Selective concentration filter for sample, method for manufacturing the concentration filter, and sample concentration device including the same

Abstract

The present invention discloses a concentration filter for selectively concentrating a sample, a method for manufacturing the concentration filter, and a sample concentration device. The selective concentration filter of the present invention includes a base having pores and a selective permeable membrane coated on the base and containing vesicles, which concentrates a target substance to be analyzed from the sample and allows at least a part of the remaining substances other than the target substance to permeate through the pore structure. According to the present invention, the target substance to be analyzed can be easily concentrated with a simplified structure.

Description

Technical Field

[0001] The technical field to which the present invention pertains relates to a selective concentration device, a selective concentration filter, and a method for manufacturing a selective concentration filter. The present invention is related to research supported by the National Research Foundation of Korea (NRF)'s Bio and Medical Technology Development Program (No. 2021M3E5E3080743) under the support of the Korean government (Ministry of Science and ICT).

Background Art

[0002] The content described in this section is merely for providing background information of the present embodiment and does not constitute prior art.

[0003] Modern medicine aims not only to extend lifespan but also to achieve an extended healthy lifespan by living a long and healthy life. Therefore, the future medical paradigm is changing from a treatment medicine-centered approach to the realization of the three Ps of preventive medicine, predictive medicine, and personalized medicine. To specifically achieve this, early detection and early treatment of diseases have become very important means, and research on biomarkers is being actively conducted as a means for this.

[0004] A biomarker refers to a marker that can distinguish a normal state from a diseased state, predict a treatment response, and be objectively measured. Nucleic acids (DNA, RNA), proteins, lipids, metabolites, and changes in their patterns are used as biomarkers. That is, from simple substances such as blood glucose levels for diabetes diagnosis to genes such as the BCR-ABL gene fusion of chronic myeloid leukemia, which is the treatment target of Gleevec, all fall within the category of biomarkers and are biomarkers actually used in clinical practice.

[0005] By analyzing nucleic acids and proteins, it is possible to identify the onset and progression of a disease. Techniques and elements for protein analysis are difficult to manufacture because they use nanotechnology, are relatively expensive, and have the problem of being difficult to spread. In addition, protein analyzers require highly sensitive sensors, and there are also drawbacks such as difficulty in accurate analysis with a small amount of sample. As a typical method for detecting nucleic acids and proteins, there is a lateral flow analysis method using chromatography. Such lateral flow analysis methods are used in various fields such as pregnancy diagnosis.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0007] An embodiment of the present invention has a problem that a filter that simply uses pore size also concentrates other unnecessary biomolecules of the same size. To solve such a problem, a main object is to provide a concentration filter that concentrates a target substance while selectively removing a specific substance by an aquaporin, a phospholipid layer, and a substance transport protein of a cell membrane component fused to a porous membrane.

[0008] Other unspecified objects of the present invention can be further considered within a range that can be easily inferred from the following detailed description of the invention and its effects.

Means for Solving the Problems

[0009] The selective concentration filter for a sample for achieving the object of the present invention described above includes a base having pores and a selective permeable membrane coated on the base and containing vesicles, which concentrates a target substance to be analyzed from a sample and allows at least a part of the remaining substances other than the target substance to permeate through the pore structure.

[0010] In the present invention, the surface of the selective permeable membrane may be hydrophilic and have a negative charge. In the present invention, the pores of the base may be selected so as to be able to concentrate the target substance in consideration of at least one attribute selected from the group consisting of the size of the target substance, the charge of the target substance, and the affinity with a solvent. The pore diameter of the base is preferably 10 nm to 150 m.

[0011] In the present invention, the remaining substances of the sample include a fixed separation substance and a dynamic separation substance, and the permeability of the dynamic separation substance may depend on the interval of phospholipids of the cell membrane component in a first pressure interval or a second pressure interval delimited according to a reference pressure applied to the selective permeable membrane.

[0012] Preferably, the remaining substances of the sample further include a buffer solution, and the permeability of the dynamic separation substance may depend on the hydrogen ion concentration of the buffer solution in a first hydrogen ion concentration interval or a second hydrogen ion concentration interval delimited according to the reference hydrogen ion concentration.

[0013] To achieve still other objects of the present invention, a sample concentration device includes a selective concentration unit including a selective concentration filter and a concentration filter holder that wraps the selective concentration filter, and a coupling unit that couples with the selective concentration filter as needed, and includes a main body in which a channel for supplying a sample to be concentrated to the selective concentration filter module is formed. Here, the selective concentration filter includes a base having pores and a selective permeation membrane coated on the base and including vesicles, which does not allow a target substance to be analyzed from the sample to permeate, and allows at least a part of the remaining substances other than the target substance to permeate through the pores.

[0014] In the present invention, the concentration filter holder may further include a first passage and a second passage formed in spaces separated from each other for introducing or discharging a sample. The first passage may be located on the selective permeation membrane side of the selective concentration filter, and the second passage may be located on the base side.

[0015] In the present invention, the concentration filter holder may further include a first holder having a first passage and a second holder having a second passage, and the selective concentration filter may be located between the first holder and the second holder.

[0016] Further, a fixing unit that is located between the first holder and the second holder and makes the selective concentration filter watertight, and a support unit that supports the selective concentration filter may be further included. The first passage is for injecting the sample, and the second passage is for discharging at least a part of the remaining substances that have permeated through the pores to the outside.

[0017] Preferably, the sample concentration device may further include a first pressure providing unit that applies pressure to the sample to inject the sample from the first passage and provides pressure for discharging at least a part of the remaining substances from the second passage to the outside, and the main body may further include a first channel in which the first pressure supply unit is movable.

[0018] Preferably, the sample concentrator may further include a second pressure supply unit that discharges the concentrated target substance from the first passage to the outside by applying pressure to the selective concentration filter through a second passage. The main body may further include a second channel through which the second pressure supply unit is movable.

[0019] Preferably, the main body may further include a coupling part that surrounds and supports the first pressure supply unit and the second pressure supply unit and can be selectively coupled to the first passage or the second passage.

[0020] Preferably, the surface of the selective permeation membrane is hydrophilic and has a negative charge, and the pores may be selected to concentrate the target substance in consideration of at least one attribute selected from the group consisting of the size of the target substance, the charge of the target substance, and the affinity with the solvent.

[0021] A method for manufacturing a selective concentration filter for achieving still another object of the present invention includes the steps of preparing a cell membrane mixture containing a cell membrane containing vesicles and a buffer solution, applying the cell membrane mixture to a base having pores, and discharging at least a part of the buffer solution to the outside through the pore structure to fix the cell membrane to the base.

[0022] The cell membrane vesicles are obtained by sonication of erythrocyte membranes, and the cell membrane vesicles may be 1 to 4% (v / v) in the cell membrane mixture. Further, after the coating step, a heat treatment step at 40 to 60 ° C may be further provided.

[0023] A sample concentration method using a sample selective concentration device including a selective concentration filter for achieving still another object of the present invention and a concentration filter holder for wrapping the selective concentration filter includes injecting the sample from a first passage and discharging at least a part of the remaining substances that have passed through the pore structure from the second passage to the outside, and applying pressure to the selective concentration filter through the second passage to discharge at least a part of the target substance concentrated on the selective permeation membrane from the first passage.

Advantages of the Invention

[0024] As described above, according to the embodiment of the present invention, there is an effect that water can be discharged through a selective permeation structure in which a porous membrane and a cell membrane are laminated, and the target substance can be concentrated while selectively removing specific substances.

[0025] According to the embodiment of the present invention, by adjusting the pressure applied to the movement path of the selective permeation structure in which the porous membrane and the cell membrane are laminated and / or the pH condition of the sample buffer, there is an effect that the permeability of substances in the concentration filter can be dynamically controlled.

[0026] Even when the effects are not explicitly described here, the effects described in the following specification expected from the technical features of the present invention and their provisional effects are treated in the same manner as those described in the specification of the present invention.

Brief Description of the Drawings

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Embodiments for Carrying Out the Invention

[0028] Hereinafter, in explaining the present invention, when it is determined that related known functions are obvious to those skilled in the art and may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted, and some embodiments of the present invention will be described in detail with reference to the exemplary drawings.

[0029] Conventional filters adjust the pore size and concentrate only by size, so there is a problem that unnecessary biomolecules are also concentrated together if the sizes are similar.

[0030] To solve such problems, in this embodiment, by coating a red blood cell membrane on a hydrophilic porous membrane, only water in the red blood cell membrane can be selectively removed and concentrated by aquaporin that selectively permeates only water. Biomolecules can be selectively separated and concentrated through aquaporin, phospholipid layer, and various transport proteins in the red blood cell membrane, and the concentration characteristics by the cell membrane can be adjusted using pressure changes or charge changes (or pH concentration). A method of adjusting the charge or pH concentration under certain pressure conditions, or a method of adjusting the pressure at a certain charge or pH concentration can be applied. It is also possible to adjust the charge or pH concentration while adjusting the pressure conditions.

[0031] FIG. 1 is an exemplary diagram of a method for manufacturing a selective concentration filter according to an embodiment of the present invention.

[0032] Step S10 is a step of preparing a cell membrane. The cell membrane is, for example, a red blood cell membrane extracted from human or animal blood. Preferably, the plasma and buffy coat are separated and removed from the red blood cell membrane, and it is preferably processed so as to contain the red blood cell membrane component from which the supernatant has been removed. The cell membrane is a membrane surrounding the cytoplasm and has a structure in which protein molecules are inserted or attached in the phospholipid bilayer. The cell membrane functions as a selective permeable membrane.

[0033] Step S20 is a step of diluting the cell membrane processed as described above with purified water to prepare a cell membrane mixture. Preferably, the cell membrane mixture contains the cell membrane obtained in step S10 and a buffer solution. The buffer solution may be purified water, phosphate buffered saline, or the like.

[0034] Step S30 is a step of applying ultrasonic waves to the cell membrane mixture. When ultrasonic waves are applied to the cell membrane mixture, cell membrane vesicles are further formed.

[0035] Step S40 is a step of applying based on the cell membrane mixture containing the cell membrane in which vesicles are formed in step S30. Here, the base preferably has a pore structure and is sheet-shaped. For example, the base may be an AAO membrane (6809-6002, Whatman, UK), but is not limited thereto. The base may apply PCTE or a PES membrane. As a method of applying the cell membrane mixture to the base, the cell membrane mixture may be sufficiently applied to at least one surface of the base by a method such as dropping the cell membrane mixture onto at least one surface of the base. Also, for the uniformity of the coating, Meniscus-coating, Spin-coating, etc. may be applied.

[0036] Step S50 is a step of fixing the cell membrane component to at least one surface of the base by subjecting the base in a state where the cell membrane mixture is applied to heat treatment. In this step, it is preferable to induce cell fusion by additional heat treatment. The coating in this step is a process in which the erythrocyte membrane existing in a vesicular form is fixed in the form of a plurality of bilayers on the base surface. In this embodiment, the cell membrane is a concept of concentrating through aquaporin, which is a water transport protein, and a transport protein that transports specific proteins, and the change in the distance between phospholipids due to pressure. As the cell membrane, not only the human cell membrane but also the cell membranes of animals, plants, bacteria, etc. are applicable. An artificial cell membrane may be applied as the cell membrane. An artificial cell membrane is not a naturally occurring cell membrane but a cell membrane formed artificially with components.

[0037] The cell membrane may be sheet-shaped or have an unspecified shape. Since the shape of the cell membrane itself is unspecified, a vesicular shape is advantageous when coating the cell membrane on a porous membrane. A cell membrane vesicle is a concept encompassing the cell membrane and means that the shape has been deformed into a small size. The cell membrane can be deformed into a vesicular shape by ultrasonic treatment. Instead of ultrasonic treatment, extrusion may be applied, but ultrasonic treatment is the simplest method.

[0038] In the process of coating the porous membrane with cell membrane vesicles, to a certain extent, the fusion of cell membrane components can occur even at room temperature, but the optimal conditions are about 50°C for 50 minutes, and heat treatment may be performed at a constant temperature (for example, 30 - 70°C) for a certain period of time (for example, 30 minutes - 2 hours) according to the environment. After heat treatment, the cell membrane vesicles fuse to form a layer.

[0039] Figure 2 is an exemplary diagram of a method for manufacturing a selective concentration filter according to still another embodiment of the present invention. This embodiment is an example in which an erythrocyte membrane is applied as the selective permeation membrane and anodic aluminum oxide (AAO) is applied as the hydrophilic porous membrane.

[0040] The method for manufacturing the selective concentration filter may perform a step of preparing an erythrocyte membrane (step S11). The method for manufacturing the selective concentration filter may perform a step of diluting the erythrocyte membrane with purified water to form a cell membrane solution (step S21). The method for manufacturing the selective concentration filter may perform a step of extracting erythrocyte membrane vesicles (step S31) by subjecting the cell membrane solution to ultrasonic treatment for a certain period of time (for example, 10 minutes, etc.). Step S41 of forming a selective permeation structure in which a selective permeation membrane based on cell membrane components and a porous membrane are laminated may be performed by pouring erythrocyte membrane vesicles onto the AAO membrane and performing heat treatment at a constant temperature (for example, 30 - 70°C) for a certain period of time (for example, 30 minutes - 2 hours).

[0041] The AAO membrane is a two-dimensional porous body having regularly sized nanopores and is widely used as a membrane, a template, a filter, etc. As the porous membrane, other membranes are also possible as long as they have pores, and a hydrophilic membrane is useful for discharging water.

[0042] In the step of diluting the cell membrane vesicles in FIGS. 1 and 2 with purified water to form a cell membrane mixture, the cell membrane components in the cell membrane mixture are preferably 1 to 4% (v / v). The cell membrane components can be adjusted within an error range based on 2%. The error range can be set to 1.9 to 2.1%, 1.8 to 2.2%, 1.5 to 2.5%, etc. considering experiments, products, and the environment.

[0043] Pouring the cell membrane solution onto the porous membrane and performing heat treatment can be carried out in a temperature range of 30 to 70°C. The temperature range and time range may be set differently considering experiments, products, and the environment.

[0044] FIGS. 3 and 4 are exemplary diagrams of the step of preparing the cell membrane in the method for manufacturing a selective concentration filter according to an embodiment of the present invention.

[0045] Cells (such as liver cells, intestinal cells, nerve cells) are grown by cell culture to obtain a sufficient amount, and then the cells are disrupted by a homogenizer or a lysis process.

[0046] In the case of cells present in blood (red blood cells, platelets, white blood cells), the blood is separated into three layers, such as plasma, white blood cells and platelets, and red blood cells, by centrifugation to separate the layer with the desired cell membrane, and then the cells are disrupted by a homogenizer or a lysis process.

[0047] Thereafter, by centrifugation, substances other than the cell membrane (substances inside the cell, for example, hemoglobin in the case of red blood cells) are separated, and a part with the cell membrane is separated.

[0048] Referring to FIG. 3, assuming the situation of extracting from whole blood, the extraction process of the cell membrane (red blood cell membrane) includes rotating the centrifuge once first to remove white blood cells, platelets, plasma, etc., disrupting the cells by a homogenizer or a lysis process, and then rotating the centrifuge again, and it is preferable to perform a total of 2 cycles.

[0049] Referring to FIG. 4, assuming the situation of obtaining other somatic cells by culturing, without using a centrifuge, the cells are immediately disrupted by a homogenizer or the like, and the centrifuge is rotated. It is also possible to perform a total of one cycle.

[0050] When the cell membrane is prepared, the cell membrane in the form of a sheet or an unspecified shape is made into vesicles by ultrasonic waves to facilitate lamination.

[0051] Since the obtained cell membrane (cell membrane component) is in a high-concentration state, it cannot be directly coated on the porous membrane, and it is necessary to adjust the concentration operable with a selective filter.

[0052] According to still another embodiment of the present invention, red blood cell membranes can be produced according to the following method. In this embodiment, the cell membrane produced by the following method is called BEETLES 2 membrane.

[0053] First, to prevent coagulation, RBCMs (Red Blood Cell Membranes) are extracted from anticoagulated human whole blood stored in K2EDTA, and a centrifuge is applied under the conditions of 800 g for 5 minutes to remove plasma and buffy coat and separate red blood cells. The supernatant is removed, and then ice-cold 1X PBS is added to the remaining red blood cells and mixed appropriately, and the washing process under the same conditions is performed 3 times. Next, the red blood cells were lysed by suspending them in cold 0.25× for 30 minutes. Then, the pre-hemoglobin was removed with a centrifuge at 20,000 g for 30 minutes. Such a washing process was repeated 3 times, and then, after obtaining a bright pink RBCMs pellet, it was stored at -80°C.

[0054] The extracted RBCM obtained in this way was in the form of negatively charged ribosomes with a size of 200 nm. The sonicated RBCM was dropped onto an AAO membrane (6809 - 6002, Whatman, UK) and incubated at 50 °C for 50 minutes to form multiple supported lipid bilayers. The microstructure of RBCMs - AAO was confirmed using SEM, AFM, and a fluorescence microscope.

[0055] Figures 5 to 9 are exemplary views of the surface according to the concentration of a selective concentration filter generated by the method for manufacturing a selective concentration filter according to an embodiment of the present invention.

[0056] Figure 5 shows an SEM (Scanning Electron Microscope) image of the surface according to the concentration. Figures 6 and 7 show fluorescence analysis images of the surface according to the concentration.

[0057] By setting the conditions of fluorescence intensity, Gain 600, and pinhole 0.2%, the cell membrane coating using fluorescence can be verified. By tagging the cell membrane with fluorescence, it can be confirmed whether it is well-coated for each concentration after coating on the AAO membrane.

[0058] Defects are observed on the surface at a concentration of 0.1%, a large number of overall vesicles are observed on the surface at a concentration of 0.25%, and it can be seen that the surface is overall coated at a concentration of 0.5%.

[0059] At concentrations of 1% and 2% of the cell membrane (for example, red blood cell membrane), which are the currently used concentrations, almost no defects are seen. On the surface at a concentration of 2%, the red blood cell membrane shows a form that spreads entirely over the gold. It can be inferred that it corresponds to dozens to hundreds of layers.

[0060] Since the cell membrane has fluidity, it is possible to confirm whether the cell membrane is well-coated by a FRAP experiment. The principle is that photobleaching is induced by irradiating a very high-energy laser beam on a predetermined part, and then the degree of coating of the cell membrane can be confirmed by observing the process of subsequent recovery.

[0061] Figures 7(a), (b), and (c) show the results of FRAP (Fluorescence Recovery After Photobleaching) for cell membranes with a concentration of 0.1%, and Figures 7(d), (e), and (f) show the results of FRAP (Fluorescence Recovery After Photobleaching) for cell membranes with a concentration of 2%.

[0062] Figures 7(a) and (d) show the state before photobleaching, Figures 7(b) and (e) show the state immediately after photobleaching, and Figures 7(c) and (f) show the state after 100 seconds. When a permeable structure formed by a 0.1% concentration cell membrane was subjected to a FRAP experiment, it was confirmed that the recovery of the photobleached area was slow. This means that the fluorescence did not spread overall. When a permeable structure formed by a 2% concentration cell membrane was subjected to a FRAP experiment, it was confirmed that the recovery of the photobleached area was fast within 100 seconds. This means that the self-recovery ability is very excellent.

[0063] Figures 8 and 9 show the AFM (Atomic Force Microscope) images and cross-sections of the surface according to the concentration. In the basic (Bare) AAO membrane (0%), AFM is measured with fluctuations caused by the pores of the AAO membrane, but as the cell membrane is gradually coated, the pores are covered, the fluctuations decrease, and the coating of the cell membrane can be confirmed even in the morphological part. From a concentration of 2% or more, a very similar pattern is shown, and it can be confirmed that a concentration of 2% is the most optimal.

[0064] Figures 10 and 11 are exemplary diagrams of the surface potential according to the concentration of a selective concentration filter generated by a method for manufacturing a selective concentration filter according to an embodiment of the present invention, showing a Kelvin probe force microscope (KPLM) image and the surface potential with respect to the surface according to the concentration. Figure 12 shows the surface potential according to the concentration. It can be confirmed that the surface potential of the AAO membrane becomes lower as the negatively charged erythrocyte membrane is coated. The surface potential of the porous membrane changes due to the concentration difference of the charged cell membrane.

[0065] When coating at a concentration of 2% or more, it can be confirmed that the tendency of the surface charge conversely becomes higher. At the reference concentration of the charged cell membrane, the change in the surface potential of the porous membrane has an inflection point. As the cell membrane is thickly coated, it has dielectric properties, and it is difficult to measure due to the principle of KPFM that measures surface charges through current, so there is a possibility of increase. It is the most optimized and well-concentrated value at a concentration of 2%. If it is lower than 2%, the concentration does not meet the reference value, and if it is higher than 2%, the water permeability decreases and the concentration time increases. The concentration range can be adjusted within an error range based on 2%. The error range can be set to 1.9 - 2.1%, 1.8 - 2.2%, 1.5 - 2.5%, etc. considering experiments, products, and the environment.

[0066] Figure 13 is an exemplary diagram of the concentration rate according to the concentration of a selective concentration filter generated by a method for manufacturing a selective concentration filter according to an embodiment of the present invention. The sample volume was 3 mL, the pressure was 3 bar, and the sample buffer regarding IgG was experimented with 1×PBS. It can be confirmed that as the concentration of the cell membrane formed on the porous membrane increases, the concentration rate sharply increases at a 2% concentration.

[0067] Figure 14 shows an image of an erythrocyte membrane formed with cell membrane vesicles according to an embodiment of the present invention. (a) and (b) in Figure 14 show cell membrane vesicles at different magnifications, and (c) shows the zeta potential.

[0068] Figures 15 to 19 are exemplary views of a sample concentration device according to another embodiment of the present invention.

[0069] The sample concentration device 10 includes a selective concentration filter 100, a concentration filter holder 200, and a sample supply unit 300. The selective concentration filter 100 is formed from a selective permeation structure in which a selective permeation membrane and a porous membrane are laminated. The concentration filter holder 200 has a structure that wraps around the selective concentration filter and forms an inlet and an outlet. The sample supply unit 300 is coupled to the inlet of the concentration filter holder and has a space for accommodating a sample. The sample supply unit 300 may be formed in a syringe shape, and may have other shapes as long as it can apply pressure. If it is in a syringe shape, it can operate sufficiently even with the force of a finger.

[0070] The sample concentration device 10 may further include a pressure supply unit 400 that presses the sample accommodated in the sample supply unit by itself or provides pressure to the sample by supplying a gas.

[0071] The pressure supply unit 400 may include a piston 410 for pressing the sample by itself and a power supply unit 420 for supplying power to the piston. It may also include a pressure measurement unit for measuring the applied pressure.

[0072] The pressure supply unit 400 may include a gas pipe 430 for supplying a gas and a gas pump 440 connected to the gas pipe. It may also include a gas measurement unit for measuring the supplied gas.

[0073] The pressure supply unit 400 may apply the operating principle of a Stirred cell to apply a constant pressure. When pressure is applied with nitrogen gas, only the buffer escapes, and only the concentrated sample remains on the selective permeation structure.

[0074] The sample concentration device 10 may further include a pH adjustment unit 500 that is coupled to the control unit and adjusts the hydrogen ion concentration (pH) by adding ions to the buffer solution contained in the sample accommodated in the sample supply unit.

[0075] The sample concentration device 10 may further include a control unit 600 that is coupled to the pressure providing unit and controls the range of the pressure provided by the pressure providing unit.

[0076] The selective concentration filter 100 forms a movement path through which some components of the sample pass through the selective permeation membrane 120 and the porous membrane 110. Due to pressure, some components of the sample move from the sample supply unit to the inlet, then move from the inlet to the selective concentration filter, and then move from the selective concentration filter to the outlet.

[0077] The concentration filter holder 200 includes a first holder 210 at the inlet and a second holder 220 at the outlet. The concentration filter holder may form a structure that can be mutually fastened via the first holder 210 and the second holder 220, or may include a separate holder fastening part 215.

[0078] The concentration filter holder 200 may arrange the selective concentration filter 100 between the first holder 210 and the second holder 220.

[0079] The concentration filter holder may include a fixing part 230 that makes the selective concentration filter watertight between the first holder and the second holder, and a supporting part 240 that supports the selective concentration filter against pressure. The fixing part 230 may be realized by a rubber ring or the like, and the supporting part 240 may be formed in a mesh structure.

[0080] The sample includes a target substance and a fixed separation substance. The sample may include a target substance, a fixed separation substance, and a dynamic separation substance. For example, the fixed separation substance may be water, and the dynamic separation substance may be a specific protein.

[0081] The control unit 600 may store an attribute-concentration data table that records (i) a first attribute regarding the size of the target substance, (ii) a second attribute regarding the positive or negative charge of the target substance, and (iii) a third attribute regarding the hydrophobicity or hydrophilicity of the target substance.

[0082] Based on the attribute-concentration data table, the control unit 600 may send an instruction to control the pressure providing unit to adjust the presence or absence of the concentration of the target substance using the first attribute, the second attribute, the third attribute, or a combination thereof.

[0083] The control unit 600 may store a pressure-permeation data table that records a first pressure interval or a second pressure interval delimited according to a reference pressure.

[0084] Based on the pressure-permeation data table, the control unit 600 may send an instruction to the pressure providing unit to control the pressure supply unit to adjust the permeability of the dynamic separation substance in the first pressure interval or the second pressure interval.

[0085] The selective concentration filter of the sample concentration device concentrates the target substance by removing the fixed separation substance in the first pressure interval. The selective concentration filter may concentrate the target substance by removing the fixed separation substance in the second pressure interval and simultaneously remove the dynamic separation substance.

[0086] The sample may contain a buffer solution, a target substance, a fixed separation substance, and a dynamic separation substance.

[0087] The control unit 600 may store a pH-permeation data table that records a first hydrogen ion concentration interval or a second hydrogen ion concentration interval delimited according to a reference hydrogen ion concentration.

[0088] Based on the pH-permeation data table, the control unit 600 may send an instruction to the pH adjustment unit to control the pH adjustment unit to adjust the permeability of the dynamic separation substance in the first hydrogen ion concentration interval or the second hydrogen ion concentration interval.

[0089] Figures 20 and 21 are exemplary diagrams of a selective concentration filter according to still other embodiments of the present invention.

[0090] The selective concentration filter 100 includes a porous membrane 110 and a selective permeation membrane 120. The selective permeation membrane 120 is formed on the porous membrane and may be formed based on cell membrane components. The selective concentration filter 100 may be manufactured using a cell membrane solution and can be optimized by adjusting the concentration of the cell membrane.

[0091] The selective concentration filter 100 forms a selective permeation structure in which the selective permeation membrane and the porous membrane are laminated.

[0092] The selective concentration filter 100 concentrates some components of the sample. The selective permeation membrane does not allow the target substance contained in the sample to pass through, and the selective concentration filter may form a movement path through which the fixed separation substance contained in the sample passes through the selective permeation membrane and the porous membrane. For example, by selectively removing only water from a solution containing a virus, the target biomolecule can be concentrated.

[0093] Even with a small force, pressure (e.g., 3 bar) can be applied through the highly permeable porous membrane and the cell membrane which is a selective permeation membrane, and a concentration result can be obtained within a few minutes (e.g., 5 minutes). By applying the concentrated sample to a commercially available LFA (Lateral Flow Immunoassay) kit, the performance (LOD (Limit Of Detection), sensitivity, etc.) may be improved.

[0094] Under the environment where the sample volume is 3 mL, the operation time is 2 minutes, and the pressure difference is 3 bar, the permeability of the basic (Bare) AAO membrane is 2050 LMH / bar, and the permeability of the AAO membrane coated with red blood cell membrane is 332.41 LMH / bar. If it is 300 or more in LMH, it corresponds to a very high level of flow rate.

[0095] Figures 22 and 23 are exemplary views of the surface of a selective concentration filter according to still other embodiments of the present invention.

[0096] Figure 22 shows a structure in which a 2% concentration of cell membrane coating is completed on a 25 mm size AAO membrane. When the cell membrane coating is completed, it becomes slightly reddish.

[0097] Figure 23(a) is an SEM image of a basic porous membrane, and Figures 23(b) and (c) are SEM images in which a 2% concentration of cell membrane coating is completed on the porous membrane. It can be confirmed that the surface is uniformly coated. The SEM image is an image of a cell membrane coated on an AAO membrane with a pore diameter of about 20 nm. It can be confirmed that the cell membrane by the method of the present invention is well coated on the AAO membrane. Therefore, it can be applied to various base layers other than AAO. The diameter of the vesicles formed in the selective permeation membrane is in the range of 100 to 300 nm, and the pore diameter of the base is preferably smaller than the diameter of the vesicles. Considering that the diameter of the vesicular red blood cell membrane in this embodiment is about 200 nm, the pore diameter of the membrane is preferably smaller than the diameter of the red blood cell membrane or 10 to 150 nm. If the pore diameter is too small, it takes a considerably long time to concentrate the target substance, and if the pore diameter is too large, there is a risk that the red blood cell membrane will be discharged from the pores even at low pressure.

[0098] Figures 24 to 28 are exemplary views of the concentration rate of a selective concentration filter according to still other embodiments of the present invention.

[0099] Referring to Figures 24 and 25, the sample volume was 3 mL, the concentration time was 3 to 6 minutes, the pressure was 3 bar, and the nucleocapsid protein was concentrated. In Figure 24, (a) shows the Ag test result of COVID-19, (b) shows the IgG rapid test result of COVID-19, and (c) is the Ag test result of COVID-19 in artificial saliva. VTM (Virus transport medium); SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BEETLES2 It is a biotechnology concentration tool for LFA of the present invention with high sensitivity and selectivity. Since IgG is also concentrated, it can be seen that the performance of the COVID-19 antibody kit is also improved. In addition, it can be seen that a sample obtained by adding a nucleocapsid protein to artificial saliva can be concentrated and the LFA performance is improved.

[0100] When comparing the kit results detected at the concentrated concentration in Fig. 25(b) with the kit results detected in the non-concentrated state in Fig. 25(c), it is confirmed that a concentration ratio of 20 times or more can be obtained by selective concentration using the cell membrane.

[0101] Referring to Figs. 26 and 27, it is confirmed that a patient sample (Nasal) with a Ct value of 24 is diluted 100-fold (Ct~30.6) and concentrated, and a concentration ratio of at least 20 times or more is obtained, and samples of patients with a Ct value of 30 or more can also be detected with an LFA kit.

[0102] Referring to Fig. 28, it is confirmed that the COVID-19 nucleocapsid protein can be concentrated by the porous membrane laminated structure coated with the cell membrane, and it can be concentrated 20 times or more by cross-verification with a nanodrop and SDS-PAGE. Fig. 29 is an exemplary diagram of the contact angle of a selective concentration filter according to still another embodiment of the present invention. It is confirmed that the porous membrane maintains hydrophilic properties even after being coated with the cell membrane.

[0103] Figs. 30 to 32 are exemplary diagrams of a concentration rate adjustable using sample attributes of a selective concentration filter according to still another embodiment of the present invention. The concentration characteristics change depending on the size, charge, and hydrophobicity of the target molecule.

[0104] The selective concentration filter can adjust the presence or absence of concentration of the target substance by using a first attribute (size attribute) related to the size of the target substance that is not permeated by the selective permeation membrane and is concentrated.

[0105] The selective concentration filter can adjust the presence or absence of the concentration of the target substance by using a second attribute (charge attribute) related to the positive or negative charge of the target substance that is not permeated by the selective permeable membrane and is concentrated. The selective concentration filter can adjust the presence or absence of the concentration of the target substance by using a third attribute (hydrophobicity / hydrophilicity attribute) related to the hydrophobicity or hydrophilicity of the target substance that is not permeated by the selective permeable membrane and is concentrated.

[0106] If the molecular weight is 100 kDa or more, it cannot pass through the cell membrane and is concentrated. Referring to FIG. 29, if it is above the reference size according to the size characteristics of the target molecule, it can be concentrated. A small sample of about several tens of kDa has a positive charge, and a sample having hydrophobicity is concentrated. Depending on the size characteristics of the target molecule, even if it is below the reference size, it can be concentrated by charge characteristics and / or hydrophobicity / hydrophilicity characteristics. Referring to FIG. 32, influenza, which is a nucleocapsid protein, can also be concentrated.

[0107] Since the cell surface having hydrophilicity forms a hydration layer, it prevents the adsorption of proteins. The negatively charged cell membrane and the positively charged protein are electrically attracted, resulting in weak adsorption. When the surface is relatively hydrophobic, weak adsorption occurs. Therefore, a protein having a positive charge and hydrophobicity cannot pass through the cell membrane and is concentrated.

[0108] FIGS. 33 to 38 are exemplary diagrams of the concentration rate that can be adjusted by using the pressure characteristics applied to the selective concentration filter according to still other embodiments of the present invention.

[0109] The sample contains a target substance, a fixed separation substance, and a dynamic separation substance. The selective permeation membrane can adjust the degree of permeation of the dynamic separation substance by adjusting the interval between the phospholipids of the cell membrane in the first pressure interval or the second pressure interval delimited according to the reference pressure.

[0110] The selective concentration filter may concentrate the target material by removing the fixed separation substance in the first pressure interval. The selective concentration filter may concentrate the target material by removing the fixed separation substance in the second pressure interval and at the same time remove the dynamic separation substance. Although BSA cannot originally pass through the cell membrane, the selective concentration filter can control the permeation of BSA by applying pressure.

[0111] Figure 36 is a graph showing the concentration of IgG and NP with the applied pressure. As can be seen from Figures 35 and 36, it can be seen that IgG and NP, different from BSA, are not affected by pressure and both have good concentration.

[0112] Figure 37(a) shows that when the pressure is almost close to 0, almost all biomolecules other than water do not permeate. (b) shows that when the pressure increases (for example, above about 0.5 bar), the phospholipids open and proteins (small negatively charged proteins) can pass through the gaps between the phospholipids. Referring to Figure 38, the pressure corresponding to the second pressure interval (for example, 1 - 3 bar) further opens the gap between the cell membrane phospholipids and BSA permeates, but when a small pressure corresponding to the first pressure interval (for example, 0.1 - 0.5 bar) is applied, the gap (d1) does not open sufficiently and BSA does not permeate.

[0113] Although BSA theoretically cannot pass through the cell membrane, a gap can be formed between the phospholipids of the cell membrane by applying pressure, allowing it to pass through the cell membrane. Using such characteristics, albumin, which is unnecessary in the diagnosis of quality diseases, can be selectively removed.

[0114] Figures 39 to 41 are exemplary diagrams of the concentration ratio adjustable using the hydrogen ion concentration characteristics of the sample buffer of the selective concentration filter according to still another embodiment of the present invention.

[0115] The sample contains a buffer solution, a target substance, a fixed separation substance, and a dynamic separation substance. The selective permeation membrane may adjust the permeation degree of the dynamic separation substance by adjusting the hydrogen ion concentration of the buffer solution in the first hydrogen ion concentration interval or the second hydrogen ion concentration interval delimited according to the reference hydrogen ion concentration.

[0116] The selective concentration filter may concentrate the target material by removing the fixed separation substance in the first hydrogen ion concentration interval. The selective concentration filter may concentrate the target material by removing the fixed separation substance in the second hydrogen ion concentration interval and at the same time remove the dynamic separation substance.

[0117] Since BSA has a pI (isoelectric point) value of about 4.9, it has a negative charge in a physiological environment. Since the cell membrane can concentrate positively charged molecules, BSA can be concentrated by lowering the pH of the buffer to make it positively charged.

[0118] Referring to Figure 40, HCl is added little by little to PBS at a pressure of 3 bar to lower the pH. When BSA becomes positively charged at a low pH, it is confirmed that it cannot pass even under pressure and is concentrated.

[0119] Referring to Figure 41, it can be confirmed that Bovine Serum Albumin (BSA) protein has an isoelectric point (pl) of 4.9 and is concentrated by the positive charge due to its acidic nature.

[0120] The selective concentration filter according to this embodiment may adjust the cell membrane permeation characteristics according to pressure and charge, and may control the concentration and permeation conditions according to pressure and charge characteristics (pH concentration).

[0121] [BEETLES 2 [Example of Membrane Characterization Method] BEETLES according to an embodiment of the present invention 2 The method for characterizing the membrane will be specifically described. The AAO-RBCM membrane was sampled for KPFM imaging on a p-type silicon wafer (ePAK Internation, USA), which is a conductive substrate. The silicon wafer was immersed in a piranha solution (H2O2 and H2SO4) for 15 minutes, washed with distilled water, and dried with N2 gas (Segion Industrial Gas, Korea). The AAO-RBCM membrane was electrically and phase-analyzed in the amplitude modulation KPFM mode (Bruker, USA) using a MultiMode VIII atomic force microscope. The KPFM measurement was performed in air at 23° using a lift scan mode based on the tapping mode. The nanoelectrical properties of the sample were analyzed using a Pt-coated conductive atomic force microscope tip (SCM-PIT-V2; Bruker, USA). The topological AFM image was captured in the first scan using the tapping mode with zero tip bias.

[0122] To detect the surface potential, the AFM tip was lifted 20 nm from the sample surface with a sample bias voltage applied during the interleaved scan. During the interleaved scan, the mechanical drive to the cantilever was turned off, and an alternating (AC) bias voltage of 1000 mV applied at the mechanical resonance (ω) of the cantilever was applied to the probe. VAC vibrated the cantilever due to the attractive and repulsive electrostatic interaction (Fes) between the probe and the sample.

[0123] [Number]

[0124] Here, VCPD is the contact potential difference between the probe and the sample, and VDC is the direct current (DC) bias voltage.

[0125] The proportional-integral-derivative feedback loop monitored and controlled the amplitude of the cantilever vibration by applying a compensating VDC to the probe to cancel the probe-sample electrostatic force (Fes). This varies with the capacitance C and height z between the probe and the sample. In KPFM operation, the scan speed was 0.6 Hz, and the scan size and amplitude set value at 12 nm were 512×512 pixels and 4 μm×4 μm, respectively. MountainsSPIP software (version 9, Digital Surf, France) was used for leveling, processing, and analysis of the AFM images. Additionally, Gwyddion (version 2.60) was used to examine the surface roughness.

[0126] FRAP was used to verify the SLB formation of RBCM for the purpose of evaluating fluidity and diffusivity. In all experiments, 1 wt% PE-CF was inserted into RBCM as a fluorescent probe. A 20-μm-diameter location in the z-plane of the bilayer was bleached for 5 s with a 150-mW, 561-nm optical pumping semiconductor laser (Coherent, Inc.). The recovery of the bleached spot was monitored over 15 min using a Zeiss LSM-800 confocal microscope. Each image reference spot was used as a standard for measuring and normalizing the spot fluorescence intensity. Next, the diffusion coefficient (D) of the dye in each SLB configuration was calculated using D = w 2 / 4t 1 / 2 where w is the radius of the photo-bleached spot and t 1 / 2 is the time required to achieve half of the maximum recovery of the fluorescence intensity.

[0127] [Examples of Reagents and Analytical Methods] We used a commercially available COVID-19 Ag Rapid Kit (Calth Inc., Korea), BEETLES 2The performance improved using [it] was tested. The COVID-19 N protein recombinant antigen (45 kDa, FPZ0516, Fapon Biotech Inc., China), known as the best COVID-19 Ag test target, was used with 1xPBS buffer (LB004, DUKSAN, Republic of Korea) to test sensitivity and LOD. Bovine serum albumin (BSA) (A7030, Sigma-Aldrich, USA) and IgG (I4506, Sigma-Aldrich, USA) were purchased from Sigma-Aldrich.

[0128] Next, COVID-19 N protein samples of seven concentrations were prepared with 1×PBS. Approximately 3 mL of each sample was dropped into a polycarbonate stirring cell (341000, STERLITECH, USA), and the reaction was waited for 3 minutes. Next, the concentrated sample was pipetted with a 100 μL volume. The protein was quantified using a NanoDrop spectrophotometer (Thermo Scientific, USA) and SDS-PAGE (KOMA precast gel BC type, Koma Biotech, Korea).

[0129] We used LabVIEW software (National Instruments Co., USA) and a custom National Instruments (NI) controlled optical system to analyze the color intensity of a commercially available LFA kit and BEETLES 2 for the corresponding LFA.

[0130] To set the LOD value (Figure 24) and the cut-off value, first the color signal was measured using a commercially available reader and a customized NI controlled optical system with LabVIEW software. Next, five individually trained engineers (Calth Inc. http: / / www.thecalth.com) observed the colorimetric signal using a standard color chart and the manufacturer's instructions to determine the LOD. The cut-off value was set to minimize false detection to obtain higher specificity according to the manufacturer's guidelines.

[0131] [Analysis: Clinical Research] We obtained approval from the Institutional Review Board (KC21TIDI0134K) and collected clinical samples of nasopharynx / mid-pharynx (NP / OP) and saliva from COVID-19 patients at Seoul St. Mary's Hospital. The NP / OP swabs were contained in viral transport medium (VTM), while the saliva samples were collected in sterile tubes and diluted with phosphate-buffered saline. To test the performance against low viral targets, OP / NP samples were spiked into PBS buffer for high viral load (Ct < 25), and then prepared as low viral samples. For saliva samples, the samples were spiked into PBS buffer. Finally, low viral samples with Ct > 30 were prepared. For the healthy control group, samples were collected (or purchased) from healthy volunteers and stored in a -20°C freezer. The obtained results were compared with the RT-qPCR SARS-CoV-2 test results using the AccuPower SARS-CoV-2 Variants ID2 kit (BIONEER, Korea) according to the manufacturer's protocol. By maximizing the sum of sensitivity and specificity, the diagnostic cut-off value for in-field COVID-19 was optimized.

[0132] To implement a prototype for preparing POCT samples, a portable device integrated with a BEETLES 2 membrane was fabricated. We designed two reservoirs, a sample reservoir and a commercially available buffer reservoir. Micro-milling technology was applied to the prototype. We calculated the average hand pressure applied by five individuals (3 males and 2 females) at 5 ± 1 bar.

[0133] A portable syringe that can be pressurized by hand can be directly applied to commercially available LFAs as an inexpensive alternative for in-field diagnosis. First, the BEETLES 2 membrane was connected to the sample reservoir and the plunger was pushed for concentration. After 3 minutes of operation, the BEETLES 2The membrane was connected reversely to the running buffer reservoir. Next, it was analyzed using a commercially available LFA according to the manufacturer's instructions. A more simplified second prototype that operates with a pressure driving force by a vacuum chamber is also possible.

[0134] Figure 42 is a flowchart showing the concentration characteristics and permeation characteristics of cell membranes according to pressure, size, and charge. The inserted figure shows the cell membrane permeation characteristics for the COVID-19 virus and various proteins. First, when the pressure is less than 0.5 bar, buffer solutions such as water can pass through the pores. Even when the pressure increases, if the size of the protein is large, it cannot permeate even when the phospholipid opens, and as a result, it is concentrated on the base surface.

[0135] Of course, small substances can escape through the gaps in the phospholipids generated according to the applied pressure. At that time, positively charged proteins are electrostatically attached to the negatively charged phospholipid surface and cannot permeate, and as a result, they are concentrated on the cell membrane surface.

[0136] Figure 43 is a result diagram comparing the sensitivity between the pre-treatment system of BEETLES 2 according to an embodiment of the present invention and a conventional LFA. Clinical samples include COVID-19 patients (n = 42) and a healthy control group (n = 20). To observe the improvement in the case of low virus load, low virus patient samples (Ct ≧ 30, n = 20: 48%) are included. All samples of NP / OP, saliva, asymptomatic, and various variants are included. Regardless of the specimen (nasopharynx, oropharynx, saliva), mutation (Delta, Omicron), and asymptomatic patients, an improvement in the performance of the LFA kit (immunodiagnosis) by pre-treatment was confirmed. The sensitivity of the conventional LFA was 14.29%, but it can be seen that the performance is improved up to 88.1% by the pre-treatment system of BETTLES 2

[0137] ​FIG. 44 is a reference diagram showing the selectivity test results of the LFA. Referring to FIG. 44, it can be seen that when other viruses other than SARS-CoV-2 were pretreated, no positive reaction occurred in the LFA kit.

[0138] FIG. 45 is a result diagram of molecular diagnosis application (RT-PCR). It is a result of comparing two types: a patient sample stored in VTM with an AAO membrane (pore size 20 nm) and an AAO membrane coated with a cell membrane (BEETLES 2 ). Since the patient sample stored in VTM has a high probability of lysis, it is highly likely to pass through the pores of the AAO membrane, so it is not concentrated, and it can be confirmed that the Ct value hardly changes compared with the reference value. In the case of BEETLES 2 , since it can concentrate even the lysed virus, it can be seen that the Ct value can be reduced to about 5 compared with the reference value.

[0139] FIG. 46 is a configuration diagram showing a sample concentration device according to still another embodiment of the present invention.

[0140] The sample concentration device shown in FIG. 46 includes a first pressure providing unit 1120, a second pressure providing unit 1110, a main body 1200, and a selective concentration unit. FIGS. 47 and 46 are cross-sectional views of the sample concentration device.

[0141] Referring to FIGS. 46 and 47, the selective concentration unit includes a selective concentration filter 1400 and a concentration filter holder 1300. The selective concentration unit is provided so that it can be removed and attached to the main body 1200, and it goes without saying that it can be produced and sold separately from the sample concentration device.

[0142] The selective concentration filter 1400 includes a base 1420 and a selective permeation membrane 1410. The base 1420 has a pore structure and may be provided in a sheet form. The selective permeation membrane 1410 is coated on the base and vesicles are formed. The selective permeation membrane 1410 does not allow the target substance to be concentrated from the sample to permeate, and allows at least a part of the remaining substances other than the target substance to permeate through the pore structure. Here, the target substance may be a virus to be diagnosed, and the remaining substances may be a buffer solution such as water and electrolytes.

[0143] The concentration filter holder 1300 includes a first holder 1310 and a second holder 1320, and an internal space for wrapping the selective concentration filter 1400 is formed. The first holder 1310 includes a first passage 1312, and the second holder 1320 includes a second passage 1322. The first passage 1312 is located on the side of the selective permeation membrane 1410 of the selective concentration filter, and the second passage 1322 is located on the side of the base 1420 of the selective concentration filter. The first passage 1312 is a passage through which the sample is injected by the pressure from the first pressure providing unit 1120. Thereby, a part of the remaining substances in the injected sample permeates through the base 1420 and is discharged to the outside from the second passage 1322. The target substance of the sample that has not been discharged to the outside is concentrated on the selective permeation membrane 1410.

[0144] In this embodiment, the pressure providing unit 1100 includes a first pressure providing unit 1120 and a second pressure providing unit 1110. The main body 1200 supports the pressure providing unit and includes a path through which the pressure providing unit can move, that is, a channel formed inside. Here, the channel may include a first channel 1124 and a second channel 1114. The space of the first channel 1124 is a space for introducing a sample and a buffer solution. When the sample and the buffer solution introduced through the first pressure providing unit 1120 are pressurized, the sample and the buffer solution are transferred to the selective concentration filter 1400 according to the pressurization. FIG. 48 is a reference diagram for explaining the concept of concentrating a sample by a sample concentration device. Referring to FIGS. 47 and 48, a first coupling part 1222 and a second coupling part 1212 are formed at the end part of the main body 1200. The first coupling part 1222 is provided so as to be able to be coupled to and separated from a first passage 1322 of the concentration filter housing 1300.

[0145] The space of the second channel 1114 is a space into which the buffer solution can be selectively introduced, and provides a path for the second pressure providing unit 1110 to move. The selective concentration parts 1300, 1400 in which the sample is concentrated by the first pressure supply unit 1120 are separated from the main body 1200, and then are re-coupled to the main body again through the second coupling part 1212. In particular, it is preferable to turn the selective concentration part over and couple it to the second coupling part 1212. At that time, the second coupling part 1212 is coupled to the second passage 1322. According to the pressurization of the second pressure providing unit 1110, the buffer solution transmits the received pressure to the selective concentration filter 1400. That is, according to the additionally applied pressure, the target substance concentrated on the selective permeation membrane 1410 of the selective concentration filter 1400 is discharged to the outside from the first passage 1312. Here, the concentrated target substance may be dropped into the reservoir of the diagnostic kit. Further, the first pressure providing unit 1120 may further include a first packing 1122, and the second pressure providing unit 1110 may further include a second packing 1112. The packing may be applied to enhance the sealing force of the channel during pressure transmission.

[0146] Figure 49 is a reference diagram showing the sample concentration and concentrated sample supply process using the sample concentration device according to an embodiment of the present invention. Step (a) is a process of coupling a selective concentration device (selective concentration unit) to the main body and introducing a sample and (optionally) a buffer solution into the first channel. Step (b) is a process of pressurizing the sample by the first pressure supply unit, discharging at least a part of the remaining substances to the outside, and concentrating the target substance on the selective permeable membrane. The pressure can be applied manually, but it can also be applied to concentration by a method using negative pressure such as a vacuum tube used for blood collection. Also, concentration using a dip stick assay or the like is possible, and it is not necessarily limited to these.

[0147] Step (c) shows a process of separating the selective concentration device from the main body, reversing the direction, and then coupling the selective concentration device to another coupling part of the main body. Step (d) shows a process of introducing a buffer solution, stationary water, etc. into the second channel and pressurizing through the second pressure providing unit to supply the target substance concentrated on the selective permeable membrane to the outside, particularly to the injection part of the LFA kit.

[0148] Figure 50 is an illustrative diagram regarding the use field of the selective concentration filter of the present invention and the sample concentration device including the same. By an effective pressurization process of BEETLES2, which is a selective concentration filter included in the sample concentration device according to an embodiment of the present invention, the sample can be concentrated, and the concentrated sample can be provided to a diagnostic kit or a molecular diagnostic device. Also, when using the sample concentration device of the present invention, specimens of nasopharynx, oropharynx, and saliva can be applied not only to immunoassay (LFA) but also to molecular diagnosis (RT-qPCR) by pretreatment.

[0149] BEETLES according to the above-described embodiment of the present invention 2 is a selective concentration filter effective for COVID-19 diagnosis, and BEETLES which is a hybrid filter composed of RBCM on an AAO membrane 2 has permeation selectivity and adjustability, and thus can effectively and selectively concentrate the biomolecules to be concentrated. Also, BEETLES2 By using the support of 2 and combining LFA with smartphone-based AI technology, clinical outcomes can be easily determined based on AI.

[0150] BEETLES according to an embodiment of the present invention 2 has permeation selectivity and, as described above, also has permeability and adjustability under a certain pressure. BEETLES 2 has permeability and adjustability according to the conditions of pressure, molecular size, charge, and buffer solution. This filter has a dependence on pressure. In particular, when using BSA (bovine serum albumin), negatively charged BSA has the advantage that its concentration can be adjusted according to pressure. Also, in the case of BSA, there is a surface charge effect (permeability). That is, at low pH, it is concentrated in positively charged BSA (pI: 4.5 - 4.8), and at higher pH, it is no longer concentrated (neutral and negative charge conditions).

[0151] This embodiment is for explaining the technical idea of this embodiment, and the scope of the technical idea of this embodiment is not limited by such an embodiment. The protection scope of this embodiment should be interpreted by the scope of the appended utility model claims, and all technical ideas within the equivalent scope should be interpreted as being included in the scope of rights of this embodiment.

Claims

1. A base having pores, A selective permeable membrane coated on the base and containing vesicles (Vesicle), which concentrates the target substance to be analyzed from the sample and allows at least a part of the remaining substances other than the target substance to permeate through the pore structure. A selective concentration filter comprising:

2. The selective concentration filter according to claim 1, wherein the surface of the selective permeable membrane is hydrophilic and has a negative charge.

3. The pores are selected so as to be able to concentrate the target substance in consideration of at least one attribute selected from the group consisting of the size of the target substance, the charge of the target substance, and the affinity with the solvent, and the diameter of the pores is 10 nm to 150 m. The selective concentration filter according to claim 1, characterized in that:

4. The remaining substances of the sample include a fixed separation substance and a dynamic separation substance, and the permeability of the dynamic separation substance is in a first pressure section or a second pressure section separated according to a reference pressure applied to the selective permeable membrane. The selective concentration filter according to claim 1, characterized in that it depends on the interval of phospholipids of the cell membrane component.

5. The remaining substances of the sample further include a buffer solution, The selective concentration filter according to claim 4, characterized in that the permeability of the dynamic separation substance depends on the hydrogen ion concentration of the buffer solution in a first hydrogen ion concentration section or a second hydrogen ion concentration section separated according to the reference hydrogen ion concentration.

6. A selective concentration section including a selective concentration filter and a concentration filter holder that wraps the selective concentration filter, A sample concentration device including a main body in which a channel for supplying a sample to be concentrated to the selective concentration filter module is formed, including a binding portion that binds to the selective concentration filter as necessary, wherein the selective concentration filter is, A base having pores, A selective permeable membrane coated on the base and containing vesicles (Vesicle), which does not allow the target substance to be analyzed from the sample to permeate, and allows at least a part of the remaining substances other than the target substance to permeate through the pores. A sample concentration device characterized by including:

7. The sample concentration device according to claim 6, wherein the concentration filter holder further includes a first passage and a second passage formed in spaces separated from each other for introducing or discharging a sample.

8. The sample concentration device according to claim 7, wherein the first passage is located on the selective permeation membrane side of the selective concentration filter, and the second passage is located on the base side.

9. The concentration filter holder further includes a first holder having the first passage, and a second holder having the second passage, The sample concentration device according to claim 6, wherein the selective concentration filter is located between the first holder and the second holder.

10. The sample concentration device according to claim 9, further including a fixing portion that is located between the first holder and the second holder and makes the selective concentration filter watertight, and a support portion that supports the selective concentration filter.

11. The first passage is for injecting the sample, and the second passage is for discharging at least a part of the remaining substances that have passed through the pores to the outside. The sample concentration device further includes a first pressure providing portion that applies pressure to the sample to inject the sample from the first passage and provides pressure for discharging at least a part of the remaining substances from the second passage to the outside. The sample concentration device according to claim 7, wherein the main body further includes a first channel in which the first pressure supply portion is movable.

12. The sample concentration device further includes a second pressure supply portion that applies pressure to the selective concentration filter through the second passage to discharge the concentrated target substance from the first passage to the outside. The sample concentration device according to claim 11, wherein the main body further includes a second channel in which the second pressure supply portion is movable.

13. The sample concentration device according to claim 12, wherein the main body surrounds and supports the first pressure supply portion and the second pressure providing portion, and further includes a coupling portion that can be selectively coupled to the first passage or the second passage.

14. The surface of the selective permeation membrane is hydrophilic and has a negative charge. The sample concentration device according to claim 6, wherein the pores are selected so as to be able to concentrate the target substance in consideration of at least one attribute selected from the group consisting of the size of the target substance, the charge of the target substance, and the affinity with the solvent.

15. The remaining substances of the sample include a fixed separation substance and a dynamic separation substance, and the permeability of the dynamic separation substance depends on the interval of phospholipids of the cell membrane components in a first pressure interval or a second pressure interval delimited according to a reference pressure applied to the selective permeable membrane. The sample concentration device according to claim 6.

16. The remaining substances of the sample further include a buffer solution, The permeability of the dynamic separation substance depends on the hydrogen ion concentration of the buffer solution in a first hydrogen ion concentration interval or a second hydrogen ion concentration interval delimited according to the reference hydrogen ion concentration. The sample concentration device according to claim 15.

17. Preparing a cell membrane mixture containing a cell membrane containing vesicles and a buffer solution; Applying the cell membrane mixture to a base having pores; A method for manufacturing a selective concentration filter, comprising evaporating at least a part of the buffer solution by heat treatment to fix the cell membrane to the base.

18. The cell membrane vesicles are obtained by sonication of erythrocyte membranes, and the cell membrane vesicles are 1 to 4% (v / v) in the cell membrane mixture. The method for manufacturing a selective concentration filter according to claim 17.

19. After the coating step, The method for manufacturing a selective concentration filter according to claim 17 further comprises the heat treatment step at 40 to 60 °C.

20. In a sample concentration method using a sample selective concentration device including a selective concentration filter and a concentration filter holder that encloses the selective concentration filter, The selective concentration filter includes a base having pores and a selective permeable membrane coated on the base and containing vesicles, which does not allow the target substance to be analyzed from the sample to permeate, and allows at least a part of the remaining substances other than the target substance to permeate through the pores. The concentration filter holder includes a first passage and a second passage for introducing or discharging the sample, The sample concentration method is Injecting the sample from the first passage and discharging at least a part of the remaining substances that have permeated through the pore structure from the second passage to the outside; A sample concentration method comprising: applying pressure to the selective concentration filter through the second passage to discharge at least a part of the target substance concentrated on the selective permeation membrane from the first passage.

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