Microchannel device, separation device, and separation method

Abstract

A microfluidic device includes a flow path rotating along a curve, in which a flow path cross-section obtained by cutting the flow path in a direction orthogonal to a direction in which a liquid in which particles are dispersed flows has an asymmetric shape. In the microfluidic device, in a flow path cross-section having an asymmetric shape, a flow path cross-section area S1 on the inner peripheral side with respect to a line segment LS that connects a point PP of an upper side edge (11) that is the maximum distance from a lower side edge (12) and the lower side edge at the shortest distance is larger than a flow path cross-section area S2 on the outer peripheral side with respect to the line segment LS.

Description

Technical Field

[0001] The present invention relates to a microfluidic device, a separation device comprising the microfluidic device, etc. Background Art

[0002] In recent years, in various fields such as engineering, chemistry, and biomedicine, there is a need for a technology for classifying particles dispersed in a liquid and a technology for separating and extracting specific blood cells (blood corpuscles) from a blood sample. To date, density gradient centrifugation for classifying, separating, and extracting a target object using a density difference is known, but in the density gradient centrifugation, a large centrifugal force is applied to the target object, and therefore, a state change (e.g., damage) may occur in the target object during the process.

[0003] Therefore, as a method for performing separation and extraction while preventing damage to a target object dispersed in a liquid, a method for making a liquid flow along a spiral flow path so as to classify or separate particles has been developed. For example, in the case of separating and extracting microparticles such as blood cells (blood corpuscles), a spiral flow path is formed by a flow path called a microflow path, which has a smaller flow path cross-sectional area. Usually, a microflow path refers to a narrow flow path, wherein the outer edge of the flow path cross section cut along a direction orthogonal to the liquid flow direction is formed by a combination of edges below 1 mm. However, it is not necessary that the microflow path is strictly limited to a flow path with a cross section formed by a combination of edges below 1 mm.

[0004] By using a spiral microchannel, miniaturization of the device and increase in processing speed (throughput) can be expected when separating and extracting minute particles such as blood cells (blood corpuscles). In addition, for example, even in applications where the parts in contact with the sample must be disposable (such as in medical examinations), if the structure of the microchannel can be simplified, mass production can be performed at low cost, and high practicality as a disposable inspection device can be expected.

[0005] Patent document 1 describes that a spiral micro-channel used in a micro-channel device has a trapezoidal cross-sectional shape defined by a radial inner side, a radial outer side, a bottom side, and an upper side of the spiral. More specifically, Patent document 1 describes a trapezoidal channel cross section in which the bottom side of the trapezoid is linear, the radial inner side and the outer side are substantially orthogonal to the bottom side, and the upper side is inclined relative to the bottom side.

[0006] Patent Document 2 describes a microchip which, instead of a spiral microchannel, is configured to move an exposed portion of a channel of an external channel device in order to switch the channel.

[0007] Patent Document 3 describes a control method for controlling the flow of a fluid by gelling a sol-gel transition substance added to the fluid by applying a stimulus to a desired portion on a microchannel of a non-spiral microchannel.

[0008] Citation list

[0009] Patent Literature

[0010] Patent Document 1: WO 2014 / 046621 A

[0011] Patent Document 2: JP 2005-214741 A

[0012] Patent Document 3: JP 2002-163022 A Summary of the invention

[0013] Technical issues

[0014] The micro-flow path device described in Patent Document 1 adopts a trapezoidal shape in which the height changes monotonically from the inside to the outside along the radial direction of the spiral. As a result, the position of the Dean vortex core can be closer to one side of the radial direction of the spiral compared to a micro-flow path with a rectangular cross section of uniform height.

[0015] Patent document 1 describes that if the cross-sectional shape of the flow path is a trapezoid (where the height of the radial inner side of the spiral is greater than the height of the radial outer side), the two Dean vortex cores are unevenly distributed on the radial inner side, thereby obtaining a micro-flow path suitable for concentrating and filtering particles. In addition, it describes that if the cross-sectional shape of the flow path is a trapezoid (where the height of the radial outer side of the spiral is greater than the height of the radial inner side of the spiral), the two Dean vortex cores are unevenly distributed on the outer side, thereby obtaining a micro-flow path suitable for separating particles by size.

[0016] However, as described in Patent Document 1, in a microchannel with a trapezoidal cross-sectional shape, the radial inner and outer sides of the spiral are basically orthogonal to the bottom side, and the height of the trapezoid changes monotonically from the radial inside to the radial outside, and the particle classification performance is not necessarily sufficient.

[0017] Therefore, there is a need for a microfluidic device having better particle classification performance than the related art.

[0018] Solution to the problem

[0019] According to one aspect of the present invention, a microfluidic device is provided, which includes a flow path rotating along a curve, wherein a flow path cross section obtained by cutting the flow path in a direction orthogonal to the direction of flow of a liquid in which particles are dispersed has an asymmetric shape, and a flow path cross-sectional area S1 on the inner peripheral side relative to a line segment LS is larger than a flow path cross-sectional area S2 on the outer peripheral side relative to the line segment LS, and the line segment LS connects a point PP on the upper side that is at a maximum distance from the lower side and the lower side at the shortest distance.

[0020] Beneficial effects of the present invention

[0021] According to the present invention, particles of different sizes contained in a liquid can be sorted with high accuracy using a microfluidic channel.

[0022] Other features and advantages of the present invention will become apparent from the following description with reference to the accompanying drawings, in which the same or similar structures are denoted by the same reference numerals. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic diagram showing the configuration of a separation device according to a first embodiment.

[0024] Figure 2 : is a schematic plan view showing a schematic configuration of a micro flow channel according to the embodiment.

[0025] Figure 3A It means along Figure 2 The schematic diagram shows a state where the plane CL cuts the microchannel.

[0026] Figure 3B is a view showing the cross-sectional shape of the flow path in the first embodiment.

[0027] Figure 4A This is a diagram for explaining the area of ​​each portion of the flow path cross section, etc.

[0028] Figure 4B This is a diagram for explaining the dimensions and the like of each portion of the flow path cross section.

[0029] Figure 5A This is a view for explaining the flow of liquid in the microchannel.

[0030] Figure 5B It is a view showing the distribution of particles in the flow path cross section of the relatively upstream portion of the spiral flow path.

[0031] Figure 5C is a view showing the distribution of particles in the flow path cross section of a relatively downstream portion of the spiral flow path.

[0032] Fig. 6A is a view showing the measurement results of the liquid collected in the container 8B.

[0033] Figure 6B is a view showing the measurement results of the liquid collected in the container 8A.

[0034] Figure 7 is an external view of a mold used to manufacture a microfluidic device.

[0035] Figure 8 is an external view of a resin molded product which is a component of a micro flow channel device.

[0036] Fig. 9 It is a schematic diagram for explaining the method of assembling the microfluidic device.

[0037] Fig.10 is an external view of the completed microfluidic device.

[0038] Fig.11A is a view showing the cross-sectional shape of the flow path, wherein, as another embodiment, the outer side 14 is curved.

[0039] Fig. 11B is a view showing the cross-sectional shape of a flow path, wherein, as another embodiment, the outer side 14 has a curved shape.

[0040] Fig. 11C 2 is a view showing the cross-sectional shape of the flow path, wherein, as another embodiment, the outer side 14 has a stepped shape.

[0041] Fig. 12A 1 is a view showing the cross-sectional shape of the flow path, wherein, as another embodiment, only one side of the lower side 12 is convex toward the outer peripheral side.

[0042] Fig. 12B : is a view showing the cross-sectional shape of the flow path, wherein, as another embodiment, only a portion within a predetermined height range protrudes toward the outer peripheral side.

[0043] Fig.13A 1 is a view showing a spiral microchannel which is three-dimensionally rotated as another embodiment.

[0044] Fig. 13B : is a view showing a spiral micro-channel which has a constant radius of curvature and rotates three-dimensionally as another embodiment.

[0045] Fig.14 is a view showing a rectangular cross-sectional shape of a spiral micro-channel serving as a comparative example.

[0046] Fig.15 : is a schematic diagram showing the configuration of a micro-flow channel system 100 according to the second embodiment.

[0047] Fig.16 is a schematic plan view showing a schematic configuration of a micro-channel according to the second embodiment.

[0048] Fig.17 It is a view showing the flow channel cross-sectional shape of the micro flow channel.

[0049] Fig.18 is a schematic diagram for describing a mechanism for separating particles having different particle diameters.

[0050] Fig.19 : is a schematic diagram showing the configuration of a micro-flow channel system 200 according to the third embodiment.

[0051] Fig. 20 : is a schematic diagram showing the configuration of a micro-flow channel system 300 according to the fourth embodiment.

[0052] Fig.21 : is a schematic diagram showing the configuration of a micro-flow channel system 400 according to the fifth embodiment.

[0053] Fig. 22 : is a schematic diagram showing the configuration of a micro-flow channel system 500 according to the sixth embodiment.

[0054] Fig.23 : is a schematic diagram showing a micro-flow channel system 91 according to a reference example.

[0055] Fig.24 This is a view showing the time-lapse of the actual flow rate in the microchannel. DETAILED DESCRIPTION

[0056] A micro-channel device, a separation device, etc. according to an embodiment of the present invention will be described with reference to the drawings. The embodiments described below are merely examples, and those skilled in the art may appropriately change and implement the detailed configuration without departing from the gist of the present invention.

[0057] In the drawings referred to in the following description of the embodiments and examples, elements denoted by the same reference numerals have the same functions unless otherwise specified.

[0058] In addition, for the convenience of illustration and description, the drawings may be schematic, and thus the shapes, sizes, arrangements, etc. of the elements in the drawings may not strictly match the shapes, sizes, arrangements, etc. of the actual elements.

[0059] In the following description, the term "particle" or "microparticle" is used to broadly include: solid particles containing organic materials, inorganic materials or both; and objects that can exist in a state of being dispersed in a liquid, such as blood cells (blood corpuscles) contained in blood.

[0060] [First embodiment]

[0061] (Structure of separation device)

[0062] Figure 1 1 is a schematic diagram showing the configuration of a separation device according to a first embodiment. The separation device 1 is a device for separating and extracting particles having a larger particle diameter and particles having a smaller particle diameter from particles dispersed in a liquid. The separation device 1 includes a liquid container 2, a pump 3, a microfluidic device 4, a connecting portion 5, a tube 6, a tube 6A, a tube 6B, a connecting portion 7A, a connecting portion 7B, a container 8A, and a container 8B.

[0063] The liquid container 2 is a container for holding a liquid (suspension) in which particles of different sizes are dispersed. As the liquid container 2, a syringe (for example, a 10 ml syringe manufactured by Terumo Corporation) can be used, but the liquid container 2 is not limited thereto.

[0064] The pump 3 is a pump for pushing the liquid (suspension) held in the liquid container 2 out of the liquid container 2. A syringe pump (for example, YSP-301 manufactured by YMC Co., Ltd.) can be used as the pump 3, but the pump 3 is not limited thereto.

[0065] The tube 6 is a tube connecting the pump 3 and the connection portion 5. The tube 6 serves as a flow path for guiding the liquid (suspension) pushed out of the liquid container 2 by the pump 3 to the micro-flow path device 4. For example, a tube having an inner diameter of 1 mm and 3mm outer diameter A silicone tube of may be used as the tube 6, but the tube 6 is not limited thereto.

[0066] The connection portion 5 is a portion that connects the tube 6 and the flow channel inlet of the micro-flow channel device 4. It is actually preferable that the connection portion 5 has a mechanism that allows the tube 6 to be easily attached to and detached from the micro-flow channel device 4.

[0067] The microfluidic device 4 is a device capable of separating particles in the liquid according to size and taking out particles from the connecting portion 7A and the connecting portion 7B respectively by allowing the liquid (suspension) introduced via the connecting portion 5 to flow along a flow path having a spiral shape. The spiral shape refers to a state along a rotation curve, and is generally a shape similar to a spiral. The details of the microfluidic device 4 will be described below.

[0068] The connection portion 7A is a portion connecting the tube 6A and the inner peripheral side of the outlet of the flow path of the micro-flow path device 4. The connection portion 7B is a portion connecting the tube 6B and the outer peripheral side of the outlet of the flow path of the micro-flow path device 4. It is actually preferable that the connection portion 7A and the connection portion 7B have a mechanism that allows the tube to be easily attached to and detached from the micro-flow path device 4.

[0069] The container 8A is a container connected to the connection portion 7A via the tube 6A, and stores a liquid containing particles having a larger particle diameter separated by the microfluidic device 4. The container 8B is a container connected to the connection portion 7B via the tube 6B, and stores a liquid containing particles having a smaller particle diameter separated by the microfluidic device 4.

[0070] (Structure of microfluidic device)

[0071] Next, the micro-channel device 4 will be described. Figure 2 1 is a schematic plan view showing the structure of the microchannel included in the microchannel device 4. Described from the upstream portion to the downstream portion of the microchannel, the microchannel starting from the connection portion 5 is connected to the outer peripheral side end portion 103 of the spiral portion through the straight portion 102, and rotates in a spiral shape to reach the inner peripheral side end portion 104. At the inner peripheral side end portion 104, the microchannel branches into two branches, the inner peripheral side branch and the outer peripheral side branch, the inner peripheral side branch extends to the connection portion 7A, and the outer peripheral side branch extends to the connection portion 7B. The outer peripheral side end portion 103 as the upstream end of the spiral portion is used as an injection portion for injecting liquid, and the inner peripheral side end portion 104 as the downstream end is used as a branch portion, at which the spiral channel branches into the inner peripheral side branch and the outer peripheral side branch.

[0072] For example, the length of the straight portion 102 may be 20 mm, the radius of curvature of the circle at the outer peripheral end portion 103 may be 24 mm, and the spiral rotates 8 times so that the radius of curvature decreases by 2 mm per turn, and the radius of curvature at the inner peripheral end portion 104 may be 8 mm. However, this is only an example, and the configuration of the microchannel is not limited to this example.

[0073] In the microfluidic device 4 according to the present embodiment, the flow path cross section of the spiral portion of the microfluidic path has a unique flow path cross section shape. The flow path cross section shape refers to the cross-sectional shape when the flow path is cut along a plane CL orthogonal to FL at an arbitrary position, wherein FL is the direction in which the liquid flows at an arbitrary position in the microfluidic path.

[0074] Figure 3A The schematic diagram shows the Figure 2 The plane CL shown cuts the state of the microchannel. The spiral microchannel is a tubular channel defined by an upper surface US, a lower surface BS, an inner peripheral side surface IS, and an outer peripheral side surface OS. The interval between the upper surface US and the lower surface BS of the channel gradually increases from the inner peripheral side toward the outer peripheral side, and the outer peripheral side surface OS is inclined relative to the inner peripheral side surface IS. As a result, the cross-sectional shape of the channel is an asymmetric quadrilateral.

[0075] The following will refer to Figures 3B to 4BThe characteristic flow path cross-sectional shape in this embodiment is described in detail. In this embodiment, the flow path cross-sectional shape has an asymmetric quadrilateral shape, which includes an upper side 11 corresponding to the upper surface US of the tubular path, a lower side 12 corresponding to the lower surface BS of the tubular path, an inner side 13 corresponding to the inner peripheral side surface IS of the tubular path, and an outer side 14 corresponding to the outer peripheral side surface OS of the tubular path.

[0076] like Figure 3B As shown, when the distance between the upper side 11 and the lower side 12 is h, h gradually increases from the inner peripheral side toward the outer peripheral side, and the distance to the lower side 12 is the maximum (hmax) at point PP, which is the outer peripheral end portion of the upper side 11. That is, the point PP at which the distance to the lower side 12 is the largest is the boundary point between the upper side 11 and the outer side 14. In other words, the boundary between the upper surface US and the outer peripheral side surface OS of the microchannel is at a position where the distance between the upper surface US and the lower surface BS becomes the largest. In the example shown, the portion of the upper side 11 from the point connected to the inner side 13 of the flow path cross section to the point PP is formed by a straight line.

[0077] Figure 4A Indicates the width of each part of the flow path cross-sectional shape. Plan view ( Figure 2 ) is W1, the width of the spiral microchannel in the upper side 11 from the inner peripheral end portion to the point PP is W2, and the width of the outer side 14 from the point PP to the outer peripheral end portion is W3. It should be noted that W2 can be interpreted as the width of the upper side 11 in a plan view, and W3 can be interpreted as the width of the outer side 14 in a plan view.

[0078] As shown in the following formula (1), W2 is greater than W3.

[0079] W2>W3···(1)

[0080] This formula means that the point PP on the upper side 11 having the largest distance to the lower side 12 (ie, the point PP which is the peripheral end portion of the upper side 11) is located on the peripheral side relative to the center in the width direction in a plan view of the microchannel.

[0081] In this example, since the width of the inner side edge 13 in the plan view is 0, W1 = W2 + W3, but even if the inner side edge 13 is inclined and has a width in the plan view, point PP (that is, the peripheral end portion of the upper side edge 11) is still located on the peripheral side relative to the center of the microchannel in the width direction.

[0082] In addition, when the point on the upper side 11 that is the longest in distance to the lower side 12 is defined as point PP, the line segment connecting point PP and the lower side 12 at the shortest distance is defined as line segment LS, as shown in the figure. When the line segment LS is used as a boundary, the flow path cross-sectional area on the inner peripheral side is represented by S1, and the area on the outer peripheral side is represented by S2, S1 is greater than S2, as shown in the following formula (2).

[0083] S1>S2···(2)

[0084] In addition, if Figure 4B As shown, when the length of the line segment LS is h1, and the angle formed by the line segment LS and the outer side 14 is θ1, the relationship of the following formula (3) is established.

[0085] 10°<θ1≤arctan(W2 / h1)···(3)

[0086] In the spiral microchannel having the above-mentioned characteristic cross-sectional shape, the Dean flow D1 and the Dean flow D2 can be formed in the microchannel, as shown in FIG. Figure 5A As shown by the dotted line in .

[0087] As described above, in the present embodiment, the point PP at which the distance between the upper side 11 and the lower side 12 is the largest is arranged on the peripheral side relative to the center of the micro-flow path in the width direction. Therefore, the vortex center of the Dean flow D1 and the Dean flow D2 is shifted from the center in the width direction to the peripheral side, and the gap between the Dean flow D1 and the Dean flow D2 expands in the vertical direction from the inner peripheral side toward the outer peripheral side. In addition, the portion protruding toward the peripheral side from the line segment LS connecting the point PP and the lower side 12 at the shortest distance in the plan view can be used as an area V, in which particles with smaller diameters guided by the combination of Dean drag and inertial lift are stably unevenly distributed on the peripheral side. If the flow path cross section is continuously observed while moving with the flow, particles with smaller particle diameters appear to gradually stagnate in the area V on the peripheral side, so the area V can also be called a stagnation area V.

[0088] For example, in the case of separating blood cells having a larger particle diameter (e.g., white blood cells) and blood cells having a smaller particle diameter (e.g., red blood cells) from blood cells contained in blood, excellent separation performance can be exhibited by forming a microchannel having a cross-sectional shape satisfying the above formulas (1) to (3). When observing the distribution of blood cells in blood in a spiral microchannel, as blood cells flow from upstream to downstream in the spiral channel, a distribution from Figure 5B The status shown is Figure 5CThe state of the state shown changes, and the blood cells with smaller particle diameters are stably unevenly distributed in the region V. When the blood cells with smaller particle diameters can stably stay in the region V (stagnation region V), it is possible to suppress the blood cells from migrating to the inner peripheral side along the Dean vortex again. As a result, it is possible to reduce the migration and mixing of blood cells with smaller particle diameters into the blood cells with larger particle diameters unevenly distributed on the inner peripheral side in the microchannel. Therefore, blood cells (such as white blood cells and red blood cells) separated with high purity can be taken out from the connection part 7A and the connection part 7B of the microchannel device 4.

[0089] (Method for manufacturing microfluidic device)

[0090] Next, a method for manufacturing the micro-channel device 4 will be described. As a manufacturing method suitable for mass production, a method for manufacturing a resin micro-channel by injection molding using a metal mold will be described. However, this is an example, and manufacturing can be performed by a manufacturing method different from the above-mentioned manufacturing method.

[0091] First, a metal mold for transfer molding of the spiral flow path is manufactured. For example, NAK 80 is used as a base material and the mold is produced by cutting using Makino's V56 vertical machining center. Figure 7 The mold 113 includes a molding surface 114 for transfer molding the spiral micro-channel and an attachment hole 115 for attaching to an injection molding machine.

[0092] Subsequently, injection molding is performed using the mold 113. For example, the mold is arranged in an injection molding machine SE-75DU manufactured by Sumitomo Heavy Industries, Ltd., and injection molding is performed using a resin material PanliteLV-2250Z manufactured by Teijin Co., Ltd. (as a molding material).

[0093] Figure 8 1 shows the appearance of a resin molded product 116, and a groove 117 as a part of a spiral micro-channel is formed on the upper surface of the resin molded product 116. Next, holes 118 penetrating the rear surface of the resin molded product 116 are formed at three locations of the end portion of the groove 117 (see FIG. Fig. 9 ) to provide a connection portion 5, a connection portion 7A and a connection portion 7B (see Figure 2 ). The size of the hole 118 may be, for example, Φ1.5 mm, but is not limited thereto.

[0094] Next, if Fig. 9As shown, the resin molded product 116 is positioned at a position where the surface where the groove 117 is formed faces the substrate 120, and the resin molded product 116 and the substrate 120 are bonded by an adhesive 119. For example, an adhesive transfer tape #9969 for microfluidic diagnostic chips manufactured by 3M Japan Co., Ltd. can be used as the adhesive 119, and a polycarbonate resin sheet of model PCTSH manufactured by MISUMI Corporation can be used as the substrate 120.

[0095] Next, the microfluidic device 4 is completed by bonding the transfer port 122 to the three holes of the resin molded product 116, in which the connection portion 5, the connection portion 7A and the connection portion 7B are provided. Fig.10 shown.

[0096] Example

[0097] A specific example according to the first embodiment will be described below. In the example, the spiral portion of the microchannel (see Figure 4B ) has a cross-sectional shape in which W1=675 μm, W2=600 μm, h1=150 μm, h2=100 μm, and θ2=60°. For the shape of the microchannel in a plan view (see Figure 2 ), the length of the straight portion 102 is 20 mm, the radius of curvature of the circle at the outer peripheral end portion 103 is 24 mm, the spiral rotates eight times so that the radius of curvature decreases by 2 mm per turn, and the radius of curvature at the inner peripheral end portion 104 is 8 mm.

[0098] In order to verify the classification performance of microparticles dispersed in a liquid, two types of samples were prepared, and the classification performance (separation performance according to particle size) was evaluated.

[0099] (Sample 1) Suspension of resin particles

[0100] Beads made of polystyrene with a particle diameter of 5 μm (particle standard 4205A) and beads with a particle diameter of 10 μm (particle standard 4210A) were dispersed in pure water to prepare a suspension. The components of the suspension were adjusted so that the total amount of the suspension was 5 mL, the number ratio of 5 μm particles was 99.85%, and the number ratio of 10 μm particles was 0.15%.

[0101] (Sample 2) Diluted blood

[0102] The blood collected from the donor was diluted 1000 times with physiological saline (Dalbec phosphate buffered saline manufactured by NACALAI TESQUE, Ca- and Mg-free). The total amount of the diluted blood was 5 mL.

[0103] (Classification performance of sample 1)

[0104] 5 mL of the suspension containing beads of sample 1 was filled into the liquid container 2, and the liquid was supplied to the microfluidic device 4 at a flow rate of 1.2 [mL / min] using the pump 3. How many particles of various sizes were contained in the liquid separated and collected in the container 8A and the container 8B by the microfluidic device 4 was measured. Specifically, the particle size distribution measuring instrument FPIA-3000 manufactured by Sysmex Corporation was used to measure the particles contained in the liquid collected in each container.

[0105] Here, the collection rate and the removal rate are used as indicators for evaluating the classification performance (separation performance according to particle size) of the separation device 1. The collection rate represents the ratio of the number of 10 μm beads collected in the container 8A to the total number of 10 μm beads collected in the container 8A and the container 8B. The removal rate represents the ratio of the number of 5 μm beads collected in the container 8B to the total number of 5 μm beads collected in the container 8A and the container 8B. The "collection rate" and "removal rate" are terms used for convenience (assuming the purpose of extracting 10 μm beads from a suspension and using the beads), and may be expressed by different names as long as the terms are used as numerical values ​​representing the performance of separating 10 μm beads and 5 μm beads contained in the suspension.

[0106] In the example, the removal rate of sample 1 was 75.7%, and the collection rate of sample 1 was 99.8%. From the value of the removal rate, it can be seen that about 70% or more of the beads with a small size of 5 μm were discharged from the connection part 7B on the outer peripheral side of the spiral microchannel to the container 8B. From the value of the collection rate, it can be seen that most of the beads with a large size of 10 μm were collected from the connection part 7A on the inner peripheral side of the spiral microchannel to the container 8A.

[0107] (Classification performance of sample 2)

[0108] Sample 2 was supplied to the micro-channel device 4 in the same procedure as sample 1, and the liquid was collected in container 8A and container 8B. Then, how many cells of various sizes were contained in the liquid separated and collected in container 8A and container 8B was analyzed. A flow cytometer SA 3800 manufactured by Sony Corporation was used, and the cell count was 50000. The ratio of lymphocytes contained in each liquid separated and collected in container 8A and container 8B was measured to grasp the separation effect of blood cells as microparticles.

[0109] Fig. 6A Graph 1 shows the measurement results of the liquid collected in the container 8B from the outlet on the outer peripheral side of the spiral microchannel. Figure 6BThe measurement results of the liquid collected in the container 8A from the outlet on the inner circumference side of the spiral microchannel are shown. The horizontal axis FSC represents the intensity of the forward scattered light of the flow cytometer and is proportional to the size of the blood cell. The vertical axis SSC represents the intensity of the side scattered light of the flow cytometer and represents the internal complexity of the blood cell. The point cloud in the grid (grid 1) shown in the figure corresponds to the point cloud of the lymphocyte. Fig. 6A As shown in FIG. 1 , the cells contained in the liquid collected from the outer peripheral side of the spiral microchannel are cells with low FSC, that is, many cells have a small size, and the proportion of lymphocytes is about 3.9%. Figure 6B As shown, among the cells contained in the liquid collected from the inner peripheral side of the spiral microchannel, the cell population with a small size decreased, and the proportion of lymphocytes was as high as 37.0%. According to such results, it was confirmed that cells with a small size were easily discharged to the outside of the spiral microchannel, and cells with a large size were easily collected from the inside.

[0110] Here, as a comparative example, a comparison with a case of using a micro-channel device in which the cross-sectional shape of a micro-channel having a spiral shape is rectangular is described. Fig.14 The rectangular cross-sectional shape 201 of the spiral micro-flow path used as a comparative example is shown. The width W4 of the flow path is 675 μm, and the height h4 is 105 μm. The spiral shape in the plan view is the same as that in the example. The flow path cross-sectional area of ​​the comparative example is different from that of the example, and the flow velocity is different when the same flow rate is set, and an appropriate comparison cannot be made. Therefore, the flow rate of the comparative example is set so that the average flow velocity in the flow path is the same as that of the example.

[0111] As described above, blood collected from two donors was diluted 1000 times and made to flow through the micro-channel devices of the example and the comparative example, and the liquid collected from the inner peripheral side outlet of the spiral micro-channel was analyzed by the above-mentioned flow cytometer. The ratio of lymphocytes was extracted as described above, and the comparison results are shown in Table 1. As shown in Table 1, for the blood cells of any donor, the results obtained show that the extraction ratio of lymphocytes in the example is higher than that of the comparative example in which the cross-sectional shape of the channel is rectangular. That is, it was confirmed that the example is suitable for separating blood cells from blood according to size and extracting blood cells of a desired type.

[0112] [Table 1]

[0113]

[0114] [Modification example]

[0115] It should be noted that the present invention is not limited to the above-mentioned embodiments and examples, and many modifications can be made within the technical concept of the present invention. The spiral microchannel of the microchannel device according to the present invention can have the following features, and is not limited to the above-mentioned embodiments and examples.

[0116] (1) The spiral portion has an asymmetric flow path cross section.

[0117] (2) The flow path cross-sectional area S1 on the inner side relative to the line segment LS is larger than the flow path cross-sectional area S2 on the outer side relative to the line segment LS, and the line segment LS connects the point PP of the upper side that is the longest distance from the lower side and the lower side at the shortest distance.

[0118] Since the flow path cross section has an asymmetric shape (wherein a stagnation area having a cross-sectional area smaller than that of the inner peripheral side is formed on the outer peripheral side relative to the line segment LS in the width direction), the center position of the Dean vortex can be positioned on the outer peripheral side relative to the center, which is different from the case where the flow path cross section is rectangular. As a result, particles with a smaller particle diameter that have moved to the outer peripheral side can be prevented from migrating to the inner peripheral side again along the Dean vortex. In other words, the mixing of particles with a smaller particle diameter into aggregates of particles with a larger particle diameter on the inner peripheral side that are unevenly distributed in the microchannel due to the Dean flow due to migration is reduced. Therefore, unlike the WO

[0119] Compared with the microfluidic device with a trapezoidal flow path cross section described in 2014 / 046621A, particles can be separated and removed with higher purity.

[0120] In the above Figure 4B In the example of FIG. 1 , the outer side 14 of the flow path cross section is a line segment along a straight line. However, for example, Fig.11A As shown, the outer side 14 may be a curved outer side 14 in which a plurality of line segments having different inclinations relative to the lower side 12 are connected. Alternatively, a portion or all of the outer side 14 may be curved. Fig. 11B The curved shape shown, or the outer side 14 can have a Fig. 11C By setting the shape of the outer side 14 using any one shape or a combination thereof according to the condition of the particles to be separated and extracted, the shape of the Dean flow in the flow path space and the position, shape and size of the stagnant area can be adjusted and high classification performance can be achieved.

[0121] In addition, Figure 4B and 11A In the examples of FIG. 11C , the region V (stagnation region) for stably unevenly distributing particles having a smaller diameter has a shape in which the outer peripheral side protrudes from the upper side 11 toward the lower side 12, but the region V may have other shapes. For example, Fig. 12A and 12B As shown, in order to form the region V (stagnation region), a protruding portion may be provided on the outer side 14, in which only a portion having a height within a predetermined range protrudes toward the outer peripheral side.

[0122] exist Figure 4B and 11A In the examples of 11C, the inner peripheral side and point PP are connected by a straight line. However, a curved line or a wavy line may be used as long as the distance between point PP and the lower side is maximized.

[0123] In addition, for example, Figure 2 In the example of , the spiral microchannel has a structure in which liquid is injected from the outer peripheral side end portion 103, and the spiral microchannel branches into two branches, an inner peripheral side branch and an outer peripheral side branch, at the inner peripheral side end portion 104, so as to separate and collect liquid, but the spiral microchannel is not limited thereto. That is, liquid may be injected from the inner peripheral side end portion of the spiral microchannel, and the spiral microchannel may branch into two branches, an inner peripheral side branch and an outer peripheral side branch, at the outer peripheral side end portion, so as to separate and collect liquid.

[0124] In addition, if Figure 8 As shown in the above-mentioned embodiment, the spiral microchannel has a structure in which the spiral rotates in a two-dimensional plane, but Fig.13A As shown in the schematic, the spiral can rotate in three-dimensional space. In the case of three-dimensional rotation of the spiral, a spiral structure rotating with a constant radius of curvature can be adopted, such as Fig. 13B shown.

[0125] In addition, the target object to be processed by the micro-channel device or separation device according to the present invention is not limited to a suspension containing polystyrene particles or blood, and various liquids containing various particles having different sizes can be processed as the target object.

[0126] As described above, according to the present invention, by having a characteristic flow path cross-sectional shape, a micro flow path device having excellent particle classification performance can be realized.

[0127] The specific examples of the methods performed in the reference embodiments will be described below to facilitate understanding of the second to sixth embodiments of the present invention. The examples of the methods performed in the reference embodiments include methods for separating cells of different sizes by using a microfluidic channel, for example, methods for separating blood cells (hereinafter referred to as blood cells) according to size. For example, red blood cells and white blood cells are separated from blood cells, and the extracted white blood cells are used for biomedical research and clinical research. In order to obtain useful research samples, it is necessary to separate and extract white blood cells from a liquid containing blood cells with high purity. Even for purposes different from the research, it is still necessary to separate and extract the target blood cells with high purity.

[0128] For example, according to Fig.23 The micro-flow channel system 91 of the illustrated reference embodiment includes a spiral flow channel 94 as a spiral micro-flow channel, and blood cells can be separated according to size differences in the spiral flow channel 94 .

[0129] Here, the operation of the microfluidic system 91 according to the reference embodiment will be briefly described. Blood diluted with physiological saline or the like is filled into a syringe 92, and the syringe 92 is attached to a syringe pump 93. The syringe 92 is connected to an injection port 95 of a spiral flow path 94 through a tube 96. The other end of the spiral flow path 94 branches into two ports: an internal discharge port 98A disposed on the inner side of the spiral and an external discharge port 98B disposed on the outer side of the spiral. The internal discharge port 98A is connected to a first collection container 911 through a tube 99A, and the external discharge port 98B is connected to a second collection container 912 through a tube 99B.

[0130] When the syringe 92 is pushed out by the syringe pump 93 so as to reach a flow rate suitable for separating blood cells in the spiral flow path 94, the diluted blood in the syringe 92 flows into the spiral flow path 94. Although the mechanism will be described below, in the cross section of the spiral flow path, blood cells with a large size (such as white blood cells) mainly flow in the radial direction inside the spiral flow path 94, and blood cells with a small size (such as red blood cells and platelets) mainly flow in the radial direction outside the spiral flow path 94. Therefore, the cell suspension containing a large number of white blood cells is discharged to the inner discharge port 98A of the spiral flow path 94, and the cell suspension containing a large number of red blood cells and platelets is discharged to the outer discharge port 98B.

[0131] The cell suspension discharged from the corresponding discharge port is collected in the above-mentioned first collection container 911 and the second collection container 912. Here, the target cells to be separated and extracted with high purity are white blood cells, and the white blood cells are collected in the first collection container 911. In addition, red blood cells and platelets are considered to be unnecessary cells, and the unnecessary cells are discharged to the second collection container 912. As described above, blood cells can be separated and collected with a simple system configuration by using the microfluidic system 91.

[0132] However, the inventors have found that when the above operation is performed, a large amount of unnecessary red blood cells and the like enter the first collection container 911 for collecting white blood cells. The details will be described below. Before starting the above operation of separating blood cells, the spiral flow path 94 is not filled with fluid but is empty. When the cell suspension suddenly flows in a state where the spiral flow path 94 is empty, the air in the flow path stagnates in the internal discharge port 98A, the external discharge port 98B, etc., and in some cases, appropriate liquid supply or separation cannot be performed. In order to prevent such a problem, physiological saline or the like that does not contain blood cells is made to flow into the spiral flow path 94 in advance so that the air in the spiral flow path is discharged before the cell suspension flows into the spiral flow path 94.

[0133] At this time, for example, if unavoidable tiny foreign matter or the like has entered the spiral flow path 94 when the spiral flow path 94 is manufactured, the foreign matter may be stored together with physiological saline or the like in the first collection container 911 for collecting target white blood cells.

[0134] Once the air in the spiral flow path 94 is eliminated, the cell suspension is supplied from the syringe 92 filled with the cell suspension to the spiral flow path 94 by the syringe pump 93 at a set flow rate suitable for separating blood cells. Fig.24 The time evolution of the actual flow rate is shown in FIG. 1 . It can be seen that a certain amount of time is required (in Fig.24 In the example shown, this is approximately 100 seconds).

[0135] Since the desired cell separation cannot be performed at a set flow rate or lower in the spiral flow path 94, unwanted red blood cells and the like are discharged into the first collection container 911 for collecting white blood cells before the set flow rate is reached. Therefore, the purity of white blood cells in the liquid entering the first collection container 911 is reduced.

[0136] As described above, in the period from when the air in the spiral flow path 94 is removed to when the flow rate reaches the set flow rate after the supply of the cell suspension is started, the liquid is discharged from the internal discharge port 98A of the spiral flow path 94 to the first collection container 911 for collecting white blood cells (as the target object), which is disadvantageous. As a method of preventing such a problem, it is conceivable to provide a means for switching the flow path so that when the cell suspension does not flow at a predetermined flow rate, the liquid discharged from the internal discharge port 98A is not stored in the first collection container 911.

[0137] The above-mentioned patent document 2 describes a microchip that is configured to move the exposed portion of the flow path of the external flow path device in order to switch the flow path, and it is conceivable that the microchip is applied to switch the flow path in the spiral micro-flow path. However, in the method of patent document 2, the flow path is also formed in a cover covering the flow path, and the liquid supply line is switched by rotating the cover. However, in this method, it is difficult to move the cover formed with the flow path in a sealed state without liquid leakage. Moreover, a drive mechanism for moving the cover is required, which may make the device complicated.

[0138] The above-mentioned patent document 3 describes a method of adding a sol-gel transition substance to a fluid and gelling the fluid at a portion of the flow path where the fluid flow is not desired so as to block the flow. It is conceivable that the method of patent document 3 is applied to the switching of the flow path in a spiral micro-flow path, but in this method, there is a possibility that the physical properties of the fluid are changed and the desired separation cannot be performed or the cells are affected. Therefore, an embodiment of the present invention that can solve this problem will be described below.

[0139] [Second embodiment]

[0140] (Structure of Microfluidic System 100)

[0141] Fig.15 1 is a schematic diagram showing the configuration of the microfluidic system 100 according to the present embodiment. The microfluidic system 100 includes a syringe 2A, a syringe 2B, a syringe pump 3A, a syringe pump 3B, a microfluidic device 4 including a spiral flow path, a first container 211, a second container 212, and a control unit CNT.

[0142] The control unit CNT is a computer for controlling the operation of each part of the microfluidic system 100, and internally includes a central processing unit (CPU), a read-only memory (ROM), a random access memory (RAM), an input / output (I / O) port, etc. The ROM stores the operation program of the microfluidic system 100. The program for performing various types of processing according to the separation method of the present embodiment can be stored in the ROM similarly to other operation programs, but can also be loaded into the RAM from the outside through a network. Alternatively, the program can be loaded into the RAM through a computer-readable recording medium that records the program.

[0143] The syringe 2A is connected to the connection part 5, which is an injection port of the spiral flow path included in the micro-flow path device 4, through the tube 26A, and the syringe 2B is connected to the connection part 5 through the tube 26B. The syringe 2A and the syringe pump 3A form a first supply part that can supply a liquid with dispersed particles to the micro-flow path. The syringe 2B and the syringe pump 3B form a second supply part that can supply a liquid without dispersed particles to the micro-flow path.

[0144] The pinch valve 27A is provided on the tube 26A, and the pinch valve 27B is provided on the tube 26B. Since the operations of the pinch valve 27A and the pinch valve 27B can be controlled by the control unit CNT, it is possible to automatically control which of the syringes 2A and 2B the liquid will be supplied to the connection portion 5 which is the injection port of the spiral flow path included in the micro-flow path device 4.

[0145] The spiral flow path included in the micro-flow path device 4 branches into two branches at the downstream end: an inner peripheral side branch including a connection portion 7A (the connection portion 7A is an internal discharge port) and an outer peripheral side branch including a connection portion 7B (the connection portion 7B is an external discharge port). The connection portion 7A as an internal discharge port is connected to the tube 9A, and the connection portion 7B as an external discharge port is connected to the tube 9B. The clamping valve 10A (first opening / closing portion) is arranged in the middle of the tube 9A (first tube). The connection portion 7A as an internal discharge port is connected to the first container 211 through the tube 9A. The connection portion 7B as an external discharge port is connected to the second container 212 through the tube 9B (second tube). The tube 9A and the tube 9B are connected by the tube 9C (third tube), and the clamping valve 10B (second opening / closing portion) is arranged in the middle of the tube 9C. Since the operations of the pinch valve 10A and the pinch valve 10B can be controlled by the control unit CNT, it is possible to automatically control to which of the first container 211 and the second container 212 the liquid discharged from the connection portion 7A as the internal discharge port flows. On the other hand, since the connection portion 7B is directly connected to the second container 212 through the tube 9B, the liquid discharged from the connection portion 7B as the external discharge port naturally flows into the second container 212.

[0146] In other words, the microfluidic system 100 includes a first flow path that can connect and disconnect the inner peripheral side branch and the first container 211 and a second flow path that connects the outer peripheral side branch and the second container 212 and can connect and disconnect the inner peripheral side branch and the second container.

[0147] The microfluidic system 100 has a function of separating blood cells contained in diluted blood into white blood cells and (red blood cells and platelets). Here, for convenience, white blood cells, which are target cells to be separated and extracted with high purity, are referred to as essential cells, and red blood cells / platelets are referred to as non-essential cells. As described below, the liquid containing high-purity essential cells separated by the spiral flow path contained in the microfluidic device 4 is stored (collected) in the first container 211, and the liquid containing low-purity essential cells or the liquid containing non-essential cells and impurities is discharged to the second container 212.

[0148] The following will refer to Figures 16 to 18The spiral flow channel included in the micro-flow channel device 4 is described. Fig.16 1 is a schematic plan view showing the structure of the spiral flow path included in the micro-flow path device 4. When describing from the upstream part to the downstream part of the spiral flow path, the micro-flow path starting from the connection part 5 (the connection part 5 is an injection port) is connected to the outer peripheral side end part 103 of the spiral part through the straight part 102, and rotates in a spiral shape so as to reach the inner peripheral side end part 104. At the inner peripheral side end part 104, the micro-flow path branches into two branches, the inner peripheral side branch and the outer peripheral side branch, and the inner peripheral side branch extends to the connection part 7A as the internal discharge port, and the outer peripheral side branch extends to the connection part 7B as the external discharge port. The outer peripheral side end part 103 as the upstream end of the spiral part is used as an injection part for injecting liquid into the spiral part, and the inner peripheral side end part 104 as the downstream end is used as a branch part, at which the spiral flow path branches into the inner peripheral side branch and the outer peripheral side branch.

[0149] For example, the length of the straight portion 102 may be 20 mm, the radius of curvature of the circumference at the outer peripheral end portion 103 may be 24 mm, the spiral rotates 8 times so that the radius of curvature decreases by 2 mm per turn, and the radius of curvature at the inner peripheral end portion 104 may be 8 mm. However, this is only an example, and the configuration of the microchannel is not limited to this example.

[0150] Fig.17 4 is a view showing the flow path cross-sectional shape of the spiral flow path included in the micro-flow path device 4. That is, the flow path cross-sectional shape refers to the cross-sectional shape when the flow path is cut along a plane CL orthogonal to FL at an arbitrary position, where FL is the direction in which the liquid flows at an arbitrary position in the micro-flow path, such as Fig.16 For example, the width 107 of the flow path is 675 μm, the height 108 of the inner circumference of the spiral is 80 μm, and the height 110 of the highest point 109 on the outer circumference is 130 μm. In addition, the distance 111 from the inner circumference end portion of the spiral to the point 109 may be 600 μm, and the angle 112 of the outer circumference end portion of the flow path may be 30°.

[0151] The following will refer to Fig.18 The mechanism by which the spiral flow path included in the micro-flow path device 4 separates blood cells into white blood cells and other blood cells (red blood cells and platelets) is briefly described. Fig.18As shown, when the cell suspension is caused to flow through a set flow path in a spiral flow path, a secondary flow is generated in the radial direction of the spiral flow path. The secondary flow is called Dean vortex. Small-sized red blood cells and platelets move outward in the radial direction of the flow path through the Dean vortex. On the other hand, large-sized white blood cells move inward in the radial direction of the flow path by inertial lift (not shown). Therefore, since red blood cells flow outward in the radial direction of the flow path and white blood cells easily flow inward in the radial direction, white blood cells and other blood cells (red blood cells and platelets) can pass through the spiral flow path as shown in FIG. Fig.16 The two branch discharge ports shown are separated.

[0152] As specific examples of the components of the microfluidic system 100, for example, a syringe with a volume of 10 mL manufactured by Terumo Corporation can be used as the syringe 2A and the syringe 2B. For example, YSP-301 manufactured by YMC Co., Ltd. can be used as the syringe pump 3A and the syringe pump 3B. For example, a syringe with an inner diameter of 1 mm and 3mm outer diameter Silicone tubes of can be used as the tube 26A, the tube 26B, the tube 9A, the tube 9B, and the tube 9C. For example, PS-1015NC manufactured by Takasago Electric Co., Ltd. can be used as the pinch valve 27A, the pinch valve 27B, the pinch valve 10A, and the pinch valve 10B.

[0153] Syringe 2A is filled with diluted blood obtained by diluting whole blood 500 times with physiological saline (e.g., Dalbec phosphate buffered saline manufactured by NACALAI TESQUE, which does not contain Ca and Mg). The dilution rate is not limited to 500 times, but if the dilution rate is too low, the blood cell separation mechanism described below becomes difficult to function, so the dilution rate is preferably 100 times or more. Alternatively, syringe 2A may be filled with a cell suspension obtained by collecting white blood cells captured by a filter using physiological saline or the like after roughly removing red blood cells using a filter or the like (without diluting the whole blood). Syringe 2B is filled with physiological saline that does not contain cells (e.g., Dalbec phosphate buffered saline manufactured by NACALAI TESQUE, which does not contain Ca and Mg).

[0154] It should be noted that the specific examples of the above-mentioned constituent elements are merely examples, and the present embodiment is not limited thereto.

[0155] (Operation of Microfluidic System 100)

[0156] Next, the operation of the microfluidic system 100 will be described.

[0157] First, a startup operation is performed. That is, the control unit CNT closes the clamping valve 27A and opens the clamping valve 27B so as to drive the syringe pump 3B. As a result, the physiological saline filled in the syringe 2B is injected into the spiral flow path contained in the microfluidic device 4, and the spiral flow path is filled with the physiological saline. The startup operation is performed so that the air in the flow path is discharged from the flow path. This is because, if the air remains in the flow path, when the cell suspension is subsequently made to flow into the flow path, the flow of the cells is disturbed by the air and the cells cannot be separated with high precision. In the present embodiment, for example, the amount of physiological saline is set to 5 [mL] at startup, and the flow rate during liquid supply is 1.0 [mL / min], but the present invention is not limited to this.

[0158] In the present embodiment, during the startup operation, the control unit CNT closes the pinch valve 10A and opens the pinch valve 10B. By doing so, even if there are minute foreign matters or the like in the flow path and the foreign matters flow out from the connection portion 7A as the internal discharge port, the foreign matters do not flow into the first container 211 for collecting necessary cells, and the foreign matters are discharged to the second container 212. In addition, since the foreign matters are discharged to the second container 212 in the case where the foreign matters flow out from the connection portion 7B as the external discharge port, the foreign matters do not flow into the first container 211 for collecting necessary cells. In the startup operation, it can be said that the first process is performed, in which the first flow path does not connect the first container to both the inner circumference side branch and the outer circumference side branch, and the second flow path connects both the inner circumference side branch and the outer circumference side branch to the second container.

[0159] When the startup operation is completed, the blood cell separation operation is started. First, the control unit CNT opens the clamp valve 27A and closes the clamp valve 27B. Then, the syringe pump 3A is driven to supply the diluted blood in the syringe 2A to the spiral flow path included in the micro-flow path device 4 at a set flow rate. For example, 1.2 [mL / min] is set as a flow rate suitable for blood cell separation, but the flow rate is not limited to this example.

[0160] At the same time, if Fig.24 As shown, even when the syringe pump 3A is driven at a set flow rate, a certain amount of time (in hours) is required before the flow rate per unit time (flow velocity) in the spiral flow path reaches a predetermined flow rate suitable for separating blood cells. Fig.24 100 seconds in the example of FIG. 1). Therefore, after the start of the driving of the syringe pump 3A, before the flow rate (flow speed) of the liquid flowing in the spiral flow path 94 reaches a predetermined flow rate, the spiral flow path cannot exhibit sufficient blood cell separation performance. Therefore, red blood cells and platelets are mixed in the liquid discharged from the connecting portion 7A as the internal discharge port, and the purity of white blood cells is reduced.

[0161] Therefore, in the present embodiment, the control unit CNT closes the pinch valve 10A so as to prevent the liquid from the connection portion 7A as the internal discharge port from flowing into the first container 211 until the flow rate per unit time (flow speed) of the liquid flowing in the spiral flow path included in the micro-flow path device 4 reaches a predetermined flow rate. At the same time, the control unit CNT opens the pinch valve 10B so that the liquid from the connection portion 7A as the internal discharge port is discharged to the second container 212. A first process may be performed in which the first flow path does not connect both the inner circumference side branch and the outer circumference side branch to the first container, and the second flow path connects both the inner circumference side branch and the outer circumference side branch to the second container until the flow rate (flow speed) reaches a predetermined flow rate.

[0162] Then, when the flow rate reaches a predetermined flow rate, the control unit CNT opens the clamping valve 10A and closes the clamping valve 10B. Through this operation, only the liquid containing high-purity essential cells (e.g., white blood cells) separated by the spiral flow path included in the micro-flow path device 4 is collected in the first container 211, and the liquid containing non-essential cells (e.g., red blood cells) is discharged to the second container 212. In other words, when the flow rate reaches a predetermined flow rate, the second process can be performed, in which the first flow path connects the inner peripheral side branch and the first container 211, and the second flow path connects the outer peripheral side branch and the second container 212.

[0163] The control unit CNT can control the opening and closing of the pinch valve 10A and the pinch valve 10B according to, for example, a measurement value of a flow measurement unit (not shown). For example, a flow meter for measuring the flow rate of the diluted blood supplied to the tube 26A may be provided, so that the control unit CNT can monitor the flow rate of the diluted blood flowing in the spiral flow path included in the micro-flow path device 4. The control unit CNT can control the opening and closing of the pinch valve 10A and the pinch valve 10B by comparing the measurement result of the flow meter with the set predetermined flow rate, as described above.

[0164] Alternatively, a predetermined time required for the flow rate of the diluted blood flowing in the spiral flow path included in the micro-flow path device 4 to reach a predetermined flow rate from the start of driving the syringe pump 3A may be measured in advance and stored in the control unit CNT. The control unit CNT may measure the time using a built-in timer while starting to drive the syringe pump 3A, and switch between opening and closing of the pinch valve 10A and the pinch valve 10B when the predetermined time has passed, as described above.

[0165] This embodiment and the reference Fig.23 The method of the reference example described is compared. Table 2 shows the ratio between essential cells (white blood cells) and non-essential cells (red blood cells) contained in the first container 211 for collecting essential cells.

[0166] Table 2

[0167]

[0168] As shown in Table 2, according to this embodiment, the proportion of white blood cells contained in the cells collected in the first container is significantly increased compared to the method according to the reference embodiment. That is, in this embodiment, the spiral microchannel can be used to classify particles of different sizes contained in the liquid with high accuracy.

[0169] [Third embodiment]

[0170] The following will refer to Fig.19 A micro-fluidic system 200 according to a second embodiment will be described. Fig.19 In the present invention, the constituent elements common to the second embodiment are denoted by the same reference numerals. The description of the contents common to the second embodiment will be simplified or omitted.

[0171] The microfluidic system 200 according to the present embodiment also includes: a first flow path that can connect and disconnect the inner side branch and the first container 211; and a second flow path that connects the outer side branch and the second container 212, and can connect and disconnect the inner side branch and the second container.

[0172] The present embodiment is different from the second embodiment in that a device is provided for switching a flow path from a spiral flow path contained in the microfluidic device 4 to a first container 211 and a second container 212. In the second embodiment, a clamping valve 10A and a clamping valve 10B are provided to switch the flow path. On the other hand, in the present embodiment, a double clamping valve 10C (third opening / closing portion) capable of clamping both tubes 9A and 9C is provided at a portion branching into tubes 9A (first tube) and tubes 9C (third tube). Since a double clamping valve is provided, tube 9C can be opened while tube 9A is closed by one valve. For example, PM-1015W manufactured by Takasago Electric Co., Ltd. can be used as a double clamping valve 10C.

[0173] In this embodiment, the control unit CNT controls the double pinch valve 10C to close the tube 9A and open the tube 9C until the flow rate reaches the set flow rate during the startup operation or the blood cell separation operation. As a result, it is possible to prevent unnecessary liquid from flowing into the first container 211 for collecting necessary cells and discharge the unnecessary liquid to the second container 212.

[0174] Then, when the conditions for obtaining the desired separation performance are met, the control unit CNT controls the double pinch valve 10C to open the tube 9A and close the tube 9C. As a result, the liquid containing the necessary cells at high purity can enter the first container 211, and the liquid containing the unnecessary cells can be discharged to the second container 212.

[0175] In this embodiment, by using a double pinch valve, the number of components can be reduced, and the discharge side flow path can be easily assembled. In this embodiment, similar to the first embodiment, the spiral micro-channel can also be used to classify particles of different sizes contained in the liquid with high accuracy.

[0176] [Fourth embodiment]

[0177] The following will refer to Fig. 20 A micro-flow channel system 300 according to a fourth embodiment will be described. Fig. 20 In the present invention, the constituent elements common to the second embodiment are denoted by the same reference numerals, and the description of the contents common to the second embodiment will be simplified or omitted.

[0178] The microfluidic system 300 according to this embodiment also includes: a first flow path, which can connect and disconnect the inner side branch and the first container 211; and a second flow path, which connects the outer side branch and the second container 212, and can connect and disconnect the inner side branch and the second container 212.

[0179] In the second embodiment, tube 9C is connected to tube 9B, and one tube is connected to the second container 212. On the other hand, in this embodiment, tube 9C (third tube) and tube 9B (second tube) are not connected, and tubes 9C and 9B can discharge liquid to the second container 212 independently.

[0180] In this embodiment, a branch portion of the tube (a connecting portion between the tubes) can be reduced, the pressure loss in the discharge path can be reduced when the liquid is supplied, and the discharge path can be easily assembled, which is very advantageous.

[0181] In this embodiment, similar to the second embodiment, particles of different sizes contained in a liquid can also be classified with high accuracy using the spiral microchannel.

[0182] [Fifth embodiment]

[0183] The following will refer to Fig.21 A micro-fluidic system 400 according to a fifth embodiment will be described. Fig.21 In the present invention, the constituent elements common to the second embodiment are denoted by the same reference numerals, and the description of the contents common to the second embodiment will be simplified or omitted.

[0184] The microfluidic system 400 according to this embodiment also includes: a first flow path, which can connect and disconnect the inner side branch and the first container 211; and a second flow path, which connects the outer side branch and the second container 212, and can connect and disconnect the inner side branch and the second container 212.

[0185] In this embodiment, similar to the fourth embodiment, the tube 9C (third tube) and the tube 9B (second tube) are not connected, and the tubes 9C and 9B can independently discharge the liquid to the second container 212. In this embodiment, similar to the third embodiment, a double pinch valve 10C is provided at a portion branching into the tube 9A (first tube) and the tube 9C. Since the double pinch valve is provided, the tube 9C can be opened while the tube 9A is closed by one valve. For example, PM-1015W manufactured by Takasago Electric Co., Ltd. can be used as the double pinch valve 10C.

[0186] In this embodiment, similar to the second embodiment, particles of different sizes contained in a liquid can also be classified with high accuracy using the spiral microchannel. In addition, the advantages of the third embodiment and the fourth embodiment can be achieved.

[0187] [Sixth embodiment]

[0188] The following will refer to Fig. 22 A micro-flow channel system 500 (separation device) according to the sixth embodiment is described. Fig. 22 In the present invention, the constituent elements common to the fourth embodiment are denoted by the same reference numerals, and the description of the contents common to the second or fourth embodiment will be simplified or omitted.

[0189] The microfluidic system 500 (separation device) according to this embodiment also includes: a first flow path, which can connect and disconnect the inner side branch and the first container 211; and a second flow path, which connects the outer side branch and the second container 212, and can connect and disconnect the inner side branch and the second container 212.

[0190] The flow path configuration according to this embodiment is the same as that of the fourth embodiment, but is different from the fourth embodiment in that a first container 211 for collecting necessary cells is installed in an incubator 213 capable of maintaining a cell culture environment.

[0191] In the present embodiment, similar to the second embodiment, the spiral microchannel can also be used to classify particles of different sizes contained in the liquid with high precision. Moreover, in the present embodiment, the necessary cells are collected in the first container 211 together with the culture medium, and the first container 211 is installed in the incubator 213, and, for example, an environment with a temperature of 37°C, a humidity of 90%, and a carbon dioxide (CO2) gas concentration of 5% is maintained. Therefore, the cells collected in the first container 211 can be cultured as they are. By culturing the cells directly after blood cell separation, the number of necessary cells can be increased according to use, while reducing the risk of contamination, cell loss when moving the cells to another container, etc.

[0192] [Modification example]

[0193] It should be noted that the present invention is not limited to the above-described embodiments, and many modifications can be made within the technical spirit of the present invention. For example, all or some of the above-described different embodiments can be implemented in combination.

[0194] For example, in the above-mentioned embodiment, the case where the necessary particles to be separated with high precision are particles with a large particle diameter (e.g., white blood cells) is described as an example. Therefore, a flow path configuration is adopted in which the liquid discharged from the inner peripheral side discharge port during the separation operation is collected in the first container 211, and the liquid is not discharged to the first container 211 during the startup operation or just after the liquid containing particles is injected. On the other hand, in the case where the necessary particles to be separated with high precision are particles with a small particle diameter, a flow path configuration is adopted in which the liquid discharged from the outer peripheral side discharge port during the separation operation is collected in the first container 211. Then, it is sufficient not to discharge the liquid to the first container 211 during the startup operation or just after the liquid containing particles is injected. That is, according to the particle diameter of the necessary particles, it is sufficient that one of the inner peripheral side branch and the outer peripheral side branch is disconnectably connected to the first container 211 through the first flow path and the other of the inner peripheral side branch and the outer peripheral side branch is always connected to the second container 212 through the second flow path.

[0195] In addition, the control unit CNT does not necessarily have to directly control all the connections and disconnections of the corresponding flow paths, and some or all of the connections and disconnections may be operated by the operator. In this case, a notification device such as a lamp, an audio device, or a display device may be provided so that the operator can appropriately operate the opening / closing device such as a valve, and the control unit CNT may notify the operator of the opening / closing timing through the notification device. Alternatively, the operator may be notified of the timing of switching from the first process to the second process.

[0196] In the sixth embodiment, the first container 211 is installed in the incubator 213 in the flow path configuration according to the fourth embodiment, but the first container 211 may be installed in the incubator 213 in the flow path configuration according to the second, third, and fifth embodiments.

[0197] In addition, it is preferable to use the spiral flow path according to the first embodiment as the spiral flow path used in each of the second to sixth embodiments.

[0198] In each embodiment, the spiral flow path is used to classify particles with different particle diameters. However, the present invention is not necessarily limited to the spiral flow path, as long as the flow path is a micro flow path along a curve having a function of performing separation according to particle diameter, micro flow paths having other forms can be used. The spiral flow path is suitable for implementation due to the advantage of high throughput.

[0199] In addition, the device for controlling the connection and disconnection of the flow path is not limited to the pinch valve or the double pinch valve, and for example, a two-way stopcock or a pipe clamp may be used.

[0200] In addition, the particles separated by the microfluidic device are not limited to blood cells, but may be various particles, such as solid particles containing organic materials, inorganic materials, or both, and cells other than blood cells.

[0201] This specification discloses at least the following items.

[0202] [Article 1]

[0203] A microfluidic device, comprising:

[0204] flow path that rotates along a curve, where

[0205] The cross section of the flow path obtained by cutting the flow path in a direction orthogonal to the direction in which the liquid in which the particles are dispersed flows has an asymmetric shape, and

[0206] The flow path cross-sectional area S1 on the inner peripheral side of the line segment LS connecting the point PP of the upper side with the maximum distance from the lower side and the lower side at the shortest distance is larger than the flow path cross-sectional area S2 on the outer peripheral side of the line segment LS.

[0207] [Article 2]

[0208] The micro-flow path device according to item 1, wherein a stagnant area is formed in the flow path space on the outer peripheral side relative to the line segment LS, and particles having a particle diameter smaller than a predetermined particle diameter among particles contained in the liquid are unevenly distributed in the stagnant area.

[0209] [Article 3]

[0210] The microfluidic device according to clause 1, wherein the outer side of the flow path cross section includes a portion along a straight line.

[0211] [Article 4]

[0212] The microfluidic device according to item 3, wherein a relationship of 10°<θ1≤arctan(W2 / h1) is established, wherein h1 represents the length of the line segment LS, and θ1 represents the angle formed by the line segment LS and the straight line.

[0213] [Article 5]

[0214] The micro-flow path device according to clause 1, wherein a portion of the upper side from a point connected to the inner side of the flow path cross section to a point PP is formed by a straight line.

[0215] [Article 6]

[0216] The micro-flow path device according to clause 1, wherein the outer side of the flow path cross section includes a portion in which a plurality of line segments having different inclinations relative to the lower side are connected.

[0217] [Article 7]

[0218] The micro-flow path device according to clause 2, wherein, among the outer sides of the flow path cross section, only a portion having a height within a predetermined range protrudes toward the outer peripheral side relative to the line segment LS.

[0219] [Article 8]

[0220] A microfluidic device according to any one of clauses 1 to 7, wherein the curve is rotated in a two-dimensional plane.

[0221] [Article 9]

[0222] The microfluidic device according to any one of clauses 1 to 8, wherein the curve is a curve whose inner radius of curvature is smaller than its outer radius of curvature.

[0223] [Article 10]

[0224] A microfluidic device, comprising:

[0225] A micro-channel that rotates along a curve and is provided with a branch portion at a downstream end, at which the micro-channel branches into an inner peripheral side branch and an outer peripheral side branch;

[0226] a first supply portion configured to supply a liquid in which particles are dispersed to the microchannel;

[0227] a first container configured to store a liquid containing particles having a predetermined particle diameter;

[0228] a second container;

[0229] a first flow path provided so that any one of the inner peripheral side branch and the outer peripheral side branch is connected to and disconnected from the first container; and

[0230] The second flow path is provided so that the other of the inner circumference side branch and the outer circumference side branch is connected to the second container, and so that either of the inner circumference side branch and the outer circumference side branch is connected to and disconnected from the second container.

[0231] [Article 11]

[0232] The microfluidic device according to clause 10, wherein

[0233] When the first supply portion starts supplying the liquid in which particles are dispersed to the microchannel,

[0234] After executing the first process, a second process is executed in which the first flow path does not connect the first container to both the inner side branch and the outer side branch, and the second flow path connects both the inner side branch and the outer side branch to the second container, and in which the first flow path connects any one of the inner side branch and the outer side branch to the first container, and the second flow path connects the other of the inner side branch and the outer side branch to the second container.

[0235] [Article 12]

[0236] The micro-fluidic channel device according to clause 11, further comprising: a flow measurement unit configured to measure a flow rate of the liquid in which particles are dispersed supplied from the first supply portion to the micro-fluidic channel,

[0237] When the measurement result of the flow measurement unit reaches a predetermined flow rate, switching from the first process to the second process is performed.

[0238] [Article 13]

[0239] The micro-fluidic device according to clause 11, wherein the switching from the first process to the second process is performed after the first process is performed for a predetermined time.

[0240] [Article 14]

[0241] The microfluidic device according to item 10, further comprising: a second supply portion configured to supply the liquid in which particles are not dispersed to the microfluidic channel,

[0242] Here, after the liquid in which particles are not dispersed is supplied from the second supply portion to the microchannel, the liquid in which particles are dispersed is supplied from the first supply portion to the microchannel.

[0243] [Article 15]

[0244] A microfluidic device according to item 14, wherein, while a liquid in which particles are not dispersed is supplied from a second supply portion to the microfluidic channel, the first flow path does not connect the first container to both the inner side branch and the outer side branch, and the second flow path connects both the inner side branch and the outer side branch to the second container.

[0245] [Article 16]

[0246] The micro-channel device according to any one of clauses 11 to 15, wherein the micro-channel discharges the liquid containing particles having a predetermined particle diameter from the inner peripheral side branch, and in the second process, the first channel connects the inner peripheral side branch and the first container.

[0247] [Article 17]

[0248] The microfluidic device according to clause 10, wherein

[0249] The first flow path includes: a first tube provided so that one of the inner peripheral side branch and the outer peripheral side branch is connected to the first container; and a first opening / closing portion provided in a middle portion of the first tube, and

[0250] The second flow path includes: a second tube, which is arranged so that the other of the inner peripheral side branch and the outer peripheral side branch is connected to the second container; a third tube, which is arranged so that the upstream part of the first tube relative to the first opening / closing part is connected to the second tube; and a second opening / closing part, which is arranged in the middle of the third tube.

[0251] [Article 18]

[0252] The microfluidic device according to clause 10, further comprising:

[0253] a first tube provided so that any one of the inner peripheral side branch and the outer peripheral side branch is connected to the first container;

[0254] a second tube disposed so that the other of the inner peripheral side branch and the outer peripheral side branch is connected to the second container;

[0255] a third tube configured to connect the first tube and the second tube; and

[0256] A third opening / closing portion is provided to control opening and closing of both the first tube and the third tube.

[0257] [Article 19]

[0258] The microfluidic device according to clause 10, wherein

[0259] The first flow path includes: a first tube provided so that one of the inner peripheral side branch and the outer peripheral side branch is connected to the first container; and a first opening / closing portion provided in a middle portion of the first tube, and

[0260] The second flow path includes: a second tube, which is arranged so that the other of the inner peripheral side branch and the outer peripheral side branch is connected to the second container; a third tube, which is arranged so that the upstream part of the first tube relative to the first opening / closing part is connected to the second container; and a second opening / closing part, which is arranged in the middle of the third tube.

[0261] [Article 20]

[0262] The microfluidic device according to clause 10, further comprising:

[0263] a first tube provided so that any one of the inner peripheral side branch and the outer peripheral side branch is connected to the first container;

[0264] a second tube disposed so that the other of the inner peripheral side branch and the outer peripheral side branch is connected to the second container;

[0265] a third tube configured to connect the first tube and the second container; and

[0266] A third opening / closing portion is provided to control opening and closing of the first tube and the third tube.

[0267] [Article 21]

[0268] The microfluidic device according to any one of clauses 1 to 20, wherein the particles are solid particles comprising organic materials, inorganic materials, or both, or are cells.

[0269] [Article 22]

[0270] A separation device, comprising:

[0271] A microfluidic device according to any one of clauses 15 to 20; and

[0272] An incubator configured to culture cells, wherein

[0273] The particles are cells, and

[0274] The first container is installed in the incubator.

[0275] [Article 23]

[0276] A separation method comprising:

[0277] By using the microfluidic device according to any one of items 1 to 20, particles having a larger particle diameter or particles having a smaller particle diameter are separated from particles dispersed in a liquid.

[0278] Industrial Applicability

[0279] The present invention can be widely applied to devices for classifying particles dispersed in a liquid and devices for separating and extracting specific blood cells (blood corpuscles) from a blood sample in various fields such as engineering, chemistry, and biomedicine.

[0280] The present invention is not limited to the above-described embodiments, and various modifications and changes may be made without departing from the spirit and scope of the present invention. Therefore, in order to disclose the scope of the present invention, the following claims are attached.

[0281] Reference numerals list

[0282] 1 Separation device

[0283] 2 Liquid Containers

[0284] 3 Pumps

[0285] 4 Microfluidic Device

[0286] 5 Connection part

[0287] 6, 6A, 6B pipe

[0288] 7A, 7B connection part

[0289] 8A, 8B Container

[0290] 11 Upper side

[0291] 12 Lower side

[0292] 13 Inside edge

[0293] 14 Outer edge

[0294] 102 Straight section

[0295] 103 Outer peripheral end portion

[0296] 104 Inner circumferential side end portion

[0297] 113 Mould

[0298] 114 Molding surface

[0299] 115 Attachment hole

[0300] 116 Resin molding products

[0301] 117 slots

[0302] 118 holes

[0303] 119 Adhesive

[0304] 120 substrate

[0305] 122 Pipetting port

[0306] 201 Rectangular cross-sectional shape

[0307] BS lower surface

[0308] D1, D2 Dean Stream

[0309] IS Inner Surface

[0310] LS Segment

[0311] OS Outer Surface

[0312] US upper surface

[0313] V Stagnation Area

Claims

1. A microfluidic device, comprising: A flow path that rotates along a curve, wherein a cross section of the flow path obtained by cutting the flow path in a direction orthogonal to a direction in which the liquid in which the particles are dispersed flows has an asymmetric shape, and The flow path cross-sectional area S1 on the inner peripheral side relative to the line segment LS connecting the point PP of the upper side with the maximum distance from the lower side and the lower side at the shortest distance is larger than the flow path cross-sectional area S2 on the outer peripheral side relative to the line segment LS.

2. The microfluidic device according to claim 1, wherein: A stagnant area in which particles having a particle diameter smaller than a predetermined particle diameter among particles contained in the liquid are unevenly distributed is formed in the flow path space on the outer peripheral side with respect to the line segment LS.

3. The microfluidic device according to claim 1, wherein: The outer side of the flow path cross section includes a portion along a straight line.

4. The microfluidic device according to claim 3, wherein: A relationship of 10°<θ1≤arctan(W2 / h1) is established, where h1 represents the length of the line segment LS and θ1 represents the angle formed by the line segment LS and the straight line.

5. The microfluidic device according to claim 1, wherein: The portion of the upper side from the point connected to the inner side of the flow path cross section to the point PP is formed by a straight line.

6. The microfluidic device according to claim 1, wherein: The outer side of the flow path cross section includes a portion in which a plurality of line segments having different inclinations with respect to the lower side are connected.

7. The microfluidic device according to claim 2, wherein: Among the outer sides of the flow path cross section, only the portion having a height within a predetermined range protrudes toward the outer peripheral side relative to the line segment LS.

8. The microfluidic device according to any one of claims 1 to 7, wherein: The curve is rotated in a two-dimensional plane.

9. The microfluidic device according to any one of claims 1 to 8, wherein: The curve is a curve whose inner radius of curvature is smaller than its outer radius of curvature.

10. A microfluidic device, comprising: a micro-flow path, the micro-flow path rotating along a curve and provided with a branch portion at a downstream end, at which the micro-flow path branches into an inner peripheral side branch and an outer peripheral side branch; a first supply portion configured to supply a liquid in which particles are dispersed to the microchannel; a first container configured to store a liquid containing particles having a predetermined particle diameter; a second container; a first flow path configured to connect and disconnect any one of the inner peripheral side branch and the outer peripheral side branch to a first container; as well as The second flow path is provided so that the other of the inner circumference side branch and the outer circumference side branch is connected to the second container, and so that any one of the inner circumference side branch and the outer circumference side branch is connected to and disconnected from the second container.

11. The microfluidic device according to claim 10, wherein When the first supply portion starts supplying the liquid in which particles are dispersed to the microchannel, After executing the first process, a second process is executed in which the first flow path does not connect the first container to both the inner side branch and the outer side branch, and the second flow path connects both the inner side branch and the outer side branch to the second container, and in the second process, the first flow path connects any one of the inner side branch and the outer side branch to the first container, and the second flow path connects the other of the inner side branch and the outer side branch to the second container.

12. The microfluidic device according to claim 11, further comprising: a flow measurement unit configured to measure a flow rate of the liquid in which particles are dispersed, supplied from the first supply portion to the microchannel, When the measurement result of the flow measurement unit reaches a predetermined flow rate, switching from the first process to the second process is performed.

13. The microfluidic device according to claim 11, wherein: After the first process is executed for a predetermined time, switching is performed from the first process to the second process.

14. The microfluidic device according to claim 10, further comprising: a second supply portion configured to supply liquid in which particles are not dispersed to the microchannel, Here, after the liquid in which particles are not dispersed is supplied from the second supply portion to the microchannel, the liquid in which particles are dispersed is supplied from the first supply portion to the microchannel.

15. The microfluidic device according to claim 14, wherein: While liquid in which particles are not dispersed is supplied from the second supply portion to the microchannel, the first channel does not connect the first container to both the inner and outer branches, and the second channel connects both the inner and outer branches to the second container.

16. The microfluidic device according to any one of claims 11 to 15, wherein: The micro channel discharges a liquid containing particles having a predetermined particle diameter from the inner peripheral side branch, and in the second process, the first channel connects the inner peripheral side branch and a first container.

17. The microfluidic device according to claim 10, wherein First flow path includes: a first tube, the first tube being arranged so that one of the inner peripheral side branch and the outer peripheral side branch is connected to a first container; and a first opening / closing portion provided in a middle portion of the first tube, and The second flow path includes: a second tube, the second tube is arranged so that the other of the inner peripheral side branch and the outer peripheral side branch is connected to the second container; a third tube, the third tube is arranged so that the upstream part of the first tube relative to the first opening / closing part is connected to the second tube; and a second opening / closing part, the second opening / closing part is arranged in the middle of the third tube.

18. The microfluidic device according to claim 10, further comprising: a first tube, the first tube being arranged so that any one of the inner peripheral side branch and the outer peripheral side branch is connected to a first container; a second tube disposed so that the other of the inner peripheral side branch and the outer peripheral side branch is connected to a second container; a third tube, the third tube being configured to connect the first tube and the second tube; as well as A third opening / closing portion configured to control opening and closing of both the first tube and the third tube.

19. The microfluidic device according to claim 10, wherein First flow path includes: a first tube, the first tube being arranged so that one of the inner peripheral side branch and the outer peripheral side branch is connected to a first container; and a first opening / closing portion provided in a middle portion of the first tube, and The second flow path includes: a second tube, which is arranged so that the other of the inner peripheral side branch and the outer peripheral side branch is connected to the second container; a third tube, which is arranged so that the upstream part of the first tube relative to the first opening / closing part is connected to the second container; and a second opening / closing part, which is arranged in the middle of the third tube.

20. The microfluidic device according to claim 10, further comprising: a first tube, the first tube being arranged so that any one of the inner peripheral side branch and the outer peripheral side branch is connected to a first container; a second tube disposed so that the other of the inner peripheral side branch and the outer peripheral side branch is connected to a second container; a third tube, the third tube being configured to connect the first tube and the second container; as well as A third opening / closing portion configured to control opening and closing of the first tube and the third tube.

21. The microfluidic device according to any one of claims 1 to 20, wherein: The particles are solid particles comprising organic material, inorganic material, or both, or are cells.

22. A separation device comprising: The microfluidic device according to any one of claims 15 to 20; as well as An incubator configured to culture cells, wherein The particles are cells, and The first container is installed in the incubator.

23. A separation method comprising: By using the micro-flow channel device according to any one of claims 1 to 20, particles having a larger particle diameter or particles having a smaller particle diameter are separated from particles dispersed in a liquid.

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