Particle separation chip and particle separation device

Abstract

The invention provides a particle separation chip and a particle separation device capable of improving flux when separating particles. A particle separation chip (1) for separating a plurality of particles contained in a sample on the basis of the size of the particles comprises an upper substrate (10) and a lower substrate (20) each having a microchannel (1a) therein, and a porous sheet (30) having a plurality of pores formed therein. The microchannel (1a) is provided with: a main channel (100) having an inlet (101) provided on the upstream side for feeding a first sample containing first particles and second particles smaller than the first particles, and an outlet (104) provided on the downstream side for taking out a second sample mainly containing the first particles; and a branch flow path (200) having, on the downstream side, an outlet (213) from which a third sample containing the second particles is taken out. At the plurality of diverging positions, the main flow path (100) and the diverging flow paths (200) communicate with each other via the pores of the porous sheet (30).

Description

[Technical field]

[0001] The 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, the following non-patent document 1 discloses a particle separation method using a hydraulic filtration method. In this separation method, a microchannel consisting of a main channel and a plurality of branch channels branching from the main channel is used. By introducing a sample into the main channel, the particles are separated between the main channel and the branch channels based on the size of the particles. This separation method can reduce the damage to the particles caused by the separation, and the structure of the channel can also be simplified.

[0003] [Prior art literature]

[0004]

Non-patent literature

[0005] [Non-patent document 1] Miyuki Matsuda, et al., “Blood cell fractionation using hydraulic filtration,” Journal of the Society of Electrical Engineers E, The Society of Electrical Engineers, 2008, Vol. 128, No. 10, p. 396-401

[0006] [Summary of the Invention]

[0007] [Problems to be solved by the invention]

[0008] The microchannel using the hydraulic filtration method as described above may be used at the laboratory level, but is not suitable for practical use, for example, at product development sites or clinical sites where high sample processing capacity (throughput) is required.

[0009] In view of the above problems, the present invention aims to provide a particle separation chip and a particle separation device that can demonstrate sufficient sample processing capacity (throughput) at a product development site or a clinical site.

[0010]

Methods for solving the problem

[0011] The particle separation chip (1, CP1 to CP5, CP11 to CP16) of the present invention comprises a substrate (10, 20) having a microchannel (1a) therein and a porous sheet (30) having a plurality of holes (31). The microchannel (1a) comprises a main channel (100) having an inlet (101) disposed on the upstream side for introducing a sample containing a first particle and a second particle smaller than the first particle and a first outlet (104) disposed on the downstream side for taking out a sample mainly containing the first particle, and a branch channel (200) connected to the main channel (100) at a plurality of branch positions (B) along the flow direction of the main channel (100) and having a second outlet (213) on the downstream side for taking out a sample containing the second particle. At the plurality of branch positions (B), the main channel (100) and the branch channel (200) are connected via the holes (31) of the porous sheet (30, PS1 to PS7).

[0012] In the particle separation chip of the present invention, the main flow path and the branch flow paths are connected at a plurality of branch positions (B) through the holes of the porous sheet, thereby achieving sufficient sample processing capacity (throughput) at the product development site or clinical site.

[0013] 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, a setting part (41) loaded with the particle separation chip (1, CP1 to CP5, CP11 to CP16), a liquid delivery part (61 to 63) for introducing the sample and the 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), a first recovery part (64) for the first particles connected to the first outlet (104), and a second recovery part (65) for the second particles connected to the second outlet (213).

[0014] According to the particle separation device of the present invention, the particle separation chip is mounted on the installation part, the liquid feeding part is driven to introduce the first sample and the sheath liquid, and the first particles and the second particles can be smoothly recovered from the first recovery part and the second recovery part, respectively.

[0015] [Effects of the Invention]

[0016] The present invention can provide a particle separation chip and a particle separation device that can demonstrate sufficient sample processing capacity (throughput) at a product development site or a clinical site.

[0017] [Brief description of the attached drawings]

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[0049] The following embodiments are to apply the present invention 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 sample (first sample) of a liquid containing a first particle and a second particle smaller than the first particle into a sample (second sample) of a liquid mainly containing the first particle and a sample (third sample) of a liquid mainly containing 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, a circulating tumor cell (CTC) in the blood, and the second particle is, for example, a white blood cell, an red blood cell, a platelet, and a nanoparticle. Nanoparticles are, for example, extracellular vesicles such as exosomes, tiny proteins, tiny fragments, and the like. The size of a particle refers to, for example, the diameter or radius of the particle, the area when viewed in a plane, or the length of the circumference. In the following description, the particle size represents the diameter of the particle.

[0050] In the following, for convenience, mutually orthogonal X, Y, and Z axes are attached in each figure. The Z axis direction is the height direction of the particle separation chip 1 and the particle separation device 2 described later. The XY plane is parallel to the horizontal plane. Furthermore, in the embodiment, the sample flows in the X axis direction, and in the figure, 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.

[0051] Reference Figures 1 to 5 , the structure of the particle separation chip 1 is described. The particle separation chip 1 comprises an upper substrate 10 (shown in Figure 1 ), lower substrate 20 (shown in Figure 2 ) and a porous sheet 30 (shown in FIG. 1 ) sandwiched between the upper substrate 10 and the lower substrate 20 Figure 3 )constitute.

[0052] Figure 1 It is a perspective view schematically showing the structure of the upper substrate 10.

[0053] The upper substrate 10 is composed of a plate-like member having an opposing surface 10a and an upper surface 10b parallel to the XY plane, and the interval between the two surfaces is D1. In addition, the upper substrate 10 is formed symmetrically in the Y-axis direction with respect to a central axis CA parallel to the X-axis direction and an imaginary vertical plane VP perpendicularly intersecting the upper substrate 10. The opposing surface 10a is a surface facing the lower substrate 20 described later, and the upper surface 10b is a surface that becomes the upper surface of the particle separation chip 1 when the particle separation chip 1 is completed.

[0054] The upper substrate 10 is made of a material that allows the blood sample to flow smoothly over the surface and can be processed with high precision, such as polydimethylsiloxane (PDMS). In addition, the upper substrate 10 can also be made of glass, cycloolefin polymer (COP), cycloolefin copolymer (COC), etc.

[0055] A main flow path 100 of a specified depth d1 is formed on the facing surface 10a. In addition, four outlets 213 consisting of through holes are formed along the right side end of the upper substrate. The main flow path 100 and the four outlets 213 are formed, for example, by a semiconductor processing process. The main flow path 100 has a recessed portion that is depressed in the Z-axis direction relative to the facing surface 10a, and the bottom surface of the recess is parallel to the XY plane. The main flow path 100 is formed by overlapping the upper substrate 10 and the lower substrate 20 to form a flow path, and the sample can be transferred therein. The four outlets 213 constitute a part of the branch flow path 200 described later.

[0056] The main channel 100 includes inlets 101 , 102 , and 103 provided on the left side thereof, channels 111 , 112 , a pair of channels 113 , a channel 120 provided in the center thereof, and an outlet 104 provided on the right side thereof.

[0057] The inlets 101 to 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 both sides along the Y-axis of the outlet 104. The four outlets 213 are formed by through holes penetrating the upper substrate 10 in the Z-axis direction.

[0058] The channels 111 and 112 extend in the X-axis direction and are arranged along the central axis CA. The channels 111 and 112 have a constant width. The upstream end of the channel 111 is connected to the inlet 101, the downstream end of the channel 111 is connected to the channel 120, and the central portion of the channel 111 is connected to the inlet 102.

[0059] The inlet 103 is located on the left side of the inlet 101, and the left end of the flow path 112 is connected to the inlet 103. A pair of flow paths 113 diverge from the flow path 112 on both sides along the Y-axis direction of the vertical plane VP. 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 to the inlet 102 and the flow path 120 in an oblique direction relative to the flow path 111. The width of the pair of flow paths 113 is constant.

[0060] The flow path 120 extends in the X-axis direction and is disposed on the central axis CA. The width of the flow path 120 in the Y-axis direction gradually increases from the connecting portion of the flow paths 111 and 120 to the central portion at the left side of the flow path 120. The width of the flow path 120 in the Y-axis direction except the left side is constant and is wider than the width of the flow paths 111 to 113. The right end of the flow path 120 is connected to the outlet 104.

[0061] The dimensions of the main flow path 100 can be set, for example, as follows.

[0062] When the particle separation chip 1 has an outer shape of 150 mm square in a plan view, the length of the flow path 120 in the X-axis direction is set to, for example, 150 mm or less. The width of the flow path 120 in the Y-axis direction is set to, for example, 50 mm or less, based on about one third of the length of one side of the outer shape.

[0063] The width of the flow path 120 in the Y-axis direction is set to, for example, 500 μm or more when 5 mL of whole blood flows to the particle separation chip 1 as a blood sample in one particle separation process. According to the present embodiment, when the width of the flow path 120 in the Y-axis direction is 500 μm, for example, the time required for 5 mL of whole blood to flow is 200 minutes, and the flux of the particle separation process becomes 25 μL / min. In addition, when the width of the flow path 120 in the Y-axis direction is 50 mm, for example, the time required for 5 mL of whole blood to flow is 2 minutes, and the flux of the particle separation process becomes 2.5 mL / min. In one particle separation process, when a further smaller amount of whole blood flows to the particle separation chip 1, the width of the flow path 120 in the Y-axis direction can also be set to, for example, 100 μm or more. Flux refers to the amount of sample provided for particle separation per unit time, and when the sample is a blood sample, it is the amount of undiluted whole blood provided for particle separation per unit time.

[0064] The depth d1 of the flow path 120 (the height of the concave portion in the Z-axis direction corresponding to the flow path 120 formed on the upper substrate 10) is set to be larger than the particle size of the first particle in such a manner that the first particle can be transferred in the flow path 120. When the first particle is a CTC, the depth d1 of the flow path 120 is set to, for example, 100 μm or more, and when the first particle is a nanoparticle, the depth d1 of the flow path 120 is set to, for example, 50 nm or more. In addition, since the flow path 120 is formed by a semiconductor processing process, the depth d1 of the flow path 120 is set to, for example, 1 mm or less.

[0065] Furthermore, the depth of the flow channels 111 to 113 is set to, for example, not less than 50 nm and not more than 1 mm for the same reason as the depth of the flow channel 120 .

[0066] Figure 2 It is a perspective view schematically showing the structure of the lower substrate 20.

[0067] The lower substrate 20 is composed of a plate-like member having an opposing surface 20a and a lower surface 20b parallel to the XY plane, and the interval between the two surfaces is D2. In addition, the lower substrate 20 is formed symmetrically in the Y-axis direction with respect to a central axis CA parallel to the X-axis direction and an imaginary vertical plane VP perpendicularly intersecting the lower substrate 20. The opposing surface 20a is a surface facing the upper substrate 10, and the lower surface 20b is a surface that becomes the lower surface of the particle separation chip 1 when the particle separation chip 1 is completed.

[0068] The lower substrate 20 is made of a material that allows the blood sample to flow smoothly over the surface and can be processed with high precision. The lower substrate 20 of this embodiment is made of polydimethylsiloxane (PDMS) as is the upper substrate 10. Furthermore, the lower substrate 20 may also be made of glass, cycloolefin polymer (COP), cycloolefin copolymer (COC), etc.

[0069] A branch flow path 200 having a predetermined depth d2 is formed on the facing surface 20a. The branch flow path 200 is formed, for example, by a semiconductor processing process. The branch flow path 200 has a concave portion that is depressed in the Z-axis direction relative to the facing surface 20a, and the bottom surface of the concave portion is parallel to the XY plane. The branch flow path 200 is formed by overlapping the upper substrate 10 and the lower substrate 20 to form a flow path, so that a sample can be transferred therein.

[0070] The branch flow path 200 includes a plurality of flow paths 201, two relay flow paths 211, and four relay flow paths 212. Figure 1 The four outlets 213 are shown in FIG. Figure 2 In the figure, for the sake of convenience, a part of the plurality of flow paths 201 and a part of the two relay flow paths 211 are omitted from illustration by dotted lines.

[0071] The plurality of flow paths 201 extend parallel to the Y-axis direction and are arranged in the X-axis direction at a predetermined interval. The length of each flow path 201 in the Y-axis direction becomes shorter as it goes to the right side in the X-axis direction. That is, the overall shape of the plurality of flow paths 201 is a trapezoid in a plan view.

[0072] The two relay channels 211 are respectively arranged at one end and the other end of the plurality of channels 201, and connected to the plurality of channels 201. The relay channels 211 are connected to the main channel 100 (see Figure 1 ) and its two ends extend in a manner close to the central axis CA. The four relay channels 212 extend parallel to the X-axis direction. The two relay channels 212 are respectively connected to the relay channel 211 at one end side of the plurality of channels 201 and the relay channel 211 at the other end side of the plurality of channels 201.

[0073] The dimensions of the branch flow path 200 can be set, for example, as follows.

[0074] When the particle separation chip 1 is formed to have a 150 mm square in plan view, the length of the multiple channels 201 in the Y-axis direction is set to be less than 150 mm. In addition, the length of the multiple channels 201 in the Y-axis direction is set to be longer than the width of the channel 120 in the Y-axis direction.

[0075] The width (length in the X-axis direction) of the flow path 201 is set to be larger than the particle size of the second particle so that the second particle can be transferred in the flow path 201. When the second particle is a white blood cell, an red blood cell, or a platelet, the width of the flow path 201 is set to, for example, 100 μm or more, and when the second particle is a nanoparticle, the width of the flow path 201 is set to, for example, 50 nm or more. The width of the flow path 201 is set to, for example, 1 mm or less in order to arrange a sufficient number of flow paths 201.

[0076] In addition, the number of the plurality of flow paths 201 is set to be not less than 2 and not more than 15,000. According to the inventor's research, when the width of the flow path 201 is set to 100 μm and the interval between adjacent flow paths 201 is set to 50 μm, about 150 flow paths 201 can be formed. In addition, according to the semiconductor processing process, since the width and interval of the above-mentioned flow path 201 can be further reduced to about 1 / 100, it becomes possible to form 15,000 flow paths 201. In addition, in order to realize the hydrodynamic filtration method (Hydrodynamic Filtration: HDF) described later, more than two flow paths 201 are configured.

[0077] The depth d2 of the flow path 201 (the height of the concave portion in the Z-axis direction corresponding to the flow path 201 formed on the lower substrate 20) is also set to be larger than the particle size of the second particle in such a manner that the second particle can be transferred in the flow path 201. When the second particle is a white blood cell, an red blood cell, or a platelet, the depth d2 of the flow path 201 is set to, for example, more than 100 μm, and when the second particle is a nanoparticle, the depth d2 of the flow path 201 is set to, for example, more than 50 nm. In addition, since the flow path 201 is formed by a semiconductor processing process, the depth d2 of the flow path 201 is set to, for example, less than 1 mm.

[0078] Furthermore, the depth of the relay channels 211 and 212 is set to, for example, not less than 50 nm and not more than 1 mm for the same reason as the depth of the channel 201 .

[0079] Figure 3 It is a perspective view and an enlarged view schematically showing the structure of the porous sheet 30.

[0080] The porous sheet 30 is a thin film sheet with a rectangular shape in a plan view. The porous sheet 30 has a plurality of holes 31 that penetrate the porous sheet 30 in the Z-axis direction. As described later, the second particle flowing through the main flow path 100 moves 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 through the surface, can form holes 31 with high precision, and is difficult to break. The porous sheet 30 of this embodiment is made of, for example, nickel. Furthermore, the porous sheet 30 is not necessarily made of metal, and can also be made of glass or PDMS, etc.

[0081] The dimensions of the porous sheet 30 can be set, for example, as follows.

[0082] The thickness of the porous sheet 30 is set to, for example, 1 μm or more and 1 mm or less in a manner to achieve separation of the second particle by the hydraulic filtration method. According to the inventor's research, even if the thickness of the porous sheet 30 is set to 1 mm, the fluid resistance to the second particle passing through the hole 31 from the porous sheet 30 can be roughly ignored. The pore size of the hole 31 is set to, for example, 30 nm or more and 100 μm or less in a manner that the first particle smoothly enters the downstream side in the flow path 120 of the main flow path 100, and more preferably 50 nm or more and 100 μm or less.

[0083] The porosity, which is the ratio of the area of ​​the holes 31 of the porous sheet 30, is set to, for example, greater than 0% and less than 50% in order to maintain the strength of the porous sheet 30. In the present embodiment, the size and shape of all the holes 31 of the porous sheet 30 are the same, and all the holes 31 are evenly arranged on the porous sheet 30. Thus, the porosity of the porous sheet 30 is set to be greater than 0% and less than 50% in order to maintain the strength of the porous sheet 30. In the present embodiment, the size and shape of all the holes 31 of the porous sheet 30 are the same, and all the holes 31 are evenly arranged on the porous sheet 30. Fig. 9 ), become substantially the same value.

[0084] Furthermore, the opening ratio of the porous sheet 30 may be different values ​​at a plurality of diverging positions B. In this case, the porous sheet 30 may have different numbers of holes 31 or different diameters of holes 31 at a plurality of diverging positions B. In addition, the porous sheet 30 may have different numbers of holes 31 or different diameters of holes 31 at a single diverging position B depending on the region.

[0085] The length of the porous sheet 30 in the Y-axis direction is set to be longer than the Y-axis width of the flow path 120 of the upper substrate 10. The length of the porous sheet 30 in the X-axis direction is set to be longer than the length of the plurality of flow paths 201 of the lower substrate 20 as a whole.

[0086] Figure 4 1 is a perspective view schematically showing the assembly of the upper substrate 10, the lower substrate 20 and the porous sheet 30. Figure 4 In FIG. 1 , for convenience, the outline of the facing surface 10 a side of the main flow channel 100 is indicated by a dotted line.

[0087] Figure 1 The upper substrate 10 shown in FIG. 1 is in a state of being turned inside out and Figure 2 That is, the upper substrate 10 and the lower substrate 20 are joined in a manner that the facing surface 10a of the upper substrate 10 and the facing surface 20a of the lower substrate 20 face each other. At this time, a Figure 3The porous sheet 30 shown in FIG. When the upper substrate 10 and the lower substrate 20 are made of PDMS, the facing surfaces 10a and 20a are bonded by surface modification of the silicon dioxide on the surfaces of the facing surfaces 10a and 20a by oxygen plasma. The surface modification can also be performed by UV ozone treatment using ultraviolet rays. Thus, Figure 5 The particle separation chip 1 shown is completed.

[0088] Figure 5 It is a perspective view schematically showing the structure of the particle separation chip 1.

[0089] The main flow path 100 is composed of a concave portion formed as the facing surface 10a of the upper substrate 10 and the facing surface 20a of the lower substrate 20 facing the concave portion. The branch flow path 200 is composed of a concave portion formed as the facing surface 20a of the lower substrate 20 and the facing surface 10a of the upper substrate 10 facing the concave portion. The micro flow path 1a is composed of the main flow path 100 and the branch flow path 200. A micro flow path generally refers to a flow path with a depth and width of about 10μm to 1mm. The outlet 213 of the upper substrate 10 overlaps with the end of the relay flow path 212 of the lower substrate 20 on the right side of the X-axis direction, and the outlet 213 is connected to the relay flow path 212.

[0090] In the X-axis direction, the porous sheet 30 is arranged so as to cover the entirety of the plurality of flow paths 201 of the branch flow path 200. In the Y-axis direction, the porous sheet 30 is arranged inside the shortest flow path 201.

[0091] Next, refer to Figures 6 to 11 , the function of the particle separation chip 1 to separate the first particles and the second particles contained in the sample will be described.

[0092] Figure 6 The diagram schematically shows the injection of the sample and the 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 .

[0093] A liquid sample containing a first particle and a second particle smaller than the first particle flows into the inlet 101 as a first sample. A sheath liquid is injected into the inlets 102 and 103. As the sheath liquid, for example, CELLPACK (registered trademark) manufactured by Sysmex Co., Ltd. can be used. A liquid sample mainly containing the first particle is taken out from the outlet 104 as a second sample. A liquid sample mainly containing the second particle is taken out from the plurality of outlets 213 as a third sample.

[0094] Figure 7 , 8 This is a diagram schematically showing a range in which the flow of the first sample is restricted by the sheath fluid. Figure 7 For convenience, the vicinity of the connection portion between the inlets 101, 102 and the flow paths 111, 113 is schematically shown. Figure 8The upper, middle and lower sections of Figure 7 C21-C22 section, C31-C32 section and C41-C42 section.

[0095] like Figure 7 As shown in FIG. 1 , the first sample flows into the flow path 111 through the inlet 101. The sheath liquid injected from the inlet 102 presses the first sample flowing through the flow path 111 downward. Figure 8 As shown in the upper part of FIG. 1 , the range of the first sample flowing through the flow channel 111 is limited to the vicinity of the bottom surface of the flow channel 111 by the sheath liquid.

[0096] like Figure 7 As shown, a pair of flow paths 113 are connected to both side surfaces of the flow path 111 between the inlet 102 and the flow path 120 in a direction inclined relative to the X-axis direction. The sheath liquid flowing into the flow path 111 from the pair of flow paths 112 will flow from the bottom of the inlet 102 to the first sample on the downstream side on a plane close to the vertical plane VP (refer to Figure 1 , 2 ) direction. Therefore, if Figure 8 As shown in the middle section of FIG. 1 , the range of the first sample flowing through the flow channel 111 is limited to the vicinity of the center of the flow channel 111 by the sheath liquid.

[0097] like Figure 7 As shown in FIG. 1 , since the width of the flow path 120 is wider than the width of the flow path 111, the first sample flowing from the flow path 111 to the flow path 120 expands in the Y-axis direction and flows toward the downstream side. Figure 8 As shown in the lower part of FIG. 1 , although the range of the first sample is expanded in the Y-axis direction, it is limited to the vicinity of the center of the flow channel 120 because it is pressed toward the center by the sheath fluid from the pair of flow channels 113 .

[0098] Fig. 9 It is a perspective view schematically showing the vicinity of a portion where the flow path 120 of the main flow path 100 and the flow path 201 of the branch flow path 200 intersect.

[0099] In the portion where the flow path 120 and the flow path 201 overlap when viewed from a plane, the recess on the flow path 120 side is connected to the recess on the flow path 201 side in the Z-axis direction. The intersection of the flow path 120 and the flow path 201 becomes an opening, but a porous sheet 30 is arranged at this opening, and the opening is blocked. The position corresponding to the opening of the intersection of the flow path 120 and the flow path 201 arranged on the porous sheet 30 is a divergence position B. Thus, the area of ​​the divergence position B is equal to the area of ​​the opening of the intersection of the flow path 120 and the flow path 201. The divergence flow path 200 is connected to the main flow path 100 at multiple divergence positions B along the flow direction (X-axis direction) of the main flow path 100. The porous sheet 30 is arranged in a manner that spans multiple divergence positions B.

[0100] Furthermore, in the present embodiment, the plurality of divergent positions B are of the same shape and are arranged at the same positions in the Y-axis direction and the Z-axis direction. In addition, the plurality of divergent positions B are arranged in such a way that the intervals in the X-axis direction become the same as each other. However, the plurality of divergent positions B only need to be arranged at intervals in the X-axis direction between the inlet 101 and the outlet 104 of the main channel 100. For example, the plurality of divergent positions B may be of different shapes or of different areas. The plurality of divergent positions B may be arranged at different positions in the Y-axis direction or at different positions in the Z-axis direction. The plurality of intervals between the divergent positions B may also be different from each other.

[0101] The first sample flowing to the downstream side through the flow path 120 passes through the branch position B, and the second particles contained in the first sample flow to the flow path 201 through the holes 31 of the porous sheet 30. Then, the third sample containing the second particles flows from the outlet 213 (see Figure 6 ) is taken out. On the one hand, the first particles contained in the first sample do not flow to the flow path 201 but flow to the downstream side through the flow path 120. Furthermore, the second sample containing the first particles is taken out from the outlet 104 (refer to Figure 6 )take out.

[0102] At this time, if Figure 8 As shown in the lower section of , since the first sample flows to the branching position B while being restricted at the upper portion in the thickness direction and at both ends in the width direction, the second particles contained in the first sample can be smoothly separated to the flow path 201 .

[0103] Fig.10 It is indicated in Fig. 9 The divergence position B shown in FIG. 1 separates the first particle and the second particle. Fig.10 show Figure 6 C11-C12 section.

[0104] exist Fig.10 For convenience, the first particle is represented by a white circle, and the second particle is represented by a black circle. The components other than the first particle and the second particle contained in the first sample are represented by dense dots, and the sheath liquid injected from the inlets 102 and 103 is represented by sparse dots. In addition, in the entire porous sheet 30, only the divergence position B (refer to Fig. 9 ) of the porous sheet 30.

[0105] As reference Figure 7 and Figure 8 As described in the upper part of the figure, since the first sample injected from the inlet 101 is pressed downward by the sheath liquid injected from the inlet 102, the first sample flows toward the downstream side along the bottom surface of the main flow path 100. The plurality of flow paths 201 of the present embodiment are configured in such a way that the first particles and the second particles can be separated by hydraulic filtration (HDF).

[0106] Fig.11 It is a schematic display Fig.10 An enlarged view of the vicinity of one of the flow channels 201 is shown.

[0107] When the flow rate in the flow path 120 flowing from the upstream side relative to a flow path 201 is set to Qm, and the flow rate flowing to the flow path 201 is set to Qs, the relationship between the flow rates Qm and Qs can be expressed by the following formula (1) using the liquid removal rate a at the branch position B.

[0108] Qs=Qm×a…(1)

[0109] The liquid removal rate a at the branch position B is set to a value in which the second particle flows to the branch flow path 200 and the first particle flows to the main flow path 100. According to the hydraulic filtration method, the height h of the liquid portion of the first sample that flows into each flow path 201 among the liquid portion of the first sample is determined by the liquid removal rate a. Therefore, in the present embodiment, the liquid removal rate a is set in such a way 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 particle is transferred to the downstream side along the flow of the first sample, and the second particle is separated from the liquid portion of the first sample in the range of the height h to each flow path 201.

[0110] In this embodiment, the liquid removal rates a are set to substantially the same value at all branch positions B. When the liquid removal rates a of the respective flow paths 201 are equal to each other, the heights h of the liquid portions of the first sample flowing into the respective flow paths 201 also become equal to each other. Thus, the second particles of a certain size can be generally separated at the plurality of branch positions B.

[0111] The liquid removal rate a at the branch position B is from the branch position B to the outlet 213 (refer to Figure 6 ) is determined by the fluid resistance of the branch flow path 200 at the upstream branch position B to the outlet 213. Therefore, in the present embodiment, the fluid resistance of the branch flow path 200 from the upstream branch position B to the outlet 213 is set to be larger than the fluid resistance of the branch flow path 200 from the downstream branch position B to the outlet 213. As a result, the fluid resistance at the upstream branch position B becomes larger than the fluid resistance at the downstream branch position B, and the liquid removal rate a at each branch position B can be set to a value that is substantially equal to each other.

[0112] In this embodiment, the relay channels 211 and 212 (see Figure 6) is larger than the flow path 201, and the liquid removal rate a at the branch position B is substantially determined by the fluid resistance of the flow path 201. Therefore, in this embodiment, the length of the flow path 201 in the Y-axis direction on the upstream side is set to be longer than the length of the flow path 201 in the Y-axis direction on the downstream side. As a result, the fluid resistance of the flow path 201 connected to the branch position B on the upstream side can be set to be larger than the fluid resistance of the flow path 201 connected to the branch position B on the downstream side.

[0113] Furthermore, the liquid removal rates a are all set to be greater than 0% and less than 50% at the plurality of branch positions B. Thus, the second particles having a particle diameter smaller than the depth d1 of the flow channel 120 can be separated into the flow channel 201 .

[0114] In addition, in this embodiment, the holes 31 of the porous sheet 30 arranged at the branch position B are as follows. Fig.11 As shown by the thick arrow at the position of the hole 31, a sample flow can be formed to move the second particle from the flow path 120 of the main flow path 100 to the flow path 201 of the branch flow path 200. Therefore, in order to increase the flux, even if the expansion Fig.11 By increasing the width of the flow path 201 in the X-axis direction as shown in the figure, the flow rate of the flow path 201 can be increased, and the flow of the sample of the second particle to the flow path 201 can be dispersed in the plurality of holes 31 of the porous sheet 30. Thus, the flow of the sample of the second particle is not formed only in a part of the flow path 201, but can be formed in the entire flow path 201. In addition, as Fig.11 As shown, since a plurality of holes 31 are formed in the porous sheet 30 , the width of the flow path 201 in the X-axis direction is wide, and turbidity in the porous sheet 30 and the flow path 201 can be suppressed.

[0115] Therefore, the second particles can be efficiently separated to the flow path 201 without being turbid, the flow rates of the main flow path 100 and the branch flow path 200 can be increased, and the flux of particle separation can be efficiently increased.

[0116] Furthermore, in the present embodiment, the particle size of the second particles separated to the flow path 201 is not determined by the size relationship between the pore size of the pore 31 of 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 for separating the second particles from the first sample through the pore 31, but is used to disperse the flow of the second particles to the flow path 201 as described above. Therefore, the pore size of the pore 31 may be, for example, larger than the particle size of the second particles to be separated.

[0117] return Fig.10 By separating the second particles at a plurality of branch positions B, the second particles and the liquid portion of the first sample flow in the flow path 201 of the branch flow path 200. Thus, the outlet 213 (refer to Figure 6) The third sample taken out from the outlet 104 mainly contains the second particles. On the other hand, the first particles and the sheath liquid remain in the flow path 120 of the main flow path 100. Thus, the second sample taken out from the outlet 104 mainly contains the first particles.

[0118] Next, the particle separation device 2 for separating the first particles from the second particles will be described.

[0119] Fig.12 It is a perspective view schematically showing the structure of the particle separation device 2.

[0120] The particle separation device 2 includes a particle separation chip 1 , a fixing unit 40 , three inlet nozzles 51 to 53 , five outlet nozzles 54 and 55 , liquid feeding units 61 to 63 , a first recovery unit 64 , and four second recovery units 65 .

[0121] The particle separation chip 1 is cleaned and reused when the particle separation process is performed. Furthermore, the particle separation chip 1 can also be configured as a replaceable particle separation chip that can be replaced with a new one when the particle separation process is performed.

[0122] The fixing part 40 includes a setting part 41 and a lifting part 42. The setting part 41 includes a flat plate 40a parallel to the XY plane and a frame part 40b provided on the upper surface of the flat plate 40a. In a plan view, an opening having a size substantially the same 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 provided in this opening. The lifting part 42 is supported on the lifting mechanism 2b (refer to FIG. 1 ) so as to be movable in the Z-axis direction. Fig.13 ) The lifting unit 42 includes an arm portion 42a extending upward from the particle separation chip 1 disposed in the frame portion 40b.

[0123] The upper ends of the inlet nozzles 51 to 53 are connected to the liquid supply units 61 to 63 via tubes. The liquid supply units 61 to 63 are, for example, syringe pumps. The liquid supply unit 61 is configured to be able to supply a specified amount of the first sample to the inlet nozzle 51 at a specified speed. The liquid supply units 62 and 63 are configured to be able to supply a specified amount of the sheath liquid to the inlet nozzles 52 and 53 at a specified speed.

[0124] The upper end of the outlet nozzle 54 is connected to the first recovery part 64 via a tube. The upper ends of the four outlet nozzles 55 are respectively connected to the second recovery part 65 via a tube. The first recovery part 64 and the four second recovery parts 65 are containers for recovering samples. Furthermore, a liquid delivery part consisting of a syringe pump for delivering the second sample to the first recovery part 64 may also be configured in the tube connecting the outlet nozzle 54 and the first recovery part 64. A liquid delivery part consisting of a syringe pump for delivering the third sample to the second recovery part 65 may also be configured in the tube connecting the outlet nozzle 55 and the second recovery part 65.

[0125] When performing particle separation processing, the particle separation chip 1 is set in the opening of the frame 40b, and the lifting part 42 moves downward, and the upper surface of the particle separation chip 1 (the upper surface 10b of the upper substrate 10) is pressed downward by the arm 42a. Thus, the particle separation chip 1 is fixed. Thereafter, the inlet nozzles 51 to 53 are respectively connected to the inlets 101 to 103 of the particle separation chip 1, and the outlet nozzles 54 and 55 are respectively connected to the outlets 104 and 213 of the particle separation chip 1.

[0126] In this state, the liquid delivery unit 62 delivers liquid to the flow path 111 (see Figure 6 ) to deliver the sheath liquid, the liquid delivery unit 63 delivers the sheath liquid to the flow paths 112 and 113 (see Figure 6 ) to deliver the sheath liquid. Then, the liquid delivery unit 61 delivers the first sample to the flow path 111 through the inlet 101. Fig.10 , 11 It is explained that the sample containing the first particle and the second particle (the first sample) is separated into the sample containing the first particle (the second sample) and the sample containing the second particle (the third sample). Furthermore, the second sample is recovered into the first recovery part 64 through the outlet 104, and the third sample is recovered into the four second recovery parts 65 through the four outlets 213. Thus, the particle separation process is completed.

[0127] Fig.13 It is a block diagram showing the functional structure of the cell detection system 5.

[0128] The cell detection system 5 includes a particle separation device 2, a staining concentration device 3, and an imaging flow cytometer 4. The cell detection system 5 is a system for detecting first particles (for example, CTCs) contained in a blood sample collected from a subject.

[0129] Fig.12 The particle separation device 2 shown in FIG. 1 includes a control unit 2a, a lifting mechanism 2b, and liquid feeding units 61 to 63. The control unit 2a includes, for example, a CPU and a memory, and controls each unit of the particle separation device 2. The lifting mechanism 2b includes a Fig.12 The lifting portion 42 is moved up and down by a motor.

[0130] 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 dye concentration device 3 performs a dyeing process for dyeing 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 photographs the particles contained in the second sample after the dyeing process to obtain a camera image. An operator such as a physician or an inspection technician refers to the camera image obtained by the imaging flow cytometer 4 to determine the presence or absence of the first particles (e.g., CTC) in the blood sample collected from the subject.

[0131] <Configuration of Example>

[0132] Next, the specific examples of the above-mentioned embodiments are described. In the following examples, the first sample is whole blood collected from a subject. The first particle is the cells of the object (target cells), circulating tumor cells (CTC) in the blood. The second particle is the cells of the object (non-target cells), white blood cells, red blood cells and platelets. The inventors will refer to Figures 14 to 18 The chip and the porous sheet described above are combined to form the particle separation chip 1 , and the particle separation process is actually carried out using the prepared particle separation chip 1 .

[0133] Fig.14 It is a diagram schematically showing the dimensions of the main flow channel 100 and the branch flow channel 200 according to the embodiment. Fig.15 It is a diagram schematically showing the arrangement and size of the holes 31 of the porous sheet 30 according to the embodiment.

[0134] like Fig.14 As shown, the depth of the main flow path 100 is 0.1 mm. The diameter of the inlets 101 to 103 is 1.5 mm. The width of the flow paths 111 to 113 (the length in the direction perpendicular to the direction of the sample in the flow path) is 0.5 mm. The width of the flow path 120 (the length in the Y-axis direction) is 4 mm. The end of the right side in the X-axis direction of the region where the width of the flow path 120 in the Y-axis direction is constant is 2 mm away from the right side in the X-axis direction relative to the end of the rightmost side in the X-axis direction of the plurality of flow paths 201.

[0135] The depth of the branch flow path 200 is 0.1 mm. The distance from the leftmost end of the plurality of flow paths 201 to the right end of the region of the flow path 120 having a constant width in the Y-axis direction is Lmain. The number of the plurality of flow paths 201 is n. The sequence number of the leftmost flow path 201 is 1, and the sequence number of the rightmost flow path 201 is n. The lengths of the first and nth flow paths 201 protruding in the Y-axis direction relative to the flow path 120 are L1 and Ln, respectively. The width of the flow path 201 (the length in the X-axis direction) is w, and the interval between two adjacent flow paths 201 is v.

[0136] 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. Fig.15 As shown, the pore diameter of the hole 31 of the porous sheet 30 is φ, and the distance between two adjacent holes 31 is p. The three straight lines connecting the centers of the three adjacent holes 31 have no mutual angle of 60°. The opening ratio as the ratio of the area of ​​the hole 31 of the porous sheet 30 is R. The thickness of the porous sheet 30 is 5 μm.

[0137] Below, the distance Lmain, the number of strips n, the length L1, Ln, the width w and the interval v are different, such as Fig.16 As shown in FIG. 1 , four types of chips C1 to C4 are made of an upper substrate 10 and a lower substrate 20. In addition, the pitch p, the aperture φ and the opening ratio R are made different, such as Fig.17 As shown, porous sheets PS1 to PS7 are made.

[0138] Furthermore, if Fig.18 As shown, four relay channels 212 are provided in chips C1 and C2, and four relay channels 211 are provided to introduce the third sample into each of the four relay channels 212. Six relay channels 212 are provided in chips C3 and C4, and six relay channels 211 are provided to introduce the third sample into each of the six relay channels 212. Fig.18 In the chips C1 to C4 shown in FIG, the third sample flowing out to one channel 201 is taken out from the outlet 213 via one relay channel 211 and one relay channel 212 corresponding to the channel 201 .

[0139] [Example 1]

[0140] In Example 1, four types of particle separation chips 1 were installed in the above-mentioned particle separation device 2, and the first sample was flowed to each particle separation chip 1 to examine the removal rates of red blood cells and white blood cells.

[0141] Fig.19 The upper part of is a table showing the configurations of four types of particle separation chips CP1 to CP4 according to Example 1. Fig.19 The lower part of is a table showing the structures of the three types of first samples involved in Example 1.

[0142] like Fig.19 As shown in the upper part of the figure, the four particle separation chips CP1 to CP4 are Fig.16 The chips C1 to C4 and Fig.17 The porous sheet PS7 combination shown in FIG. Fig.19 As shown in the lower section of the figure, the three first samples have different whole blood volumes and diluent volumes. The first sample of "whole blood without dilution" is a 5 mL sample consisting only of whole blood. The first sample of "whole blood diluted 1.5 times" is a 5 mL sample made by diluting 3.33 mL of whole blood with 1.67 mL of diluent. The first sample of "whole blood diluted 2 times" is a 5 mL sample made by diluting 2.5 mL of whole blood with 2.5 mL of diluent. The whole blood used in the three first samples was collected from healthy people.

[0143] In Example 1, 1 mL of the above three first samples are made to flow through the particle separation chips CP1 to CP4 at inlets 101 to 100 μL / min, respectively. At this time, the fluxes of whole blood of the three first samples (undiluted whole blood, whole blood diluted 1.5 times, and whole blood diluted 2 times) are 100 μL / min, 67 μL / min, and 50 μL / min, respectively. In addition, the sheath liquid is made to flow at inlets 102 to 900 μL / min, and the sheath liquid is made to flow at inlets 103 to 2000 μL / min. The liquid delivery time of the first sample to inlets 101 to 103 is set to 10 minutes for any first sample. Furthermore, on each particle separation chip CP1 to CP4, the first recovery unit 64 (refer to Fig.12 ) to recover the second sample from the plurality of second recovery sections 65 (refer to Fig.12 )Recover the third sample.

[0144] Next, the number of cells (red blood cells and white blood cells) contained in the three types of first samples and the second samples collected by the particle separation chips CP1 to CP4 is obtained from the following formula (2).

[0145] Number of cells = cell concentration (cells / mL) × liquid volume (mL)…(2)

[0146] The cell concentration was measured using a blood cell counting device (XN series) or a blood cell counting chamber manufactured by Sysmex Co., Ltd. The liquid volume was measured by weight using an electronic balance.

[0147] Next, for each combination of the first sample and the particle separation chips CP1 to CP4, the red blood cell removal rate and the white blood cell removal rate were calculated by the following equations (3) and (4) based on the number of cells (red blood cells and white blood cells) obtained by the above equation (2).

[0148] Red blood cell removal rate = [1-(the number of red blood cells contained in the second sample / the number of red blood cells contained in the first sample)] × 100 (%) (3)

[0149] Leukocyte removal rate = [1-(the number of leukocytes contained in the second sample / the number of leukocytes contained in the first sample)] × 100 (%) (4)

[0150] Fig. 20 It 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.

[0151] exist Fig. 20 In the coordinate diagram of , for convenience, the type removal rate of each first sample is connected by a straight line. The liquid removal rates of the particle separation chips CP1 to CP4 are respectively Fig.16This corresponds to the liquid removal rates (0.73%, 1.12%, 1.31%, 1.50%) of chips C1 to C4 shown in FIG.

[0152] like Fig. 20 As shown, a high red blood cell removal rate and a high white blood cell removal rate can be achieved in any case. In addition, it is known that the higher the liquid removal rate, Fig.11 The higher the height h shown in becomes, the more reliably red blood cells and white blood cells can be removed. Furthermore, the higher the dilution rate, the higher the red blood cell removal rate and the white blood cell removal rate become. This reason is expected to be because the higher the dilution rate, the lower the density and viscosity of the cells in the first sample, and the easier it is to separate the red blood cells and white blood cells into the branch flow path 200.

[0153] [Example 2]

[0154] In Example 2, a particle separation chip 1 was installed in the above-mentioned particle separation device 2, and a first sample was flowed into the particle separation chip 1 to examine the removal rate of red blood cells and white blood cells and the recovery rate of CTCs.

[0155] Fig.21 The upper part of is a table showing the structure of the particle separation chip CP5 involved in Example 2. Fig.21 The lower part of is a table showing the structure of the first sample involved in Example 2.

[0156] like Fig.21 As shown in the upper part of the figure, the particle separation chip CP5 is Fig.16 The chip C4 and Fig.17 The porous sheet PS6 combination shown in FIG. Fig.21 As shown in the lower section of , the first sample of "whole blood diluted 2 times" involved in Example 2 is a 2.5 mL sample obtained by diluting 1.25 mL of whole blood collected from a healthy person with 1.25 mL of diluent. In addition, in Example 2, A549 cells obtained from the A549 cell line are added to the first sample as CTCs. Specifically, the A549 cells are stained with a pre-specified reagent, and about 400,000 stained A549 cells are added to the first sample. Furthermore, the size of the above-mentioned A549 cells, according to the inventor's measurement, is above 11 μm and below 20 μm, with an average of about 17 μm.

[0157] In Example 2, the total amount (2.5 mL) of the first sample is allowed to flow through the particle separation chip CP5 three times at inlets 101 to 100 μL / min. At this time, the flux of the whole blood of the first sample is 50 μL / min at any time. In addition, the sheath liquid is allowed to flow at inlets 102 to 900 μL / min, and the sheath liquid is allowed to flow at inlets 103 to 2000 μL / min. The liquid delivery time of the first sample to inlets 101 to 103 is set to 25 minutes at any time. Furthermore, on the particle separation chip CP5, the second sample is recovered from the first recovery section 64, and the third sample is recovered from multiple second recovery sections 65.

[0158] Next, the same procedure as in Example 1 is used to obtain the cells (red blood cells and white blood cells) contained in the first sample and the second sample by the above formula (2), and based on the obtained number of red blood cells and white blood cells, the red blood cell removal rate and the white blood cell removal rate are calculated by the above formulas (3) and (4).

[0159] In addition, the A549 cells contained in the recovered second sample and the third sample were obtained by the above formula (2), and the recovery rate of A549 cells was calculated by the following formula (5) based on the number of A549 cells obtained.

[0160] A549 cell recovery rate = the number of A549 cells contained in the second sample / (the number of A549 cells contained in the second sample + the number of A549 cells contained in the third sample) × 100 (%) (5)

[0161] Fig. 22 It 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.

[0162] like Fig. 22 As shown in the upper part of , in any of the cases where the first sample is passed through for the first to third times, the red blood cell removal rate is about 99% or more, and the white blood cell removal rate is about 90% or more, and a high red blood cell removal rate and a high white blood cell removal rate can be achieved. Fig. 22 As shown in the lower part of FIG, in any of the cases where the first sample was passed through for the first to third times, the recovery rate of A549 cells was about 90% or more, and a high recovery rate of A549 cells was achieved. From the above results, it is known that blood cell components (red blood cells and white blood cells) and A549 cells can be appropriately separated from the first sample based on the difference in particle size.

[0163] [Example 3]

[0164] In Example 3, six types of particle separation chips 1 were set in the above-mentioned particle separation device 2, and the first sample was flowed to 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.

[0165] Fig.23 The upper part of is a table showing the configurations of six types of particle separation chips CP11 to CP16 according to the third embodiment. Fig.23 The lower part of is a table showing the structure of the first sample involved in Example 3.

[0166] like Fig.23 As shown in the upper part of the figure, the six particle separation chips CP11 to CP16 are Fig.16 The chip C4 and Fig.17 The porous sheets PS1 to PS6 shown in FIG. Fig.23 As shown in the lower part of the figure, the first sample involved in Example 3 is a cell suspension. This cell suspension contains 2 mL of 1% polyvinyl pyrrolidone solution, 1×10 6 Jurkat cells and 3×10 5 A549 cells stained with a specified reagent in advance. Jurkat cells correspond to blood cell components (red blood cells or white blood cells) present in normal blood, and A549 cells correspond to CTCs. Furthermore, the inventors used imaging flow cytometry to investigate the particle sizes of Jurkat cells and A549 cells contained in the first sample (cell suspension) in advance.

[0167] Fig.24 Graph showing the particle size distribution of Jurkat cells and A549 cells contained in the first sample. Fig.24 In the graph, the horizontal axis shows the cell diameter, and the vertical axis shows the frequency (%) corresponding to the diameter. The frequency corresponding to a specified diameter corresponds to the existence probability of the specified diameter.

[0168] In Example 3, the first sample is allowed to flow through the particle separation chips CP11 to CP16 at inlets 101 to 110 μL / min. In addition, the sheath liquid is allowed to flow at inlets 102 to 1400 μL / min, and the sheath liquid is allowed to flow at inlets 103 to 1510 μL / min. The liquid delivery time to the inlets 101 to 103 is set to 15 minutes. Furthermore, on the particle separation chips CP11 to CP16, the second sample is recovered from the first recovery section 64, and the third sample is recovered from the plurality of second recovery sections 65.

[0169] Next, the number of cells (Jurkat cells and A549 cells) contained in each of the second sample and the third sample was calculated by the following formula (6).

[0170] The number of cells of a specified size in the sample = the probability of existence of cells of a specified size in the sample × the total number of cells in the sample (6)

[0171] In the above formula (6), the probability of cell existence is given by Fig.24 The total cell number was obtained by the above formula (2) using the cell concentration measured by a hemacytometer.

[0172] 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 specified particle size was calculated by the following formula (7).

[0173] Recovery rate of cells of a specified particle size = [number of cells of a specified particle size in the second sample / (number of cells of a specified particle size in the second sample + number of cells of a specified particle size in the third sample)] × 100 (%) (7)

[0174] Furthermore, the numbers of Jurkat cells and A549 cells contained in the recovered second and third samples were obtained by the above formula (2), and the Jurkat cell removal rate was calculated by the following formula (8) based on the obtained numbers of Jurkat cells.

[0175] Jurkat cell removal rate = the number of Jurkat cells contained in the third sample / (the number of Jurkat cells contained in the second sample + the number of Jurkat cells contained in the third sample) × 100 (%) (8)

[0176] Furthermore, the A549 cells contained in the recovered second sample and the third sample were obtained by the above formula (2), and the recovery rate of A549 cells was calculated by the above formula (5) based on the number of A549 cells obtained.

[0177] Fig.25 It is a graph showing the removal rate of Jurkat cells and the recovery rate of A549 cells of particle separation chips CP11 to CP16.

[0178] like Fig.25 As shown in the above paragraph, it is known that Jurkat cells corresponding to the usual blood cell components (red blood cells, white blood cells, platelets, etc.) can be separated on any particle separation chip and removed as the third sample. Fig.23 As shown in the upper part of FIG, in the particle separation chips CP11 to CP16, only the structure of the porous sheet is different. Fig.17 As shown in FIG. 1 , the diameters of the pores 31 of the porous sheets PS1 to PS6 increase in approximately this order. Fig.25 The results in the upper section show that the larger the pore size of the hole 31 is, the more Jurkat cells corresponding to normal blood cell components can be removed.

[0179] like Fig.25 As shown in the lower part of the figure, A549 cells corresponding to CTC can be separated and recovered as the second sample on any particle separation chip. In addition, the pore sizes of the porous sheets PS1 to PS6 increase in this order. Fig.25 The results in the lower section show that the smaller the pore size of hole 31 becomes, the more A549 cells corresponding to CTCs can be recovered.

[0180] <Investigation on Flux>

[0181] Next, the inventors investigated to what extent the throughput was improved compared with the conventional particle separation method (comparative example) described in the above-mentioned Non-Patent Document 1 by using the particle separation chips having the configurations shown in Examples 1 to 3.

[0182] In the comparative example, 1 mL of whole blood diluted 10 times with PBS is introduced into the flow path at a flow rate of 25 μL / min, and the flux of the whole blood is 2.5 μL / min. Therefore, according to the comparative example, the time consumed for the particle separation treatment of 5 mL of whole blood is 2000 minutes (about 33 hours). In comparison, on the particle separation chip 1 of the structure shown in Examples 1 to 3, the flux 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 consumed for the particle separation treatment of 5 mL of whole blood becomes less than 100 minutes. In this way, according to Examples 1 to 3, the flux can be greatly improved compared with the comparative example.

[0183] Fig.26 It is a graph showing the flow rate to each outlet of the particle separation chips CP11 to CP16.

[0184] exist Fig.26 , waste liquids 1 to 3, 5 to 7 are respectively discharged from six outlets 213 arranged in the Y-axis direction (see Fig.18 That is, waste liquid 1 is the third sample obtained from the outlet 213 at the bottom of the Y-axis direction among the three outlets 213 located below the main channel 100 in the Y-axis direction, waste liquid 3 is the third sample obtained from the outlet 213 at the top of the Y-axis direction among the three outlets 213 located below the main channel 100 in the Y-axis direction, waste liquid 5 is the third sample obtained from the outlet 213 at the bottom of 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 the third sample obtained from the outlet 213 at the top of the Y-axis direction among the three outlets 213 located above the main channel 100 in the Y-axis direction. Recovery liquid 4 represents the second sample obtained from outlet 104. Fig.26The vertical axis shows the weight ratio of the samples obtained from the waste liquids 1 to 3, the recovery liquid 4, and the waste liquids 4 to 7 of each particle separation chip CP11 to CP16.

[0185] Referring to the weight ratios of 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 flow path 100 (i.e., the outlet 213 connected to the flow path 201 located on the downstream side of the main flow path 100) becomes smaller. In addition, although different porous sheets PS1 to PS6 are provided 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 roughly the same. Therefore, it is known that when the porous sheets PS1 to PS6 are constructed as in Example 3, the difference in fluid resistance caused by the porous sheets can be roughly ignored. In other words, it is known that at this time, the flow rate of the flow path 201 to the branch flow path 200 is dominated by the fluid resistance of the flow path 201.

[0186] Fig. 27 It is a graph showing the recovery rate for each particle size of the particle separation chips CP11 to CP16.

[0187] exist Fig. 27 In the figure, the horizontal axis shows the cell size, and the vertical axis shows the recovery rate calculated by the above formula (7). Fig. 27 In the figure, for convenience, the cutoff value of 70% recovery is shown as a dotted line.

[0188] As described above, in the porous sheets PS1 to PS6, the pores 31 have pore sizes that increase in approximately this order. Fig. 27 According to the coordinate diagram, on the particle separation chips CP11 to CP16, the larger the pore size becomes in this order, the larger the particle size of the cells above the cutoff value becomes. For example, on the particle separation chip CP11, the particle size of the cells above the cutoff value is about 11.5μm, and on the particle separation chip CP16, the particle size of the cells above the cutoff value is about 16.3μm. Therefore, it is known that as the pore size of the porous sheet becomes larger, even if the configuration of the main flow path 100 and the branch flow path 200 is the same, the recovery rate changes, and it becomes easier to recover cells with relatively large particle sizes.

[0189] That is, it is known that although the liquid removal rate is basically determined by the particle separation chip, the particle size of the particles not separated into the branch flow channel 200 but recovered through the main flow channel 100 and the proportion of particles of a specified particle size recovered through the main flow channel 100 also change due to the pore size of the porous sheet.

[0190] Furthermore, when the porous sheet PS1 provided in the particle separation chip CP11 and the porous sheet PS2 provided in the particle separation chip CP12 are compared, as shown in FIG. Fig.17As shown in FIG. 1 , the pore diameters are the same as 12 μm, and the porosity of the porous sheet PS2 is 20.9%, which is smaller than the porosity of the porous sheet PS1, which is 36.9%. That is, the number of pores 31 of the porous sheet PS2 is less than the number of pores 31 of the porous sheet PS1. Fig.26 As shown in FIG. 1 , the weight ratio of the samples obtained from each outlet of the particle separation chips CP11 and CP12 is approximately the same. Therefore, the flow rate of the third sample flowing from one hole 31 to the branch flow path 200 is larger in the particle separation chip CP12 than in the particle separation chip CP11. Thus, when the flow rate flowing to one hole 31 is large, the force of introducing cells into the hole 31 becomes larger. For this reason, it is believed that, Fig. 27 As shown in the recovery rate, the recovery rate of cells with relatively large particle sizes (for example, cells with a particle size of 12.5 μm to 18 μm) in the particle separation chip CP12 is smaller than that in the particle separation chip CP11.

[0191] <Simulation of the amount of discharge to the branch flow path>

[0192] Next, the inventors investigated the flow rate (discharge rate) of the sample flowing from the main flow channel 100 to the branch flow channel 200 by simulation.

[0193] Fig.28 The upper stage of FIG. 1 is a diagram schematically showing the configuration of the flow channel 120 of the main flow channel 100 and the flow channel 201 of the branch flow channel 200 involved in the present simulation.

[0194] In this simulation, only one flow path 201 is arranged for the flow path 120, and the porous sheet 30 is arranged at the branch position B between the flow paths 120 and 201. In this simulation, the sample is made to flow in the X-axis direction relative to the flow path 120, and the flow rate of the sample discharged to the flow path 201 through the porous sheet 30 is calculated by the simulation.

[0195] Fig.28 The lower stage is a graph showing the results of the discharge ratio of the sample to the branch flow channel 200 involved in the present simulation.

[0196] exist Fig.28 In the coordinate graph in the lower section of FIG. 1 , the horizontal axis shows the position of the flow path 201 in the Y-axis direction, the center corresponds to the center position of the flow path 120 in the Y-axis direction, and the left end and the right end correspond to one end and the other end of the flow path 120 in the Y-axis direction, respectively. The vertical axis shows the discharge ratio (%) of the sample of the flow path 201 to the branch flow path 200.

[0197] like Fig.28 The solid line in the lower coordinate diagram of shows that the flow rate of the sample flowing from the flow path 120 to the flow path 201 is greater at the left and right ends than at the center at the divergence position B. Thus, when the height of the sample at the left and right ends ( Fig.11When the height h) of the sample increases, when the sample contains the first particles (e.g., CTC), the first particles are unexpectedly discharged to the branch flow path 200 at both ends. In addition, since the height of the sample at the center becomes extremely small, when the sample contains the second particles (e.g., red blood cells, white blood cells, and platelets), it becomes difficult for the second particles at the center to be discharged to the branch flow path 200. According to the inventor's research, in order to make the second particles with a particle size of less than 14.5 μm flow to the branch flow path 200, for example, it is necessary to increase the discharge ratio Fmin near the center to about the cutoff value (1.8%).

[0198] For this reason, in the above-mentioned embodiment, the first sample is placed near the center of the flow path 120 by the sheath liquid flowing from the inlet 103 in such a manner that the first particles contained in the first sample do not flow to the branch flow path 200, and the second particles contained in the first sample flow to the branch flow path 200. Thus, the first particles and the second particles can be appropriately separated. In addition, since the first sample is suppressed from flowing to both ends of the flow path 120, the particles can be generally separated at the branch position B and the second particles can be efficiently separated.

[0199] <Effects of particle separation chip and particle separation device>

[0200] like Figure 6 As shown, the particle separation chip 1 includes an upper substrate 10 and a lower substrate 20 (substrate) having a microchannel 1a therein, and a porous sheet 30 formed with a plurality of holes 31. The microchannel 1a includes a main channel 100 and a branch channel 200. The main channel 100 includes an inlet 101 for a first sample containing a first particle and a second particle smaller than the first particle disposed on the upstream side, and an outlet 104 (first outlet) disposed on the downstream side for taking out a second sample mainly containing the first particle. The branch channel 200 has a plurality of branch positions B (refer to Fig. 9 ) is connected to the main flow path 100, and has an outlet 213 (second outlet) on the downstream side for taking out the third sample containing the second particles. At a plurality of branching positions B, the main flow path 100 and the branch flow path 200 are connected via the holes 31 of the porous sheet 30.

[0201] According to this configuration, the main channel 100 and the branch channel 200 communicate with each other through the holes 31 of the porous sheet 30 at a plurality of branch positions B. Thus, sufficient sample processing capacity (throughput) can be demonstrated at a product development site or a clinical site.

[0202] In addition, if Fig.11As shown, the flow of the sample that moves the second particles from the main channel 100 to the branch channel 200 can be formed by the respective holes 31 of the porous sheet 30. Therefore, the flow of the sample of the second particles to the branch channel 200 can be dispersed in the plurality of holes 31 of the porous sheet 30. Thus, the second particles can be efficiently separated to the branch channel 200. Thus, the flow rates of the main channel 100 and the branch channel 200 can be increased, and the flux of particle separation can be efficiently increased.

[0203] In addition, since the width of the branch flow path 200 can be expanded, even when the blood sample contains cell aggregates, particle separation can be performed without turbidity. For example, when CTC is estimated as the first particle, there is a case where CTC forms aggregates. Even in such a case, since the CTC aggregates can be recovered without turbidity, appropriate diagnosis can be performed based on the CTCs contained in the blood sample.

[0204] In addition, the whole blood can be used as the first sample without hemolyzing the unnecessary blood cells (e.g., red blood cells) in the whole blood. Thus, the process of preparing the first sample using a hemolytic agent can be omitted, and the damage of the particles to be obtained by the hemolytic agent can be avoided. In addition, since there is no need to excessively dilute the whole blood in order to reliably perform the particle separation process, the throughput of the particle separation can be increased.

[0205] like Figure 6 As shown, the main flow path 100 and the branch flow path 200 are constructed in such a manner that a portion of the first sample introduced from the inlet 101 flows to the branch flow path 200, the remaining sample flows to the main flow path 100, the first particle is discharged from the outlet 104 (first outlet), and the second particle is discharged from the outlet 213 (second outlet).

[0206] 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 recovered.

[0207] The width of the flow path 120 of the main flow path 100 in the Y-axis direction (flow path width) is 500 μm or more and 50 mm or less. In addition, for example, when the amount of whole blood contained in the first sample is small, the width of the flow path 120 of the main flow path 100 in the Y-axis direction (flow path width) may also be 100 μm or more and 50 mm or less.

[0208] By setting the channel width of the main channel 100 in this way, a high flux of particle separation can be achieved. In addition, when the width of the channel 120 in the Y-axis direction is 100 μm or more, first particles of 100 μm or less can flow into the channel 120 .

[0209] like Fig. 9As shown, the branch flow path 200 includes a flow path 201 that communicates with the main flow path 100 at a branch position B and is arranged non-parallel to the main flow path 100 in a plan view.

[0210] According to this configuration, the lengths of the plurality of channels 201 of the branch channel 200 can be smoothly adjusted in the in-plane direction of the lower substrate 20. Therefore, the lengths of the respective channels 201 can be smoothly set to lengths suitable for the hydraulic filtration method.

[0211] like Fig. 9 As shown, the flow channel 201 communicating with the main flow channel 100 at the branching position B is arranged to intersect the main flow channel 100 in a plan view, so that the third sample flows to both sides of the main flow channel 100 .

[0212] According to this configuration, the sample of the main channel 100 near the inlet (branch position B) of the channel 201 of the branch channel 200 can flow to the channel 201 without being biased 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 properly function for particle separation. Therefore, the particles can be separated efficiently.

[0213] The branch flow path 200 includes not less than 2 and not more than 15,000 flow paths 201 that communicate with the main flow path 100 at the branch position B.

[0214] 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 the particle separation chip 1 having a four-side area of ​​about 150 mm while at least achieving hydraulic filtration.

[0215] like Figure 4 , 5 As shown, the branch channel 200 is arranged below the main channel 100 when the particle separation chip 1 is in use.

[0216] According to this configuration, the third sample containing the second particles can be introduced into the branch channel 200 using gravity. Therefore, the particle separation efficiency can be smoothly improved.

[0217] In addition, the particle separation chip 1 can be formed by repeatedly bonding the upper substrate 10 having a recessed portion on the lower surface for forming the main flow path 100 and the lower substrate 20 having a recessed portion on the upper surface for forming the branch flow path 200 via the porous sheet 30. Thus, the particle separation chip 1 can be easily formed.

[0218] like Figure 6 As shown, the main flow path 100 also has a branch point B (see Fig. 9 ) The inlets 102, 103 of the sheath liquid on the upstream side.

[0219] According to this composition, Figure 7 , 8As shown, the sheath liquid can limit the range through which the first sample flows to a range suitable for particle separation, thereby enabling efficient particle separation.

[0220] like Figure 6 As shown, the sheath liquid inlet 102 is an inlet for introducing the sheath liquid from the upper side of the first sample flowing through the main channel 100 .

[0221] According to this composition, Figure 8 As shown, since the first sample flows to the inlet (branch position B) of the branch flow path 200 in a state where the upper portion in the thickness direction is restricted, overflow of the second particles can be suppressed.

[0222] like Figure 6 As shown, 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 .

[0223] According to this composition, Figure 8 As shown in the middle and lower sections of FIG. , the first sample flows to the inlet (branch position B) of the branch flow path 200 while being restricted at both ends in the width direction. Fig.28 The simulation shows that the first particle and the second particle can be appropriately separated, the particles can be generally separated at the diverging position B, and the second particle can be efficiently separated.

[0224] The fluid resistance of the branch channel 200 from the upstream branch position B of the main channel 100 to the outlet 213 (second outlet) is greater than the fluid resistance of the branch channel 200 from the downstream branch position B of the main channel 100 to the outlet 213 (second outlet).

[0225] According to this configuration, the liquid removal rate, which is the ratio of the flow rate to the branch flow channel 200 to the flow rate of the main channel 100 at each branch position B, can be made close to each other at each branch position B. Thus, the second particles of a certain size can be separated.

[0226] 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 and connected to each of the plurality of branch positions B with the main flow path 100, and relay flow paths 211 and 212 connecting the plurality of flow paths 201 with an outlet 213 (second outlet). The length of the flow path 201 connected to the main flow path 100 at the branch position B on the upstream side of the main flow path 100 is longer than the length of the flow path 201 connected to the main flow path 100 at the branch position B on the downstream side of the main flow path 100.

[0227] According to this configuration, the fluid resistance at the branching point B on the upstream side of the main channel 100 can be set larger than the fluid resistance at the branching point B on the downstream side of the main channel 100 with a simple configuration.

[0228] The microchannel 1 a 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 of the main channel 100 at the branch position B, becomes a value at which the second particles flow to the branch channel 200 and the first particles flow to the main channel 100 .

[0229] According to this configuration, the first particles and the second particles can be separated by hydrodynamic filtration.

[0230] 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 of the main channel 100 at the branch position B, has substantially the same value at the plurality of branch positions B.

[0231] According to this configuration, second particles of a certain size can be separated at a plurality of branch positions B in general.

[0232] 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 of the main channel 100 at the branch position B, is greater than 0% and less than 50% at a plurality of branch positions B.

[0233] According to this configuration, the second particles below the depth of the main channel 100 can be separated into the branch channel.

[0234] The porous sheets PS1 to PS6 are formed so that the porosity, which is the ratio of the area of ​​the holes 31 to the area of ​​the opening at the intersection of the main channel 100 and the branch channel 200 (the area of ​​the branch position B), becomes a value at which the first particles of a specified size are recovered from the outlet 104 (first outlet) at a specified recovery rate.

[0235] According to this configuration, as shown in Examples 2 and 3, first particles of a desired size can be recovered at a predetermined recovery rate.

[0236] The pore diameter of the pores 31 of the porous sheets 30 and PS1 to PS7 is not less than 30 nm and not more than 100 μm.

[0237] According to this configuration, when the particle diameter of the first particles is about 50 nm to 100 μm, the porous sheet can be configured so as to suppress the first particles from flowing into the branch flow channel 200 .

[0238] The porous sheets 30 and PS1 to PS7 are formed so that the opening ratio is greater than 0% and less than 50% at the plurality of branch positions.

[0239] According to this structure, the strength of the porous sheet can be maintained.

[0240] The particle size of the first particles is not less than 30 nm and not more than 100 μm.

[0241] According to this configuration, first particles of various sizes can be separated, ranging from nanoparticles such as exosomes to relatively large circulating tumor cells.

[0242] The first sample introduced from 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.

[0243] Circulating tumor cells (CTCs) in the blood that leak from tumors into the blood have been reported to be useful as a factor for cancer diagnosis and are expected to be examined as non-invasive cancers. However, since the presence rate of CTCs in the blood is several to dozens per 10 mL, it is extremely difficult to separate CTCs from a blood sample and take them out. However, according to the above-mentioned configuration, CTCs, which are rare cells, can be efficiently separated. In addition, since no reagents are used in the particle separation process, CTCs can be separated without damaging them.

[0244] like Fig.12 As shown, the particle separation device 2 includes a particle separation chip 1, a mounting portion 41 on which the particle separation chip 1 is mounted, an inlet 101 for the first sample of the particle separation chip 1, and inlets 102 and 103 for the sheath liquid, respectively, for introducing the first sample and the sheath liquid, and a microchannel 1a (see Figure 6 ) in the liquid delivery section 61 to 63, a first recovery section 64 for the first particles communicating with the outlet 104 (first outlet), and a second recovery section 65 for the second particles communicating with the outlet 213 (second outlet).

[0245] According to this configuration, by placing the particle separation chip 1 on the installation portion 41 and driving the liquid supply portions 61 to 63 to introduce the first sample and the sheath liquid, the first particles and the second particles can be smoothly recovered from the first recovery portion 64 and the second recovery portion 65 .

[0246] <Other changes>

[0247] In order to make the fluid resistance of the branched flow path 200 from the upstream branch position B to the outlet 213 larger than the fluid resistance of the branched flow path 200 from the downstream branch position B to the outlet 213, the length of the flow path 201 in the Y-axis direction on the upstream side is set to be longer than the length of the flow path 201 in the Y-axis direction on the downstream side. However, the present invention is not limited to this, and the cross-sectional area of ​​the flow path 201 on the upstream side may also be set to be smaller than the cross-sectional area of ​​the flow path 201 on the downstream side.

[0248] Fig.29 2 is a diagram schematically showing the configuration of the branch flow channel 200 according to the modification example at this time. Fig.29 The upper part is a plan view showing the structure near the branch flow path 200. Fig.29 The lower stage of FIG. 2 is a cross-sectional view showing the structure of the recessed portion corresponding to the flow path 201 formed in the lower substrate 20 .

[0249] like Fig.29 As shown in the upper part of FIG. 1 , the lengths of the plurality of flow paths 201 in the Y-axis direction are the same as each other. Fig.29 As shown in the lower section of FIG. 1 , the widths of the plurality of flow paths 201 in the X-axis direction are the same, and the height of the flow path 201 in the Z-axis direction on the upstream side is set smaller than the height of the flow path 201 in the Z-axis direction on the downstream side. Thus, the cross-sectional area of ​​the flow path 201 on the upstream side is set smaller than the cross-sectional area of ​​the flow path 201 on the downstream side. In this configuration, the fluid resistance at the branching position B on the upstream side of the main flow path 100 can also be set larger than the fluid resistance at the branching position B on the downstream side of the main flow path 100 by a simple configuration.

[0250] Furthermore, 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 path 201 may be adjusted. In setting the fluid resistance, both the length in the Y-axis direction and the cross-sectional area of ​​the plurality of flow paths 201 may be adjusted.

[0251] The plurality of flow paths 201 are formed to extend to both sides of the flow path 120 of the main flow path 100 , but the present invention is not limited thereto and the plurality of flow paths 201 may be formed to extend to only one side of the flow path 120 .

[0252] Fig.30 2 is a plan view showing the structure of the vicinity of the branched flow path 200 according to the modification example at this time. In this modification example, one end of the plurality of flow paths 201 coincides with one end of the flow path 120 in the Y-axis direction in a plan view.

[0253] At this time, the second particle separated at the branch position B moves in one direction along the flow path 201. At this time, it becomes difficult to generate a flow of the sample in the one direction in the flow path 201 at the end of the flow path 201 in the direction opposite to the Y axis. Therefore, it becomes difficult to exert the separation function of the second particle at the branch position B, and as a result, the separation efficiency of the second particle decreases. In contrast, as in the above-mentioned embodiment, when the flow path 201 extends to both sides of the flow path 120, the separation function at the branch position B is appropriately exerted, and the separation efficiency of the second particle is improved.

[0254] like Figure 1As shown, in order to inject the sheath liquid into the flow path 111 from the side of the flow path 111, one inlet 103 is arranged on the central axis CA, but it is not limited to this, and two inlets 103 may be arranged at both sides of the Y-axis direction relative to the central axis CA. In this case, the two flow paths connected to the two inlets 103 are connected to the two side surfaces of the flow path 111 in the Y-axis direction. Thus, as in the above-mentioned embodiment and example, the sheath liquid can be injected into the flow path 111 from the side of the flow path 111. However, in this case, two liquid supply parts 63 for injecting the sheath liquid into the two inlets 103 respectively become necessary, and since it is necessary to make the liquid supply amounts of the two liquid supply parts 63 equal to each other, the structure and control of the particle separation device 2 become complicated. Therefore, as described above, it is preferable to set only one inlet 103 along the central axis CA.

[0255] Although one porous sheet 30 is arranged so as to overlap with all the branching positions B, the present invention is not limited thereto, and a plurality of porous sheets 30 may be arranged so as to overlap with one or more branching positions B, respectively.

[0256] The plurality of channels 201 are formed to extend perpendicularly to the channel 120 of the main channel 100 in a plan view, but are not limited thereto and may be arranged obliquely to the channel 120. In this case, the lengths of the plurality of channels 201 can be smoothly adjusted in the in-plane direction of the lower substrate 20.

[0257] In the above embodiment, if Figure 2 As shown, each relay channel 211 is provided at both ends of the plurality of channels 201 in the Y-axis direction. Fig.18 As shown, two or three relay channels 211 are provided at both ends of the plurality of channels 201 in the Y-axis direction. The present invention is not limited thereto, and four or more relay channels 211 may be provided at both ends of the relay channels 211 in the Y-axis direction.

[0258] In the above embodiment, if Figure 2 As shown, one relay channel 211 is connected to one end of the entire channel 201 in the Y-axis direction, and two outlets 213 are connected to one relay channel 211. This is not limited to this, and one or more than three outlets 213 may be connected to one relay channel 211. In the above embodiment, Fig.18 As shown, one outlet 213 is connected to one relay flow path 211. However, the present invention is not limited thereto, and a plurality of outlets 213 may be connected to one relay flow path 211, or a single outlet 213 may be connected to a plurality of relay flow paths 211.

[0259] In the above embodiment, if Figure 1 As shown, one outlet 104 is arranged on the downstream side of the main flow path 100 , but a plurality of outlets 104 may be arranged.

[0260] In the above embodiment, the first particle is CTC, and the second particle is red blood cells, white blood cells, and platelets, but the combination of the first particle and the second particle is not limited thereto. For example, the first particle may be an extracellular vesicle such as an exosome, and the second particle may be a protein or fragment smaller than an extracellular vesicle.

[0261] The first particle is a particle to be obtained, and the second particle is a particle to be removed, but the present invention is not limited thereto, and the first particle may be a particle to be removed, and the second particle may be a particle to be obtained. In this case, the first particle and the second particle may be separated at a high flux based on the size of the particles to obtain the second particle.

[0262] The first sample introduced from the inlet 101 is a blood sample, but is not limited thereto. When there are two kinds of particles of different sizes contained in the first sample, and these two kinds of particles can be separated by the particle separation chip 1, the first sample can be a body fluid other than blood (cerebrospinal fluid, ascites, pleural effusion, synovial fluid, peritoneal dialysis drainage, etc.) or a sample prepared from a body fluid, and can also be urine or a sample prepared from urine.

[0263] The embodiments of the present invention can be appropriately and variously modified within the scope of the technical concept shown in the claims.

[0264]

Explanation of symbols

[0265] 1: Particle separation chip

[0266] 1a: Microfluidics

[0267] 2: Particle separation device

[0268] 10: Upper base plate (base plate)

[0269] 20: Lower base plate (base plate)

[0270] 30: Porous sheet

[0271] 31: Hole

[0272] 41: Settings

[0273] 61~63: Liquid delivery part

[0274] 64: 1st Recovery Department

[0275] 65: Second Recovery Department

[0276] 100: Mainstream Road

[0277] 101: Entrance

[0278] 102, 103: Inlet (inlet of sheath fluid)

[0279] 104: Exit (Exit 1)

[0280] 200: Branch flow path

[0281] 201: flow path

[0282] 211, 212: Relay flow path

[0283] 213: Exit (Exit 2)

[0284] B: Divergence position

[0285] CP1~CP5, CP11~CP16: Particle separation chips

[0286] SP1~SP7: porous sheet

Claims

1. A particle separation chip for separating a plurality of particles contained in a sample based on the size of the particles, comprising a substrate having a microchannel therein and a porous sheet having a plurality of holes formed therein, The microchannel comprises: The main flow path has: an inlet provided on the upstream side for feeding a sample containing a first particle and a second particle smaller than the first particle, and a first outlet provided on the downstream side for taking out a sample mainly containing the first particles, and a branch flow path connected to the main flow path at a plurality of branch positions along the flow direction of the main flow path, and having a second outlet on the downstream side for taking out a sample containing the second particles, At the plurality of branching positions, the main flow path and the branch flow paths are communicated with each other through the holes of the porous sheet.

2. The particle separation chip according to claim 1, wherein the main flow path and the branch flow path are configured in such a manner that a portion of the sample introduced from the inlet flows into the branch flow path, and the remaining sample flows into the main flow path, the first particle is discharged from the first outlet, and the second particle is discharged from the second outlet. 3 . The particle separation chip according to claim 1 , wherein a flow channel width of the main flow channel is not less than 100 μm and not more than 50 mm. 4 . The particle separation chip according to claim 1 , wherein a flow path width of the main flow path is not less than 500 μm and not more than 50 mm. 5 . The particle separation chip according to claim 1 , wherein the branch flow path includes a flow path that communicates with the main flow path at the branch position and is arranged non-parallel to the main flow path in a plan view. 6 . The particle separation chip according to claim 5 , wherein the flow channel communicating with the main flow channel at the branch position is arranged to intersect the main flow channel in a plan view, so that the sample containing the second particles flows to both sides of the main flow channel. 7 . The particle separation chip according to claim 1 , wherein the branch flow path includes not less than 2 and not more than 15,000 flow paths connected to the main flow path at the branch position. 8 . The particle separation chip according to claim 1 , wherein the branch flow path is arranged below the main flow path when the particle separation chip is in use. 9 . The particle separation chip according to claim 1 , wherein the main channel further includes an inlet for a sheath liquid provided on an upstream side of the branching position. 10 . The particle separation chip according to claim 9 , wherein the inlet of the sheath liquid is an inlet for introducing the sheath liquid from an upper side of the sample flowing through the main channel. 11 . The particle separation chip according to claim 9 , wherein the inlet of the sheath liquid is an inlet for introducing the sheath liquid from both sides of the sample flowing through the main channel.

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

13. The particle separation chip according to claim 1, The branched flow path comprises: a plurality of flow paths which are connected to the main flow path at the plurality of branching positions and are arranged non-parallel to the main flow path in a plan view, and a relay flow path connecting the plurality of flow paths and the second outlet, The length of the flow path connected to the main flow path at the branching position on the upstream side of the main flow path is longer than the length of the flow path connected to the main flow path at the branching position on the downstream side of the main flow path.

14. The particle separation chip according to claim 1, The branched flow path comprises: a plurality of flow paths which are connected to the main flow path at the plurality of branching positions and are arranged non-parallel to the main flow path in plan view, and a relay flow path connecting the plurality of flow paths and the second outlet, The cross-sectional area of ​​the flow path communicating with the main flow path at the branch position on the upstream side of the main flow path is smaller than the cross-sectional area of ​​the flow path communicating with the main flow path in the branch flow path on the downstream side of the main flow path.

15. The particle separation chip of claim 1, wherein the microchannel is formed in such a manner that a liquid removal rate, which is a ratio of a flow rate to the branch channel to a flow rate in the main channel at the branch position, becomes a value at which the second particles flow to the branch channel and the first particles flow to the main channel.

16. 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 position, becomes substantially the same value at the plurality of branch positions.

17. 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 position, is greater than 0% and less than 50% at the plurality of branch positions.

18. The particle separation chip of claim 1, wherein the porous sheet is formed in such a way that the porosity, which is the ratio of the area of ​​the holes of the porous sheet to the area of ​​the opening at the intersection of the main flow path and the branch flow path, becomes a value at which the first particles of a specified size are recovered from the first outlet at a specified recovery rate.

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

20. The particle separation chip according to claim 1, wherein the porous sheet is formed so that an opening ratio, which is a ratio of an area of ​​holes of the porous sheet to an area of ​​an opening at an intersection of the main channel and the branch channel, is greater than 0% and less than 50% at the plurality of branch positions.

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

23. A particle separation device comprising: The particle separation chip according to any one of claims 1 to 22, a setting part equipped with the particle separation chip, a liquid feeding section for feeding the liquid in the microchannel by introducing the sample and the sheath liquid into the sample inlet and the sheath liquid inlet of the particle separation chip, respectively; a first recovery portion for the first particles communicating with the first outlet, and A second recovery portion for the second particles communicated with the second outlet.

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