Particle separation chip and particle separation device

Abstract

To provide a particle separation chip and a particle separation device which enable improvement of throughput when separating particles.SOLUTION: A particle separation chip 1 for separating a plurality of particles included in a sample on the basis of the sizes of the particles includes an upper substrate 10 and a lower substrate 20 having micro flow channels 1a therein, and a porous sheet 30 formed with a plurality of holes. The micro flow channels 1a includes: a main flow channel 100 having an inlet 101 provided on the upstream side, to which a first sample including first particles and second particles smaller than the first particles is sent, and an outlet 104 provided on the downstream side, from which a second sample mainly including the first particles is taken; and a branch flow channel 200 which communicates with the main flow channel 100 in a plurality of branch places in a flow direction of the main flow channel 100, and has an outlet 213 for taking out a third sample including the second particles on the downstream side. In the plurality of branch places, the main flow channel 100 and the branch flow channel 200 communicate with each other through the holes of the porous sheet 30.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to a particle separation chip and a particle separation device. [Background technology]

[0002] In recent years, various particle separation methods using microchannels have been developed. For example, Non-Patent Document 1 below discloses a particle separation method using hydrodynamic filtration. In this separation method, a microchannel consisting of a main channel and a plurality of branch channels branching off from the main channel is used. A sample is introduced into the main channel, and the particles are separated between the main channel and the branch channels based on the particle size. This separation method causes less damage to the particles due to separation, and also allows the structure of the channel to be simple. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Miyuki Matsuda and 2 others, "Classification of Blood Cells Using Hydrodynamic Filtration," IEEJ Transactions on Electrical Engineering, Vol. 128, No. 10, pp. 396-401, 2008. Summary of the Invention [Problem to be solved by the invention]

[0004] Microchannels utilizing the above-mentioned hydrodynamic filtration method may be usable at the laboratory level, but they do not satisfy the high sample processing capacity (throughput) required at a practical level, for example, in product development and clinical settings.

[0005] In view of the above problems, an object of the present invention is to provide a particle separation chip and a particle separation device that can provide sufficient sample processing capacity (throughput) in product development and clinical settings. [Means for solving the problem]

[0006] The particle separation chip (1, CP1 to CP5, CP11 to CP16) of the present invention includes a substrate (10, 20) having a microchannel (1a) therein and a porous sheet (30) having a plurality of holes (31). The microchannel (1a) includes a main channel (100) having an inlet (101) provided on the upstream side through which a sample containing a first particle and a second particle smaller than the first particle is fed, and a first outlet (104) provided on the downstream side through which a sample mainly containing the first particle is taken out, and a branch channel (200) that communicates with the main channel (100) at a plurality of branch points (B) along the flow direction of the main channel (100) and has a second outlet (213) on the downstream side through which a sample containing the second particle is taken out. At the plurality of branch points (B), the main channel (100) and the branch channel (200) communicate with each other via holes (31) in the porous sheet (30, PS1 to PS7).

[0007] According to the particle separation chip of the present invention, the main channel and the branch channels communicate with each other through holes in the porous sheet at a plurality of branch points (B), thereby providing sufficient sample processing capacity (throughput) in product development and clinical settings.

[0008] The particle separation device (2) of the present invention comprises the particle separation chip (1, CP1 to CP5, CP11 to CP16) of the above invention, an installation section (41) on which the particle separation chip (1, CP1 to CP5, CP11 to CP16) is placed, a liquid delivery section (61 to 63) that introduces a sample and sheath liquid into the sample inlet (101) and the sheath liquid inlet (102, 103) in the particle separation chip (1, CP1 to CP5, CP11 to CP16), respectively, and delivers the liquid in the microchannel (1a), a first collection section (64) for first particles that communicates with a first outlet (104), and a second collection section (65) for second particles that communicates with a second outlet (213).

[0009] According to the particle separation device of the present invention, the particle separation chip is placed on the installation section, and the liquid delivery section is driven to introduce the first sample and sheath liquid, thereby smoothly recovering the first particle and the second particle from the first recovery section and the second recovery section, respectively. Effect of the Invention

[0010] According to the present invention, it is possible to provide a particle separation chip and a particle separation device that can exhibit sufficient sample processing capacity (throughput) in product development sites and clinical sites. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a perspective view illustrating a schematic configuration of an upper substrate according to an embodiment. [Diagram 2] FIG. 2 is a perspective view illustrating a schematic configuration of a lower substrate according to the embodiment. [Diagram 3] FIG. 3 is a perspective view and an enlarged view showing a schematic configuration of a porous sheet according to an embodiment. [Figure 4] FIG. 4 is a perspective view that illustrates the assembly of an upper substrate, a lower substrate, and a porous sheet according to an embodiment. [Diagram 5] FIG. 5 is a perspective view that illustrates a schematic configuration of a particle separation chip according to an embodiment. [Figure 6] FIG. 6 is a diagram showing a schematic diagram of injection of a sample and a sheath liquid into an inlet of a particle separation chip and dispensing of the sample from an outlet according to an embodiment. [Figure 7] FIG. 7 is a schematic diagram illustrating that the range in which the first sample flows is limited by the sheath liquid according to the embodiment. [Figure 8] FIG. 8 is a schematic diagram illustrating that the range in which the first sample flows is limited by the sheath liquid according to the embodiment. [Figure 9] FIG. 9 is a perspective view that illustrates a portion near an intersection of a main flow path and a branch flow path according to an embodiment. [Figure 10] FIG. 10 is a schematic diagram illustrating separation of a first particle and a second particle at a branch point according to an embodiment. [Figure 11] FIG. 11 is an enlarged view that illustrates a schematic view of the vicinity of one of the branched flow paths according to the embodiment. [Figure 12]FIG. 12 is a perspective view that illustrates a configuration of a particle separator according to an embodiment. [Figure 13] FIG. 13 is a block diagram showing the functional configuration of the cell detection system according to the embodiment. [Figure 14] FIG. 14 is a diagram showing a schematic diagram of the sizes of the main channel and the branch channel in the embodiment. [Figure 15] FIG. 15 is a diagram showing a schematic diagram of the arrangement and size of holes in a porous sheet according to an embodiment. [Figure 16] FIG. 16 is a table showing the configurations of four types of chips according to an embodiment. [Figure 17] FIG. 17 is a table showing the configurations of seven types of porous sheets according to the embodiment. [Figure 18] FIG. 18 is a plan view showing a schematic configuration of four types of chips according to an embodiment. [Figure 19] FIG. 19 is a table showing the configurations of four types of particle separation chips and a table showing the configurations of three types of first samples according to the first embodiment. [Figure 20] FIG. 20 is a graph showing the red blood cell removal rate and the white blood cell removal rate of the four types of particle separation chips according to Example 1. [Figure 21] FIG. 21 is a table showing the configuration of the particle separation chip and a table showing the configuration of the first sample according to the second embodiment. [Figure 22] FIG. 22 is a graph showing the red blood cell removal rate, the white blood cell removal rate, and the A549 cell recovery rate of the particle separation chip according to Example 2. [Figure 23] FIG. 23 is a table showing the configurations of six types of particle separation chips and a table showing the configuration of the first sample according to the third embodiment. [Figure 24] FIG. 24 is a graph showing particle size distribution of Jurkat cells and A549 cells contained in the first sample according to Example 3. [Diagram 25] FIG. 25 is a graph showing the removal rate of Jurkat cells and the recovery rate of A549 cells for six types of particle separation chips according to Example 3. [Figure 26]FIG. 26 is a graph showing the flow rates to each outlet of six types of particle separation chips according to Example 3. [Figure 27] FIG. 27 is a graph showing the recovery rate for each particle size of six types of particle separation chips according to Example 3. [Figure 28] FIG. 28 is a diagram showing a schematic configuration of the main channel and the branch channel in a simulation of the amount of sample discharged into the branch channel, and a graph showing the results of the discharge ratio of the sample into the branch channel. [Figure 29] FIG. 29 is a diagram showing a schematic configuration of a branch flow path according to a modified example of the branch flow path. [Diagram 30] FIG. 30 is a plan view showing a configuration in the vicinity of the branch flow path according to a modified example of the branch flow path. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] <Embodiment> In the following embodiments, the present invention is applied to a particle separation chip and a particle separation device for separating a plurality of particles contained in a liquid sample based on the size of the particles. In the following embodiments, the particle separation chip separates a liquid sample (first sample) containing a first particle and a second particle smaller than the first particle into a liquid sample (second sample) containing mainly the first particle and a liquid sample (third sample) containing mainly the second particle. The first sample is a blood sample. The blood sample is, for example, whole blood or a sample prepared from whole blood. The first particle is, for example, circulating tumor cells (CTCs), and the second particle is, for example, white blood cells, red blood cells, platelets, and nanoparticles. The nanoparticles are, for example, extracellular vesicles such as exosomes, minute proteins, minute debris, and the like. The size of a particle is, for example, the diameter or radius of the particle, the area or perimeter when viewed in a plane, and the like. In the following description, the particle size indicates the diameter of the particle.

[0013] For convenience, each figure below is labeled with mutually orthogonal X, Y, and Z axes. The Z axis direction is the height direction of a particle separation chip 1 and a particle separation device 2, which will be described later. The XY plane is parallel to the horizontal plane. In the embodiment, the sample flows in the X axis direction, and in the figures, the left side of the flow path is called the upstream side, and the right side of the flow path is called the downstream side.

[0014] The configuration of the particle separation chip 1 will be described with reference to Figures 1 to 5. The particle separation chip 1 is composed of an upper substrate 10 (shown in Figure 1), a lower substrate 20 (shown in Figure 2), and a porous sheet 30 (shown in Figure 3) sandwiched between the upper substrate 10 and the lower substrate 20.

[0015] FIG. 1 is a perspective view that diagrammatically illustrates the configuration of the upper substrate 10. As shown in FIG.

[0016] The upper substrate 10 is configured as a plate-like member having an opposing surface 10a and an upper surface 10b parallel to the XY plane, with the distance between the two surfaces being D1. The upper substrate 10 is configured to be plane-symmetrical in the Y-axis direction with respect to a virtual vertical plane VP that perpendicularly intersects with the upper substrate 10 at a central axis CA parallel to the X-axis direction. The opposing surface 10a is a surface that faces the lower substrate 20, which will be described later, and the upper surface 10b is a surface that will become the upper surface of the particle separation chip 1 when the particle separation chip 1 is completed.

[0017] The upper substrate 10 is made of a material that allows the blood sample to flow smoothly over its surface and that can be processed with high precision, such as polydimethylsiloxane (PDMS). The upper substrate 10 may also be made of glass, cycloolefin polymer (COP), cyclic olefin copolymer (COC), or the like.

[0018] A main channel 100 having a predetermined depth d1 is formed in the opposing surface 10a. Four outlets 213 consisting of through holes are formed along the right end of the upper substrate. The main channel 100 and the four outlets 213 are formed, for example, by a semiconductor processing process. The main channel 100 has a recess recessed in the Z-axis direction relative to the opposing surface 10a, and the bottom surface of this recess is parallel to the XY plane. The main channel 100 is formed by overlapping the upper substrate 10 and the lower substrate 20, and a sample can be transported through the main channel 100. The four outlets 213 form a part of the branch channel 200 described later.

[0019] The main flow channel 100 includes inlets 101, 102, and 103 provided on the left side thereof, flow channels 111 and 112, a pair of flow channels 113, a flow channel 120 provided in the center thereof, and an outlet 104 provided on the right side thereof.

[0020] The inlets 101-103 and the outlet 104 are formed by through holes penetrating the upper substrate 10 in the Z-axis direction, and are arranged in a row along the vertical plane VP and the central axis CA. Two outlets 213 are arranged in a row on each side of the outlet 104 along the Y-axis. The four outlets 213 are formed by through holes penetrating the upper substrate 10 in the Z-axis direction.

[0021] The flow channels 111 and 112 extend in the X-axis direction and are disposed along a central axis CA. The widths of the flow channels 111 and 112 are constant. An upstream end of the flow channel 111 is connected to the inlet 101, a downstream end of the flow channel 111 is connected to the flow channel 120, and a center portion of the flow channel 111 is connected to the inlet 102.

[0022] The inlet 103 is located to the left of the inlet 101, and the left end of the flow path 112 is connected to the inlet 103. The pair of flow paths 113 branch off from the flow path 112 along both sides of the vertical plane VP in the Y-axis direction. The pair of flow paths 113 bypass the inlets 101 and 102 and connect the flow path 112 and the flow path 111. The right end of the pair of flow paths 113 is connected obliquely to the flow path 111 between the inlet 102 and the flow path 120. The width of the pair of flow paths 113 is constant.

[0023] The flow channel 120 extends in the X-axis direction and is disposed on a central axis CA. The width of the flow channel 120 in the Y-axis direction gradually increases from the connection portion of the flow channels 111, 120 to the center portion in the left portion of the flow channel 120. The width of the flow channel 120 in the Y-axis direction excluding the left portion is constant and is wider than the widths of the flow channels 111 to 113. The right end portion of the flow channel 120 is connected to the outlet 104.

[0024] The size of the main flow channel 100 can be set, for example, as follows.

[0025] When the particle separation chip 1 has an outer shape of 150 mm square in a plan view, the length of the flow channel 120 in the X-axis direction is set to, for example, 150 mm or less. The width of the flow channel 120 in the Y-axis direction is set to, for example, 50 mm or less, with approximately one-third of the length of one side of the outer shape as a guide.

[0026] The width of the flow channel 120 in the Y-axis direction is set to, for example, 500 μm or more when 5 mL of whole blood is flowed as a blood sample in the particle separation chip 1 in one particle separation process. According to this embodiment, when the width of the flow channel 120 in the Y-axis direction is 500 μm, for example, the time required to flow 5 mL of whole blood is 200 minutes, and the throughput of the particle separation process is 25 μL / min. When the width of the flow channel 120 in the Y-axis direction is 50 mm, for example, the time required to flow 5 mL of whole blood is 2 minutes, and the throughput of the particle separation process is 2.5 mL / min. When an even smaller amount of whole blood is flowed in the particle separation chip 1 in one particle separation process, the width of the flow channel 120 in the Y-axis direction may be set to, for example, 100 μm or more. The throughput is the amount of sample subjected to the particle separation process per unit time, and when the sample is a blood sample, it is the amount of undiluted whole blood subjected to the particle separation process per unit time.

[0027] The depth d1 of the flow channel 120 (the height in the Z-axis direction of the recess corresponding to the flow channel 120 formed in the upper substrate 10) is set to be larger than the particle size of the first particle so that the first particle can be transported within the flow channel 120. When the first particle is a CTC, the depth d1 of the flow channel 120 is set to, for example, 100 μm or more, and when the first particle is a nanoparticle, the depth d1 of the flow channel 120 is set to, for example, 50 nm or more. Since the flow channel 120 is formed by a semiconductor processing process, the depth d1 of the flow channel 120 is set to, for example, 1 mm or less.

[0028] For the same reason as the depth of the flow channel 120, the depth of the flow channels 111 to 113 is set to, for example, 50 nm or more and 1 mm or less.

[0029] FIG. 2 is a perspective view that illustrates a schematic configuration of the lower substrate 20. As shown in FIG.

[0030] The lower substrate 20 is configured as a plate-like member having an opposing surface 20a and a lower surface 20b parallel to the XY plane, with the distance between the two surfaces being D2. The lower substrate 20 is configured to be plane-symmetrical in the Y-axis direction with respect to a virtual vertical plane VP that perpendicularly intersects with the lower substrate 20 at a central axis CA parallel to the X-axis direction. The opposing surface 20a is a surface that faces the upper substrate 10, and the lower surface 20b is a surface that will become the lower surface of the particle separation chip 1 when the particle separation chip 1 is completed.

[0031] The lower substrate 20 is made of a material that allows the blood sample to flow smoothly over its surface and that can be processed with high precision. The lower substrate 20 of this embodiment is made of, for example, polydimethylsiloxane (PDMS) like the upper substrate 10. The lower substrate 20 may also be made of glass, cycloolefin polymer (COP), cyclic olefin copolymer (COC), or the like.

[0032] A branch channel 200 having a predetermined depth d2 is formed in the opposing surface 20a. The branch channel 200 is formed, for example, by a semiconductor processing process. The branch channel 200 has a recess recessed in the Z-axis direction with respect to the opposing surface 20a, and the bottom surface of the recess is parallel to the XY plane. The branch channel 200 is formed by overlapping the upper substrate 10 and the lower substrate 20, and is configured so that a sample can be transported through the channel.

[0033] The branch flow path 200 includes a plurality of flow paths 201, two relay flow paths 211, and four relay flow paths 212. The branch flow path 200 also includes the four outlets 213 shown in Fig. 1. In Fig. 2, a portion of the plurality of flow paths 201 and a portion of the two relay flow paths 211 are omitted by dotted lines for convenience.

[0034] The multiple flow paths 201 extend in parallel in the Y-axis direction and are arranged in the X-axis direction at predetermined intervals. The length of each flow path 201 in the Y-axis direction becomes shorter toward the right in the X-axis direction. That is, the overall outer shape of the multiple flow paths 201 is trapezoidal in a plan view.

[0035] The two relay flow paths 211 are disposed at one end and the other end of the multiple flow paths 201, respectively, and are connected to the multiple flow paths 201. The relay flow paths 211 extend so that both ends approach the central axis CA as they move to the right of the main flow path 100 (see FIG. 1). The four relay flow paths 212 extend parallel to the X-axis direction. Two relay flow paths 212 are connected to each of the relay flow path 211 on one end side of the multiple flow paths 201 and the relay flow path 211 on the other end side of the multiple flow paths 201.

[0036] The size of the branch flow path 200 can be set, for example, as follows.

[0037] When the particle separation chip 1 is formed to have an outer shape of 150 mm square in a plan view, the length of the multiple flow paths 201 in the Y-axis direction is set to, for example, 150 mm or less. Also, the length of each of the multiple flow paths 201 in the Y-axis direction is set to be longer than the width of the flow path 120 in the Y-axis direction.

[0038] The width (length in the X-axis direction) of the flow channel 201 is set to be larger than the particle size of the second particles so that the second particles can be transported within the flow channel 201. When the second particles are white blood cells, red blood cells, and platelets, the width of the flow channel 201 is set to, for example, 100 μm or more, and when the second particles are nanoparticles, the width of the flow channel 201 is set to, for example, 50 nm or more. The width of the flow channel 201 is set to, for example, 1 mm or less so that a sufficient number of flow channels 201 can be arranged.

[0039] The number of the multiple flow channels 201 is set to 2 or more and 15,000 or less. According to the inventors' study, when the width of the flow channel 201 is 100 μm and the interval between adjacent flow channels 201 is 50 μm, about 150 flow channels 201 can be formed. Furthermore, according to a semiconductor processing process, the width and interval of the flow channels 201 can be further reduced to about 1 / 100, so that 15,000 flow channels 201 can be formed. Furthermore, two or more flow channels 201 are arranged to realize a hydrodynamic filtration (HDF) method described later.

[0040] The depth d2 of the flow channel 201 (the height in the Z-axis direction of a recess formed in the lower substrate 20 corresponding to the flow channel 201) is also set to be larger than the particle size of the second particle so that the second particle can be transported within the flow channel 201. When the second particles are white blood cells, red blood cells, and platelets, the depth d2 of the flow channel 201 is set to, for example, 100 μm or more, and when the second particles are nanoparticles, the depth d2 of the flow channel 201 is set to, for example, 50 nm or more. Moreover, since the flow channel 201 is formed by a semiconductor processing process, the depth d2 of the flow channel 201 is set to, for example, 1 mm or less.

[0041] For the same reason as the depth of the flow channel 201, the depth of the relay flow channels 211 and 212 is set to, for example, 50 nm or more and 1 mm or less.

[0042] FIG. 3 is a perspective view and an enlarged view showing a schematic configuration of the porous sheet 30. As shown in FIG.

[0043] The porous sheet 30 is a thin-film sheet having a rectangular shape in a plan view. The porous sheet 30 has a plurality of holes 31 penetrating the porous sheet 30 in the Z-axis direction. As described later, the second particles flowing through the main flow path 100 move to the branch flow path 200 through the holes 31 of the porous sheet 30. The porous sheet 30 is made of a material that allows the blood sample to flow smoothly over the surface, can form the holes 31 with high accuracy, and is not easily broken. The porous sheet 30 of this embodiment is made of nickel, for example. The porous sheet 30 does not necessarily have to be made of metal, and may be made of glass, PDMS, or the like.

[0044] The size of the porous sheet 30 can be set, for example, as follows.

[0045] The thickness of the porous sheet 30 is set to, for example, 1 μm or more and 1 mm or less so that separation of the second particles by hydrodynamic filtration is realized. According to the study by the inventors, even if the thickness of the porous sheet 30 is set to 1 mm, the fluid resistance applied to the second particles passing through the holes 31 by the porous sheet 30 can be almost negligible. The pore size of the holes 31 is set to, for example, 30 nm or more and 100 μm or less, more preferably 50 nm or more and 100 μm or less so that the first particles smoothly proceed downstream in the flow path 120 of the main flow path 100.

[0046] The aperture ratio, which is the ratio of the area of ​​the holes 31 in the porous sheet 30, is set to, for example, greater than 0% and not greater than 50% in order to maintain the strength of the porous sheet 30. In this embodiment, all of the holes 31 formed in the porous sheet 30 have the same size and shape, and all of the holes 31 are evenly arranged in the porous sheet 30. Therefore, the aperture ratio of the porous sheet 30 is substantially the same at a plurality of branch points B (see FIG. 9) where the main flow path 100 and the branch flow paths 200 communicate with each other, which will be described later.

[0047] The porosity of the porous sheet 30 may be different at the branching points B. In this case, the number of holes 31 in the porous sheet 30 may be different at the branching points B, and the diameters of the holes 31 may be different. Furthermore, the number of holes 31 in the porous sheet 30 may be different depending on the region within one branching point B, and the diameters of the holes 31 may be different.

[0048] The length in the Y-axis direction of the porous sheet 30 is set to be longer than the width in the Y-axis direction of the constant-width portion of the flow channel 120 of the upper substrate 10. The length in the X-axis direction of the porous sheet 30 is set to be longer than the length in the X-axis direction of the entire plurality of flow channels 201 of the lower substrate 20.

[0049] Fig. 4 is a perspective view that shows a schematic diagram of the assembly of the upper substrate 10, the lower substrate 20, and the porous sheet 30. For convenience, in Fig. 4, the outline of the main channel 100 on the opposing surface 10a side is shown by a dashed line.

[0050] The upper substrate 10 shown in FIG. 1 is bonded to the lower substrate 20 shown in FIG. 2 in an inverted state. That is, the upper substrate 10 and the lower substrate 20 are bonded so that the opposing surface 10a of the upper substrate 10 and the opposing surface 20a of the lower substrate 20 face each other. At this time, the porous sheet 30 shown in FIG. 3 is placed between the upper substrate 10 and the lower substrate 20. When the upper substrate 10 and the lower substrate 20 are made of PDMS, the opposing surfaces 10a and 20a are bonded by surface modification of silicon dioxide on the surfaces of the opposing surfaces 10a and 20a with oxygen plasma. The surface modification may be performed by UV ozone treatment using ultraviolet light. In this way, the particle separation chip 1 is completed as shown in FIG. 5.

[0051] FIG. 5 is a perspective view that illustrates a schematic configuration of the particle separation chip 1. As shown in FIG.

[0052] The main channel 100 is constituted by a recess formed on the facing surface 10a of the upper substrate 10 and the facing surface 20a of the lower substrate 20 facing the recess. The branch channel 200 is constituted by a recess formed on the facing surface 20a of the lower substrate 20 and the facing surface 10a of the upper substrate 10 facing the recess. The main channel 100 and the branch channel 200 constitute a microchannel 1a. A microchannel is generally a channel with a depth and width of about 10 μm to 1 mm. An outlet 213 of the upper substrate 10 overlaps with an end portion on the right side in the X-axis direction of a relay channel 212 of the lower substrate 20, and the outlet 213 is connected to the relay channel 212.

[0053] In the X-axis direction, the porous sheet 30 is disposed so as to entirely cover the multiple flow paths 201 of the branch flow path 200. In addition, in the Y-axis direction, the porous sheet 30 is disposed inside the shortest flow path 201.

[0054] Next, the operation of separating first particles and second particles contained in a sample by the particle separating chip 1 will be described with reference to FIGS.

[0055] FIG. 6 is a diagram showing a schematic diagram of the injection of a sample and sheath liquid into the inlets 101 to 103 of the particle separation chip 1, and the ejection of the sample from the outlets 104 and 213. As shown in FIG.

[0056] A liquid sample containing first particles and second particles smaller than the first particles is poured into the inlet 101 as a first sample. A sheath liquid is injected into the inlets 102 and 103. For example, CellPack (registered trademark) manufactured by Sysmex Corporation can be used as the sheath liquid. A liquid sample containing mainly the first particles is taken out from the outlet 104 as a second sample. A liquid sample containing mainly the second particles is taken out from the multiple outlets 213 as a third sample.

[0057] 7 and 8 are diagrams showing the flow range of the first sample being restricted by the sheath liquid. For convenience, Fig. 7 shows the vicinity of the connection between the inlets 101 and 102 and the channels 111 and 113. The upper, middle and lower parts of Fig. 8 show the C21-C22 cross section, the C31-C32 cross section and the C41-C42 cross section of Fig. 7, respectively.

[0058] As shown in Fig. 7, the first sample is caused to flow through the channel 111 via the inlet 101. The sheath liquid injected from the inlet 102 presses the first sample flowing through the channel 111 downward. As a result, the range of the first sample flowing through the channel 111 is restricted to the vicinity of the bottom surface of the channel 111 by the sheath liquid, as shown in the upper part of Fig. 8.

[0059] As shown in Fig. 7, the pair of channels 113 are connected to both side surfaces of the channel 111 in a direction inclined with respect to the X-axis direction between the inlet 102 and the channel 120. The sheath liquid flowing from the pair of channels 112 into the channel 111 presses the first sample flowing from below the inlet 102 to the downstream side in a direction approaching the vertical plane VP (see Figs. 1 and 2). As a result, as shown in the middle part of Fig. 8, the range of the first sample flowing through the channel 111 is limited to the vicinity of the center of the channel 111 by the sheath liquid.

[0060] 7, the width of flow channel 120 is wider than the width of flow channel 111, so the first sample flowing from flow channel 111 to flow channel 120 spreads in the Y-axis direction and flows downstream. At this time, as shown in the lower part of FIG. 8, although the range of the first sample spreads in the Y-axis direction, it is restricted to the vicinity of the center of flow channel 120 because it is pressed toward the center by the sheath liquid from the pair of flow channels 113.

[0061] FIG. 9 is a perspective view that shows a schematic view of the vicinity of a portion where the flow channel 120 of the main flow channel 100 and the flow channel 201 of the branch flow channel 200 intersect.

[0062] In a plan view, in a portion where the flow channel 120 and the flow channel 201 overlap, the recess on the flow channel 120 side and the recess on the flow channel 201 side are connected in the Z-axis direction. The crossing surface between the flow channel 120 and the flow channel 201 is an opening, and the porous sheet 30 is placed in this opening to close the opening. The location corresponding to the opening of the crossing surface between the flow channel 120 and the flow channel 201 where the porous sheet 30 is placed is the branch point B. Therefore, the area of ​​the branch point B is equal to the area of ​​the opening of the crossing surface between the flow channel 120 and the flow channel 201. The branch flow channel 200 communicates with the main flow channel 100 at a plurality of branch points B along the flow direction (X-axis direction) of the main flow channel 100. The porous sheet 30 is placed so as to straddle the plurality of branch points B.

[0063] In this embodiment, the branch points B have the same shape and are arranged at the same Y-axis and Z-axis positions. The branch points B are arranged so that the intervals in the X-axis direction are the same. However, it is sufficient that the branch points B are arranged at intervals in the X-axis direction between the inlet 101 and the outlet 104 of the main flow path 100. For example, the branch points B may have different shapes or different areas. The branch points B may be arranged at different Y-axis positions or different Z-axis positions. The intervals between the branch points B may be different.

[0064] When the first sample flowing downstream through the flow channel 120 passes through the branch point B, the second particles contained in the first sample flow into the flow channel 201 through the holes 31 of the porous sheet 30. Then, a third sample containing the second particles is taken out from the outlet 213 (see FIG. 6). On the other hand, the first particles contained in the first sample flow downstream through the flow channel 120 without flowing into the flow channel 201. Then, the second sample containing the first particles is taken out from the outlet 104 (see FIG. 6).

[0065] At this time, as shown in the lower part of Figure 8, the first sample flows to branch point B with its upper part in the thickness direction and both ends in the width direction restricted, so that the second particles contained in the first sample can be smoothly separated into flow path 201.

[0066] Fig. 10 is a diagram showing a schematic diagram of the first particle and the second particle being separated at the branch point B shown in Fig. 9. Fig. 10 shows a cross section taken along C11-C12 in Fig. 6.

[0067] In Fig. 10, for the sake of convenience, the first particles are indicated by white circles, and the second particles are indicated by black circles. Components other than the first and second particles contained in the first sample are indicated by dark dots, and the sheath liquid injected from the inlets 102 and 103 is indicated by light dots. Of the entire porous sheet 30, only the porous sheet 30 at the position of the branch point B (see Fig. 9) is illustrated.

[0068] 7 and the upper part of FIG. 8, the first sample injected from the inlet 101 is pushed downward by the sheath fluid injected from the inlet 102, and therefore the first sample flows downstream along the bottom surface of the main channel 100. The multiple channels 201 of this embodiment are configured to enable separation of the first particles and the second particles by hydrodynamic filtration (HDF).

[0069] FIG. 11 is an enlarged view that illustrates a schematic view of the vicinity of one of the flow channels 201 in FIG.

[0070] If the flow rate in a flow path 120 flowing from the upstream side to one flow path 201 is Qm and the flow rate flowing into the flow path 201 is Qs, the relationship between the flow rate Qm and the flow rate Qs can be expressed by the following equation (1) using the liquid removal rate a at the branch point B.

[0071] Qs = Qm × a … (1)

[0072] The liquid removal rate a at the branch point B is set to a value at which the second particles flow into the branch channel 200 and the first particles flow into the main channel 100. According to the hydrodynamic filtration method, the height h of the liquid portion of the first sample that flows into each channel 201 is determined by the liquid removal rate a. Therefore, in this embodiment, the liquid removal rate a is set so that the height h of the liquid portion of the first sample is smaller than the radius of the first particle and larger than the radius of the second particle. As a result, the first particles are transported downstream along the flow of the first sample, and the second particles are separated into each channel 201 by the liquid portion in the range of the height h of the first sample.

[0073] In this embodiment, the liquid removal rates a at all branch points B are set to substantially the same value. When the liquid removal rates a at the respective channels 201 are equal to each other, the heights h of the liquid portions of the first sample flowing into the respective channels 201 are also equal to each other. This allows the second particles of a uniform size to be separated evenly at the multiple branch points B.

[0074] Here, the liquid removal rate a at the branch point B is determined by the fluid resistance of the branch flow path 200 from the branch point B to the outlet 213 (see FIG. 6). Therefore, in this embodiment, the fluid resistance of the branch flow path 200 from the branch point B on the upstream side to the outlet 213 is set to be greater than the fluid resistance of the branch flow path 200 from the branch point B on the downstream side to the outlet 213. This makes the fluid resistance at the upstream branch point B greater than the fluid resistance at the downstream branch point B, and the liquid removal rates a at each branch point B can be set to substantially equal values.

[0075] In this embodiment, since the size of the relay flow paths 211, 212 (see FIG. 6) is larger than that of the flow path 201, the liquid removal rate a at the branch point B is substantially determined by the fluid resistance of the flow path 201. Therefore, in this embodiment, the length of the flow path 201 on the upstream side in the Y-axis direction is set to be longer than the length of the flow path 201 on the downstream side in the Y-axis direction. This allows the fluid resistance of the flow path 201 connected to the branch point B on the upstream side to be set to be larger than the fluid resistance of the flow path 201 connected to the branch point B on the downstream side.

[0076] Moreover, the liquid removal rate a at each of the branch points B is set to be greater than 0% and equal to or less than 50%. This allows the second particles having a particle size smaller than the depth d1 of the flow path 120 to be separated into the flow path 201.

[0077] In this embodiment, each hole 31 of the porous sheet 30 arranged at the branch point B can form a flow of the sample that moves the second particles from the flow path 120 of the main flow path 100 to the flow path 201 of the branch flow path 200, as shown by the thick arrow at the position of the hole 31 in FIG. 11. Therefore, even if the flow rate of the flow path 201 is increased by widening the width of the flow path 201 in the X-axis direction as shown in FIG. 11 to increase the throughput, the flow of the sample of the second particles toward the flow path 201 can be dispersed to the multiple holes 31 of the porous sheet 30. This allows the flow of the sample of the second particles to be formed throughout the entire flow path 201, rather than being formed only in a part of the flow path 201. In addition, as shown in FIG. 11, multiple holes 31 are formed in the porous sheet 30 and the width of the flow path 201 in the X-axis direction is wide, so that clogging in the porous sheet 30 and the flow path 201 can be suppressed.

[0078] Therefore, the second particles can be separated into the flow channel 201 efficiently without clogging, and the flow rates in the main flow channel 100 and the branch flow channels 200 can be increased, thereby efficiently increasing the throughput of particle separation.

[0079] In this embodiment, the particle size of the second particles separated into the flow path 201 is not determined by the pore size of the holes 31 in the porous sheet 30 and the particle size of the second particles, but is determined by the height h based on the liquid removal rate a. That is, the porous sheet 30 is not used as a filter that separates the second particles from the first sample by the holes 31, but is used to disperse the flow of the second particles toward the flow path 201, as described above. Therefore, the pore size of the holes 31 may be larger than the particle size of the second particles to be separated, for example.

[0080] Returning to Fig. 10, by separating the second particles at the multiple branch points B, the second particles and the liquid portion of the first sample flow in the channel 201 of the branch channel 200. As a result, the third sample extracted from the outlet 213 (see Fig. 6) mainly contains the second particles. Meanwhile, the first particles and sheath liquid are left in the channel 120 of the main channel 100. As a result, the second sample extracted from the outlet 104 mainly contains the first particles.

[0081] Next, the particle separating device 2 for separating the first particles and the second particles will be described.

[0082] FIG. 12 is a perspective view that illustrates a schematic configuration of the particle separator 2. As shown in FIG.

[0083] The particle separation device 2 includes a particle separation chip 1, a fixing section 40, three inlet nozzles 51-53, five outlet nozzles 54, 55, liquid delivery sections 61-63, a first recovery section 64, and four second recovery sections 65.

[0084] The particle separation chip 1 is cleaned and reused every time a particle separation process is performed. Note that the particle separation chip 1 may be configured as an exchangeable particle separation chip that is replaced with a new one every time a particle separation process is performed.

[0085] The fixed part 40 includes an installation part 41 and a lifting part 42. The installation part 41 includes a flat plate 40a parallel to the XY plane and a frame part 40b installed on the upper surface of the flat plate 40a. In a plan view, an opening of approximately the same size as the outer shape of the particle separation chip 1 is formed in the center of the frame part 40b, and the particle separation chip 1 is installed in this opening. The lifting part 42 is supported by a lifting mechanism 2b (see FIG. 13) so as to be movable in the Z-axis direction. The lifting part 42 includes an arm part 42a extending above the particle separation chip 1 arranged on the frame part 40b.

[0086] The upper ends of the inlet nozzles 51-53 are connected to liquid delivery units 61-63 via tubes, respectively. The liquid delivery units 61-63 are, for example, syringe pumps. The liquid delivery unit 61 is configured to be able to deliver a predetermined amount of the first sample to the inlet nozzle 51 at a predetermined speed. The liquid delivery units 62 and 63 are configured to be able to deliver a predetermined amount of sheath liquid to the inlet nozzles 52 and 53, respectively, at a predetermined speed.

[0087] The upper end of the outlet nozzle 54 is connected to the first recovery section 64 via a tube. The upper ends of the four outlet nozzles 55 are each connected to the second recovery section 65 via a tube. The first recovery section 64 and the four second recovery sections 65 are containers for recovering samples. Note that a liquid delivery section constituted by a syringe pump for delivering the second sample to the first recovery section 64 may be disposed in the tube connecting the outlet nozzle 54 and the first recovery section 64. A liquid delivery section constituted by a syringe pump for delivering the third sample to the second recovery section 65 may be disposed in the tube connecting the outlet nozzle 55 and the second recovery section 65.

[0088] When a particle separation process is performed, the particle separation chip 1 is placed in the opening of the frame portion 40b, and the lifting portion 42 moves downward, so that the upper surface of the particle separation chip 1 (upper surface 10b of the upper substrate 10) is pressed downward by the arm portion 42a. This fixes the particle separation chip 1. Thereafter, the inlet nozzles 51-53 are connected to the inlets 101-103 of the particle separation chip 1, respectively, and the outlet nozzles 54 and 55 are connected to the outlets 104 and 213 of the particle separation chip 1, respectively.

[0089] In this state, the liquid delivery unit 62 delivers sheath liquid to the flow path 111 (see FIG. 6) via the inlet 102, and the liquid delivery unit 63 delivers sheath liquid to the flow paths 112 and 113 (see FIG. 6) via the inlet 103. Then, the liquid delivery unit 61 delivers the first sample to the flow path 111 via the inlet 101. As a result, as described with reference to FIGS. 10 and 11, the sample (first sample) containing the first particles and the second particles is separated into a sample (second sample) containing the first particles and a sample (third sample) containing the second particles. Then, the second sample is collected in the first collection unit 64 via the outlet 104, and the third sample is collected in the four second collection units 65 via the four outlets 213. In this way, the particle separation process is completed.

[0090] FIG. 13 is a block diagram showing the functional configuration of the cell detection system 5.

[0091] The cell detection system 5 includes a particle separation device 2, a stain concentration device 3, and an imaging flow cytometer 4. The cell detection system 5 is a system for detecting a first particle (e.g., CTC) contained in a blood sample collected from a subject.

[0092] The particle separator 2 shown in Fig. 12 includes a control unit 2a, a lifting mechanism 2b, and liquid sending units 61 to 63. The control unit 2a includes, for example, a CPU and a memory, and controls each unit of the particle separator 2. The lifting mechanism 2b includes a motor for moving the lifting unit 42 in Fig. 12 up and down.

[0093] The particle separation device 2 separates the blood sample using the particle separation chip 1 to obtain a second sample containing the first particles. The staining and concentrating device 3 performs a staining process to stain the first particles contained in the second sample based on the second sample obtained by the particle separation device 2. The imaging flow cytometer 4 captures images of the particles contained in the second sample that has undergone the staining process, and obtains the captured images. An operator such as a doctor or a medical technician refers to the captured images obtained by the imaging flow cytometer 4 to determine the presence or absence of first particles (e.g., CTCs) in the blood sample collected from the subject.

[0094] <Configuration of the embodiment> Next, a specific example of the above embodiment will be described. In the following example, the first sample is whole blood collected from a subject. The first particles are cells to be acquired (target cells), and are circulating tumor cells (CTCs). The second particles are cells to be removed (non-target cells), and are white blood cells, red blood cells, and platelets. The inventors created a particle separation chip 1 by combining a chip and a porous sheet, which will be described with reference to Figs. 14 to 18, and actually performed particle separation processing using the created particle separation chip 1.

[0095] Fig. 14 is a diagram showing the size of the main channel 100 and the branch channel 200 according to an embodiment. Fig. 15 is a diagram showing the arrangement and size of the holes 31 of the porous sheet 30 according to an embodiment.

[0096] 14, the depth of main channel 100 is 0.1 mm. The diameter of inlets 101-103 is 1.5 mm. The width (length in the direction perpendicular to the direction of travel of the sample in the channel) of channels 111-113 is 0.5 mm. The width (length in the Y-axis direction) of channel 120 is 4 mm. The right end of the region of channel 120 with a constant width in the Y-axis direction is 2 mm to the right in the X-axis direction from the rightmost end of multiple channels 201 in the X-axis direction.

[0097] The depth of the branch flow channel 200 is 0.1 mm. The distance from the leftmost end of the multiple flow channels 201 to the right end of the region of the flow channel 120 where the width in the Y-axis direction is constant is Lmain. The number of the multiple flow channels 201 is n. The serial number of the leftmost flow channel 201 is 1, and the serial number of the rightmost flow channel 201 is n. The lengths by which the first and nth flow channels 201 protrude in the Y-axis direction from the flow channel 120 are L1 and Ln, respectively. The width of the flow channel 201 (length in the X-axis direction) is w, and the distance between two adjacent flow channels 201 is v.

[0098] The length of the porous sheet 30 in the X-axis direction is 30 mm, and the width of the porous sheet 30 in the Y-axis direction is 6 mm. As shown in Fig. 15, the hole diameter of the hole 31 in the porous sheet 30 is φ, and the pitch between two adjacent holes 31 is p. Three straight lines connecting the centers of three adjacent holes 31 form an angle of 60° with each other. The porosity, which is the ratio of the area of ​​the hole 31 in the porous sheet 30, is R. The thickness of the porous sheet 30 is 5 µm.

[0099] Below, by varying the distance Lmain, the number n, the lengths L1, Ln, the width w and the interval v, four types of chips C1 to C4 each consisting of an upper substrate 10 and a lower substrate 20 were created as shown in Fig. 16. In addition, by varying the pitch p, the hole diameter φ and the opening rate R, porous sheets PS1 to PS7 were created as shown in Fig. 17.

[0100] 18, in chips C1 and C2, four relay channels 212 are provided, and four relay channels 211 are provided for guiding the third sample to the four relay channels 212, respectively. In chips C3 and C4, six relay channels 212 are provided, and six relay channels 211 are provided for guiding the third sample to the six relay channels 212, respectively. According to chips C1 to C4 shown in FIG. 18, the third sample flowing out into one channel 201 passes through one relay channel 211 and one relay channel 212 corresponding to the channel 201, and is taken out from outlet 213.

[0101] [Example 1] In Example 1, four types of particle separation chips 1 were installed in the above-mentioned particle separation device 2, and a first sample was passed through each particle separation chip 1 to investigate the removal rates of red blood cells and white blood cells.

[0102] 19 is a table showing the configurations of four types of particle separation chips CP1 to CP4 according to Example 1. The lower part of FIG.

[0103] As shown in the upper part of FIG. 19, the four types of particle separation chips CP1 to CP4 are combinations of the chips C1 to C4 shown in FIG. 16 and the porous sheet PS7 shown in FIG. 17. As shown in the lower part of FIG. 19, the three types of first samples have different amounts of whole blood and diluent. The first sample of "no whole blood dilution" is a 5 mL sample consisting of whole blood only. The first sample of "1.5-fold diluted whole blood" is a 5 mL sample obtained by diluting 3.33 mL of whole blood with 1.67 mL of diluent. The first sample of "2-fold diluted whole blood" is a 5 mL sample obtained by diluting 2.5 mL of whole blood with 2.5 mL of diluent. The whole blood used for the three types of first samples was collected from a healthy subject.

[0104] In Example 1, in the particle separation chips CP1 to CP4, 1 mL of the above three types of first samples were separately flowed from the inlet 101 at 100 μL / min. At this time, the throughputs of whole blood for the three types of first samples (no whole blood dilution, 1.5-fold diluted whole blood, and 2-fold diluted whole blood) were 100 μL / min, 67 μL / min, and 50 μL / min, respectively. In addition, sheath liquid was flowed from the inlet 102 at 900 μL / min, and sheath liquid was flowed from the inlet 103 at 2000 μL / min. The time for sending the first sample to the inlets 101 to 103 was set to 10 minutes for each of the first samples. Then, in each of the particle separation chips CP1 to CP4, the second sample was collected from the first collection unit 64 (see FIG. 12), and the third sample was collected from a plurality of second collection units 65 (see FIG. 12).

[0105] Next, the numbers of cells (red blood cells and white blood cells) contained in the three types of first samples and the second sample collected by each of the particle separation chips CP1 to CP4 were obtained by the following formula (2).

[0106] Number of cells = cell concentration (cells / mL) × volume (mL) … (2)

[0107] Here, the cell concentration was measured using a hemocytometer (XN series) or a hemocytometer manufactured by Sysmex Corp. The liquid volume was measured from the weight using an electronic balance.

[0108] Next, for each combination of the first sample and particle separation chips CP1 to CP4, the red blood cell removal rate and white blood cell removal rate were calculated using the following formulas (3) and (4) based on the number of cells (red blood cells and white blood cells) obtained using the above formula (2).

[0109] Red blood cell removal rate = {1 - (number of red blood cells in the second sample / number of red blood cells in the first sample)} x 100 (%) ... (3) Leukocyte removal rate = {1 - (number of leukocytes in the second sample / number of leukocytes in the first sample)} x 100 (%) ... (4)

[0110] FIG. 20 is a graph showing the red blood cell removal rate and the white blood cell removal rate of the particle separation chips CP1 to CP4.

[0111] For convenience, the removal rates for each type of first sample are connected by a straight line in the graph of Fig. 20. The liquid removal rates of the particle separation chips CP1 to CP4 correspond to the liquid removal rates (0.73%, 1.12%, 1.31%, and 1.50%) of the chips C1 to C4 shown in Fig. 16, respectively.

[0112] As shown in Fig. 20, in all the examples, a high red blood cell removal rate and a high white blood cell removal rate were achieved. In addition, it was found that the higher the liquid removal rate, the higher the height h shown in Fig. 11, and therefore the more reliably the red blood cells and white blood cells could be removed. Furthermore, the higher the dilution rate, the higher the red blood cell removal rate and the white blood cell removal rate. The reason for this is expected to be that the higher the dilution rate, the lower the density and viscosity of the cells in the first sample, making it easier to separate the red blood cells and white blood cells into the branch channel 200.

[0113] [Example 2] In Example 2, one type of particle separation chip 1 was installed in the above-mentioned particle separation device 2, and a first sample was passed through the particle separation chip 1 to investigate the removal rates of red blood cells and white blood cells, and the recovery rate of CTCs.

[0114] 21 is a table showing the configuration of the particle separation chip CP5 according to Example 2. The lower part of FIG.

[0115] As shown in the upper part of FIG. 21, the particle separation chip CP5 is a combination of the chip C4 shown in FIG. 16 and the porous sheet PS6 shown in FIG. 17. As shown in the lower part of FIG. 21, the first sample of "two-fold dilution of whole blood" according to Example 2 is a 2.5 mL sample obtained by diluting 1.25 mL of whole blood collected from a healthy subject with 1.25 mL of diluent. In Example 2, A549 cells obtained from the A549 cell line were added to the first sample as CTCs. Specifically, the A549 cells were stained in advance with a predetermined reagent, and about 400,000 of the stained A549 cells were added to the first sample. According to measurements by the inventors, the size of the A549 cells is 11 μm or more and 20 μm or less, with an average of about 17 μm.

[0116] In Example 2, in the particle separation chip CP5, the entire amount (2.5 mL) of the first sample was flowed from the inlet 101 at 100 μL / min three times separately. At this time, the throughput of the whole blood of the first sample was 50 μL / min in each time. In addition, the sheath liquid was flowed from the inlet 102 at 900 μL / min, and the sheath liquid was flowed from the inlet 103 at 2000 μL / min. The time for sending the first sample to the inlets 101 to 103 was 25 minutes in each time. Then, in the particle separation chip CP5, the second sample was collected from the first collection part 64, and the third sample was collected from the multiple second collection parts 65.

[0117] Next, using the same procedure as in Example 1, the cells (red blood cells and white blood cells) contained in the first sample and the second sample were obtained using the above formula (2), and based on the obtained numbers of red blood cells and white blood cells, the red blood cell removal rate and the white blood cell removal rate were calculated using the above formulas (3) and (4).

[0118] In addition, the A549 cells contained in the recovered second and third samples were obtained using the above formula (2), and the recovery rate of A549 cells was calculated based on the number of A549 cells obtained using the following formula (5).

[0119] A549 cell recovery rate = number of A549 cells contained in the second sample / (number of A549 cells contained in the second sample + number of A549 cells contained in the third sample) × 100 (%) ... (5)

[0120] FIG. 22 is a graph showing the red blood cell removal rate, the white blood cell removal rate, and the A549 cell recovery rate of the particle separation chip CP5.

[0121] As shown in the upper part of FIG. 22, in each of the first to third runs in which the first sample was run, the red blood cell removal rate was about 99% or more, and the white blood cell removal rate was about 90% or more, so that a high red blood cell removal rate and a high white blood cell removal rate were realized. Also, as shown in the lower part of FIG. 22, in each of the first to third runs in which the first sample was run, the A549 cell recovery rate was about 90% or more, so that a high A549 cell recovery rate was realized. From the above results, it was found that blood cell components (red blood cells and white blood cells) and A549 cells could be appropriately separated from the first sample based on the difference in particle size.

[0122] [Example 3] In Example 3, six types of particle separation chips 1 were installed in the above-mentioned particle separation device 2, and a first sample was passed through each particle separation chip 1 to investigate the cell removal rate, cell recovery rate, weight ratio to the flow path 201, and recovery rate and removal rate for each particle size.

[0123] 23 is a table showing the configurations of six types of particle separating chips CP11 to CP16 according to Example 3. The lower part of FIG.

[0124] As shown in the upper part of Fig. 23, the six types of particle separation chips CP11 to CP16 are combinations of the chip C4 shown in Fig. 16 and the porous sheets PS1 to PS6 shown in Fig. 17. As shown in the lower part of Fig. 23, the first sample according to Example 3 is a cell suspension. This cell suspension is prepared by dissolving 2 mL of a 1% polyvinylpyrrolidone solution and 1 x 10 Jurkat cells. 6 Approximately 3 × 10 cells were stained with a given reagent. 5 The Jurkat cells correspond to blood cell components (red blood cells and white blood cells) present in normal blood, and the A549 cells correspond to CTCs. The inventors previously investigated the particle sizes of the Jurkat cells and A549 cells contained in the first sample (cell suspension) using an imaging flow cytometer.

[0125] Fig. 24 is a graph showing the particle size distribution of Jurkat cells and A549 cells contained in the first sample. In Fig. 24, the horizontal axis shows the particle size of the cells, and the vertical axis shows the frequency (%) corresponding to the particle size. The frequency corresponding to a given particle size corresponds to the probability of the presence of the given particle size.

[0126] In Example 3, in the particle separation chips CP11 to CP16, the first sample was flowed from the inlet 101 at 110 μL / min. Further, the sheath liquid was flowed from the inlet 102 at 1400 μL / min, and from the inlet 103 at 1510 μL / min. The time for sending the liquid to the inlets 101 to 103 was set to 15 minutes. Then, in the particle separation chips CP11 to CP16, the second sample was collected from the first collection section 64, and the third sample was collected from the multiple second collection sections 65.

[0127] Next, the number of cells (Jurkat cells and A549 cells) contained in each of the second and third samples was calculated according to the following formula (6).

[0128] Number of cells of a given particle size in a sample = Probability of cells of a given particle size in a sample × Total number of cells in a sample … (6)

[0129] In the above formula (6), the probability of the presence of a cell is obtained from the frequency in Fig. 24. The total cell number is obtained from the above formula (2) using the cell concentration measured by a hemocytometer.

[0130] Next, based on the number of cells (Jurkat cells and A549 cells) for each particle size obtained by the above formula (6), the recovery rate at a given particle size was calculated by the following formula (7).

[0131] Recovery rate of cells of a given particle size = {number of cells of a given particle size in the second sample / (number of cells of a given particle size in the second sample + number of cells of a given particle size in the third sample)} × 100 (%) ... (7)

[0132] In addition, the numbers of Jurkat cells and A549 cells contained in the collected second and third samples were obtained using the above formula (2), and the Jurkat cell removal rate was calculated based on the obtained numbers of Jurkat cells using the following formula (8).

[0133] Jurkat cell removal rate = number of Jurkat cells contained in the third sample / (number of Jurkat cells contained in the second sample + number of Jurkat cells contained in the third sample) × 100 (%) ... (8)

[0134] Furthermore, the A549 cells contained in the recovered second and third samples were obtained using the above formula (2), and the recovery rate of A549 cells was calculated using the above formula (5) based on the number of A549 cells obtained.

[0135] FIG. 25 is a graph showing the removal rate of Jurkat cells and the recovery rate of A549 cells for the particle separation chips CP11 to CP16.

[0136] As shown in the upper part of Fig. 25, it was found that in all particle separation chips, Jurkat cells corresponding to normal blood cell components (red blood cells, white blood cells, platelets, etc.) could be separated and removed as a third sample. Also, as shown in the upper part of Fig. 23, the particle separation chips CP11 to CP16 differ only in the configuration of the porous sheet. As shown in Fig. 17, the pore size of the holes 31 in the porous sheets PS1 to PS6 increases in the order given. Therefore, it was found from the results in the upper part of Fig. 25 that the larger the pore size of the holes 31, the more Jurkat cells corresponding to normal blood cell components can be removed.

[0137] As shown in the lower part of Figure 25, it was found that in all particle separation chips, A549 cells corresponding to CTCs could be separated and collected as a second sample. In addition, since the pore size of the porous sheets PS1 to PS6 increases in the order of approximately, the results in the lower part of Figure 25 show that the smaller the pore size of the hole 31, the more A549 cells corresponding to CTCs can be collected.

[0138] <Throughput Survey> Next, the inventors investigated to what extent the throughput could be improved by using the particle separation chips having the configurations shown in Examples 1 to 3, compared to the conventional particle separation method (comparative example) described in Non-Patent Document 1 above.

[0139] In the comparative example, 1 mL of whole blood diluted 10-fold with PBS is introduced into the flow channel at a flow rate of 25 μL / min, and the throughput of the whole blood is 2.5 μL / min. Therefore, according to the comparative example, the time required for particle separation processing of 5 mL of whole blood is 2000 minutes (about 33 hours). In contrast, in the particle separation chip 1 having the configuration shown in Examples 1 to 3, the throughput of whole blood (Examples 1 to 2) and cell suspension (Example 3) can be set to 50 μL / min or more. Therefore, according to the embodiment, the time required for particle separation processing of 5 mL of whole blood is 100 minutes or less. Thus, according to Examples 1 to 3, the throughput can be significantly improved compared to the comparative example.

[0140] FIG. 26 is a graph showing the flow rate to each outlet of the particle separation chips CP11 to CP16.

[0141] In FIG. 26, waste liquids 1 to 3 and 5 to 7 respectively indicate third samples obtained from six outlets 213 arranged in the Y-axis direction (see the lower right of FIG. 18). That is, waste liquid 1 is a third sample obtained from the outlet 213 located most downstream in the Y-axis direction among the three outlets 213 located below the main channel 100 in the Y-axis direction, waste liquid 3 is a third sample obtained from the outlet 213 located most upstream in the Y-axis direction among the three outlets 213 located below the main channel 100 in the Y-axis direction, waste liquid 5 is a third sample obtained from the outlet 213 located most downstream in the Y-axis direction among the three outlets 213 located above the main channel 100 in the Y-axis direction, and waste liquid 7 is a third sample obtained from the outlet 213 located most upstream in the Y-axis direction among the three outlets 213 located above the main channel 100 in the Y-axis direction. The collected liquid 4 indicates a second sample obtained from the outlet 104. The vertical axis of FIG. 26 indicates the weight ratio of samples obtained from waste liquids 1 to 3, recovered liquid 4, and waste liquids 4 to 7 for each of the particle separation chips CP11 to CP16.

[0142] With reference to the weight ratios of the waste liquids 1 to 3 and 5 to 7, the weight ratio of the third sample obtained from the outlet 213 closer to the main channel 100 (i.e., the outlet 213 connected to the channel 201 located downstream of the main channel 100) is smaller. In addition, although different porous sheets PS1 to PS6 are installed in the particle separation chips CP11 to CP16, the weight ratios of the waste liquid and the recovered liquid obtained from the same outlet are almost the same. From this, it can be seen that when the porous sheets PS1 to PS6 are configured as in Example 3, the difference in the fluid resistance due to the porous sheets can be almost ignored. In other words, in this case, it can be seen that the flow rate of the branch channel 200 to the channel 201 is dominated by the fluid resistance due to the channel 201.

[0143] FIG. 27 is a graph showing the recovery rate for each particle size of the particle separation chips CP11 to CP16.

[0144] In Fig. 27, the horizontal axis represents the particle size of the cells, and the vertical axis represents the recovery rate calculated by the above formula (7). For convenience, a cutoff indicating a recovery rate of 70% is indicated by a dashed line in Fig. 27.

[0145] As described above, the pore size of the pores 31 of the porous sheets PS1 to PS6 increases in the order shown. Also, referring to the graph in FIG. 27, in the particle separation chips CP11 to CP16, the particle size of the cells exceeding the cutoff increases as the pore size increases in the order shown. For example, in the particle separation chip CP11, the particle size of the cells exceeding the cutoff is approximately 11.5 μm, and in the particle separation chip CP16, the particle size of the cells exceeding the cutoff is approximately 16.3 μm. This shows that when the pore size of the porous sheet increases, the recovery rate changes even if the configurations of the main channel 100 and the branch channel 200 are the same, and it becomes easier to mainly recover cells with relatively large particle sizes.

[0146] In other words, although the particle size of the particles that are not separated into the branch flow path 200 and are recovered through the main flow path 100 is basically determined by the liquid removal rate of the particle separation chip, it can be seen that the proportion of particles of a given particle size that are recovered through the main flow path 100 also changes depending on the pore size of the porous sheet.

[0147] In addition, when the porous sheet PS1 installed in the particle separation chip CP11 and the porous sheet PS2 installed in the particle separation chip CP12 are compared, as shown in FIG. 17, the pore diameters are the same, 12 μm, but the porosity of the porous sheet PS2, 20.9%, is smaller than the porosity of the porous sheet PS1, 36.9%. That is, the number of holes 31 in the porous sheet PS2 is smaller than the number of holes 31 in the porous sheet PS1. In addition, as shown in FIG. 26, the weight ratios of the samples obtained from the outlets of the particle separation chips CP11 and CP12 are almost the same. From this, the flow rate of the third sample flowing from one hole 31 to the branch flow channel 200 is larger in the particle separation chip CP12 than in the particle separation chip CP11. In this way, when the flow rate flowing through one hole 31 is large, the force that draws the cells into the hole 31 becomes large. For these reasons, as shown in the recovery rates in FIG. 27, it is believed that the recovery rate of cells with relatively large particle sizes (for example, cells with particle sizes of 12.5 μm to 18 μm) is smaller in the particle separation chip CP12 than in the particle separation chip CP11.

[0148] <Simulation of discharge into branch flow path> Next, the inventors investigated the flow rate (discharge amount) of the sample flowing from the main channel 100 to the branch channel 200 by simulation.

[0149] The upper part of FIG. 28 is a diagram showing a schematic configuration of the channel 120 of the main channel 100 and the channel 201 of the branch channel 200 according to this simulation.

[0150] In this simulation, only one flow path 201 is arranged for the flow path 120, and a porous sheet 30 is arranged at a branch point B between the flow path 120 and the flow path 201. In this simulation, a sample is caused to flow in the X-axis direction for the flow path 120, and the flow rate of the sample discharged into the flow path 201 through the porous sheet 30 is calculated by simulation.

[0151] The lower part of FIG. 28 is a graph showing the results of the discharge ratio of the sample to the branch channel 200 in accordance with this simulation.

[0152] 28, the horizontal axis indicates the position in the Y-axis direction of flow channel 201, the center corresponds to the center position in the Y-axis direction of flow channel 120, and the left end and the right end correspond to one end and the other end, respectively, in the Y-axis direction of flow channel 120. The vertical axis indicates the discharge ratio (%) of the sample from branch flow channel 200 to flow channel 201.

[0153] As shown by the solid line in the graph at the bottom of FIG. 28, the flow rate of the sample flowing from the flow channel 120 to the flow channel 201 is higher at the left and right ends than at the center at the branch point B. In this way, when the height of the sample (height h in FIG. 11) is large at the left and right ends, if the sample contains a first particle (e.g., CTC), the first particle is unintentionally discharged at both ends into the branch flow channel 200. In addition, since the height of the sample becomes extremely small at the center, if the sample contains a second particle (e.g., red blood cells, white blood cells, and platelets), the second particle is difficult to discharge at the center into the branch flow channel 200. According to the inventors' study, in order to flow a second particle having a particle size of 14.5 μm or less into the branch flow channel 200, for example, the discharge ratio Fmin near the center needs to be increased to about the cutoff (1.8%).

[0154] For this reason, in the above embodiment, the first sample is brought to the center of the flow channel 120 by the sheath liquid flowing from the inlet 103 so that the first particles contained in the first sample do not flow into the branch flow channel 200 and the second particles contained in the first sample flow into the branch flow channel 200. This allows the first particles and the second particles to be separated appropriately. In addition, since the first sample is prevented from flowing excessively to both ends of the flow channel 120, the particles are separated evenly at the branch point B, and the second particles can be separated efficiently.

[0155] <Effects of particle separation chip and particle separation device> As shown in FIG. 6, the particle separation chip 1 includes an upper substrate 10 and a lower substrate 20 (substrates) having a microchannel 1a therein, and a porous sheet 30 having a plurality of holes 31 formed therein. The microchannel 1a includes a main channel 100 and a branch channel 200. The main channel 100 has an inlet 101 for a first sample including a first particle and a second particle smaller than the first particle provided on the upstream side, and an outlet 104 (first outlet) provided on the downstream side from which a second sample including mainly the first particle is taken out. The branch channel 200 communicates with the main channel 100 at a plurality of branch points B (see FIG. 9) along the X-axis direction (flow direction of the main channel 100), and has an outlet 213 (second outlet) on the downstream side from which a third sample including the second particle is taken out. At the plurality of branch points B, the main channel 100 and the branch channel 200 communicate with each other via the holes 31 in the porous sheet 30.

[0156] According to this configuration, the main channel 100 and the branch channels 200 communicate with each other through the holes 31 of the porous sheet 30 at a plurality of branch points B. This allows for sufficient sample processing capacity (throughput) to be achieved in product development and clinical settings.

[0157] 11, each of the holes 31 in the porous sheet 30 can form a sample flow that moves the second particles from the main channel 100 to the branch channel 200. Therefore, the sample flow of the second particles toward the branch channel 200 can be dispersed into the multiple holes 31 in the porous sheet 30. This allows the second particles to be efficiently separated into the branch channel 200. Therefore, the flow rates of the main channel 100 and the branch channel 200 can be increased, and the throughput of particle separation can be efficiently increased.

[0158] In addition, since the width of the branch channel 200 can be widened, particle separation can be performed without clogging even if the blood sample contains cell aggregates. For example, when CTCs are assumed to be the first particles, the CTCs may form clusters. Even in such a case, the CTC clusters can be collected without clogging, so that diagnosis can be performed appropriately based on the CTCs contained in the blood sample.

[0159] Also, whole blood can be used as the first sample without hemolyzing unnecessary blood cells (e.g., red blood cells) in the whole blood. This eliminates the need to prepare the first sample using a hemolyzing agent, and prevents damage to the particles to be obtained by the hemolyzing agent. Also, since there is no need to excessively dilute the whole blood to ensure particle separation processing, the throughput of particle separation can be increased.

[0160] As shown in FIG. 6, the main channel 100 and the branch channel 200 are configured such that a portion of a first sample introduced from the inlet 101 flows into the branch channel 200 and the remaining sample flows into the main channel 100, thereby causing the first particles to be discharged from the outlet 104 (first outlet) and the second particles to be discharged from the outlet 213 (second outlet).

[0161] According to this configuration, the separated first particles and second particles can be collected at the outlet 104 and the outlet 213, respectively. Therefore, the first particles and the second particles can be smoothly collected.

[0162] The width in the Y-axis direction (channel width) of the channel 120 of the main channel 100 is 500 μm or more and 50 mm or less. Also, for example, when the amount of whole blood contained in the first sample is small, the width in the Y-axis direction (channel width) of the channel 120 of the main channel 100 may be 100 μm or more and 50 mm or less.

[0163] Thus, a high throughput of particle separation can be achieved by setting the channel width of the main channel 100. Furthermore, if the width of the channel 120 in the Y-axis direction is 100 μm or more, first particles having a size of 100 μm or less can be caused to flow through the channel 120.

[0164] As shown in FIG. 9, the branch flow path 200 includes a flow path 201 that communicates with the main flow path 100 at a branch point B and is disposed non-parallel to the main flow path 100 in a plan view.

[0165] According to this configuration, the lengths of the multiple channels 201 of the branch channel 200 can be smoothly adjusted in the in-plane direction of the lower substrate 20. Therefore, the length of each channel 201 can be smoothly set to a length suitable for the hydrodynamic filtration method.

[0166] As shown in FIG. 9, channel 201 that communicates with main channel 100 at branch point B is disposed so as to intersect with main channel 100 in a plan view, and causes the third sample to flow on both sides of main channel 100.

[0167] According to this configuration, the sample in the main channel 100 near the inlet (branch point B) of the channel 201 of the branch channel 200 can be made to flow into the channel 201 without bias in the width direction of the main channel 100. Therefore, all the holes 31 of the porous sheet 30 within the range of the inlet of the channel 201 can be made to properly perform the function of particle separation. Therefore, the separation of particles can be performed efficiently.

[0168] The branch flow paths 200 include flow paths 201 that communicate with the main flow path 100 at the branch point B, the number of which is 2 or more and 15,000 or less.

[0169] According to this configuration, by providing two or more flow paths 201, it is possible to provide a large number of flow paths 201 in a particle separation chip 1 having four sides of approximately 150 mm while realizing at least hydrodynamic filtration.

[0170] As shown in FIGS. 4 and 5, the branch channel 200 is disposed below the main channel 100 when the particle separation chip 1 is in use.

[0171] According to this configuration, the third sample containing the second particles can be introduced into the branch channel 200 by utilizing gravity. Therefore, the efficiency of particle separation can be smoothly improved.

[0172] Moreover, the particle separation chip 1 can be formed by stacking and bonding an upper substrate 10 having a recess on its lower surface for forming the main flow path 100 and a lower substrate 20 having a recess on its upper surface for forming the branch flow path 200 via a porous sheet 30. Thus, the particle separation chip 1 can be easily formed.

[0173] As shown in FIG. 6, the main channel 100 further has inlets 102 and 103 for sheath liquid provided upstream of the branch point B (see FIG. 9).

[0174] 7 and 8, this configuration allows the sheath liquid to restrict the range in which the first sample flows to a range suitable for particle separation, thereby enabling efficient particle separation.

[0175] As shown in FIG. 6, the sheath liquid inlet 102 is an inlet for introducing the sheath liquid from above the first sample flowing through the main channel 100.

[0176] According to this configuration, as shown in FIG. 8, the first sample flows to the inlet (branch point B) of the branch channel 200 with its upper portion in the thickness direction being restricted, so that the second particles can be prevented from being left behind.

[0177] As shown in FIG. 6, the sheath liquid inlet 103 is an inlet for introducing the sheath liquid from both sides of the first sample flowing through the main channel 100.

[0178] According to this configuration, as shown in the middle and lower parts of Fig. 8, the first sample flows into the inlet (branch point B) of the branch flow channel 200 with both ends in the width direction restricted. This allows the first particles and the second particles to be properly separated, as explained in the simulation of Fig. 28, and allows the particles to be separated evenly at the branch point B, allowing the second particles to be separated efficiently.

[0179] The fluid resistance of the branch channel 200 from the branch point B on the upstream side of the main channel 100 to the outlet 213 (second outlet) is greater than the fluid resistance of the branch channel 200 from the branch point B on the downstream side of the main channel 100 to the outlet 213 (second outlet).

[0180] According to this configuration, the liquid removal rate, which is the ratio of the flow rate to the branch channel 200 to the flow rate of the main channel 100 at each branch point B, can be made closer to each other for each branch point B. Therefore, second particles of a certain size can be separated.

[0181] The branch flow path 200 includes a plurality of flow paths 201 arranged non-parallel to the main flow path 100 in a plan view, each of which communicates with the main flow path 100 at a plurality of branch points B, and relay flow paths 211, 212 connecting the plurality of flow paths 201 with an outlet 213 (second outlet). The length of the flow path 201 communicating with the main flow path 100 at the branch point B on the upstream side of the main flow path 100 is longer than the length of the flow path 201 communicating with the main flow path 100 at the branch point B on the downstream side of the main flow path 100.

[0182] According to this configuration, the fluid resistance at the branch point B on the upstream side of the main flow path 100 can be set to be greater than the fluid resistance at the branch point B on the downstream side of the main flow path 100 with a simple configuration.

[0183] The microchannel 1a is formed so that the liquid removal rate, which is the ratio of the flow rate to the branch channel 200 to the flow rate to the main channel 100 at the branch point B, is a value at which the second particles flow to the branch channel 200 and the first particles flow to the main channel 100.

[0184] According to this configuration, the first particles and the second particles can be separated by hydrodynamic filtration.

[0185] The microchannel 1a is formed so that the liquid removal rate, which is the ratio of the flow rate to the branch channels 200 to the flow rate to the main channel 100 at the branch points B, is substantially the same value at each of the branch points B.

[0186] According to this configuration, second particles of a uniform size can be separated evenly at the multiple branching points B.

[0187] The microchannel 1a is formed so that the liquid removal rate, which is the ratio of the flow rate to the branch channels 200 to the flow rate of the main channel 100 at the branch points B, is greater than 0% and equal to or less than 50% at the multiple branch points B.

[0188] According to this configuration, the second particles that are equal to or less than the depth of the main channel 100 can be separated into the branch channels.

[0189] The porous sheets PS1 to PS6 are formed so that the aperture ratio, which is the ratio of the area of ​​the hole 31 to the area of ​​the opening of the intersection between the main flow path 100 and the branch flow path 200 (the area of ​​the branch point B), is a value that allows first particles of a predetermined size to be recovered from the outlet 104 (first outlet) at a predetermined recovery rate.

[0190] According to this configuration, as shown in the second and third embodiments, the first particles of a desired size can be collected at a predetermined collection rate.

[0191] The pore diameter of the pores 31 in the porous sheets 30 and PS1 to PS7 is 30 nm or more and 100 μm or less.

[0192] According to this configuration, when the particle size of the first particles is about 50 nm to 100 μm, the porous sheet can be configured to prevent the first particles from flowing into the branch flow channel 200.

[0193] The porous sheets 30, PS1 to PS7 are formed so that the porosity is greater than 0% and equal to or less than 50% at the branching points.

[0194] This configuration allows the strength of the porous sheet to be maintained.

[0195] The particle size of the first particles is not less than 30 nm and not more than 100 μm.

[0196] This configuration allows the separation of first particles of various sizes, from nanoparticles such as exosomes to relatively large circulating tumor cells.

[0197] The first sample introduced through the inlet 101 is a blood sample, the first particles are circulating tumor cells, and the second particles are white blood cells, red blood cells and platelets.

[0198] Circulating tumor cells (CTCs) that leak from tumors into the blood have been reported to be useful as a factor for cancer diagnosis, and are expected to be a non-invasive cancer test. However, since the presence rate of CTCs in blood is several to several tens of CTCs per 10 mL, it is extremely difficult to separate and extract CTCs from blood samples. However, according to the above configuration, CTCs, which are rare cells, can be efficiently separated. In addition, since no reagents or the like are used in the particle separation process, CTCs can be separated without being damaged.

[0199] As shown in Figure 12, the particle separation device 2 includes a particle separation chip 1, an installation section 41 on which the particle separation chip 1 is placed, liquid delivery sections 61-63 which introduce a first sample and sheath liquid into a first sample inlet 101 and a sheath liquid inlet 102, 103 in the particle separation chip 1, respectively, and deliver the liquid in the microchannel 1a (see Figure 6), a first collection section 64 for the first particles which is connected to an outlet 104 (first outlet), and a second collection section 65 for the second particles which is connected to an outlet 213 (second outlet).

[0200] According to this configuration, by placing the particle separation chip 1 on the installation section 41 and driving the liquid delivery sections 61-63 to introduce the first sample and sheath liquid, the first particles and the second particles can be smoothly recovered from the first recovery section 64 and the second recovery section 65, respectively.

[0201] <Other changes> In order to make the fluid resistance of the branch flow passage 200 from the branch point B on the upstream side to the outlet 213 larger than the fluid resistance of the branch flow passage 200 from the branch point B on the downstream side to the outlet 213, the length of the flow passage 201 on the upstream side in the Y-axis direction is set to be longer than the length of the flow passage 201 on the downstream side in the Y-axis direction. However, without being limited thereto, the cross-sectional area of ​​the flow passage 201 on the upstream side may be set to be smaller than the cross-sectional area of ​​the flow passage 201 on the downstream side.

[0202] Fig. 29 is a diagram showing a schematic configuration of a branch flow path 200 according to a modified example of this case. The upper part of Fig. 29 is a plan view showing the configuration in the vicinity of the branch flow path 200, and the lower part of Fig. 29 is a cross-sectional view showing the configuration of a recess corresponding to the flow path 201 formed in the lower substrate 20.

[0203] As shown in the upper part of Fig. 29, the lengths in the Y-axis direction of the multiple flow paths 201 are the same. As shown in the lower part of Fig. 29, the widths in the X-axis direction of the multiple flow paths 201 are the same, and the height in the Z-axis direction of the flow paths 201 on the upstream side is set smaller than the height in the Z-axis direction of the flow paths 201 on the downstream side. As a result, the cross-sectional area of ​​the flow paths 201 on the upstream side is set smaller than the cross-sectional area of ​​the flow paths 201 on the downstream side. Even with this simple configuration, the fluid resistance at the branch point B on the upstream side of the main flow path 100 can be set larger than the fluid resistance at the branch point B on the downstream side of the main flow path 100.

[0204] In setting the cross-sectional area, both the width in the X-axis direction and the height in the Z-axis direction of the flow channel 201 may be adjusted. In setting the fluid resistance, both the length in the Y-axis direction and the cross-sectional area of ​​the multiple flow channels 201 may be adjusted.

[0205] Although the multiple flow channels 201 are formed to extend on both sides of the flow channel 120 of the main flow channel 100, the present invention is not limited to this, and the multiple flow channels 201 may be formed to extend on only one side of the flow channel 120.

[0206] 30 is a plan view showing a configuration in the vicinity of the branch flow path 200 according to a modified example of this case. In this modified example, one end of each of the multiple flow paths 201 coincides with one end of the flow path 120 in the Y-axis direction in plan view.

[0207] In this case, the second particles separated at the branch point B move in one direction along the flow channel 201. At this time, the flow of the sample in that one direction is difficult to occur in the flow channel 201 at the end of the flow channel 201 in the opposite direction to the Y axis. Therefore, the separation function of the second particles is difficult to exert at the branch point B, and as a result, the separation efficiency of the second particles is reduced. In contrast, when the flow channel 201 extends to both sides of the flow channel 120 as in the above embodiment, the separation function at the branch point B is properly exerted, and the separation efficiency of the second particles is improved.

[0208] As shown in FIG. 1, one inlet 103 is disposed on the central axis CA in order to inject the sheath liquid into the flow channel 111 from the side of the flow channel 111. However, this is not limited to the above, and two inlets 103 may be disposed on both sides in the Y-axis direction with respect to the central axis CA. In this case, two flow channels connected to the two inlets 103 are connected to both side surfaces of the flow channel 111 in the Y-axis direction. This allows the sheath liquid to be injected into the flow channel 111 from the side of the flow channel 111, as in the above embodiment and example. However, in this case, two liquid delivery units 63 are required to inject the sheath liquid into the two inlets 103, respectively, and the liquid delivery amounts of the two liquid delivery units 63 must be equal to each other, which makes the configuration and control of the particle separator 2 complicated. Therefore, as described above, it is preferable that only one inlet 103 is provided along the central axis CA.

[0209] Although one porous sheet 30 is disposed so as to overlap all of the branching points B, this is not limiting, and a plurality of porous sheets 30 may be disposed so as to overlap one or more branching points B, respectively.

[0210] The multiple flow channels 201 are formed to extend perpendicularly to the flow channels 120 of the main flow channel 100 in a plan view, but are not limited thereto, and may be disposed in an oblique direction with respect to the flow channels 120. In this case as well, the length of the multiple flow channels 201 can be smoothly adjusted in the in-plane direction of the lower substrate 20.

[0211] In the above embodiment, as shown in Fig. 2, one relay flow path 211 is provided at each end in the Y-axis direction of the multiple flow paths 201, and in the above example, as shown in Fig. 18, two or three relay flow paths 211 are provided at each end in the Y-axis direction of the multiple flow paths 201. Without being limited to this, four or more relay flow paths 211 may be provided at each end in the Y-axis direction of the relay flow paths 211.

[0212] In the above embodiment, as shown in Fig. 2, one relay flow path 211 is connected to one end of all the flow paths 201 in the Y-axis direction, and two outlets 213 are connected to one relay flow path 211. This is not limited to the above, and one or three or more outlets 213 may be connected to one relay flow path 211. In the above example, as shown in Fig. 18, one outlet 213 is connected to one relay flow path 211. This is not limited to the above, and multiple outlets 213 may be connected to one relay flow path 211, or one outlet 213 may be connected to multiple relay flow paths 211.

[0213] In the above embodiment, as shown in FIG. 1, one outlet 104 is disposed on the downstream side of the main channel 100, but a plurality of outlets 104 may be disposed.

[0214] In the above embodiment, the first particle is a CTC, and the second particle is a red blood cell, a white blood cell, and a platelet, but the combination of the first particle and the second particle is not limited to this. For example, the first particle may be an extracellular vesicle such as an exosome, and the second particle may be a protein or debris smaller than the extracellular vesicle.

[0215] In the above embodiment, the first particle is a particle to be acquired and the second particle is a particle to be removed, but the present invention is not limited to this. The first particle may be a particle to be removed and the second particle may be a particle to be acquired. In this case, the first particle and the second particle can be separated with high throughput based on the size of the particles, and the second particle can be acquired.

[0216] The first sample introduced from the inlet 101 is a blood sample, but is not limited to this. If the first sample contains two types of particles with different sizes and these two types of particles can be separated by the particle separation chip 1, the first sample may be a body fluid other than blood (cerebrospinal fluid, ascites, pleural fluid, synovial fluid, peritoneal dialysis effluent, etc.), a sample prepared from a body fluid, or urine or a sample prepared from urine.

[0217] The embodiments of the present invention can be modified in various ways as appropriate within the scope of the technical ideas defined in the claims. [Explanation of symbols]

[0218] 1. Particle separation chip 1a Microchannel 2 Particle separator 10 Upper board (board) 20 Lower board (board) 30 Perforated Sheet 31 holes 41 Installation part 61~63 Liquid delivery section 64 First Recovery Section 65 2nd Recovery Section 100 Main channel 101 Entrance 102, 103 inlet (sheath liquid inlet) Exit 104 (Exit 1) 200 Branching Channel 201 Flow Path 211, 212 Relay channel Exit 213 (Second Exit) B Branch point CP1~CP5, CP11~CP16 Particle separation chips SP1~SP7 Perforated Sheet

Claims

1. A particle separation chip for separating a plurality of particles contained in a sample based on the size of the particles, The device includes a substrate having a microchannel therein and a porous sheet having a plurality of holes formed therein, The microchannel is a main flow path having an inlet provided on an upstream side into which a sample containing first particles and second particles smaller than the first particles is fed, and a first outlet provided on a downstream side from which a sample mainly containing the first particles is taken out; a branch flow path that communicates with the main flow path at a plurality of branch points along a flow direction of the main flow path and has a second outlet on a downstream side from which a sample containing the second particles is taken out; A particle separation chip, wherein the main flow path and the branch flow paths communicate with each other through the holes in the porous sheet at the plurality of branch points.

2. The particle separation chip according to claim 1, wherein the main channel and the branch channel are configured such that a portion of the sample introduced from the inlet flows into the branch channel and the remaining sample flows into the main channel, thereby causing the first particle to be discharged from the first outlet and the second particle to be discharged from the second outlet.

3. The particle separation chip according to claim 1 , wherein the width of the main channel is 100 μm or more and 50 mm or less.

4. The particle separation chip according to claim 1 , wherein the width of the main channel is 500 μm or more and 50 mm or less.

5. The particle separation chip according to claim 1 , wherein the branch channel includes a channel that communicates with the main channel at the branch point and is arranged non-parallel to the main channel in a plan view.

6. 6. The particle separation chip according to claim 5, wherein the channel that communicates with the main channel at the branch point is arranged to intersect with the main channel in a planar view, and the sample containing the second particles is caused to flow on both sides of the main channel.

7. The particle separation chip according to claim 1 , wherein the branch flow paths include 2 to 15,000 flow paths communicating with the main flow path at the branch points.

8. The particle separation chip according to claim 1 , wherein the branch channel is disposed below the main channel when the particle separation chip is in a used state.

9. 2. The particle separation chip according to claim 1, wherein the main flow path further has an inlet for a sheath liquid provided upstream of the branch point.

10. 10. The particle separation chip according to claim 9, wherein the sheath fluid inlet is an inlet for introducing the sheath fluid from above the sample flowing through the main channel.

11. 10. The particle separation chip according to claim 9, wherein the sheath fluid inlets are inlets for introducing the sheath fluid from both sides of the sample flowing through the main channel.

12. The particle separation chip according to claim 1 , wherein the fluid resistance of the branched flow path from the branching point on the upstream side of the main flow path to the second outlet is greater than the fluid resistance of the branched flow path from the branching point on the downstream side of the main flow path to the second outlet.

13. The branch flow path is A plurality of flow paths that are connected to the main flow path at the plurality of branch points and that are arranged non-parallel to the main flow path in a plan view; a relay flow path connecting the plurality of flow paths and the second outlet, The particle separation chip according to claim 1 , wherein a length of the flow channel that communicates with the main flow channel at the branch point on the upstream side of the main flow channel is longer than a length of the flow channel that communicates with the main flow channel at the branch point on the downstream side of the main flow channel.

14. The branch flow path is A plurality of flow paths that are connected to the main flow path at the plurality of branch points and that are arranged non-parallel to the main flow path in a plan view; a relay flow path connecting the plurality of flow paths and the second outlet, The particle separation chip according to claim 1 , wherein a cross-sectional area of ​​the flow channel that communicates with the main flow channel at the branch point upstream of the main flow channel is smaller than a cross-sectional area of ​​the flow channel that communicates with the main flow channel at the branch flow channel downstream of the main flow channel.

15. 2. The particle separation chip according to claim 1, wherein the microchannel is formed so that a liquid removal rate, which is a ratio of a flow rate to the branch channel to a flow rate of the main channel at the branch point, is a value at which the second particles flow to the branch channel and the first particles flow to the main channel.

16. 2. The particle separation chip according to claim 1, wherein the microchannel is formed so that a liquid removal rate, which is a ratio of a flow rate into the branch channel to a flow rate of the main channel at the branch point, is substantially the same value at each of the multiple branch points.

17. 2. The particle separation chip according to claim 1, wherein the microchannel is formed so that a liquid removal rate, which is a ratio of a flow rate to the branch channel to a flow rate of the main channel at the branch point, is greater than 0% and less than 50% at the multiple branch points.

18. The particle separation chip according to claim 1, wherein the porous sheet is formed so that an opening rate, which is the ratio of the area of ​​the holes in the porous sheet to the area of ​​the opening of the intersection plane between the main flow path and the branch flow path, is a value that allows the first particles of a predetermined size to be recovered from the first outlet at a predetermined recovery rate.

19. 2. The particle separation chip according to claim 1, wherein the pores of the porous sheet have a diameter of 30 nm or more and 100 μm or less.

20. The particle separation chip according to claim 1, wherein the porous sheet is formed so that an opening rate, which is the ratio of the area of ​​the holes in the porous sheet to the area of ​​the opening of the intersection surface between the main flow path and the branch flow path, is greater than 0% and less than 50% at the multiple branching points.

21. The particle separation chip according to claim 1 , wherein the particle diameter of the first particles is not less than 30 nm and not more than 100 μm.

22. the sample introduced through the inlet is a blood sample; the first particle is a circulating tumor cell; The particle separation chip according to claim 1 , wherein the second particles are white blood cells, red blood cells and platelets.

23. A particle separation chip according to any one of claims 1 to 22; a mounting portion on which the particle separation chip is mounted; a liquid delivery unit that introduces the sample and the sheath liquid into an inlet for the sample and an inlet for the sheath liquid, respectively, of the particle separation chip, and delivers liquid within the microchannel; a first collection section for the first particles communicating with the first outlet; a second collection section for the second particles in communication with the second outlet.

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