is defined as the space on the downstream side based on the porous membrane, that is, the space on the downstream side of the porous membrane.

[0054] In addition, the forward direction (or first direction) is defined as a direction proceeding from the upper space to the lower space based on the porous membrane, and the reverse direction (or second direction) is defined as a direction proceeding from the lower space to the upper space based on the porous membrane in the direction opposite to the first direction.

[0055] The method for separating and concentrating particles based on a porous membrane using a two-way membrane permeation flow control and air bubbles according to the present invention is a porous membrane-based particle separation and concentration method for separating and concentrating a liquid sample introduced into a separation and concentration chamber using a separation and concentration chamber whose interior is configured to be divided into an upper space and a lower space by a porous membrane.

[0056] As shown in FIGS. 1 to 3, it largely includes a forward flow formation step (S100) for particle separation, and a reverse flow formation step (S200) for pore blockage release.

[0057] Specifically, a porous membrane-based particle separation-concentration method using a two-way membrane permeation flow control and air bubbles according to an embodiment of the present invention is a porous membrane-based particle separation-concentration method for separating and concentrating first particles of a predetermined size and second particles of a predetermined size smaller than the size of the first particles contained in a liquid sample introduced into a separation-concentration chamber, the interior of which is configured to be divided into an upper space and a lower space by a porous membrane, and the method includes, as shown in FIGS. 1 to 3, a forward flow forming step (S100) of applying a first force to the upper space of the separation-concentration chamber to form a forward flow from the upper space to the lower space; and a reverse flow forming step (S200) of applying a second force to the lower space of the separation-concentration chamber to form a reverse flow from the lower space to the upper space, wherein, during the reverse flow, pores of the porous membrane are unclogged by a pore unclogging means.

[0058] In a porous membrane-based particle separation-concentration method using a two-way membrane permeation flow control and air bubbles according to the present invention, the porous membrane is formed with holes (pores) that prevent first particles of a liquid sample from flowing from an upper space to a lower space and allow second particles of the liquid sample to flow from an upper space to a lower space.

[0059] The above forward flow formation step (S100) can be performed by a first force applied by a physical device such as pneumatic pressure or a piston when the upper space of the separation and concentration chamber is filled (full) with a liquid sample.

[0060] Of course, in the above forward flow formation step (S100), the upper space does not need to be filled with a liquid sample.

[0061] And, in the forward flow forming step (S100), the reverse flow forming step (S200) for releasing the blocked pores of the porous membrane by the first particle may be performed by injecting gas, specifically air or air bubbles, in the reverse direction as a pore releasing means.

[0062] In other words, the reverse flow forming step (S200) may be performed by supplying air bubbles, which are a means for releasing pore blockages, in the reverse direction from the lower side of the porous membrane, after the forward flow forming step (S100), while a space of a predetermined size is formed on the upper side (or the lower side of the porous membrane) of the lower space portion.

[0063] Meanwhile, in the description of the porous membrane-based particle separation-concentration method according to the above-described embodiment, it is described that a first force is applied to the upper space of the separation-concentration chamber to form a forward flow for separation and / or concentration of a liquid sample, and a second force is applied to the lower space of the separation-concentration chamber to form a reverse flow for unclogging the pores of the porous membrane.

[0064] As a method of applying force for forming a flow in another embodiment, positive pressure (+ pressure) and negative pressure (- pressure) can be applied to the upper space of the separation and concentration chamber to cause forward flow and reverse flow.

[0065] At this time, the container (or lower chamber) of the lower space of the separation concentration chamber is made of a flexible material, and a predetermined space is secured at the lower part of the porous membrane (i.e., the upper side of the lower space) (i.e., a state in which air is filled to a predetermined volume).

[0066] And the upper side of the upper space can be in a state where a predetermined space is formed or filled with a liquid sample.

[0067] Here, when the negative pressure is formed (i.e., when a reverse flow is generated), air may be injected into the upper space of the lower space through an air injection device.

[0068] The porous membrane-based particle separation-concentration method of these other embodiments is performed by applying positive pressure (pressurization) to the upper space of the separation-concentration chamber to form a forward flow, thereby performing separation and / or concentration of a liquid sample through the porous membrane, and by applying negative pressure (suction pressure or vacuum pressure) to the upper space of the separation-concentration chamber to form a reverse flow, thereby unclogging the pores of the porous membrane.

[0069] The operational effects of the two-way membrane permeation flow control and the particle separation-concentration method based on a porous membrane using air bubbles according to the present invention are described with reference to FIGS. 2 and 3.

[0070] For example, exosomes are particles of the order of 100 nm, and are mixed with soluble proteins (~10 nm), cells (>1 μm), and apoptotic bodies (>200 nm) in blood, urine, cell culture media, saliva, breast milk, etc.

[0071] As shown in Fig. 2, by filtering out particles larger than 200 nm with a porous membrane having pores of 200 nm in size, a sample containing exosomes and water-soluble proteins can be obtained.

[0072] If this is filtered again through a porous membrane with 50 nm pore size, an exosome sample with water-soluble proteins removed can be obtained.

[0073] During this process, if the sample is pushed forward through the porous membrane, the holes will quickly become clogged, so it is necessary to create a reverse flow to periodically clear them.

[0074] Accordingly, in the present invention, when there is air in the upper space and the lower space (when vertically arranged), a forward flow can be created through the first force (P1) and a reverse flow can be created through the second force (P2).

[0075] At this time, the air bubble injection, which is a flow by the second force (P2), causes the air bubbles to rise in the reverse direction and clear the blocked hole, and in addition, the liquid sample in the upper space is evenly mixed, which can increase the separation and concentration efficiency.

[0076] If this process is repeated, as shown in Fig. 3, the amount of liquid sample in the upper space decreases, so particles A larger than the pore diameter of the porous membrane are concentrated on the upper space side.

[0077] In this way, the present invention quickly clears a blocked hole by injecting air instead of liquid in the reverse flow, and as air bubbles rise toward the upper space, the liquid sample within the upper space is evenly distributed, which enables more efficient separation and concentration to be continuously performed.

[0078] Next, a two-way membrane permeation flow control and a porous membrane-based particle separation-concentration device using air bubbles according to an embodiment of the present invention will be described in detail with reference to FIGS. 4 to 7.

[0079]

[0080] FIG. 4 is a drawing schematically showing the configuration of a particle separation-concentration device based on a porous membrane using a two-way membrane permeation flow control and air bubbles (air) according to one embodiment of the present invention.

[0081] FIG. 5 is a drawing showing the configuration of a particle separation-concentration device unit included in a porous membrane-based particle separation-concentration device using a two-way membrane permeation flow control and air bubbles according to one embodiment of the present invention.

[0082] FIG. 6 is a drawing showing a porous membrane module included in a porous membrane-based particle separation-concentration device using a two-way membrane permeation flow control and air bubbles according to one embodiment of the present invention.

[0083] FIG. 7 is a schematic diagram illustrating the operation of a porous membrane-based particle separation-concentration device using a two-way membrane permeation flow control and air bubbles according to one embodiment of the present invention.

[0084] A porous membrane-based particle separation-concentration device using a two-way membrane permeation flow control and air bubbles according to one embodiment of the present invention is a porous membrane-based particle separation-concentration device for separating and concentrating two or more different-sized particles contained in a liquid sample using a porous membrane, and as shown in FIGS. 4 to 7, largely includes a particle separation-concentration device section (100), a flow force applying device section (200), and a control device section (300).

[0085] The porous membrane-based particle separation-concentration device using a two-way membrane permeation flow control and air bubbles according to the present invention is a porous membrane-based particle separation-concentration device for separating and concentrating two or more different-sized particles contained in a liquid sample using a porous membrane (132).

[0086] As shown in FIGS. 4 to 7, the device comprises: a particle separation-concentration device unit (100) configured to separate and concentrate a liquid sample through a porous membrane (132); a flow force application device unit (200) configured to provide a force for generating a forward flow and a reverse flow to separate particles of different sizes contained in a liquid sample introduced into the particle separation-concentration device unit (100) and to unclog pores of the porous membrane (132) through a pore unclogging means during reverse flow; and a control device unit (300) configured to control the flow force application device unit (200).

[0087] The above particle separation-concentration device unit (100) is a device unit configured to separate and concentrate particles contained in a liquid sample while the liquid sample flows from one side to the other by using a porous membrane (132) and a force applied by the flow force application device unit (200).

[0088] In one embodiment, the particle separation-concentration device unit (100) includes an upper chamber (110) in which a liquid sample before separation is introduced and stored and a force application unit (first force application unit) (111) to which the force of the flow force application unit (200) is applied is formed, a lower chamber (120) in which separated particles of the liquid sample separated through the porous membrane are received and a force application unit (second force application unit) (121) to which the force of the flow force application unit (200) is applied is provided, and a connecting unit (130) that connects the upper chamber (110) and the lower chamber (120) in a communicable manner and has a porous membrane for separating particles of the liquid sample.

[0089] The upper chamber (110) and the lower chamber (120) may be formed of a cylindrical body part (112, 122) with one side open and the other side closed, and a horn-shaped or cone-shaped connecting part (113, 123) integrally formed at the open end of the body part (112, 122).

[0090] The upper chamber (110) and the lower chamber (120) are connected and integrated by the connecting unit (130) with the connecting parts (113, 123) facing each other.

[0091] The force application portion (first force application portion) (111) formed in the upper chamber (110) may be formed integrally with the bottom portion, i.e., the bottom center portion of the body portion (112), and may be formed as a protruding connection portion connected to apply the force from the flow force application device portion (200).

[0092] And the force application part (second force application part) (121) formed in the lower chamber (120) may be formed integrally on the upper side, i.e., one side of the connection part (123), and may be formed as a protruding connection part connected to apply the force from the flow force application device part (200).

[0093] The upper chamber (110) and the lower chamber (120) may be formed of a synthetic resin material such as PET, but are not limited thereto and may be formed of various materials such as metal materials.

[0094] Continuing, the connecting unit (130) is a component that connects the upper chamber (110) and the lower chamber (120) in a communicative manner and has a porous membrane (132) having predetermined pores for separating particles of a liquid sample.

[0095] Specifically, the connecting unit (130) includes a connecting bracket module (131) configured to connect and seal the connecting portion (113) of the upper chamber (110) and the connecting portion (123) of the lower chamber (120) while allowing communication between them, and a porous membrane (132) mounted within the connecting bracket module (131).

[0096] The above connecting bracket module (131) may be configured to include a cylindrical portion (131a) into which the connecting portions (113, 123) of each chamber (110, 120) are inserted, a coupling flange portion (131b) extending outward from one end of the cylindrical portion (131a), and a fixing piece (bolt / nut) (131c) that couples and fixes the coupling flange portions (131b).

[0097] These connecting bracket modules (131) are detachably connected to each chamber (110, 120) so that the porous membrane (132) provided on the inside thereof can be replaced.

[0098] In addition, the connecting unit (130) may further include a drain disc (133) provided on the upper and lower sides of the porous membrane (132).

[0099] Drawing symbol 134 is an O-ring provided on the flange portion (131b) of the connecting bracket module (131) to ensure airtightness between the flange portions (131b).

[0100] And the porous film (132) is formed with holes (pores) that prevent the first particles of the liquid sample from flowing from the upper chamber (110) to the lower chamber (120) and allow the second particles of the liquid sample to flow from the upper chamber (110) to the lower chamber (120).

[0101] These porous membranes (132) are provided so as to be replaceable depending on the particles of the liquid sample to be separated. Of course, the connecting unit (130) itself, which is provided with porous membranes (132) having different pore sizes depending on the particles of the liquid sample to be separated, may be separately installed in each chamber (110, 120).

[0102] Meanwhile, the porous membrane (132) may be configured as a porous membrane module in which multiple porous membranes are provided instead of a single porous membrane so that parallel processing can be performed.

[0103] Specifically, as shown in FIG. 6, the porous membrane module includes a module case (410) having an inlet (411) formed in the upper center and an outlet (not shown) formed in the lower center, a plurality of porous membranes (132) arranged and provided in a compartmentalized manner within the module case (410), an upper flow path (420) formed on the upper inner surface of the module case (410) so that a fluid flowing in from the inlet (411) flows to each of the porous membranes (132), and a lower flow path (not shown) formed on the bottom surface of the module case (410) so that a fluid passing through each of the porous membranes (132) is discharged through the outlet (not shown).

[0104] The above module case (410) is configured such that a pair of cases, each having a flow path (upper flow path, lower flow path) formed on opposing surfaces, are joined together through a fixed piece (412), so that the sides on which each porous membrane (132) is provided are isolated and communicated only by the flow path.

[0105] Here, on the side where the porous membrane (132) is provided, a receiving portion in which the porous membrane (132) can be provided is formed, and an O-ring is provided at the edge of the receiving portion to ensure airtightness.

[0106] The above porous membrane may be provided with multiple groups arranged concentrically around the center, with the inlet (411) as the center and the group arranged around the center.

[0107] And the flow path connected to the inlet (411) and the outlet can be formed by branching out to extend to the center of each group, and being positioned at the center of each porous membrane (132) at the extension end.

[0108] These porous modules can be processed in parallel through multiple porous membranes (132) to significantly increase processing speed.

[0109] Although the porous module is shown in Fig. 6 as having a pentagonal shape, it may be formed in a polygonal shape including a square or a circle, and may be formed in a shape that takes into consideration the shape of the connecting bracket module (131) of the connecting unit (130).

[0110] Next, the flow force application device unit (200) provides a force to create forward flow and reverse flow in order to separate particles of different sizes contained in the liquid sample introduced into the particle separation-concentration device unit (100).

[0111] This is a device configured to release the pores of a porous membrane (132) from blockage through a pore release means during reverse flow.

[0112] As an example, the flow force application device (200) may be configured as a device configured to supply pneumatic pressure, i.e. air or air bubbles, as the hole blockage release means.

[0113] Specifically, the flow force application device (200) includes an air supply device (210) that generates compressed air, an air supply line (air supply pipe) (220) that is connected to supply air (compressed air) generated from the air supply device (210) to the force application device (111) of the upper chamber (110) and the force application device (121) of the lower chamber (120), a regulator (230) that is provided on the air supply line (220) to set and control the pressure of the air (air pressure), and a check valve (solenoid valve) (240) that is provided on the air supply line (220) on the downstream side of the regulator (230). Reference numeral 250 denotes a pressure gauge.

[0114] The above air supply device (210) may be composed of an air cylinder or a compressor, and may be composed of a bubble generator.

[0115] The above air supply line (220) may be branched from a main line (221) connected to an air supply device (210) and may be composed of a first branch line (222) connected to a force application unit (111) of an upper chamber (110) and a second branch line (223) connected to a force application unit (121) of a lower chamber (120).

[0116] The above check valve (240) includes a first solenoid valve (241) provided in the first branch line (222) and a second solenoid valve (242) provided in the second branch line (223).

[0117] Continuing, the control device unit (300) is a device unit configured to control the flow force application device unit (200), specifically, to control the opening and closing of the first and second solenoid valves (241, 242).

[0118] The above control device unit (300) is configured to control the opening and closing of the solenoid valve (240) so that the action force supplied from the flow action force application device unit (220) is applied to the upper chamber (110) and the lower chamber (120), respectively.

[0119] The above control device unit (300) can be configured integrally with a control device that controls the on / off and driving operation of the air cylinder (210).

[0120] In the present invention, when the force of the flow force applying device (200) is applied by the control of the control device (300), it is preferable to apply the force while a space of a predetermined size is secured on the upper side of the lower chamber (120), i.e., the lower side of the porous membrane (132), but the present invention is not limited thereto.

[0121] The above-described two-way membrane permeation flow control and porous membrane-based particle separation-concentration device using air bubbles according to the present invention opens the first solenoid valve (241) and closes the second solenoid valve (242) under the control of the control device unit (300), and compressed air is supplied to the upper chamber (110) through the first solenoid valve (241) to apply a forward flow (or a first force (P1)) to separate and / or concentrate a liquid sample through the porous membrane, and when the first solenoid valve (242) is closed and the second solenoid valve (242) is opened, a reverse flow (or a second force (P2)) by air or air bubbles is created to rise in the reverse direction and open the blocked hole of the porous membrane (132), and in addition, the liquid sample in the upper space is evenly mixed, thereby increasing the separation and concentration efficiency.

[0122] And when this process is repeated, the amount of liquid sample in the upper chamber (110) decreases, so particles larger than the hole diameter of the porous membrane (132) are concentrated on the upper chamber (110) side.

[0123] Meanwhile, a two-way membrane permeation flow control and a porous membrane-based particle separation-concentration device using air bubbles (air) according to another embodiment of the present invention will be described with reference to FIGS. 8 to 10.

[0124] In the description of other embodiments below, the same components as those in the above-described embodiment are given the same reference numerals and detailed descriptions thereof are brief or omitted.

[0125]

[0126] FIG. 8 is a schematic diagram showing the configuration of a porous membrane-based particle separation-concentration device using air bubbles and a two-way membrane permeation flow control according to another embodiment of the present invention.

[0127] FIG. 9 is a schematic diagram illustrating the operation of one embodiment of a porous membrane-based particle separation-concentration device using air bubbles (air) and a two-way membrane permeation flow control according to another embodiment of the present invention.

[0128] FIG. 10 is a schematic diagram illustrating the operation of another embodiment of a porous membrane-based particle separation-concentration device using air bubbles (air) and a two-way membrane permeation flow control according to another embodiment of the present invention.

[0129] According to another embodiment of the present invention, a porous membrane-based particle separation-concentration device using air bubbles (air) and a two-way membrane permeation flow control can be configured to generate forward and reverse flows by applying a positive pressure (+ pressure) or negative pressure (- pressure) force (P1, P2) to only one of the upper chamber (110) and the lower chamber (120), as shown in FIGS. 8 to 10.

[0130] Specifically, a particle separation-concentration device based on a porous membrane using a two-way membrane permeation flow control and air bubbles (air) according to another embodiment of the present invention comprises: a particle separation-concentration device unit (100) configured to separate and concentrate a liquid sample through a porous membrane (132); a flow force application device unit configured to provide forces (P1, P2) for generating forward and reverse flows to separate particles of different sizes contained in a liquid sample introduced into the particle separation-concentration device unit (100), and to unclog pores of the porous membrane (132) through a pore unclogging means during reverse flow; And it includes a control device unit (300) configured to control the flow force application device unit; and the flow force application device unit is configured with a positive pressure-negative pressure application device unit (510) that can apply positive pressure (+ pressure) and negative pressure (- pressure or vacuum pressure) to the force application unit (111) of the upper chamber (110) or the force application unit (121) of the lower chamber (120).

[0131] The above-mentioned positive pressure-negative pressure application device (510) is configured as a known device capable of injecting air with + pressure and also applying - pressure or vacuum pressure by sucking air within the upper chamber (110) or lower chamber (120).

[0132] Here, an exhaust hole (511) may be formed in the chamber to which no force (P1, P2) is applied, or the body part may be made of a flexible material.

[0133] In (A) of FIG. 9, a case is shown where an action force application portion to which action forces (P1, P2) are applied is formed in the upper chamber (110), and an exhaust hole (511) is formed in the lower chamber (120), and in (B) of FIG. 9, a case is shown where an action force application portion to which action forces (P1, P2) are applied is formed in the lower chamber (120), and an exhaust hole (511) is formed in the upper chamber (110).

[0134] In addition, in (A) of FIG. 10, a case is shown where a force application portion to which force (P1, P2) is applied is formed in the upper chamber (110), and the body portion (122) of the lower chamber (120) is formed flexibly.

[0135] In (B) of Fig. 10, a case is shown where a force application portion to which force (P1, P2) is applied is formed in the lower chamber (120), and the body portion (112) of the upper chamber (110) is formed flexibly.

[0136] When the above exhaust hole (511) is formed in the chamber, it is provided so that the sample (solution) accommodated therein does not flow out through the exhaust hole (511), for example, so that the opening direction is directed upward and positioned on the upper side of the chamber.

[0137] The porous membrane-based particle separation-concentration device of this other embodiment applies positive pressure (P1) toward the chamber (110 or 120) equipped with a force applying unit (pressurization) to form a forward flow, thereby performing separation and / or concentration of a liquid sample through the porous membrane (132).

[0138] In addition, by applying negative pressure (P2) to the chamber (110 or 120) equipped with the force application unit to form a reverse flow, the pores of the porous membrane (132) are unclogged.

[0139] At this time, when + pressure (P1) or - pressure (P2) is applied to the chamber equipped with the force application unit, the chamber not equipped with the force application unit is connected to the outside through the exhaust hole (511), or the flexible body part expands or contracts to form smooth forward and reverse flows according to the application of the force.

[0140] Meanwhile, in a porous membrane-based particle separation-concentration device of another embodiment, a chamber not equipped with a force application unit may further include an air injection device (520) for injecting air into a space (upper space) of the chamber.

[0141] That is, an air injection device (520) for injecting air into the exhaust hole (511) may be connected and configured.

[0142] Here, the lower portion of the porous membrane (132), i.e., the upper portion of the lower chamber (120), is configured to have a predetermined space (i.e., a state in which a predetermined volume of air is filled). In addition, the upper portion of the upper chamber (110) can have a predetermined space formed therein or be filled with a liquid sample.

[0143] Meanwhile, the inventor of the present invention conducted an experiment to confirm the results of separation and concentration of a liquid sample using a porous membrane-based particle separation and concentration method using air bubbles and a two-way membrane permeation flow control according to the present invention, and the present invention is described herein.

[0144] Figure 11 is a graph showing the particle size distribution analyzed by NTA (Nano particle Tracking Analysis) as a result of separating 140 and 400 nm beads through a 200 nm porous membrane using a porous membrane-based particle separation-concentration method using a two-way membrane permeation flow control and air bubbles according to the present invention. The result after separation (red) confirmed that only small-sized particles existed.

[0145] Figure 12 (A) is a scanning electron microscope (SEM) photograph of a 200 nm porous membrane after a separation experiment, in which 140 and 400 nm beads are present together, and Figure 12 (B) is a scanning electron microscope (SEM) photograph of a sample separated with a 200 nm porous membrane after concentrating it with a 50 nm porous membrane, in which only 140 nm beads were found.

[0146] Fig. 13 is a graph(s) showing the results of separating exosomes from a biological sample. As a result of comparison with existing exosome separation technologies such as UC (ultra centrifuge) and DFF (direct flow filtration), it was confirmed that the concentration of exosomes obtained by the BFF (bidirectional flow filtration), which is the separation method of the present invention, is higher than that of UC and DFF (Fig. 13 (A)), and the amount of soluble protein in the solution is lower (Fig. 13 (C)), and as a result, the purity ratio of the exosomes is significantly higher (Fig. 13 (D)). Fig. 13 (B) is a graph showing the average size.

[0147] In particular, in the case of BFF+wash, which concentrates exosomes through a 50 nm porous membrane without performing a single concentration process, and then repeats filling and concentrating with a buffer solution, the purity ratio was dozens of times higher than that of the existing gold standard method, UC, confirming the effectiveness of the separation technology of the present invention.

[0148] Figure 14 is a drawing showing the results confirmed by western blot after exosome separation using the present invention and a conventional separation method.

[0149] Among the exosome markers (CD9, TSG101, CD81) and the apoptotic body marker (calreticulin), if the exosome markers are abundant and the apoptotic body marker is absent, it means that the exosome sample is pure without contamination. As shown in the results of Fig. 14, it was confirmed that the separation according to the present invention can obtain a large amount of pure exosomes.

[0150] Figure 15 is a transmission electron microscope (TEM) photograph of exosomes separated using the present invention and a conventional separation method.

[0151] UC and DFF were found to have damaged exosomes due to high pressure, but BFF was performed under low pressure, so it was confirmed that the original shape of the exosomes was well maintained.

[0152] That is, it was confirmed that by using the separation and concentration method of the present invention, intact exosomes can be obtained in large quantities with high purity (purity ratio).

[0153] According to the method and device for particle separation and concentration based on a porous membrane using a bidirectional membrane permeation flow control and air bubbles according to the present invention as described above, unlike the conventional one-way forward porous separation method, the congestion of a porous membrane clogged with large particles or impurities can be continuously relieved through repeated bidirectional flow in the forward and reverse directions through membrane permeation flow control, thereby greatly increasing the treatment efficiency per unit pore.

[0154] In addition, the present invention can further increase the separation and concentration efficiency by dropping large particles accumulated at the pore inlet by injecting air bubbles in the reverse movement in the two-way flow control and mixing the sample by causing the air bubbles to rise through the one-side space (the space upstream of the membrane permeation).

[0155] By using a biomolecule separation and concentration device through continuous membrane permeation flow control, there is an advantage in that high-efficiency separation and concentration can be achieved through full automation even by non-trained experimenters.

[0156] In addition, unlike existing methods, the present invention can perform continuous separation or concentration of a high-concentration sample without dilution and without depending on the concentration of the original material in separating a specific biomolecule from a biological sample containing various biomolecules.

[0157] It has the advantage of being able to selectively and efficiently separate only biomolecules of a desired size within a relatively short period of time, and can contribute to the improvement of porous membrane-based technology that can separate and concentrate not only macro- and micro-scale particles and samples, but also nano- (nm)-scale particles or biomolecules in complex biological samples.

[0158] The above-described two-way membrane permeation flow control and porous membrane-based particle separation-concentration method and particle separation-concentration device using air bubbles according to the present invention can be applied to the exosome in vitro diagnosis / treatment market and the stem cell treatment market.

Claims

1. A porous membrane-based particle separation-concentration method for separating and concentrating first particles of a predetermined size contained in a liquid sample introduced into a separation-concentration chamber, the interior of which is configured to be divided into an upper space and a lower space by a porous membrane, and second particles of a predetermined size smaller than the size of the first particles, A forward flow forming step of applying a first force to form a forward flow so that the liquid sample flows from the upper space to the lower space; and After the forward flow forming step, a reverse flow forming step is characterized in that it includes a second force for forming a reverse flow so that a pore unclogging means for unclogging the pores of the porous membrane flows from the lower space to the upper space; A particle separation-concentration method based on a porous membrane.

2. In paragraph 1, The hole blocking release means used in the reverse flow forming step is characterized in that it is air or air bubble. A particle separation-concentration method based on a porous membrane.

3. In paragraph 1, The above reverse flow formation step is It is characterized in that air or air bubbles are injected in the reverse direction from the lower side of the porous membrane. A particle separation-concentration method based on a porous membrane.

4. In any one of paragraphs 1 to 3, The above forward flow forming step is performed by applying air pressure from the upper side while the upper space is filled with a liquid sample or while a space is formed on the upper side. The above reverse flow forming step is characterized in that it is performed by applying air or air bubbles toward a space in a state where there is a space at the bottom of the porous membrane. A particle separation-concentration method based on a porous membrane.

5. In any one of paragraphs 1 to 3, One of the chambers having the upper space portion and the chamber having the lower space portion is provided with a force application portion, and the other has an exhaust hole or is formed flexibly, The above forward flow forming step and the reverse flow forming step are characterized in that positive or negative pressure is applied to the force applying section in a state where a liquid sample is filled in the upper space of each chamber or a space is formed on the upper side. A particle separation-concentration method based on a porous membrane.

6. In any one of paragraphs 1 to 3, The above porous membrane-based particle separation-concentration method is, It is characterized by using a porous membrane formed with pores that prevent the first particles of the liquid sample from flowing from the upper space to the lower space and allow the second particles of the liquid sample to flow from the upper space to the lower space. A particle separation-concentration method based on a porous membrane.

7. A porous membrane-based particle separation-concentration device for separating and concentrating two or more different-sized particles contained in a liquid sample using a porous membrane, A particle separation-concentration device configured to separate and concentrate particles of different sizes in a liquid sample through a porous membrane; A flow force applying device configured to apply a force for forward flow from the upper side to the lower side and reverse flow from the lower side to the upper side based on the porous membrane in the particle separation-concentration device section, and to release the pore blockage of the porous membrane through a pore blockage releasing means when the reverse flow force is applied; and A control device configured to control the above-mentioned flow force application device unit; characterized in that it includes: A particle separation-concentration device based on a porous membrane.

8. In paragraph 7, The above particle separation-concentration device section An upper chamber in which a liquid sample is introduced and a force application unit is formed to which a first force of the flow force application unit is applied; A lower chamber having a force application unit to which a second force of the flow force application unit is applied, wherein the second force of the flow force application unit is applied, and the lower chamber receives separated particles of the liquid sample separated through the porous membrane; and A connecting unit that connects the upper chamber and the lower chamber in a communication manner and has a porous membrane in the communication path; characterized by including: A particle separation-concentration device based on a porous membrane.

9. In paragraph 8, The upper chamber and the lower chamber include a body portion having a one-sided open barrel shape, and a connecting portion formed at the open end of the body portion, The force application portion of the upper chamber is formed by a protruding connecting portion formed at the bottom of the body portion, The force application part of the lower chamber is characterized by being formed by a protruding connection hole formed on one side of the connection part. A particle separation-concentration device based on a porous membrane.

10. In paragraph 9, The above connecting unit A connecting bracket module coupled between the above connecting parts; and It is characterized by including a porous membrane mounted within the above connecting bracket module. A particle separation-concentration device based on a porous membrane.

11. In paragraph 10, The above connecting bracket module includes a cylindrical portion into which the connecting portion is inserted and coupled, a coupling flange portion extending outward from one end of the cylindrical portion, and a fixing piece that couples and fixes the coupling flange portion on the upper chamber side and the coupling flange portion on the lower chamber side. The above porous membrane is characterized in that it is detachably provided on the inside of the connecting bracket module. A particle separation-concentration device based on a porous membrane.

12. In paragraph 8 or 9, The above flow force application device part It is characterized in that it is composed of a pneumatic generating device that applies pneumatic pressure as the above hole blockage release means. A particle separation-concentration device based on a porous membrane.

13. In paragraph 8 or 9, The above flow force application device part An air supply device configured to supply air; An air supply line connected to supply air generated from the air supply device to the force application portions of each of the upper and lower chambers; and A check valve provided in each of the air supply line connected to the upper chamber side and the air supply line connected to the lower chamber side, and the opening and closing of which is controlled by the control device unit; characterized in that it includes; A particle separation-concentration device based on a porous membrane.

14. In paragraph 13, The above control device unit It is characterized in that the check valve is configured to control the forward flow in a state where the upper chamber is filled with a liquid sample or a space is formed on the upper side of the upper chamber, and to control the reverse flow in a state where there is a space on the upper side of the lower chamber. A particle separation-concentration device based on a porous membrane.

15. In paragraph 7, The particle separation-concentration device section includes an upper chamber into which a liquid sample is introduced, a lower chamber for receiving separated particles of the liquid sample separated through a porous membrane, and a connecting unit that connects the upper chamber and the lower chamber in a communication manner and has a porous membrane in the communication path. One of the upper chamber and the lower chamber is provided with a force application portion, and the other is formed to have an exhaust hole or to be formed flexibly, The above-mentioned flow force application device unit is characterized in that it is configured as a positive pressure-negative pressure application device unit configured to apply positive pressure and negative pressure to the above-mentioned force application unit. A particle separation-concentration device based on a porous membrane.

16. In paragraph 15, It is characterized by further including an air injection device configured to inject air into the exhaust hole. A particle separation-concentration device based on a porous membrane.

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