Particle separation device and particle separation method

Abstract

The present invention relates to a particle separation device and method that can minimize damage to particles during the particle separation process and separate particles by size without using complex equipment. The present invention provides a particle separation device including: a first input flow path into which a first fluid containing particles of different sizes is input; a second input flow path into which a second fluid containing no particles is input; a connecting flow path having one end connected to the first input flow path and the second input flow path and through which a third fluid, a mixture of the first and second fluids, flows; a plurality of discharge flow paths through which the particles discharged through the other end of the connecting flow path are separated according to particle size and discharged; and branch flow paths through which at least a portion of the third fluid discharged through the other end of the connecting flow path is discharged, wherein the sum of the widths of the plurality of discharge flow paths is 500 to 1000 μm.

Description

[Technical Field]

[0001] The present invention relates to a particle separation device and a particle separation method, and more particularly to a particle separation device and a particle separation method that can minimize damage to particles during the particle separation process and can separate particles by size without using complex equipment. [Background technology]

[0002] Particulate matter has a significant impact on the human body and the global environment through various forms and pathways. Its importance is gradually emerging with the development of related industries and growing interest in the environment. From measuring various molecules that can obtain biological information in biological fluids to discovering low-concentration environmental substances distributed in the air, the molecules that affect us vary in size and concentration. A variety of sensors and reactors have been developed to efficiently measure and analyze them.

[0003] These sensors and reactors are designed to operate in a state where they are separated from other substances within a specific concentration range. The sensors and reactors are able to perform stable analysis through a pretreatment process that separates and concentrates particles and molecules to a desired level. The pretreatment process mainly involves separating substances according to their size, density, and charge, and concentrating the substances to a measurable concentration range. The pretreatment process requires a certain level of high-quality equipment and skilled personnel.

[0004] In addition, the separation process is generally a temporary separation due to differences in the mobility of materials. To maintain high separation, it is necessary to minimize the working time, dispersion and diffusion, and isolate the separated molecules. This is because dispersion and diffusion progress as time passes during the process. To compensate for this, a method has been proposed in which the concentration is increased before separation so that a concentration higher than necessary can be maintained even after separation.

[0005] Recently, the increasing demand for diagnostics and synthesis using minute amounts of materials has led to a growing demand for microscale systems such as lab-on-a-chip. Technologies for separating and controlling minute amounts of materials are extremely important, especially in the fields of medicine, chemistry, and biology.

[0006] Lab-on-a-chip means a laboratory inside a chip or on a chip, and was developed to speed up existing experiments and research processes by creating microchannels of less than a nanoliter size on a substrate made mainly of glass, polymer resin, silicon, etc. and moving liquid samples of about a few nanoliters through the microchannels.

[0007] Based on this lab-on-chip technology, particle separation systems that can separate small amounts of particles with high precision are being researched. Such particle separation technology using lab-on-chips is capable of separating small amounts of particles and maintaining the separated particles at a high concentration, so it is widely used for pre-processing of other lab-on-chips or for separating particles that flow into sensors. However, conventional lab-on-chip particle separation systems have a complex structure and separation mechanism, which limits their separation precision.

[0008] Furthermore, the structure of particles separated through the particle separation system may become disordered, resulting in loss of functionality after separation.

[0009] Therefore, there is a need for a particle separator that has a simple structure and can minimize damage to particles. Summary of the Invention [Problem to be solved by the invention]

[0010] To solve the above problems, the present invention aims to provide a particle separation device and a particle separation method that can minimize damage to particles during the particle separation process and can separate particles by size without using complicated equipment. [Means for solving the problem]

[0011] In order to solve the above-mentioned problems, the present invention provides a particle separation device that includes an input flow path into which one or more fluids flow; a plurality of discharge flow paths through which a plurality of particles contained in the fluids flowing in from the input flow path are separated and discharged according to size; and branch flow paths through which a portion of the one or more fluids are discharged, wherein, based on the amount of movement of the fluids, the discharge flow paths formed close to the input flow path discharge relatively small particles, and the discharge flow paths formed away from the input flow path discharge relatively large particles.

[0012] In one embodiment, the particle separator has a hydraulic resistance (R) of 6 to 10E+12 Ns / m, as expressed by the following equation: 5 It could be.

[0013]

number

[0014] (Here, R is the fluid resistance, h is the height of the discharge channel, w is the sum of the widths of the discharge channel, μ is the kinematic viscosity of the first fluid, and L is the length of the channel through which the particles pass.)

[0015] In one embodiment, the sum of the widths of the plurality of discharge channels may be 500 to 1100 μm.

[0016] In one embodiment, the input flow path may include a first input flow path into which a first fluid and a plurality of particles having different sizes are mixed and flow in; and a second input flow path into which a second fluid not containing particles flows in. Between the input flow path and the discharge flow path, there may be a connecting flow path having one end connected to the first input flow path and the second input flow path and the other end connected to the discharge flow path, through which a third fluid composed of the first fluid and the second fluid flows.

[0017] In one embodiment, the connection channel may include a substrate; a first side formed on the substrate and connected to the first input channel; and a second side formed on the substrate opposite the first side and connected to the second input channel.

[0018] In one embodiment, the plurality of particles may move by sliding along the first side surface.

[0019] In one embodiment, the first side surface may be inclined toward the second side surface relative to the substrate.

[0020] In one embodiment, the first side may be flat, stepped, or arched.

[0021] In an embodiment, the plurality of discharge channels may be spaced apart from one another in a direction intersecting a flow direction of the third fluid on the other end side of the connecting channel.

[0022] In one embodiment, the flow rate ratio of the first fluid and the second fluid flowing into the first input channel and the second input channel, respectively, may be 0.1:99.9 to 50:50.

[0023] In one embodiment, the flow rate of the third fluid discharged through the branch channel may be 0% to 99.9% of the flow rate of the third fluid discharged through the connecting channel.

[0024] In one embodiment, the second input channel may be inclined at a predetermined angle relative to the first input channel.

[0025] The present invention also provides a particle separation device that includes an input flow path into which one or more fluids flow; a plurality of discharge flow paths through which particles contained in the fluids flowing from the input flow path are separated and discharged according to their sizes; and branch flow paths through which a portion of the one or more fluids is discharged. The particle separation device has a hydraulic resistance (R) of 6 to 10E+12 Ns / m, as expressed by the following equation 1: 5 The present invention provides a particle separation device.

[0026]

number

[0027] (Here, R is the fluid resistance, h is the height of the discharge channel, w is the sum of the widths of the discharge channel, μ is the kinematic viscosity of the first fluid, and L is the length of the channel through which the particles pass.)

[0028] The present invention also provides a particle separation method using a particle separator, the particle separator including an input flow path into which one or more fluids flow; a plurality of discharge flow paths through which particles contained in the fluids flowing in from the input flow path are separated and discharged according to size; and branch flow paths through which a portion of the one or more fluids are discharged, wherein, based on the amount of movement of the fluids, the discharge flow path formed close to the input flow path discharges relatively small particles, and the discharge flow path formed away from the input flow path discharges relatively large particles.

[0029] In one embodiment, the particle separation method may include the steps of: supplying particles and a first fluid mixture through a first flow path among the input flow paths; supplying a second fluid through a second flow path among the input flow paths; discharging particles separated by size through discharge flow paths; and discharging excess fluid through branch flow paths. [Effects of the Invention]

[0030] The particle separation device and particle separation method according to the present invention can separate particles of different sizes by particle size. The particle separation device and particle separation method according to the present embodiment can separate microparticles and nanoparticles, and can also separate nanoparticles of different sizes.

[0031] In addition, the use of physical, chemical, and electrical energy can be minimized to prevent damage to the particles during the particle separation process.

[0032] In addition, the particle separation device and particle separation method of this embodiment can separate semiconductor nanoparticles, bionanoparticles, and nanoparticles such as blood, umbilical cord blood, exosomes, and fine dust. [Brief explanation of the drawings]

[0033] [Figure 1] 1 shows a photograph of a particle separator according to an embodiment of the present invention. [Figure 2] 1 is a plan view of a particle separator according to an embodiment of the present invention; [Figure 3] 4 is a diagram illustrating a process of separating particles that have passed through a connecting channel according to an embodiment of the present invention. [Figure 4] 3 is a cross-sectional view of a connecting channel taken along line III-III in FIG. 1 according to one embodiment of the present invention. [Figure 5] 4 is a first modification of the connecting channel of FIG. 3 according to an embodiment of the present invention. [Figure 6] 4 is a second modification of the connecting channel of FIG. 3 according to an embodiment of the present invention. [Figure 7] 4 is a third modification of the connecting channel of FIG. 3 according to an embodiment of the present invention. [Figure 8] 10 is a graph showing the degree of particle separation depending on the change in flow rate of a branch flow path according to an embodiment of the present invention. [Figure 9] 10 is a photograph showing actual particle separation of a particle separator having three discharge channels according to an embodiment of the present invention. [Figure 10] 10 is a photograph showing actual particle separation of a particle separator having nine discharge channels according to an embodiment of the present invention. [Figure 11] 1 illustrates simulation results for a particle separator having three exhaust channels according to one embodiment of the present invention. [Figure 12] 10 shows simulation results of a particle separator having nine exhaust channels according to one embodiment of the present invention. [Figure 13] 10 is a simulation result of particle separation depending on the number of discharge channels according to an embodiment of the present invention. [Figure 14] 10 is a simulation result of particle separation depending on the difference in the sum of the widths of the discharge channels according to an embodiment of the present invention. [Figure 15] 10 shows particle separation ratio results according to an embodiment of the present invention. [Figure 16] 1 is a schematic diagram illustrating various methods for collecting saliva and isolating exosomes from saliva according to one embodiment of the present invention. [Figure 17] 1 shows the results of separating exosomes from saliva according to one embodiment of the present invention. [Figure 18] 1 shows the results of peptide separation from saliva according to one embodiment of the present invention. [Figure 19] 1 is a simplified illustration of a method for separating particles from urine according to one embodiment of the present invention. [Figure 20] 1 shows the results of exosome isolation from a normal subject, a glomerulonephritis patient, and a preeclampsia patient according to one example of the present invention. [Figure 21] 1 shows the results of exosome isolation from a patient with preeclampsia using each isolation method according to one example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0034] Preferred embodiments of the present invention will be described in detail below. When describing the present invention, detailed descriptions of related known technologies may obscure the gist of the present invention, such detailed descriptions will be omitted. Throughout the specification, singular terms should be understood to include plural terms unless the context clearly dictates otherwise. Terms such as "comprise" or "have" should be understood to specify the presence of stated features, numerals, steps, operations, components, parts, or combinations thereof, but should not be understood to preclude the presence or addition of one or more other features, numerals, steps, operations, components, parts, or combinations thereof. Furthermore, in performing a method or manufacturing process, the steps of the method may occur in a different order than specified unless the context clearly dictates a specific order. That is, the steps may occur in the same order as specified, may be performed substantially simultaneously, or may be performed in the reverse order.

[0035] The technology disclosed in this specification is not limited to the exemplary embodiments described herein and may be embodied in other forms. However, the exemplary embodiments described herein are provided so that the disclosed content will be thorough and complete and so that the technical concept of the present technology can be fully conveyed to those skilled in the art. In the drawings, the dimensions of the width, thickness, etc. of each device are slightly exaggerated to clearly show the components of the device. The drawings are generally described from the perspective of the viewer, and when an element is referred to as being located above another element, this means that the element may be located directly above the other element or that additional elements may be interposed between the elements. Furthermore, those skilled in the art will understand that the concept of the present invention can be embodied in various different forms without departing from the technical concept of the present invention. The same reference numerals in multiple drawings refer to substantially the same elements.

[0036] As used herein, the term "and / or" includes a combination of two or more listed items or any of two or more listed items. As used herein, "A or B" can include "A," "B," or "both A and B."

[0037] The present invention relates to a particle separation device that includes an input flow path into which one or more fluids flow; a plurality of discharge flow paths through which particles contained in the fluids flowing in from the input flow path are separated and discharged according to size; and branch flow paths through which some of the one or more fluids are discharged, and the discharge flow paths formed close to the input flow path discharge relatively small particles, and the discharge flow paths formed away from the input flow path discharge relatively large particles, based on the amount of movement of the fluids.

[0038] Hereinafter, a particle separator according to one embodiment of the present invention will be described with reference to FIGS.

[0039] Fig. 1 shows a photograph of a particle separator according to an embodiment of the present invention, Fig. 2 is a plan view of the particle separator according to an embodiment of the present invention, Fig. 3 is a diagram illustrating the process of separating particles that have passed through a connecting channel, and Fig. 4 is a cross-sectional view of the connecting channel taken along III-III in Fig. 2.

[0040] Referring to Figures 1 to 4, a particle separation device according to one embodiment of the present invention may include an input flow path, multiple discharge flow paths, and branch flow paths, and the input flow path may include a first input flow path In_P1 and a second input flow path In_P2, and the input flow path may be connected to multiple discharge flow paths EX1 to EX3 and a branch flow path DP through a connecting flow path CP.

[0041] That is, the input flow path may include a first input flow path into which a first fluid and a plurality of particles having different sizes are mixed and flow in; and a second input flow path into which a second fluid not containing particles flows in. Between the input flow path and the discharge flow path, there may be a connecting flow path having one end connected to the first input flow path and the second input flow path and the other end connected to the discharge flow path, through which a third fluid composed of the first fluid and the second fluid flows.

[0042] According to this embodiment, the particles A, B, C, and D passing through the connecting flow path CP connected to the first and second input flow paths In_P1 and In_P2 are separated and discharged into the discharge flow paths Ex according to the particle size. At this time, the particles are discharged in the following order based on the fluid movement amount: relatively small particles are discharged into the discharge flow paths formed close to the input flow paths, and relatively large particles are discharged into the discharge flow paths formed away from the input flow paths.

[0043] In the present invention, the outlet flow channel formed close to the input flow channel based on the amount of fluid movement refers to a flow channel in a portion where the amount of movement of the particle-containing fluid is small, and in the present invention, this refers to the uppermost outlet flow channel (Ex1 in FIG. 2) based on FIGS. 1 and 2. That is, in the present invention, relatively small particles are discharged through the uppermost flow channel, and relatively larger particles may be discharged toward the lower flow channels. In this case, the particle size may be between 10 nm and 20 μm. In the present invention, small particles, i.e., particles close to 10 nm in size, are discharged through the upper outlet flow channel, and larger particles may be discharged toward the lower outlet flow channel. In addition, particles that are not separated by size may be discharged through the lowermost outlet flow channel or a branch flow channel.

[0044] In addition, a portion of the fluid discharged through the connecting flow path CP is discharged to the outside through the branch flow path DP, and the branch flow path DP controls the flow rate of the fluid discharged to the outside through the branch flow path DP, thereby controlling the separation of multiple particles through multiple discharge flow paths Ex according to particle size.

[0045] Referring to FIG. 2, a first fluid L1 may be input through a first input flow path In_P1. At this time, the first fluid L1 may contain a plurality of particles having different sizes. That is, the first fluid L1 containing a plurality of particles may be input through the first input flow path In_P1. At this time, the first fluid may act as a carrier for moving the plurality of particles. Furthermore, the plurality of particles input through the first input flow path In_P1 may pass through the particle separator according to this embodiment and be separated from one another by particle size, as will be described later.

[0046] In this case, the particles may be nanoparticles, microparticles, quantum dots, organelles, etc.

[0047] Here, nanoparticles refer to particles with a size of several nanometers, and microparticles refer to particles with a size of several micrometers. The particles used in the particle separation device according to this embodiment may be various particles with sizes of several nanometers or several micrometers. Furthermore, quantum dots refer to nanometer-sized semiconductor crystals manufactured through a chemical synthesis process. Quantum dots can emit light of various colors by generating light wavelengths of different lengths depending on the particle size without changing the type of material. Finally, organelles refer to organelles present within cells, and representative examples include nuclei, mitochondria, and lysosomes. Since these organelles are mostly spherical or spherical-like in shape, they can be separated in the same way as the nanoparticles discussed above. Furthermore, since they vary in size, each organelle can be separated by separating them according to size.

[0048] A second fluid L2 containing no particles is introduced into the second input flow path In_P2. Unlike the first fluid L1 described above, the second fluid L2 is composed of only liquid. That is, a first input flow path In_P1 and a second input flow path In_P2 are disposed at the inlet side of the particle separation device according to this embodiment. Here, a plurality of particles of different sizes to be separated are introduced into one input flow path, i.e., the first input flow path In_P1, via the first fluid L1, and a second fluid L2 containing no particles is introduced into the other input flow path, i.e., the second input flow path In_P2. Here, the second fluid can act as a momentum to separate the plurality of nanoparticles contained in the first fluid. The first fluid simply acts as a carrier to move the plurality of particles. Here, when the second fluid is supplied from the side of the movement direction of the first fluid, a momentum is applied to the plurality of particles in the first fluid, causing them to move according to their size (see FIG. 3).

[0049] In this case, the first fluid L1 and the second fluid L2 are preferably hydrophilic fluids, and may include, for example, any one of an organic solvent containing any one of polyetheramine, hexane, and toluene, distilled water, an aqueous solution containing Tween 20, saline, a culture medium, and PBS (Phosphate Buffer Saline). For example, in this embodiment, a culture medium containing a plurality of particles having different sizes may be introduced into the first input flow path In_P1.

[0050] The first input flow path In_P1 and the second input flow path In_P2 may be connected to a connecting flow path CP. As shown in FIG. 2, the first input flow path In_P1 and the second input flow path In_P2 may be coupled to one end of the connecting flow path CP.

[0051] At this time, the first input flow path In_P1 and the second input flow path In_P2 are connected to one end of the connection flow path CP at a certain angle to each other. That is, the first input flow path In_P1 and the second input flow path In_P2 are arranged parallel to each other and are not connected to one end of the connection flow path CP, but the second input flow path In_P2 is connected to the connection flow path CP at a certain angle based on the first input flow path In_P1. As a result, as described above, a certain momentum can be applied to a plurality of particles contained in the first fluid using the second fluid.

[0052] For example, as shown in FIG. 2, the first input flow path In_P1 is arranged parallel to the connecting flow path CP to supply a plurality of particles, while the second input flow path In_P2 is arranged at a certain angle relative to the first input flow path In_P2 to apply a moment to the side of the first fluid.

[0053] 3, when the first input flow path In_P1 and the second input flow path In_P2 are angled with each other, particles contained in the first fluid L1 may be moved along the first side W1 of the connecting flow path CP by the supply of the second fluid, where the first side W1 refers to one side of the connecting flow path CP connected to the first input flow path In_P1.

[0054] For example, the first fluid L1 passing through the first input flow path In_P1 is slid by the second fluid L2 onto the first side surface W1 of the connecting flow path CP and moves along the connecting flow path CP.

[0055] In this case, assuming that the first fluid L1 contains a plurality of particles A, B, C, and D of different sizes, the particles of each size may be arranged on the first side surface according to their sizes. At the point where the first input flow path In_P1 and the second input flow path In_P2 meet, the pressure of the second fluid L2 supplied through the second input flow path In_P2 causes the particles A, B, C, and D of the first fluid L1 to move toward the first side surface W1 of the connecting flow path CP. This is because the second fluid L2 input into the connecting flow path CP is input at an angle relative to the first fluid L1. That is, the second fluid L2 pressurizes the side surfaces of the particles A, B, C, and D flowing into the connecting flow path CP, generating a moment, causing the particles to slide and move toward the first side surface W1 of the connecting flow path CP.

[0056] As described above, the first side W1 of the connection channel CP may be inclined at a first angle θ1 with respect to the substrate Su (see FIG. 4). In this case, the first side W1 is inclined toward the second side W2 opposite the first side W1. Here, the second side W2 refers to one side of the connection channel CP connected to the second input channel In_P2. When the first side is inclined at a certain angle in this manner, as shown in FIG. 4, particles of various sizes may be positioned to a certain depth on the first side by the supply of the second fluid. Furthermore, in the case of such particles, the point at which they contact the first side is determined by their size, which may result in separation according to their size.

[0057] More specifically, the particles A, B, C, and D may be spaced from the first side W1 in order of particle size. For example, the particles may be spaced from the first side W1 in the order of increasing particle size: particle A, particle B, particle C, and particle D (for convenience, it is assumed that particles A, B, C, and D are spherical and that the particle sizes are determined by the diameters of particles A, B, C, and D). As a result, particles A, B, C, and D may be spaced from the first side W1 in the order of decreasing diameter, and positioned at the center of the connecting channel.

[0058] The second side surface W2 may also be inclined at the second angle θ2 with respect to the substrate Su. However, the present invention is not limited to this, and the second side surface W2 may be perpendicular to the substrate Su as shown in the first modified example of Fig. 5. This is because the particles A, B, C, and D are moved along the first side surface W1 by the second fluid L2, but not along the second side surface W2.

[0059] In this case, the first angle θ1 and the second angle θ2 may be less than 90 degrees. Preferably, the first angle θ1 and the second angle θ2 may be between 35 degrees and 55 degrees. In particular, when the first side W1 is inclined at the first angle θ1, particles contained in the first fluid L1 may be separated from the first side W1 in order of particle size. If the first angle is 90 degrees or more, it is impossible to separate the plurality of particles by size. In particular, if the first angle is less than 35 degrees, the angle of the first side is too low, which may result in a lack of space inside the flow path, and therefore a decrease in the overall flow rate. Furthermore, if the first angle exceeds 55 degrees, the positional change according to size may be small, which may decrease the separation efficiency of the plurality of particles.

[0060] That is, in this embodiment, the first side W1 is tilted toward the second side W2, so that the distances between the particles A, B, C, and D can be increased compared to when the first side W1 is not tilted and is arranged perpendicular to the substrate Su. Here, the distances between the particles A, B, C, and D refer to the distances between the centers of the particles A, B, C, and D.

[0061] 4, the first side W1 may be flat, and the particles A, B, C, and D are positioned in the connecting channel CP while contacting the substrate Su and the flat first side W1.

[0062] Meanwhile, the first side W1 may have a stepped shape as shown in the second modified example of Fig. 6. When the first side W1 has a stepped shape, the distance between the particles A, B, C, and D can be further increased compared to when the first side W1 has a flat shape. In this case, the second side W2 may also have a stepped shape like the first side W1 as shown in Fig. 6, or the second side may have a simple vertical wall shape as shown in Fig. 7.

[0063] Meanwhile, the first side may be arched (FIG. 8). As described above, the first side may be formed in a straight line, but may also have an arched shape for ease of manufacturing and processing. In this case, the first side W1 may have an independent arched shape, or may be connected to the second side W2 to form an arched shape. In this case, the inside of the flow path may have a hemispherical arch shape.

[0064] 1 and 2, particles A, B, C, and D are contained in a third fluid L3 composed of a first fluid L1 and a second fluid L2 and pass through the connecting channel CP. As described above, particles A, B, C, and D move while sliding along the first side W1. After passing through the connecting channel CP, particles A, B, C, and D are separated from each other according to their size and move as shown in FIG.

[0065] In addition, in this case, the first fluid L1 and the second fluid L2 may be mixed to form a third fluid L3, but due to the characteristics of the second fluid supplied from the side of the first fluid, they may not be mixed but may form a laminar flow to form the third fluid, or only a portion of them may be mixed to form the third fluid.

[0066] In Figure 3, multiple particles A, B, C, and D are slid along the first side W1 by the second fluid L2, and when they pass through the connecting flow path CP due to inertial force, they move diagonally toward the first side W1.

[0067] At this time, particles A, B, C, and D move along different paths depending on their size. In more detail, particles A, B, C, and D move along paths P1, P2, P3, and P4, respectively. That is, particle A moves along path P1, particle B moves along path P2, particle C moves along path P3, and particle D moves along path P4.

[0068] 3, the smaller the particle size, the more it can bend toward the first side surface W1. That is, particles D, C, B, and A can bend more toward the first side surface W1 in this order, which is the order of particle size.

[0069] Meanwhile, according to this embodiment, as described above, when the first side W1 is tilted toward the second side W2, the distance between particles A, B, C, and D increases, and thus the distance between paths P1, P2, P3, and P4 along which particles A, B, C, and D move after passing through the connecting channel CP increases. When the distance between adjacent pairs of paths P1, P2, P3, and P4 increases, particles moving along the paths can be easily separated.

[0070] For example, when particle A moves along path P1 and particle B moves along path P2, the distance between path P1 and path P2 increases when the first side W1 is tilted compared to when the first side W1 is not tilted. This is because the tilt of the first side W1 increases the distance between particle A and particle B in the connecting channel CP.

[0071] 2, a plurality of discharge channels Ex are disposed at the other end of the connecting channel CP, and the plurality of discharge channels Ex can separate and discharge the particles discharged through the connecting channel CP according to their sizes.

[0072] As described above, the particles and fluid passing through the connecting channel are separated according to size and then fed into the plurality of discharge channels EX1 to EX3. At this time, the plurality of discharge channels Ex are disposed at the other end of the connecting channel CP. The plurality of discharge channels Ex can separate and discharge the plurality of particles discharged through the connecting channel CP according to particle size.

[0073] As described above, a plurality of discharge channels Ex1, Ex2, and Ex3 may be formed at the other end of the connecting channel CP. Although three discharge channels are illustrated in FIG. 2, the number of discharge channels is not limited thereto and may be less than or greater than three. According to this embodiment, particles may be separated and discharged into the plurality of discharge channels Ex1, Ex2, and Ex3 depending on their sizes.

[0074] At this time, the particles can be separated by size, and this separation can be performed by the pressure and velocity of the fluid. However, in the case of a separation chip such as the present invention, hydraulic resistance (R) can exist against the fluid, and this fluid resistance can lead to a pressure drop of the fluid. If the chip has a structure with high fluid resistance, a high pressure drop occurs, which can prevent smooth separation by size or prevent particles from being discharged to the top discharge channel (Ex1 in FIG. 2). Furthermore, if the fluid resistance is too low, all particles can be discharged to one discharge channel, which can reduce the separation effect.

[0075] In the case of the particle separation chip of the present invention, the fluid resistance can be defined by the following Equation 1.

[0076]

number

[0077] (Here, R is the fluid resistance, h is the height of the discharge channel, w is the sum of the widths of the discharge channel, μ is the kinematic viscosity of the first fluid, and L is the length of the channel through which the particles pass.)

[0078] In the present invention, since a lab-on-a-chip is used, the channel height (h), dynamic viscosity (μ), and channel length (L) can be maintained constant. However, the width of each discharge channel can be varied to design and manufacture under various conditions, and in this process, a lab-on-a-chip with various fluid resistances can be manufactured. However, as explained above, the particles can be separated by size and discharged into each discharge channel only when the fluid resistance is within a certain range.

[0079] In the present invention, the hydraulic resistance (R) expressed by Equation 1 is 6 to 10E+12 Ns / m 5 It could be.

[0080] The fluid resistance is 5.5E+12Ns / m 5 If the fluid resistance is less than 3.5E+13Ns / m, the width of the discharge passage must be increased excessively to form a low fluid resistance, making it impossible to form a large number of discharge passages, and excessive pressure may cause multiple particles to be discharged without being separated by size. 5 If the pressure exceeds 1000 psi, the particles may not be separated due to an excessive pressure drop, and may not be discharged to the top discharge channel.

[0081] To achieve this fluid resistance, the sum of the widths of the discharge channels may be 500 to 1100 μm, preferably 750 to 1000 μm, and more preferably 900 μm. As discussed above, the fluid resistance can be adjusted by adjusting the width of the discharge channels. That is, increasing the sum of the widths of the discharge channels decreases the fluid resistance, while decreasing the sum of the widths increases the fluid resistance. Therefore, it is preferable that the sum of the discharge channels be within a certain range. If the sum of the discharge channels is less than 500 μm, high fluid resistance may occur, resulting in excessive pressure drop. If it exceeds 1100 μm, excessive pressure must be maintained continuously, which may result in particle breakage or damage to the separation chip. Furthermore, as shown in FIG. 16, particle separation was observed in the discharge channel direction when the sum of the widths exceeded 500 μm. Only when the width was 750 μm or greater did particles separate and be discharged into the first and second discharge channels. Therefore, if the sum of the widths of the discharge channels is less than 500 μm, excessive pressure drop may cause particles to be separated improperly. Also, if the sum of the widths of the discharge channels is more than 1100 μm, the width of the first discharge channel may be too wide, causing particles to be discharged through the first discharge channel without being separated.

[0082] The Hagen-Poisson equation, rewritten to resemble Ohm's law, is given by Equation 2 below.

[0083]

number

[0084] (P is pressure, R is fluid resistance, Q is average flow velocity)

[0085] At the same flow rate, the pressure will differ by approximately 100 times due to the R value, so the fluid resistance will be 6E+12Ns / m 5 If the pressure is less than 1000 kJ / cm, it may be impossible to discharge the particles into the discharge flow path Ex1 at the top stage, and separation according to size may not be easy due to the low pressure.

[0086] In Equation 1, h represents the height of the discharge channel. However, since the channels of the lab-on-a-chip generally have the same or similar height, h may be defined as the average height of the entire channel. In the present invention, h is 10 to 100 μm, and preferably 40 μm. If h is less than 10 μm, the cross-sectional area is reduced, resulting in high fluid resistance, making it difficult to discharge particles through the discharge channel. If h exceeds 100 μm, it is inefficient because it is difficult to maintain the channel shape in high-pressure sections.

[0087] The μ is the kinematic viscosity of the first fluid, but in the present invention, since the first fluid and the second fluid are made of the same material, the μ may be the kinematic viscosity of the first fluid, the second fluid, and the third fluid. In this case, the kinematic viscosity may have a different value depending on the material, but in the present invention, it is 0.0001 to 0.005 Ns / m 2 The fluid used in the present invention may be a hydrophilic fluid. For example, polyetheramine, an organic solvent containing one of hexane and toluene, distilled water, an aqueous solution containing Tween 20, saline, culture medium, and PBS (Phosphate Buffer Saline) may be used. Such fluids have a dynamic viscosity within the above range, and substances with a similar dynamic viscosity can be separated using the lab-on-a-chip of the present invention. The viscosity is 0.0001 Ns / m. 2 If the viscosity is less than 0.005 Ns / m, the fluid resistance will be excessively reduced due to the low viscosity, making it difficult to separate particles by size. 2 If the kinematic viscosity exceeds 100%, the high viscosity may cause a lot of fluid resistance, making particle separation difficult.

[0088] The L represents the channel length, and the lab-on-chip of the present invention having the structure of Figure 2 may have a channel length of 10,000 to 20,000 μm, preferably 15,450 μm. If the channel length is less than 10,000 μm, the migration path for particle alignment inside the lab-on-chip may be reduced, resulting in insufficient separation. If the channel length exceeds 20,000 μm, the pressure drop due to fluid resistance may increase, resulting in reduced separation efficiency.

[0089] Furthermore, by precisely controlling the fluid resistance in this manner, it is possible to separate multiple particles with high efficiency when using the lab-on-a-chip of the present invention.

[0090] There are a variety of commonly used particle separation methods. However, among the existing methods, physical methods such as centrifugation, filtering, and chromatography have very poor separation efficiency (less than 10%), while chemical separation methods such as precipitation, magnetic particle attachment, and immunological separation methods can achieve high separation efficiency but may cause deformation or damage to the particles themselves. However, in the present invention, a moment is applied to the particles and then separation is performed based on the particle size, which allows for high-efficiency particle separation while minimizing particle damage and deformation (separation efficiency of 90% or more).

[0091] The plurality of discharge channels Ex1, Ex2, and Ex3 may be arranged in a direction intersecting the flow direction of the third fluid L3 exiting the connecting channel CP, i.e., the plurality of discharge channels Ex1, Ex2, and Ex3 may be spaced apart from one another in a direction intersecting the flow direction.

[0092] In more detail, the third fluid moves upward (toward Ex1 in FIG. 2) upon exiting the connecting channel due to the supply momentum of the second fluid. Since the particles are arranged according to size, small particles D flow closer to the wall, while large particles A flow closer to the center. Furthermore, since the third fluid moves upward and toward the lower separation channel, size separation of the particles can be further accelerated by inertia and centrifugal force. Large particles A are located at the center and therefore move less outward because of their relatively large volume and weight, while small particles D move more outward because of their relatively small volume and light weight. Since discharge channels are formed on the outside, small particles D are separated and discharged to the upper discharge channel (e.g., Ex1), while large particles A are separated and discharged to the lower discharge channel Ex3.

[0093] According to this embodiment, the plurality of discharge channels Ex1, Ex2, and Ex3 are arranged at equal intervals in the intersecting direction. More specifically, the intervals between adjacent pairs of discharge channels among the plurality of discharge channels Ex1, Ex2, and Ex3 are the same. For example, the interval between discharge channel Ex1 and discharge channel Ex2 and the interval between discharge channel Ex2 and discharge channel Ex3 are the same. This can be applied not only when there are three discharge channels as shown in the drawings of the present invention, but also when there are multiple discharge channels. That is, the interval between discharge channel Ex2 and discharge channel Ex3 and the interval between discharge channel Ex3 and discharge channel Ex4 can be the same.

[0094] In the present invention, the plurality of discharge channels may include 2 to 5 discharge channels. In the previous invention, the width of the discharge channel was reduced (about 10 μm) to secure a large number of discharge channels, but this may result in a high pressure drop and improper fluid movement. To improve this, multiple fluids may be discharged into branch channels, but discharging multiple branch channels has the disadvantage that unseparated particles may be discharged into the branch channels.

[0095] Therefore, in the present invention, the particle size distribution is 2 to 5, preferably 3, and the sum of the widths of the discharge channels is preferably 500 to 1000 μm, as explained above. If there are fewer than 2 discharge channels, particle size separation cannot be performed, and if there are more than 5, pressure drop may occur due to the large number of discharge channels. In addition, the pressure drop may increase because the sum of the widths of the discharge channels is reduced to maintain the spacing between each discharge channel.

[0096] Meanwhile, according to this embodiment, a branch flow path DP is disposed to discharge at least a portion of the third fluid L3 discharged through the connection flow path CP. The branch flow path DP may be located at the outlet side of the connection flow path CP and adjacent to the plurality of discharge flow paths Ex.

[0097] At this time, the branch flow path DP discharges a portion of the third fluid L3 to the outside, allowing the plurality of particles to be separated and discharged to the plurality of discharge flow paths Ex, each corresponding to a particle size. When the branch flow path DP discharges a portion of the third fluid L3 to the outside, the distance between the paths along which the particles travel may increase. This makes it easier to separate particles of different sizes according to particle size.

[0098] According to this embodiment, the flow rate of the third fluid L3 discharged through the branch flow path DP can be adjusted. By adjusting the flow rate of the third fluid L3 discharged through the branch flow path DP, the particles discharged through the connecting flow path CP can be more easily separated and discharged according to particle size.

[0099] In the present invention, particles can be easily separated if the flow rate of the third fluid flowing out through the branch flow channel DP is equal to or greater than a certain ratio of the flow rate of the third fluid flowing out through the connecting flow channel CP. As the ratio of the flow rate of the third fluid flowing out through the branch flow channel DP increases, the number of discharge flow channels Ex through which the third fluid flows out through the connecting flow channel CP increases. For example, if the flow rate discharged into the branch flow channel DP is 0% of the flow rate of the connecting flow channel CP, i.e., if there is no flow rate at all flowing out into the branch flow channel DP, the third fluid is discharged along one discharge flow channel Ex. On the other hand, as the ratio (%) of the flow rate discharged into the branch flow channel DP increases, the number of discharge flow channels Ex, which are the routes through which the third fluid is discharged, increases.

[0100] In particular, if the flow rate discharged into the branch channel DP exceeds 99.5% of the flow rate of the connecting channel CP, particles are discharged into the branch channel without being separated, which may reduce overall efficiency.

[0101] Meanwhile, according to this embodiment, the flow rate ratio of the first fluid L1 and the second fluid L2 flowing into the first input flow path In_P1 and the second input flow path In_P2, respectively, is different from each other. In particular, when the flow rate ratio of the first fluid L1 and the second fluid L2 is 0.1:99.9 to 50:50, the particles flowing out through the connecting flow path CP can be easily separated and discharged according to their particle size. More preferably, when the flow rate ratio of the first fluid L1 and the second fluid L2 is 5:95, the particles can be easily separated according to their particle size through the multiple discharge flow paths. When the flow rate ratio of the first fluid to the second fluid is less than 0.1:99.9, the ratio of the second fluid becomes too high, resulting in a decrease in the ratio of the first fluid, which reduces the amount of particles that can be injected and may decrease overall efficiency. When the flow rate ratio exceeds 50:50, the ratio of the second fluid decreases, which may prevent sufficient momentum from being transmitted to the particles. Such insufficient momentum can reduce the size-dependent separation efficiency of the separator, thereby reducing the particle separation efficiency.

[0102] Hereinafter, a particle separation method according to an embodiment of the present invention will be described. In describing the particle separation method of this embodiment, detailed description of the same components as those of the particle separation apparatus described above will be omitted.

[0103] 2, first, a first fluid L1 is input into a first input flow path In_P1. At this time, the first fluid L1 contains a plurality of particles of different sizes to be separated by particle size. That is, the first fluid L1 containing the plurality of particles is input through the first input flow path In_P1.

[0104] On the other hand, a second fluid L2 that does not contain particles is introduced into the second input channel In_P2. Unlike the first fluid L1 described above, the second fluid L2 is made up of only liquid.

[0105] That is, in this embodiment, a first fluid L1 containing particles and a second fluid L2 not containing particles are input into different input channels In_P1 and In_P2. At this time, the first fluid L1 and the second fluid L2 may be input simultaneously, or the first fluid may be input after the second fluid for smooth particle movement.

[0106] At this time, the first input flow path In_P1 and the second input flow path In_P2 are connected to the inlet of the connection flow path CP at a certain angle to each other. The first input flow path In_P1 and the second input flow path In_P2 are arranged parallel to each other and are not connected to one end of the connection flow path CP. That is, the second input flow path In_P2 is connected to the connection flow path CP at a certain angle based on the first input flow path In_P1.

[0107] 2, the first input flow path In_P1 may be arranged parallel to the connecting flow path CP, and the second input flow path In_P2 may be arranged inclined at a certain angle relative to the first input flow path In_P2, so that the second fluid supplied through the second flow path can apply a momentum to the particles contained in the first fluid, and the distance from the first side may be determined depending on the size of the particles due to the momentum.

[0108] Meanwhile, according to this embodiment, the flow rate ratio of the first fluid L1 and the second fluid L2 flowing into the first input flow path In_P1 and the second input flow path In_P2, respectively, is different from each other. In particular, when the flow rate ratio of the first fluid L1 and the second fluid L2 is 0.1:99.9 to 50:50, a plurality of particles flowing out through the connecting flow path CP can be easily separated and discharged according to their sizes.

[0109] More preferably, when the flow rate ratio of the first fluid L1 and the second fluid L2 is 5:95, the particles can be easily separated according to particle size through the multiple discharge channels.

[0110] Next, after the first fluid L1 and the second fluid L2 are input, the first fluid L1 and the second fluid L2 may pass through the first input flow path In_P1 and the second input flow path In_P2, respectively, and then flow into the connecting flow path CP.

[0111] 3, when the first input flow path In_P1 and the second input flow path In_P2 are angled with each other, particles contained in the first fluid L1 may be moved along a first side W1 of the connecting flow path CP by the pressure of the second fluid, where the first side W1 refers to one side of the connecting flow path CP connected to the first input flow path In_P1.

[0112] For example, assume that the first fluid L1 contains a plurality of particles A, B, C, and D of different sizes. After passing through the first input flow path In_P1, the first fluid L1 is slid to the first side surface W1 of the connecting flow path CP by the second fluid L2 and moves along the connecting flow path CP.

[0113] At the point where the first input flow path In_P1 and the second input flow path In_P2 meet, the second fluid L2 in the second input flow path In_P2 causes the particles A, B, C, and D in the first fluid L1 to move toward the first side surface W1 of the connecting flow path CP. This is because the second fluid L2 is introduced into the connecting flow path CP at a certain angle relative to the first fluid L1. That is, when a momentum is transferred to the particles by the supply of the second fluid L2, the particles A, B, C, and D flowing into the connecting flow path CP move while sliding against the first side surface W1 of the connecting flow path CP.

[0114] 4, the first side W1 of the connection channel CP may be inclined at a first angle θ1 with respect to the substrate Su. In this case, the first side W1 is preferably inclined toward the second side W2 opposite to the first side W1. Here, the second side W2 refers to one side of the connection channel CP connected to the second input channel In_P2.

[0115] The second side W2 may also be inclined at the second angle θ2 with respect to the substrate Su. However, the present invention is not limited to this, and the second side W2 may be perpendicular to the substrate Su as shown in the first modified example of Fig. 5. This is because the particles A, B, C, and D are moved along the first side W1 by the second fluid L2, but not along the second side W2.

[0116] In this case, the first angle θ1 and the second angle θ2 may be less than 90 degrees. Preferably, the first angle θ1 and the second angle θ2 may be 35 degrees to 55 degrees. When the first side surface W1 is inclined at the first angle θ1, particles contained in the first fluid W1 may be separated from the first side surface W1 in order of particle size.

[0117] More specifically, the particles A, B, C, and D may be spaced from the first side W1 in order of particle size. For example, the particles may be spaced from the first side W1 in the order of increasing particle size: particle A, particle B, particle C, and particle D. For convenience, it is assumed that the particles A, B, C, and D are spherical and the particle sizes are determined by the diameters of the particles A, B, C, and D.

[0118] As a result, particles A, B, C, and D may be separated from the first side surface W1 in the order of increasing diameter.

[0119] In this embodiment, the first side W1 is tilted toward the second side W2, which increases the distances between the particles A, B, C, and D compared to when the first side W1 is not tilted and is disposed perpendicular to the substrate Su. Here, the distances between the particles A, B, C, and D refer to the distances between the centers of the particles A, B, C, and D.

[0120] Next, particles A, B, C, and D that pass through the connecting flow path CP are separated from one another according to their size and then fed into a plurality of discharge flow paths EX1 to EX3. At this time, a plurality of discharge flow paths Ex are disposed at the other end of the connecting flow path CP. The plurality of discharge flow paths Ex can separate and discharge the plurality of particles that have passed through the connecting flow path CP according to their size.

[0121] As described above, a plurality of discharge channels Ex1, Ex2, and Ex3 may be formed at the other end of the connecting channel CP. Although three discharge channels are illustrated in FIG. 2, the number of discharge channels is not limited thereto and may be less than or greater than three. According to this embodiment, particles may be separated and discharged into the plurality of discharge channels Ex1, Ex2, and Ex3 depending on their sizes.

[0122] Meanwhile, according to this embodiment, at least a portion of the third fluid L3 discharged through the connection passage CP may be discharged to the branch passage DP. The branch passage DP is located at the outlet side of the connection passage CP and adjacent to the plurality of discharge passages Ex.

[0123] The branch flow paths DP discharge a portion of the third fluid L3 to the outside, allowing the particles to be separated and discharged to the corresponding discharge flow paths Ex, based on their particle sizes. When the branch flow paths DP discharge a portion of the third fluid L3 to the outside, the distance between the paths along which the particles travel can increase. This makes it easier to separate particles of different sizes.

[0124] According to this embodiment, the flow rate of the third fluid L3 discharged through the branch flow path DP can be adjusted. By adjusting the flow rate of the third fluid L3 discharged through the branch flow path DP, the particles discharged through the connecting flow path CP can be more easily separated and discharged according to particle size.

[0125] The flow rate of the third fluid L3 discharged through the branch flow channel DP can be adjusted. By adjusting the flow rate of the third fluid L3 discharged through the branch flow channel DP, the particles discharged through the connecting flow channel CP can be more easily separated and discharged according to particle size.

[0126] For example, when the flow rate discharged into the branch flow path DP is 0% of the flow rate of the connecting flow path CP, i.e., when there is no flow rate at all flowing into the branch flow path DP, the third fluid is discharged along one discharge flow path Ex. On the other hand, as the ratio (%) of the flow rate discharged into the branch flow path DP increases, it can be seen that the number of discharge flow paths Ex, which are the routes through which the third fluid is discharged, increases.

[0127] In particular, when the flow rate discharged into the branch flow path DP is 0% to 99.9%, preferably 0% to 97.5%, of the flow rate of the connecting flow path CP, the number of discharge flow paths Ex, which are the paths through which the third fluid is discharged, is maximized. In an embodiment of the particle separation device and particle separation method of the present invention, a branch flow path DP is disposed on the outlet side of the connecting flow path CP to discharge a portion of the fluid flowing out through the connecting flow path CP, and the multiple particles discharged through the connecting flow path CP can be easily separated by particle size.

[0128] Meanwhile, according to the particle separation device and particle separation method of the present embodiment, it is possible to separate microparticles and nanoparticles from each other, and also possible to separate nanoparticles of different sizes from each other.

[0129] In addition, this embodiment may be applied to a non-destructive, high-efficiency exosome precision separation system, a blood analysis chip, a DNA array, a microsensor, or a precision separation system, and may be used, for example, to analyze saliva, sweat, tears, nasal mucus, or seminal plasma.

[0130] The present invention also provides a particle separation device that includes an input flow path into which one or more fluids flow; a plurality of discharge flow paths through which particles contained in the fluids flowing from the input flow path are separated and discharged according to their sizes; and branch flow paths through which a portion of the one or more fluids is discharged. The particle separation device has a hydraulic resistance (R) of 6 to 10E+12 Ns / m, as expressed by the following equation 1: 5 The present invention relates to a particle separator.

[0131]

number

[0132] (Here, R is the fluid resistance, h is the height of the discharge channel, w is the sum of the widths of the discharge channel, μ is the kinematic viscosity of the first fluid, and L is the length of the channel through which the particles pass.)

[0133] The present invention also relates to a particle separation method using a particle separator, the particle separator including an input flow path into which one or more fluids flow; a plurality of discharge flow paths through which particles contained in the fluids flowing in from the input flow path are separated and discharged according to size; and branch flow paths through which a portion of the one or more fluids are discharged, the discharge flow paths formed close to the input flow path discharging relatively small particles and the discharge flow paths formed away from the input flow path discharging relatively large particles based on the amount of movement of the fluids.

[0134] The particle separation method may include the steps of: supplying particles and a first fluid mixture through a first flow path among the input flow paths; supplying a second fluid through a second flow path among the input flow paths; discharging the particles separated by size through discharge flow paths; and discharging excess fluid through branch flow paths.

[0135] In addition, the particle separation device and particle separation method of the present invention can be used to separate particles such as exosomes from human secretions such as saliva, blood, urine, sweat, breast milk, etc.

[0136] When the particle separation device is used in a method for separating exosomes from saliva, the present invention can provide a method for separating and pre-treating exosomes from saliva, including the steps of: (a) collecting saliva from a subject; (b) centrifuging the collected saliva to separate and concentrate cellular material; and (c) classifying the cellular material by size to obtain desired biological microparticles.

[0137] The step (a) may include: i) a step in which the subject spits saliva into a collection container to collect saliva; or ii) a step in which the subject gargles with the collection gargle liquid and then spits the gargle liquid into a collection container to collect saliva (see FIG. 16).

[0138] Step i) or step ii) may be repeated 1 to 5 times.

[0139] Step (c) may be performed using the particle separator.

[0140] In the present invention, the cellular material refers to cellular particles contained in the saliva, and the terms "cellular material" and "particles" may be used interchangeably in the present invention.

[0141] The present invention also provides a method for treating a blood vessel comprising: an input flow path into which one or more fluids including saliva flow; a plurality of discharge flow paths through which a plurality of cellular substances contained in the fluids flowing in from the input flow path are separated and discharged according to size; and a branch flow path through which a portion of the one or more fluids including saliva is discharged, wherein the cellular substances are biological particles including exosomes, and the hydraulic resistance (R) expressed by the following formula 1 is 6 to 10E+12 Ns / m 5 The present invention provides an apparatus for isolating and pre-treating exosomes from saliva.

[0142]

number

[0143] (Here, R is the fluid resistance, h is the height of the discharge channel, w is the sum of the widths of the discharge channel, μ is the kinematic viscosity of the first fluid, and L is the length of the channel through which the particles pass.)

[0144] Furthermore, when used to separate exosomes from urine, the present invention can provide a device for separating particles from urine, which includes a centrifuge that separates cells and foreign matter from urine; and a particle separation device that separates exosomes from urine after cells and foreign matter have been separated, wherein the particle separation device includes an input flow path into which one or more types of fluid including urine flows in; a plurality of discharge flow paths in which multiple cellular substances contained in the fluid that flows in from the input flow path are separated and discharged based on size; and a branch flow path from which a portion of the one or more types of fluid including urine is discharged, wherein the cellular matter is biological microparticles including exosomes, and based on the amount of movement of the fluid, the discharge flow path formed close to the input flow path discharges relatively small cellular matter, and the discharge flow path formed away from the input flow path discharges relatively large cellular matter.

[0145] The present invention also provides an apparatus for separating particles from urine, which includes a centrifuge for separating cells and foreign matter from urine; and a particle separation device for separating exosomes from urine from which cells and foreign matter have been separated, the particle separation device including an input flow path into which one or more fluids including urine flow in; a plurality of discharge flow paths through which a plurality of cellular substances contained in the fluids that flow in from the input flow path are separated and discharged according to size; and branch flow paths through which a portion of the one or more fluids including urine are discharged, the cellular substances being biological particles including exosomes, and the particle separation device having a hydraulic resistance (R) expressed by the following formula 1 of 6 to 10E+12 Ns / m 5 The present invention provides a device for isolating and pre-treating exosomes from urine.

[0146]

number

[0147] (Here, R is the fluid resistance, h is the height of the discharge channel, w is the sum of the widths of the discharge channel, μ is the kinematic viscosity of the first fluid, and L is the length of the channel through which the particles pass.)

[0148] The present invention also provides a method for separating particles from urine using a device for separating particles from urine, wherein the device for separating particles from urine includes: a centrifuge that separates cells and foreign matter from urine; and a particle separation device that separates exosomes from the urine from which the cells and foreign matter have been separated, wherein the particle separation device includes: an input flow path into which one or more types of fluid including urine flows in; a plurality of discharge flow paths through which multiple cellular materials contained in the fluid that flows in from the input flow path are separated and discharged based on size; and branch flow paths through which some of the one or more types of fluid including urine are discharged, wherein, based on the amount of movement of the fluid, the discharge flow path formed close to the input flow path discharges relatively small cellular materials, and the discharge flow path formed away from the input flow path discharges relatively large cellular materials.

[0149] The method for separating particles from urine includes the steps of: supplying a first fluid containing urine through a first flow path among the input flow paths; supplying a second fluid through a second flow path among the input flow paths; discharging cellular material separated by size through discharge flow paths; and discharging excess fluid through branch flow paths, wherein the cellular material may include exosomes.

[0150] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings so that those skilled in the art can easily understand them. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations will be omitted if it is determined that such descriptions may unnecessarily obscure the gist of the present invention. Furthermore, some features shown in the drawings may be enlarged, reduced, or simplified for ease of explanation, and the drawings and their components may not necessarily be drawn to scale. However, such details will be readily apparent to those skilled in the art.

[0151] Examples 1 to 4

[0152] Since there are limitations to actually fabricating and experimenting with all shapes of flow channels, we first fabricated a particle separator with three discharge channels and one with nine discharge channels (see Figures 1 and 2) and compared them with the simulation data.

[0153] In the case of Example 1, as shown in FIG. 9, the particle separator has three discharge channels, and in Example 2, the particle separator has nine discharge channels (FIG. 10).

[0154] Example 3 is simulation data (FIG. 11) in which the same conditions as Example 1 are input, and Example 4 is simulation data (FIG. 12) in which the same conditions as Example 2 are input.

[0155] The height of each channel was set to 40 μm, and the total channel length was set to 15,450 μm. Each outlet channel was set to have a width of 300 μm, and since there were three outlet channels, the total width of the outlet channels was set to 900 μm.

[0156] Experimental Example 1

[0157] The flow rate of the first fluid was set to 5 μL / min, and the flow rate of the second fluid was set to 95 μL / min. After setting the flow rate of the branched channel to 75 μL / min, the experiment was conducted by mixing the same weight of 100 nm and 600 nm particles into the first fluid at a ratio of 1 wt%.

[0158] In this case, green fluorescent particles were used for the 100 nm particles and red fluorescent particles for the 600 nm particles. Also, fluorescent particles were used and the background was black to effectively confirm the behavior.

[0159] As shown in Figures 9 and 11, it was confirmed that the actual experiment data and the simulation data matched when there were three exhaust flow paths, and as shown in Figures 10 and 12, it was confirmed that the simulation data and the actual experiment data also matched when there were nine exhaust flow paths.

[0160] For the following experiments, simulation experiments were carried out based on the above data.

[0161] Examples 5 to 13

[0162] An experiment was conducted to confirm the separation efficiency and discharge pressure depending on the number of discharge channels. Specifically, a simulation experiment using Ansys was used for visualization, and a particle separator was created as shown in Figure 1. At this time, the height of each channel was set to 40 μm, and the total channel length was set to 15,450 μm.

[0163] The sum of the total discharge flow paths was set to 900 μm, the same as in Examples 1 and 2, and Examples 5 to 13 were prepared so that there were 1 to 9 discharge flow paths, respectively.

[0164] Experimental Example 2

[0165] Particles having a size of 100 nm and particles having a size of 600 nm were mixed at the same mixing ratio, and then mixed into the first fluid at a ratio of 1 wt % to conduct the experiment.

[0166] As shown in Figure 11, the flow rate of the first fluid was set to 5 μl / min and the flow rate of the second fluid was set to 95 μl / min. When the flow rate discharged from the branch channel was set to 75 μl / min, the pressure of the first fluid, the second fluid, and the pressure of the branch channel were measured to operate normally. The pressure (dyne / cm2) and fluid resistance (E+12Ns / m) of each channel were calculated based on the number of discharge channels (units) and the sum of the channel widths (μm). 5 ) were measured respectively.

[0167] [Table 1]

[0168] As shown in Figure 13, it was confirmed that proper particle separation occurred when the first and second fluids were supplied at the pressures listed in Table 1 above and the fluids were discharged through the branch flow paths. However, as shown in Table 1, in the case of Example 7 of the present invention, i.e., when there are three discharge flow paths, it was confirmed that normal operation was possible even at the lowest pressure (Figure 13(c)).

[0169] This is the case when there are three flow paths, that is, when the fluid resistance is 6 to 10E+12Ns / m 5This is because, when the number of fluid channels is 4 to 9, the internal pressure increases due to the increase in internal pressure, and proper operation is impossible unless the supply pressure of the first and second fluids is increased to ensure a proper flow rate and flow velocity. In the following examples, experiments were conducted to optimize Example 7, which has three fluid channels.

[0170] Examples 14 to 19

[0171] In Experimental Example 2, it was confirmed that the driving pressure was lowest when there were three discharge channels. Below, the separation efficiency depending on the width of each channel was confirmed based on the case where there were three discharge channels. Each discharge channel was numbered from the top to bottom, and the ratio of particles discharged to each discharge channel was measured. The weight ratio of particles discharged from each channel depending on the number of discharge channels (number) and the sum of the channel widths (μm) is shown in Table 2 below.

[0172] [Table 2]

[0173] As shown in Table 2, when the sum of the channel widths was 900 μm (Example 19, FIG. 14(f)), a separation efficiency of over 90% was achieved. Furthermore, as shown in Examples 17 and 18 (FIGS. 14(d) and (e)), it was confirmed that when the channel width was reduced, particles with a size of 100 nm could be separated from the first channel with high purity. However, when the sum of the channel widths was less than 500 μm (Example 16, FIG. 14(c)), it was confirmed that the separation efficiency dropped sharply. In particular, it was confirmed that the proportion of particles discharged to the third channel also decreased, resulting in the loss of particles to the branch channel. In other words, in the present invention, the maximum separation efficiency was only achieved when there were three channels and the sum of the channel widths was 500 μm or more, and the separation efficiency dropped sharply when it was less than 500 μm.

[0174] In addition, when the total width of the channels was set to 1100 μm, it was confirmed that particles of 600 nm size were separated with high purity in the second channel. However, when the total width exceeded 1300 μm, the amount of particles discharged without being separated into the first channel increased, and the separation efficiency decreased.

[0175] Experimental Example 2

[0176] Practical particle separation experiments were conducted using Examples 5 to 13. 100 nm particles and 600 nm particles were mixed at the same weight ratio, and then mixed into the first fluid at a ratio of 1 wt% to conduct the experiment. The remaining conditions were the same as in Experimental Example 1. Each discharge channel was numbered from the top, and the ratio of particles discharged into each discharge channel was measured. If the ratio exceeded 80%, the particle with the largest amount (100 or 600) was recorded. Discharge channels where particles were mixed and discharged at a ratio of less than 80% were marked "mixed."

[0177] [Table 3]

[0178] As shown in Table 3, in Example 7 of the present invention, 100 nm particles and 600 nm particles were properly separated, and it was confirmed that the remaining particles were discharged through the third discharge channel (see FIG. 15). In Examples 5 and 6, which have one and two discharge channels, it was confirmed that the particles were not separated and discharged, and were mixed and discharged. Also, in Examples 8 to 13, which have four or more discharge channels, although separated particles were discharged through some discharge channels, most channels discharged mixed particles, confirming that the efficiency was very poor.

[0179] When this is combined with Experimental Example 1, it has three discharge flow paths and the fluid resistance is 6 to 10E+12 Ns / m. 5 It was confirmed that the highest efficiency can be achieved when

[0180] Experimental Example 3

[0181] We verified whether the particle separation device of the present invention can be used to separate exosomes and peptides from saliva.

[0182] The subjects gargled with the sample gargle solution and then spat the gargle solution into a collection container to collect saliva. The collected saliva was centrifuged at 2000 G for 10 minutes to remove cellular material and cell debris. For comparison, normal subjects (Example 22) and xerostomia patients (Example 23) were selected as subjects for the experiment.

[0183] Thereafter, it was confirmed whether exosomes (average size 100 μm) could be separated using the same particle separation device as in Example 19.

[0184] As shown in Figure 17, it was confirmed that large particles were successfully removed from both the samples of Examples 22 and 23. In particular, when using the particle separation device of the present invention, it was confirmed that exosomes could be separated at a high rate of 95% in normal subjects and 72% in patients with xerostomia.

[0185] Furthermore, to confirm the effectiveness of the particle separator of the present invention, an experiment was conducted to separate peptides from saliva in Example 23. As shown in Figure 18, when using an existing peptide separation kit (manufactured by Core Science), the separation efficiency was confirmed to be about 30%, but when using the particle separator of the present invention, the separation efficiency was confirmed to increase to 70%.

[0186] Experimental Example 4

[0187] An experiment was conducted to confirm the separation efficiency when using the separation device of Example 19 to separate exosomes from actual urine.

[0188] Urine samples were collected from a healthy subject (Example 24), a patient with glomerulonephritis (Example 25), and a patient with preeclampsia (Example 26), and then centrifuged at 400 G for 3 minutes to remove cellular material. The urine samples from which cellular material had been removed were centrifuged at 3,000 G for 15 minutes to remove cellular debris ( FIG. 19 ).

[0189] Thereafter, it was confirmed whether exosomes (average size 150 μm) could be separated using the same particle separator as in Example 19.

[0190] As shown in FIG. 20, it was confirmed that the samples of Examples 24, 25 and 26 all successfully removed large particles.

[0191] Hereinafter, for comparison with existing separation methods, the separation results (BEST), ultracentrifugation (UC) and precipitation results of Example 26 are plotted in one graph.

[0192] As shown in FIG. 21, in the case of Example 26 of the present invention, it was shown that exosomes could be isolated at a high rate, and the isolation efficiency was also confirmed to be superior to that of the conventional UC and Precipitation.

[0193] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. an input flow path through which one or more fluids enter; a plurality of discharge flow paths through which a plurality of particles contained in the fluid flowing in from the input flow path are separated and discharged according to their sizes; a branch flow path through which a portion of the one or more fluids is discharged, The particle separation device, wherein the discharge flow path formed close to the input flow path discharges relatively small particles based on the amount of movement of the fluid, and the discharge flow path formed away from the input flow path discharges relatively large particles.

2. The particle separator has a hydraulic resistance (R) of 6 to 10E+12 Ns / m, as expressed by the following equation: ５ 2. The particle separator of claim 1, wherein: [Equation 1] (where R is the fluid resistance, h is the height of the discharge flow path, w is the sum of the widths of the discharge flow path, μ is the kinematic viscosity of the first fluid, and L is the length of the flow path through which the particles pass.)

3. 3. The particle separator according to claim 2, wherein the sum of the widths of the plurality of discharge channels is 500 to 1100 μm.

4. The input flow path is a first input flow path into which a first fluid and a plurality of particles having different sizes are mixed and flow; 2. The particle separation device of claim 1, further comprising: a second input flow path into which a second fluid not containing particles flows; and a connecting flow path between the input flow path and the discharge flow path, the connecting flow path having one end connected to the first input flow path and the second input flow path and the other end connected to the discharge flow path, through which a third fluid composed of the first fluid and the second fluid flows.

5. The connecting flow path is A substrate; a first side formed on the substrate and connected to the first input channel; The particle separation device of claim 4 , further comprising: a second side surface on the substrate, the second side surface facing the first side surface and connected to the second input channel.

6. The particle separation device according to claim 5 , wherein the particles move by sliding against the first side surface.

7. The particle separator according to claim 6 , wherein the first side surface is inclined toward the second side surface relative to the substrate.

8. The particle separator of claim 7 , wherein the first side is flat, stepped, or arched.

9. The plurality of discharge channels include: The particle separator according to claim 1 , wherein the third fluid is spaced apart from the first fluid in a direction intersecting with a flow direction of the third fluid on the other end side of the connecting channel.

10. 2. The particle separator of claim 1, wherein a flow rate ratio of the first fluid to the second fluid flowing into the first input passage and the second input passage is 0.1:99.9 to 50:

50.

11. 2. The particle separator of claim 1, wherein the flow rate of the third fluid discharged through the branch channel is 0% to 99.9% of the flow rate of the third fluid discharged through the connecting channel.

12. 2. The particle separator of claim 1, wherein the second input channel is inclined at a predetermined angle relative to the first input channel.

13. an input flow path through which one or more fluids enter; a plurality of discharge flow paths through which a plurality of particles contained in the fluid flowing in from the input flow path are separated and discharged according to their sizes; a branch flow path through which a portion of the one or more fluids is discharged; Including, The particle separator has a hydraulic resistance (R) of 6 to 10E+12 Ns / m, as expressed by the following equation: ５ A particle separator. [Equation 1] (where R is the fluid resistance, h is the height of the discharge flow path, w is the sum of the widths of the discharge flow path, μ is the kinematic viscosity of the first fluid, and L is the length of the flow path through which the particles pass.)

14. In a method for separating particles by size using a particle separator, The particle separation device includes an input flow path through which one or more types of fluid flow in, a plurality of discharge flow paths through which a plurality of particles contained in the fluid flowing in from the input flow path are separated and discharged according to size, and a branch flow path through which a portion of the one or more types of fluid is discharged, wherein, based on the amount of movement of the fluid, the discharge flow path formed close to the input flow path discharges relatively small particles, and the discharge flow path formed away from the input flow path discharges relatively large particles.

15. The particle separation method includes: supplying particles and a first fluid mixture through a first one of the input channels; supplying a second fluid through a second one of the input channels; discharging the particles separated by size through a discharge channel; Discharging excess fluid through a branch channel; 15. The method of particle separation of claim 14, comprising:

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