WO2020255753A1 - Separation device and separation method - Google Patents

Separation device and separation method Download PDF

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Publication number
WO2020255753A1
WO2020255753A1 PCT/JP2020/022295 JP2020022295W WO2020255753A1 WO 2020255753 A1 WO2020255753 A1 WO 2020255753A1 JP 2020022295 W JP2020022295 W JP 2020022295W WO 2020255753 A1 WO2020255753 A1 WO 2020255753A1
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flow path
separation
cross
sectional area
recovery
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PCT/JP2020/022295
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French (fr)
Japanese (ja)
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憲彰 新井
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凸版印刷株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting

Definitions

  • the present invention relates to a separation device and a separation method.
  • the present application claims priority with respect to Japanese Patent Application No. 2019-115558 filed in Japan on June 21, 2019, the contents of which are incorporated herein by reference.
  • the deterministic lateral displacement is a method in which large particles are placed around the pillars when a laminar flow of fluid containing particles is passed through an array of minute pillars arranged with slight displacement. While the small particles flow diagonally due to the change in the flow that occurs, the small particles are a method of separating the particles according to their size by utilizing the property of traveling linearly on the laminar flow (see, for example, Non-Patent Document 1). ).
  • Patent Document 1 describes a method for separating cells using the principle of DLD.
  • an object of the present invention is to provide a technique for accurately separating target particles in a fluid sample.
  • a separation device for separating the target particles from a fluid sample containing target particles having a size equal to or larger than the threshold value and non-target particles having a size smaller than the threshold value, and separating the particles in the fluid according to the size.
  • the separation flow path is connected to the upstream side of the separation flow path, the fluid introduction section for introducing the fluid sample and the buffer into the separation flow path, and the target particle connected to the downstream side of the separation flow path. It has a recovery unit for collecting the fluid containing the fluid and the fluid containing the non-target particles, respectively, and the fluid introduction unit is connected to a first introduction port for introducing the buffer and the first introduction port.
  • a first flow path through which the fluid flows a first branch portion that branches the first flow path into a first bypass flow path and a second bypass flow path, and a downstream side of the first branch portion.
  • a second inlet for introducing the fluid sample a second flow path connected to the second inlet and for flowing the fluid sample, the first bypass flow path, and the second.
  • the cross-sectional area b on the plane perpendicular to the flow direction of the second bypass flow path at the confluence part satisfies the following formula (1), and the recovery part is the first one through which the fluid containing the non-target particles flows. It has a recovery flow path and a second recovery flow path through which a fluid containing the target particles flows, and the first recovery flow path is connected to the first bypass flow path side of the separation flow path.
  • the separation device in which the second recovery flow path is connected to the second bypass flow path side of the separation flow path (A / B) ⁇ (a / b) ... (1) [2] The separation device according to [1], wherein the cross-sectional area a and the cross-sectional area b satisfy the following formula (2). a ⁇ b ... (2) [3]
  • the recovery section has a second branch section that branches the separation flow path into a first recovery flow path and a second recovery flow path, and the first recovery section in the second branch section.
  • the cross-sectional area ⁇ on the plane perpendicular to the flow direction of the flow path and the cross-sectional area ⁇ on the plane perpendicular to the flow direction of the second recovery flow path at the second branch portion satisfy the following equation (3).
  • the cross-sectional area of the confluence portion on the plane perpendicular to the flow direction of the second flow path is 5 to 30% of the cross-sectional area of the plane perpendicular to the flow direction of the separation flow path [1].
  • the separation device according to any one of [3].
  • [6] A method for separating the target particles from the fluid sample containing the target particles having a size equal to or larger than the threshold value and the non-target particles having a size smaller than the threshold value, [1] to [5].
  • the buffer is introduced from the first introduction port of the separation device according to any one of the above, the fluid sample is introduced from the second introduction port, and the fluid containing the target particles or the said from the recovery unit.
  • a method comprising recovering a fluid containing non-target particles.
  • FIG. 3 is a cross-sectional view taken along the line bb'of FIG. 1A. It is a figure explaining the basic principle of the deterministic transverse substitution method (DLD). It is a figure which shows the simulation result in Experimental Example 1.
  • FIG. It is a graph which shows the moving distance of the laminar flow of a fluid sample calculated by the simulation in Experimental Example 1. In Experimental Example 2, it is a photograph which photographed the head part, the middle part, and the tail part of the separation flow path of a separation device.
  • the present invention is a separation device that separates the target particles from a fluid sample containing a target particle having a size equal to or more than a threshold value and a non-target particle having a size less than the threshold value, and is in a fluid.
  • a separation flow path that separates particles according to size, a fluid introduction section that is connected to the upstream side of the separation flow path and introduces the fluid sample and buffer into the separation flow path, and a downstream side of the separation flow path.
  • the fluid introduction unit has a first introduction port for introducing the buffer and the first introduction unit, which has a recovery unit for collecting the fluid containing the target particles and the fluid containing the non-target particles, respectively.
  • a first flow path connected to an introduction port and through which the buffer flows, a first branch portion that branches the first flow path into a first bypass flow path and a second bypass flow path, and the first branch portion.
  • the cross-sectional area a in the above and the cross-sectional area b in the plane perpendicular to the flow direction of the second bypass flow path at the confluence satisfy the following formula (1), and the recovery unit contains the non-target particles. It has a first recovery flow path through which a fluid flows and a second recovery flow path through which a fluid containing the target particles flows, and the first recovery flow path is the first bypass flow of the separation flow path.
  • the second recovery flow path which is connected to the roadside, provides a separation device which is connected to the second bypass flow path side of the separation flow path.
  • FIG. 1A is a top view illustrating the structure of the separation device of the present embodiment.
  • the arrows indicate the direction in which the fluid flows.
  • FIG. 1B is a cross-sectional view taken along the line bb'of FIG. 1A.
  • the separation device 100 includes a separation flow path 110, a fluid introduction unit 120, and a recovery unit 130.
  • the fluid introduction unit 120 is connected to the upstream side of the separation flow path 110, and introduces the fluid sample and the buffer into the separation flow path 110.
  • the recovery unit 130 is connected to the downstream side of the separation flow path 110, and recovers the fluid containing the target particles and the fluid containing the non-target particles, respectively.
  • the fluid introduction section 120 includes a first introduction port 121, a first flow path 122, a first branch section 123, a second introduction port 124, a second flow path 125, and a confluence section 126.
  • the buffer is introduced from the first introduction port 121.
  • the first flow path 122 is connected to the first introduction port 121, and the buffer flows in the first flow path 122.
  • the first branch portion 123 branches the first flow path 122 into the first bypass flow path 1221 and the second bypass flow path 1222.
  • the second introduction port 124 is located on the downstream side of the first branch portion 123.
  • the fluid sample is introduced from the second introduction port 124.
  • the second flow path 125 is connected to the second introduction port 124, and the fluid sample flows in the second flow path 125.
  • the first bypass flow path 1221, the second flow path 125, and the second bypass flow path 1222 merge at the merging portion 126.
  • the recovery unit 130 has a first recovery flow path 131 through which a fluid containing non-target particles flows, and a second recovery flow path 132 through which a fluid containing target particles flows.
  • the first recovery flow path 131 is connected to the first bypass flow path 1221 side in a direction perpendicular to the flow direction of the separation flow path 110. That is, the first recovery flow path 131 is arranged closer to the first bypass flow path 1221 than the second bypass flow path 1222 in the direction perpendicular to the flow direction of the separation flow path 110.
  • the second recovery flow path 132 is connected to the second bypass flow path 1222 side in a direction perpendicular to the flow direction of the separation flow path 110.
  • the second recovery flow path 132 is arranged closer to the second bypass flow path 1222 than the first bypass flow path 1221 in the direction perpendicular to the flow direction of the separation flow path 110. Further, the first recovery flow path 131 is connected to the discharge port 134. The second recovery flow path 132 is connected to the discharge port 135.
  • the velocity of the fluid discharged from the second bypass flow path 1222 to the separation flow path 110 is the velocity of the fluid discharged from the first bypass flow path 1221 to the separation flow path 110. It is considered that the speed is the same as or higher than the speed of the fluid discharged from the first bypass flow path 1221 to the separation flow path 110.
  • the laminar flow formed by the fluid sample merged from the second flow path 125 becomes difficult to flow to the second bypass flow path 1222 side, that is, the second recovery flow path 132 side. That is, the fluid sample is less likely to be mixed into the second recovery flow path 132 in which the target particles are recovered, and the target particles can be recovered with high accuracy.
  • the ability to accurately recover the target particles means that the target particles have a high separation ability.
  • the separation device of this embodiment can accurately separate the target particles in the fluid sample.
  • the cross-sectional areas A and B refer to the cross-sectional areas of the branch portion 123 at the positions where the first flow path branches to the first bypass flow path 1221 and the second bypass flow path for the first time.
  • the cross-sectional areas A and B are the upstream ends of the partition wall 1223 for branching the first flow path 122 into the first bypass flow path 1221 and the second bypass flow path 1222 in the branch portion 123. It is the cross-sectional area in the part.
  • the cross-sectional areas a and b refer to the cross-sectional areas at the positions where the first bypass flow path 1221 and the second bypass flow path 1222 meet for the first time in the merging portion 126.
  • the cross-sectional areas a and b are the cross-sectional areas at the downstream end of the partition wall 1223.
  • the collection unit 130 has a second branch unit 133 that branches the separation flow path 110 into the first collection flow path 131 and the second collection flow path 132.
  • the principle of separation in the separation flow path 110 is not particularly limited as long as it is a separation method for forming a laminar flow.
  • it may be a deterministic transverse substitution method (hereinafter, may be referred to as DLD), separation by size, separation by magnetism, or the like.
  • DLD deterministic transverse substitution method
  • the principle of separation in the separation flow path 110 is DLD. DLD will be described later.
  • the buffer introduced from the first introduction port 121 and the fluid sample introduced from the second introduction port 124 form a laminar flow on the downstream side of the confluence portion 126.
  • This laminar flow passes through an array of tiny pillars 111 formed inside the separation channel 110.
  • particles having a size equal to or larger than the threshold value flow diagonally in the direction toward the bypass flow path 1222.
  • the collection unit 130 passes through the second collection flow path 132 and is collected from the discharge port 135.
  • particles having a size less than the threshold value travel linearly on the laminar flow.
  • the collection unit 130 passes through the first collection flow path 131 and is collected from the discharge port 134.
  • the buffer and the fluid sample can be fed by, for example, a syringe pump, a diaphragm pump, a roller tube pump, or the like.
  • the cross-sectional area a and the cross-sectional area b preferably satisfy the following formula (2). a ⁇ b ... (2)
  • the cross-sectional area a and the cross-sectional area b satisfy the above equation (2) means that if the heights of the first bypass flow path 1221 and the second bypass flow path 1222 in the merging portion 126 are constant, they merge. It means that the opening of the second flow path 125 in the portion 126 is located at the center in the width direction (direction perpendicular to the fluid flow direction) of the separation flow path 110 or on the side of the first bypass flow path 1221. That is, it means that the opening of the second flow path 125 in the merging portion 126 is located at the center in the width direction of the separation flow path 110 or closer to the first bypass flow path 1221 than the second bypass flow path 1222. .. Generally, the height of each flow path constituting the separation device is constant.
  • the laminar flow of the fluid sample moves to the first bypass flow path side, and the fluid sample is easily collected in the first recovery flow path 131 in the recovery unit 130. Therefore, the fluid sample is less likely to be mixed into the second recovery flow path 132 in which the target particles are recovered, and the target particles can be recovered with high accuracy.
  • the ratio of the cross-sectional area a to the cross-sectional area b is preferably 0.05 or more and 0.8 or less, more preferably 0.08 or more and 0.75 or less, and 0.1 or more and 0. It is more preferably 0.7 or less, and further preferably 0.15 or more and 0.65 or less.
  • a / b is 0.05 or more and 0.8 or less, it becomes easy to control the laminar flow of the fluid sample to move to the first bypass flow path side. As a result, the fluid sample is easily collected in the first collection flow path 131 in the collection unit 130.
  • the fluid sample is less likely to be mixed into the second recovery flow path 132 in which the target particles are recovered, and the target particles can be recovered with high accuracy.
  • the cross-sectional area ⁇ in the plane perpendicular to the flow direction of the first recovery flow path 131 in the second branch portion 133 and the second recovery flow path 132 in the second branch portion 133 preferably satisfies the following equation (3). (A / b) ⁇ ( ⁇ / ⁇ ) ... (3)
  • the cross-sectional areas ⁇ and ⁇ refer to the cross-sectional areas at the positions where the separation flow path 110 first branches into the first recovery flow path 131 and the second recovery flow path 132 in the branch portion 133.
  • the fact that the cross-sectional areas a, b, ⁇ , and ⁇ satisfy the above equation (3) is more than the ratio of the width of the first bypass flow path 1221 to the width of the second bypass flow path 1222 at the merging portion 126. It means that the ratio of the width of the first recovery flow path 131 to the width of the second recovery flow path 132 in the merging portion 133 is larger.
  • the fluid sample is easily collected in the first collection flow path 131 in the collection unit 130. Therefore, the fluid sample is less likely to be mixed into the second recovery flow path 132 in which the target particles are recovered, and the target particles can be recovered with high accuracy.
  • ⁇ may have a size of ⁇ or more regardless of the sizes of the cross-sectional areas a and b. That is, it is preferable that ⁇ and ⁇ satisfy the following formula (4). ⁇ ⁇ ⁇ ... (4)
  • the cross-sectional area C of the merging portion 126 on the plane perpendicular to the flow direction of the second flow path 125 is 5 with respect to the cross-sectional area D on the plane perpendicular to the flow direction of the separation flow path 110. It is preferably about 30%.
  • the fluid sample can be reliably recovered in the first recovery flow path 131 in the recovery unit 130.
  • the larger the cross-sectional area C the larger the volume of the fluid sample that can be introduced into the separation device per unit time, and the faster the processing speed for separating the target particles can be improved.
  • the cross-sectional area D of the separation flow path 110 is not constant, the average value of the cross-sectional areas on the plane perpendicular to the flow direction of the separation flow path 110 may be set as the cross-sectional area D.
  • the average value of the cross-sectional areas on the plane perpendicular to the flow direction of the separation flow path 110 in the present specification is calculated by the following method. Ten points between the smallest cross-sectional area and the largest cross-sectional area in the plane perpendicular to the flow direction of the separation flow path 110 are selected at equal intervals, and the respective cross-sectional areas are measured. The sum of the cross-sectional area values of 10 points is taken as the measurement point, that is, the value divided by 10 is taken as the average value.
  • examples of particles to be separated include beads, cells, and the like.
  • the beads may be those in which a specific binding substance that specifically recognizes a target protein or the like is bound to the surface thereof.
  • Specific binding substances include antibodies, antibody fragments, aptamers and the like.
  • the material of the beads is not particularly limited, and examples thereof include silica, polystyrene, latex, and metal.
  • the beads may be magnetic particles.
  • the target protein or the like in the fluid sample can be bound to the beads. Further, by recovering the beads with the separation device described above, the target protein or the like in the fluid sample can be recovered.
  • the cell or the exosome or the like is bound to the bead. Can be done. Then, by recovering such beads with the separation device described above, specific cells, exosomes, etc. in the fluid sample can be recovered.
  • the cell is not particularly limited, and examples thereof include any cell expressing a specific marker protein on the cell surface.
  • the cell may be, for example, a circulating tumor cell in blood (hereinafter, may be referred to as CTC).
  • the size of the target cell and the size of the non-target cell are similar, it may be difficult to separate the two.
  • beads of an appropriate size are specifically bound only to the target cells to make the target cells apparently large particles, thereby increasing the size difference from the non-target cells and separating the device. Can be easily separated with.
  • the size of the particle when the particle is a sphere, the size of the particle may be the diameter of the sphere.
  • the particle when the particle is not a sphere, a sphere having the same volume as the particle may be assumed, and the diameter of the sphere may be the size of the particle.
  • DLD is small when a laminar flow of fluid containing particles is passed through an array of minute pillars arranged with a slight shift, while large particles flow diagonally due to changes in the flow that occur around the pillars.
  • Particles are a method of separating particles according to their size by utilizing the property of traveling linearly on a laminar flow.
  • the threshold value (Dc) of the particle diameter displaced in the oblique direction can be set according to the particle size of the particles to be separated, that is, the target particles. More specifically, the setting of the threshold value Dc can be obtained from the following equation (5).
  • Dc 2 ⁇ G ⁇ ... (5)
  • ⁇ is a variable
  • G is the gap between pillars
  • ⁇ the deviation angle of pillars (tan ⁇ ).
  • the inter-pillar gap G is calculated based on the threshold value Dc of the particle diameter displaced in the oblique direction. Then, from the above equation (7), the inter-pillar gap G is calculated based on the threshold value Dc of the particle diameter displaced in the oblique direction. Then, a cell separation device having a basic structural portion in which the pillar group of the inter-pillar gap G (hereinafter, may be referred to as an obstacle structure) is installed can be produced. In this way, a separation device having a DLD microchannel having a desired threshold Dc can be made.
  • FIG. 2 is a diagram illustrating the basic principle of the deterministic transverse substitution method (DLD).
  • FIG. 2 shows the basic structure 20 of the DLD microchannel (hereinafter, may be referred to as a separation area).
  • an obstacle structure hereinafter, may be referred to as a pillar 21 arranged diagonally according to a certain rule with respect to the flow direction of the fluid in the direction of the arrow is provided.
  • the flow velocity changes around the obstacle structure 21.
  • the particles 22 having a size equal to or larger than the threshold value are displaced in the oblique direction according to the arrangement of the obstacle structures 21 arranged continuously.
  • the particles 23 having a size less than the threshold value generally travel straight along the flow direction while bypassing the obstacle structure 21.
  • Non-Patent Document 1 a known method
  • the target contained in the fluid It can be separated depending on the size of the particles.
  • the separated particles can be recovered respectively.
  • the shape of the obstacle structure 21 is not limited to the cylindrical structure as shown in FIG.
  • the obstacle structure 21 may have a polygonal prism structure such as a triangular prism or a quadrangular prism as long as it has a shape capable of causing a desired change in flow velocity.
  • Each flow path of the separation device of the present embodiment and an array of minute pillars of the DLD can be produced by appropriately selecting a known method.
  • the material of the separation device for example, glass, silicone, dimethylpolysiloxane, plastic or the like can be used.
  • the present invention is a method for separating the target particles from a fluid sample containing target particles having a size equal to or larger than a threshold and non-target particles having a size smaller than the threshold, and any of the above-mentioned ones.
  • the buffer is introduced from the first inlet of the separation device, the fluid sample is introduced from the second inlet, and the fluid containing the target particles or the non-target particles is contained from the recovery unit.
  • a method including recovering a fluid is provided.
  • the recovery unit has a first recovery flow path and a second recovery flow path.
  • the first recovery flow path is connected to the first bypass flow path side of the separation flow path.
  • the second recovery flow path is connected to the second bypass flow path side of the separation flow path.
  • the non-target particles are recovered from the outlet to which the first recovery channel is connected.
  • the target particles are collected from the discharge port to which the second collection channel is connected.
  • the target particles can be accurately separated from the fluid sample containing the target particles having a size equal to or larger than the threshold value and the non-target particles having a size smaller than the threshold value.
  • the particles are similar to those described above.
  • a separation device that separates the target particles from a fluid sample containing target particles having a size equal to or larger than the threshold and non-target particles having a size smaller than the threshold, and separates the particles in the fluid according to the size.
  • the separation flow path is connected to the upstream side of the separation flow path, the fluid introduction section for introducing the fluid sample and the buffer into the separation flow path, and the target particle connected to the downstream side of the separation flow path. It has a recovery unit for recovering the containing fluid and the fluid containing the non-target particles, respectively, and the fluid introduction unit is connected to a first introduction port for introducing the buffer and the first introduction port.
  • the cross-sectional area b on the plane perpendicular to the flow direction of the second bypass flow path at the confluence satisfies the following formula (1), and a / b which is the ratio of the cross-sectional area a to the cross-sectional area b is , 0.05 or more and 0.8 or less
  • the recovery unit includes a first recovery flow path through which the fluid containing the non-target particles flows, and a second recovery flow path through which the fluid containing the target particles flows.
  • the first recovery flow path is connected to the first bypass flow path side of the separation flow path, and the second recovery flow path is the second bypass flow of the separation flow path.
  • a separate device connected to the roadside. (A / B) ⁇ (a / b) ... (1)
  • a / b which is the ratio of the cross-sectional area a to the cross-sectional area b, is 0.08 or more and 0.75 or less.
  • a / b which is the ratio of the cross-sectional area a to the cross-sectional area b, is 0.1 or more and 0.7 or less.
  • the recovery unit has a second branching portion that branches the separation flow path into a first recovery flow path and a second recovery flow path, and the first recovery section in the second branching section.
  • the cross-sectional area ⁇ on the plane perpendicular to the flow direction of the flow path and the cross-sectional area ⁇ on the plane perpendicular to the flow direction of the second recovery flow path at the second branch portion satisfy the following equation (3).
  • the cross-sectional area of the confluence portion on the plane perpendicular to the flow direction of the second flow path is 5 to 30% of the cross-sectional area of the plane perpendicular to the flow direction of the separation flow path [7].
  • the separation device according to any one of [11].
  • the buffer is introduced from the first introduction port of the separation device according to any one of the above, the fluid sample is introduced from the second introduction port, and the target particles are introduced from the recovery unit.
  • a method comprising recovering a fluid comprising or a fluid comprising said non-target particles.
  • Example 1 The laminar flow flow of the fluid sample when the fluid sample and the buffer are introduced into the separation devices of Test Examples 1 to 8 having the structures shown in FIGS. 1A and 1B and having the respective cross-sectional areas shown in Table 1 below. I simulated it.
  • A is a cross-sectional area ( ⁇ m 2 ) in a plane perpendicular to the flow direction of the first bypass flow path 1221 in the first branch portion 123 shown in FIGS. 1A and 1B.
  • B is a cross-sectional area ( ⁇ m 2 ) in a plane perpendicular to the flow direction of the second bypass flow path 1222 in the first branch portion 123 shown in FIGS. 1A and 1B.
  • a is a cross-sectional area ( ⁇ m 2 ) in a plane perpendicular to the flow direction of the first bypass flow path 1221 in the confluence portion 126 shown in FIGS. 1A and 1B.
  • b is a cross-sectional area ( ⁇ m 2 ) in a plane perpendicular to the flow direction of the second bypass flow path 1222 at the confluence portion 126 shown in FIGS. 1A and 1B.
  • C is a cross-sectional area ( ⁇ m 2 ) in a plane perpendicular to the flow direction of the second flow path 1222 in the confluence portion 126 shown in FIGS. 1A and 1B.
  • D is a cross-sectional area ( ⁇ m 2 ) in a plane perpendicular to the flow direction of the separation flow path 110 shown in FIGS. 1A and 1B.
  • FloDEF manufactured by Mentor Graphics Corporation
  • a simulation was performed in which a fluid having a specific gravity of 1 was sent as a fluid sample at the flow velocity shown in Table 2 below, and a fluid having a specific gravity of 1 was sent as a buffer at the flow velocity shown in Table 2 below.
  • the length of the separation flow path was set to infinity.
  • FIG. 4 shows the trace line at the highest position (position on the plane perpendicular to the flow direction) among the trace lines at the position 1.8 mm from the confluence in the separation flow path calculated by the simulation.
  • Example 2 The separation devices of Test Examples 1, 4, 5, and 7 simulated in Experimental Example 1 were actually manufactured. In addition, the separation device of Test Example 8 was manufactured for comparison. Table 3 below shows the cross-sectional areas of the separated devices of each of the manufactured test examples. In Table 3, A, B, a, b, ⁇ , ⁇ , C, and D represent the cross-sectional areas ( ⁇ m 2 ) of the positions similar to those in Table 1 in the separation device, respectively. Table 3 also shows the values of a / b, A / B, and C / D. In all separation devices, the length of the separation channel was 12 mm.
  • a fluid sample and a buffer were introduced into these separation devices, and the laminar flow of the fluid sample was observed.
  • a fluid sample a sample obtained by diluting blood collected from a human with a PBS buffer containing 1% BSA and 5 mM EDTA 2-fold was sent.
  • a buffer a PBS buffer containing 1% BSA and 5 mM EDTA was sent.
  • the fluid sample and the buffer were fed at the same flow rates as in Table 2 above.
  • the separation device of Test Example 8 the fluid sample was fed at a flow rate of 100 ⁇ L / min, and the buffer was fed at a flow rate of 1,000 ⁇ L / min.
  • FIG. 5 are photographs of the head portion, the middle portion, and the tail portion of the separation flow path of each of the separation devices of Test Examples 1, 4, 5, 7, and 8, respectively.
  • the laminar flow of the fluid sample is the first laminar flow of the fluid sample. It became clear that it moved to the bypass flow path side. As described above, when the laminar flow of the fluid sample moves to the first bypass flow path side, the fluid sample is easily collected in the first recovery flow path in the recovery unit. Therefore, the fluid sample is less likely to be mixed into the second recovery flow path in which the target particles are recovered, and the target particles can be recovered with high accuracy.

Abstract

This separation device is for separating target particles from a fluid sample and comprises a separation flow path, a fluid introduction section, and a recovery section. The fluid introduction section comprises: a first introduction port for introducing a buffer; a first flow path through which the buffer flows; a first branching section at which the first flow path is made to branch into a first bypass flow path and a second bypass flow path; a second introduction port that is positioned downstream from the first branching section and that is for introducing a fluid sample; a second flow path that is connected to the second introduction port and through which the fluid sample flows; and a merging section at which the first bypass flow path, the second flow path, and the second bypass flow path merge. The cross-sectional area A of the first bypass flow path and the cross-sectional area B of the second bypass flow path in the first branching section and the cross-sectional area a of the first bypass flow path and the cross-sectional area b of the second bypass flow path in the merging section satisfy formula (1).　Formula (1): (A/B) ≤ (a/b)

Description

分離デバイス及び分離方法Separation device and separation method
　本発明は、分離デバイス及び分離方法に関する。
　本願は、２０１９年６月２１日に日本に出願された特願２０１９－１１５５５８号について優先権を主張し、その内容をここに援用する。 The present invention relates to a separation device and a separation method.
The present application claims priority with respect to Japanese Patent Application No. 2019-115558 filed in Japan on June 21, 2019, the contents of which are incorporated herein by reference.
　決定論的横置換法（Ｄｅｔｅｒｍｉｎｉｓｔｉｃ　Ｌａｔｅｒａｌ　Ｄｉｓｐｌａｃｅｍｅｎｔ、ＤＬＤ）とは、わずかにずらしながら配置された微小なピラーのアレイに粒子を含む流体の層流を通過させた際に、大きい粒子は、ピラー周囲に生じる流れの変化により斜めに流れるのに対し、小さい粒子は、層流に乗って直線的に進む性質を利用して、粒子を大きさにより分離する方法である（例えば、非特許文献１を参照）。例えば、特許文献１には、ＤＬＤの原理を利用した細胞の分離方法が記載されている。 The deterministic lateral displacement (DLD) is a method in which large particles are placed around the pillars when a laminar flow of fluid containing particles is passed through an array of minute pillars arranged with slight displacement. While the small particles flow diagonally due to the change in the flow that occurs, the small particles are a method of separating the particles according to their size by utilizing the property of traveling linearly on the laminar flow (see, for example, Non-Patent Document 1). ). For example, Patent Document 1 describes a method for separating cells using the principle of DLD.
国際公開第２０１６／１３６２７３号International Publication No. 2016/136273
　発明者らは、ＤＬＤにより粒子を分離する際に、粒子を含む流体の層流が意図しない位置に移動してしまい、粒子の分離能が低下してしまう場合があることを見出した。そこで、本発明は、流体試料中の標的粒子を精度よく分離する技術を提供することを目的とする。 The inventors have found that when separating particles by DLD, the laminar flow of the fluid containing the particles moves to an unintended position, and the separation ability of the particles may decrease. Therefore, an object of the present invention is to provide a technique for accurately separating target particles in a fluid sample.
　本発明は以下の態様を含む。
［１］閾値以上の大きさを有する標的粒子及び閾値未満の大きさを有する非標的粒子を含む流体試料から、前記標的粒子を分離する分離デバイスであって、流体中の粒子を大きさにより分離する分離流路と、前記分離流路の上流側に接続され、前記分離流路に前記流体試料及びバッファーを導入する流体導入部と、前記分離流路の下流側に接続され、前記標的粒子を含む流体及び前記非標的粒子を含む流体をそれぞれ回収する回収部と、を有し、前記流体導入部は、前記バッファーを導入する第１の導入口と、前記第１の導入口に接続され、前記バッファーが流れる第１の流路と、前記第１の流路を第１のバイパス流路及び第２のバイパス流路に分岐する第１の分岐部と、前記第１の分岐部の下流側に位置し、前記流体試料を導入する第２の導入口と、前記第２の導入口に接続され、前記流体試料が流れる第２の流路と、前記第１のバイパス流路、前記第２の流路及び前記第２のバイパス流路が合流する合流部と、を有し、前記第１の分岐部における前記第１のバイパス流路の流れ方向に垂直な面における断面積Ａと、前記第１の分岐部における前記第２のバイパス流路の流れ方向に垂直な面における断面積Ｂと、前記合流部における前記第１のバイパス流路の流れ方向に垂直な面における断面積ａと、前記合流部における前記第２のバイパス流路の流れ方向に垂直な面における断面積ｂとが、下記式（１）を満たし、前記回収部は、前記非標的粒子を含む流体が流れる第１の回収流路と、前記標的粒子を含む流体が流れる第２の回収流路と、を有し、前記第１の回収流路は前記分離流路の前記第１のバイパス流路側に接続しており、前記第２の回収流路は、前記分離流路の前記第２のバイパス流路側に接続している、分離デバイス。
　（Ａ／Ｂ）≦（ａ／ｂ）…（１）
［２］前記断面積ａ及び前記断面積ｂが、下記式（２）を満たす、［１］に記載の分離デバイス。
　ａ≦ｂ…（２）
［３］前記回収部は、前記分離流路を第１の回収流路及び第２の回収流路に分岐する第２の分岐部を有し、前記第２の分岐部における前記第１の回収流路の流れ方向に垂直な面における断面積αと、前記第２の分岐部における前記第２の回収流路の流れ方向に垂直な面における断面積βが、下記式（３）を満たす、［１］又は［２］に記載の分離デバイス。
　（ａ／ｂ）＜（α／β）…（３）
［４］前記合流部における前記第２の流路の流れ方向に垂直な面における断面積が、前記分離流路の流れ方向に垂直な面における断面積の５～３０％である、［１］～［３］のいずれか一つに記載の分離デバイス。
［５］前記分離流路は、決定論的横置換法により前記標的粒子を分離する、［１］～［４］のいずれか一つに記載の分離デバイス。
［６］前記閾値以上の大きさを有する標的粒子及び前記閾値未満の大きさを有する非標的粒子を含む前記流体試料から、前記標的粒子を分離する方法であって、［１］～［５］のいずれかに記載の分離デバイスの前記第１の導入口から前記バッファーを導入し、前記第２の導入口から前記流体試料を導入することと、前記回収部から前記標的粒子を含む流体又は前記非標的粒子を含む流体を回収することと、を含む、方法。 The present invention includes the following aspects.
[1] A separation device for separating the target particles from a fluid sample containing target particles having a size equal to or larger than the threshold value and non-target particles having a size smaller than the threshold value, and separating the particles in the fluid according to the size. The separation flow path is connected to the upstream side of the separation flow path, the fluid introduction section for introducing the fluid sample and the buffer into the separation flow path, and the target particle connected to the downstream side of the separation flow path. It has a recovery unit for collecting the fluid containing the fluid and the fluid containing the non-target particles, respectively, and the fluid introduction unit is connected to a first introduction port for introducing the buffer and the first introduction port. A first flow path through which the fluid flows, a first branch portion that branches the first flow path into a first bypass flow path and a second bypass flow path, and a downstream side of the first branch portion. A second inlet for introducing the fluid sample, a second flow path connected to the second inlet and for flowing the fluid sample, the first bypass flow path, and the second. A cross-sectional area A in a plane perpendicular to the flow direction of the first bypass flow path in the first branch portion, and the cross-sectional area A, which has a flow path and a confluence portion where the second bypass flow path merges. A cross-sectional area B on a plane perpendicular to the flow direction of the second bypass flow path at the first branch portion, and a cross-sectional area a on a plane perpendicular to the flow direction of the first bypass flow path at the confluence portion. The cross-sectional area b on the plane perpendicular to the flow direction of the second bypass flow path at the confluence part satisfies the following formula (1), and the recovery part is the first one through which the fluid containing the non-target particles flows. It has a recovery flow path and a second recovery flow path through which a fluid containing the target particles flows, and the first recovery flow path is connected to the first bypass flow path side of the separation flow path. , The separation device in which the second recovery flow path is connected to the second bypass flow path side of the separation flow path.
(A / B) ≤ (a / b) ... (1)
[2] The separation device according to [1], wherein the cross-sectional area a and the cross-sectional area b satisfy the following formula (2).
a ≦ b ... (2)
[3] The recovery section has a second branch section that branches the separation flow path into a first recovery flow path and a second recovery flow path, and the first recovery section in the second branch section. The cross-sectional area α on the plane perpendicular to the flow direction of the flow path and the cross-sectional area β on the plane perpendicular to the flow direction of the second recovery flow path at the second branch portion satisfy the following equation (3). The separation device according to [1] or [2].
(A / b) <(α / β) ... (3)
[4] The cross-sectional area of the confluence portion on the plane perpendicular to the flow direction of the second flow path is 5 to 30% of the cross-sectional area of the plane perpendicular to the flow direction of the separation flow path [1]. The separation device according to any one of [3].
[5] The separation device according to any one of [1] to [4], wherein the separation channel separates the target particles by a deterministic transverse substitution method.
[6] A method for separating the target particles from the fluid sample containing the target particles having a size equal to or larger than the threshold value and the non-target particles having a size smaller than the threshold value, [1] to [5]. The buffer is introduced from the first introduction port of the separation device according to any one of the above, the fluid sample is introduced from the second introduction port, and the fluid containing the target particles or the said from the recovery unit. A method comprising recovering a fluid containing non-target particles.
　本発明によれば、流体試料中の標的粒子を精度よく分離する技術を提供することができる。 According to the present invention, it is possible to provide a technique for accurately separating target particles in a fluid sample.
本発明の一態様における分離デバイスの構造を説明する上面図である。It is a top view explaining the structure of the separation device in one aspect of this invention. 図１Ａのｂ－ｂ’線における矢視断面図である。FIG. 3 is a cross-sectional view taken along the line bb'of FIG. 1A. 決定論的横置換法（ＤＬＤ）の基本原理を説明する図である。It is a figure explaining the basic principle of the deterministic transverse substitution method (DLD). 実験例１におけるシミュレーション結果を示す図である。It is a figure which shows the simulation result in Experimental Example 1. FIG. 実験例１におけるシミュレーションにより算出された、流体試料の層流の移動距離を示すグラフである。It is a graph which shows the moving distance of the laminar flow of a fluid sample calculated by the simulation in Experimental Example 1. 実験例２において、分離デバイスの分離流路の先頭部分、中間部分、及び尾部部分を撮影した写真である。In Experimental Example 2, it is a photograph which photographed the head part, the middle part, and the tail part of the separation flow path of a separation device.
　以下、場合により図面を参照しつつ、本発明の実施形態について詳細に説明する。なお、図面中、同一又は相当部分には同一又は対応する符号を付し、重複する説明は省略する。なお、各図における寸法比は、説明のため誇張している部分があり、必ずしも実際の寸法比とは一致しない。 Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings in some cases. In the drawings, the same or corresponding parts are designated by the same or corresponding reference numerals, and duplicate description will be omitted. The dimensional ratio in each figure is exaggerated for explanation and does not necessarily match the actual dimensional ratio.
［分離デバイス］
　一実施形態において、本発明は、閾値以上の大きさを有する標的粒子及び閾値未満の大きさを有する非標的粒子を含む流体試料から、前記標的粒子を分離する分離デバイスであって、流体中の粒子を大きさにより分離する分離流路と、前記分離流路の上流側に接続され、前記分離流路に前記流体試料及びバッファーを導入する流体導入部と、前記分離流路の下流側に接続され、前記標的粒子を含む流体及び前記非標的粒子を含む流体をそれぞれ回収する回収部と、を有し、前記流体導入部は、前記バッファーを導入する第１の導入口と、前記第１の導入口に接続され、前記バッファーが流れる第１の流路と、前記第１の流路を第１のバイパス流路及び第２のバイパス流路に分岐する第１の分岐部と、前記第１の分岐部の下流側に位置し、前記流体試料を導入する第２の導入口と、前記第２の導入口に接続され、前記流体試料が流れる第２の流路と、前記第１のバイパス流路、前記第２の流路及び前記第２のバイパス流路が合流する合流部と、を有し、前記第１の分岐部における前記第１のバイパス流路の流れ方向に垂直な面における断面積Ａと、前記第１の分岐部における前記第２のバイパス流路の流れ方向に垂直な面における断面積Ｂと、前記合流部における前記第１のバイパス流路の流れ方向に垂直な面における断面積ａと、前記合流部における前記第２のバイパス流路の流れ方向に垂直な面における断面積ｂとが、下記式（１）を満たし、前記回収部は、前記非標的粒子を含む流体が流れる第１の回収流路と、前記標的粒子を含む流体が流れる第２の回収流路と、を有し、前記第１の回収流路は前記分離流路の前記第１のバイパス流路側に接続しており、前記第２の回収流路は、前記分離流路の前記第２のバイパス流路側に接続している、分離デバイスを提供する。
　（Ａ／Ｂ）≦（ａ／ｂ）…（１） [Separation device]
In one embodiment, the present invention is a separation device that separates the target particles from a fluid sample containing a target particle having a size equal to or more than a threshold value and a non-target particle having a size less than the threshold value, and is in a fluid. A separation flow path that separates particles according to size, a fluid introduction section that is connected to the upstream side of the separation flow path and introduces the fluid sample and buffer into the separation flow path, and a downstream side of the separation flow path. The fluid introduction unit has a first introduction port for introducing the buffer and the first introduction unit, which has a recovery unit for collecting the fluid containing the target particles and the fluid containing the non-target particles, respectively. A first flow path connected to an introduction port and through which the buffer flows, a first branch portion that branches the first flow path into a first bypass flow path and a second bypass flow path, and the first branch portion. A second inlet for introducing the fluid sample, a second flow path connected to the second inlet and flowing the fluid sample, and the first bypass, which are located on the downstream side of the branch portion of the above. A plane having a flow path, a confluence portion where the second flow path and the second bypass flow path merge, and a plane perpendicular to the flow direction of the first bypass flow path in the first branch portion. The cross-sectional area A, the cross-sectional area B on the surface perpendicular to the flow direction of the second bypass flow path at the first branch portion, and the surface perpendicular to the flow direction of the first bypass flow path at the confluence portion. The cross-sectional area a in the above and the cross-sectional area b in the plane perpendicular to the flow direction of the second bypass flow path at the confluence satisfy the following formula (1), and the recovery unit contains the non-target particles. It has a first recovery flow path through which a fluid flows and a second recovery flow path through which a fluid containing the target particles flows, and the first recovery flow path is the first bypass flow of the separation flow path. The second recovery flow path, which is connected to the roadside, provides a separation device which is connected to the second bypass flow path side of the separation flow path.
(A / B) ≤ (a / b) ... (1)
　図１Ａは、本実施形態の分離デバイスの構造を説明する上面図である。図１Ａ中、矢印は、流体が流れる方向を示す。図１Ｂは、図１Ａのｂ－ｂ’線における矢視断面図である。図１Ａ及び図１Ｂに示すように、分離デバイス１００は、分離流路１１０と、流体導入部１２０と、回収部１３０とを有する。流体導入部１２０は、分離流路１１０の上流側に接続され、分離流路１１０に流体試料及びバッファーを導入する。回収部１３０は、分離流路１１０の下流側に接続され、標的粒子を含む流体及び非標的粒子を含む流体をそれぞれ回収する。 FIG. 1A is a top view illustrating the structure of the separation device of the present embodiment. In FIG. 1A, the arrows indicate the direction in which the fluid flows. FIG. 1B is a cross-sectional view taken along the line bb'of FIG. 1A. As shown in FIGS. 1A and 1B, the separation device 100 includes a separation flow path 110, a fluid introduction unit 120, and a recovery unit 130. The fluid introduction unit 120 is connected to the upstream side of the separation flow path 110, and introduces the fluid sample and the buffer into the separation flow path 110. The recovery unit 130 is connected to the downstream side of the separation flow path 110, and recovers the fluid containing the target particles and the fluid containing the non-target particles, respectively.
　流体導入部１２０は、第１の導入口１２１と、第１の流路１２２と、第１の分岐部１２３と、第２の導入口１２４と、第２の流路１２５と、合流部１２６とを有する。第１の導入口１２１から、バッファーが導入される。第１の流路１２２は、第１の導入口１２１に接続されており、バッファーが第一の流路１２２内を流れる。第１の分岐部１２３は、第１の流路１２２を第１のバイパス流路１２２１及び第２のバイパス流路１２２２に分岐する。第２の導入口１２４は、第１の分岐部１２３の下流側に位置する。第２の導入口１２４から、流体試料が導入される。第２の流路１２５は、第２の導入口１２４に接続されており、流体試料が第２の流路１２５内を流れる。第１のバイパス流路１２２１、第２の流路１２５及び第２のバイパス流路１２２２は、合流部１２６で合流する。 The fluid introduction section 120 includes a first introduction port 121, a first flow path 122, a first branch section 123, a second introduction port 124, a second flow path 125, and a confluence section 126. Has. The buffer is introduced from the first introduction port 121. The first flow path 122 is connected to the first introduction port 121, and the buffer flows in the first flow path 122. The first branch portion 123 branches the first flow path 122 into the first bypass flow path 1221 and the second bypass flow path 1222. The second introduction port 124 is located on the downstream side of the first branch portion 123. The fluid sample is introduced from the second introduction port 124. The second flow path 125 is connected to the second introduction port 124, and the fluid sample flows in the second flow path 125. The first bypass flow path 1221, the second flow path 125, and the second bypass flow path 1222 merge at the merging portion 126.
　第１の分岐部１２３における第１のバイパス流路１２２１の流れ方向に垂直な面における断面積Ａと、第１の分岐部１２３における第２のバイパス流路１２２２の流れ方向に垂直な面における断面積Ｂと、合流部１２６における第１のバイパス流路１２２１の流れ方向に垂直な面における断面積ａと、合流部１２６における第２のバイパス流路１２２２の流れ方向に垂直な面における断面積ｂとは、下記式（１）を満たす。
　（Ａ／Ｂ）≦（ａ／ｂ）…（１） The cross-sectional area A in the plane perpendicular to the flow direction of the first bypass flow path 1221 in the first branch portion 123 and the disconnection in the plane perpendicular to the flow direction of the second bypass flow path 1222 in the first branch portion 123. The area B, the cross-sectional area a of the merging portion 126 in the plane perpendicular to the flow direction of the first bypass flow path 1221, and the cross-sectional area b of the merging portion 126 in the plane perpendicular to the flow direction of the second bypass flow path 1222. Satisfies the following equation (1).
(A / B) ≤ (a / b) ... (1)
　回収部１３０は、非標的粒子を含む流体が流れる第１の回収流路１３１と、標的粒子を含む流体が流れる第２の回収流路１３２と、を有している。第１の回収流路１３１は、分離流路１１０の流れ方向に垂直な方向において、第１のバイパス流路１２２１側に接続している。つまり、第１の回収流路１３１は、分離流路１１０の流れ方向に垂直な方向において、第２のバイパス流路１２２２より第１のバイパス流路１２２１の近くに配置されている。第２の回収流路１３２は、分離流路１１０の流れ方向に垂直な方向において、第２のバイパス流路１２２２側に接続している。つまり、第２の回収流路１３２は、分離流路１１０の流れ方向に垂直な方向において、第１のバイパス流路１２２１より第２のバイパス流路１２２２の近くに配置されている。また、第１の回収流路１３１は、排出口１３４に接続している。第２の回収流路１３２は、排出口１３５に接続している。 The recovery unit 130 has a first recovery flow path 131 through which a fluid containing non-target particles flows, and a second recovery flow path 132 through which a fluid containing target particles flows. The first recovery flow path 131 is connected to the first bypass flow path 1221 side in a direction perpendicular to the flow direction of the separation flow path 110. That is, the first recovery flow path 131 is arranged closer to the first bypass flow path 1221 than the second bypass flow path 1222 in the direction perpendicular to the flow direction of the separation flow path 110. The second recovery flow path 132 is connected to the second bypass flow path 1222 side in a direction perpendicular to the flow direction of the separation flow path 110. That is, the second recovery flow path 132 is arranged closer to the second bypass flow path 1222 than the first bypass flow path 1221 in the direction perpendicular to the flow direction of the separation flow path 110. Further, the first recovery flow path 131 is connected to the discharge port 134. The second recovery flow path 132 is connected to the discharge port 135.
　上記式（１）を満たすことにより、第２のバイパス流路１２２２から分離流路１１０に吐出される流体の速度が、第１のバイパス流路１２２１から分離流路１１０に吐出される流体の速度と同じ、若しくは、第１のバイパス流路１２２１から分離流路１１０に吐出される流体の速度よりも速くなると考えられる。この結果、第２の流路１２５から合流した流体試料が形成する層流が、第２のバイパス流路１２２２側つまり、第２の回収流路１３２側に流れにくくなる。すなわち、流体試料が、標的粒子が回収される第２の回収流路１３２に混入しにくくなり、標的粒子の回収を精度よく行うことができる。本明細書において、標的粒子の回収を精度よく行うことができるとは、標的粒子の分離能が高いことを意味する。 By satisfying the above formula (1), the velocity of the fluid discharged from the second bypass flow path 1222 to the separation flow path 110 is the velocity of the fluid discharged from the first bypass flow path 1221 to the separation flow path 110. It is considered that the speed is the same as or higher than the speed of the fluid discharged from the first bypass flow path 1221 to the separation flow path 110. As a result, the laminar flow formed by the fluid sample merged from the second flow path 125 becomes difficult to flow to the second bypass flow path 1222 side, that is, the second recovery flow path 132 side. That is, the fluid sample is less likely to be mixed into the second recovery flow path 132 in which the target particles are recovered, and the target particles can be recovered with high accuracy. In the present specification, the ability to accurately recover the target particles means that the target particles have a high separation ability.
　実施例において後述するように、本実施形態の分離デバイスによれば、流体試料中の標的粒子を精度よく分離することができる。断面積Ａ及びＢは、分岐部１２３において、第１の流路が第１のバイパス流路１２２１及び第２のバイパス流路に初めて分岐する位置における断面積をいう。いいかえると、断面積Ａ及びＢは、分岐部１２３において、第１の流路１２２を第１のバイパス流路１２２１及び第２のバイパス流路１２２２に分岐させるための隔壁１２２３の、上流側の端部における断面積である。 As will be described later in the examples, the separation device of this embodiment can accurately separate the target particles in the fluid sample. The cross-sectional areas A and B refer to the cross-sectional areas of the branch portion 123 at the positions where the first flow path branches to the first bypass flow path 1221 and the second bypass flow path for the first time. In other words, the cross-sectional areas A and B are the upstream ends of the partition wall 1223 for branching the first flow path 122 into the first bypass flow path 1221 and the second bypass flow path 1222 in the branch portion 123. It is the cross-sectional area in the part.
　同様に、断面積ａ及びｂは、合流部１２６において、第１のバイパス流路１２２１及び第２のバイパス流路１２２２が初めて合流する位置における断面積をいう。いいかえると、断面積ａ及びｂは、隔壁１２２３の下流側の端部における断面積である。 Similarly, the cross-sectional areas a and b refer to the cross-sectional areas at the positions where the first bypass flow path 1221 and the second bypass flow path 1222 meet for the first time in the merging portion 126. In other words, the cross-sectional areas a and b are the cross-sectional areas at the downstream end of the partition wall 1223.
　本実施形態の分離デバイスにおいて、回収部１３０は、分離流路１１０を第１の回収流路１３１及び第２の回収流路１３２に分岐する第２の分岐部１３３を有する。 In the separation device of the present embodiment, the collection unit 130 has a second branch unit 133 that branches the separation flow path 110 into the first collection flow path 131 and the second collection flow path 132.
　分離流路１１０における分離の原理は、層流を形成する分離方法であれば特に限定されない。例えば、決定論的横置換法（以降、ＤＬＤと称することがある）、サイズによる分離、又は磁気による分離等であってよい。図１Ａ及び図１Ｂに示す分離デバイス１００の例では、分離流路１１０における分離の原理は、ＤＬＤである。ＤＬＤについては後述する。 The principle of separation in the separation flow path 110 is not particularly limited as long as it is a separation method for forming a laminar flow. For example, it may be a deterministic transverse substitution method (hereinafter, may be referred to as DLD), separation by size, separation by magnetism, or the like. In the example of the separation device 100 shown in FIGS. 1A and 1B, the principle of separation in the separation flow path 110 is DLD. DLD will be described later.
　第１の導入口１２１から導入されたバッファー及び第２の導入口１２４から導入された流体試料は、合流部１２６の下流側において層流を形成する。この層流は、分離流路１１０の内部に形成された微小なピラー１１１のアレイを通過する。その結果、ＤＬＤの原理により、閾値以上の大きさを有する粒子は、バイパス流路１２２２側の方向に斜めに流れる。そして、回収部１３０において第２の回収流路１３２を通過し、排出口１３５から回収される。一方、閾値未満の大きさを有する粒子は、層流に乗って直線的に進む。そして、回収部１３０において第１の回収流路１３１を通過し、排出口１３４から回収される。バッファー及び流体試料の送液は、例えば、シリンジポンプ、ダイヤフラムポンプ、又はローラーチューブポンプ等により行うことができる。 The buffer introduced from the first introduction port 121 and the fluid sample introduced from the second introduction port 124 form a laminar flow on the downstream side of the confluence portion 126. This laminar flow passes through an array of tiny pillars 111 formed inside the separation channel 110. As a result, according to the principle of DLD, particles having a size equal to or larger than the threshold value flow diagonally in the direction toward the bypass flow path 1222. Then, the collection unit 130 passes through the second collection flow path 132 and is collected from the discharge port 135. On the other hand, particles having a size less than the threshold value travel linearly on the laminar flow. Then, the collection unit 130 passes through the first collection flow path 131 and is collected from the discharge port 134. The buffer and the fluid sample can be fed by, for example, a syringe pump, a diaphragm pump, a roller tube pump, or the like.
　本実施形態の分離デバイスにおいて、上記の断面積ａ及び上記の断面積ｂは、下記式（２）を満たすことが好ましい。
　ａ≦ｂ…（２） In the separation device of the present embodiment, the cross-sectional area a and the cross-sectional area b preferably satisfy the following formula (2).
a ≦ b ... (2)
　断面積ａ及び上記の断面積ｂが上記式（２）を満たすということは、合流部１２６における第１のバイパス流路１２２１及び第２のバイパス流路１２２２の高さが一定であれば、合流部１２６における第２の流路１２５の開口部が分離流路１１０の幅方向（流体の流れ方向に垂直な方向）における中心又は第１のバイパス流路１２２１側に位置することを意味する。つまり、合流部１２６における第２の流路１２５の開口部が分離流路１１０の幅方向における中心又は第２のバイパス流路１２２２より第１のバイパス流路１２２１の近くに位置することを意味する。一般的に、分離デバイスを構成する各流路の高さは、一定である。 The fact that the cross-sectional area a and the cross-sectional area b satisfy the above equation (2) means that if the heights of the first bypass flow path 1221 and the second bypass flow path 1222 in the merging portion 126 are constant, they merge. It means that the opening of the second flow path 125 in the portion 126 is located at the center in the width direction (direction perpendicular to the fluid flow direction) of the separation flow path 110 or on the side of the first bypass flow path 1221. That is, it means that the opening of the second flow path 125 in the merging portion 126 is located at the center in the width direction of the separation flow path 110 or closer to the first bypass flow path 1221 than the second bypass flow path 1222. .. Generally, the height of each flow path constituting the separation device is constant.
　この結果、流体試料の層流が第１のバイパス流路側に移動し、流体試料が、回収部１３０において第１の回収流路１３１に回収されやすくなる。このため、流体試料が、標的粒子が回収される第２の回収流路１３２に混入しにくくなり、標的粒子の回収を精度よく行うことができる。 As a result, the laminar flow of the fluid sample moves to the first bypass flow path side, and the fluid sample is easily collected in the first recovery flow path 131 in the recovery unit 130. Therefore, the fluid sample is less likely to be mixed into the second recovery flow path 132 in which the target particles are recovered, and the target particles can be recovered with high accuracy.
　断面積ｂに対する断面積ａの比であるａ／ｂは、０．０５以上０．８以下であることが好ましく、０．０８以上０．７５以下であることがより好ましく、０．１以上０．７以下であることがより好ましく、０．１５以上０．６５以下であることが更に好ましい。ａ／ｂが０．０５以上０．８以下であると、流体試料の層流が第１のバイパス流路側に移動するよう制御しやすくなる。その結果、流体試料が、回収部１３０において第１の回収流路１３１に回収されやすくなる。また、流体試料が、標的粒子が回収される第２の回収流路１３２に混入しにくくなり、標的粒子の回収を精度よく行うことができる。 The ratio of the cross-sectional area a to the cross-sectional area b, which is a / b, is preferably 0.05 or more and 0.8 or less, more preferably 0.08 or more and 0.75 or less, and 0.1 or more and 0. It is more preferably 0.7 or less, and further preferably 0.15 or more and 0.65 or less. When a / b is 0.05 or more and 0.8 or less, it becomes easy to control the laminar flow of the fluid sample to move to the first bypass flow path side. As a result, the fluid sample is easily collected in the first collection flow path 131 in the collection unit 130. In addition, the fluid sample is less likely to be mixed into the second recovery flow path 132 in which the target particles are recovered, and the target particles can be recovered with high accuracy.
　本実施形態の分離デバイスにおいて、第２の分岐部１３３における第１の回収流路１３１の流れ方向に垂直な面における断面積αと、第２の分岐部１３３における第２の回収流路１３２の流れ方向に垂直な面における断面積βは、下記式（３）を満たすことが好ましい。
　（ａ／ｂ）＜（α／β）…（３） In the separation device of the present embodiment, the cross-sectional area α in the plane perpendicular to the flow direction of the first recovery flow path 131 in the second branch portion 133 and the second recovery flow path 132 in the second branch portion 133. The cross-sectional area β on the plane perpendicular to the flow direction preferably satisfies the following equation (3).
(A / b) <(α / β) ... (3)
　断面積α及びβは、分岐部１３３において、分離流路１１０が、第１の回収流路１３１及び第２の回収流路１３２に初めて分岐する位置における断面積をいう。断面積ａ、ｂ、α、及びβが上記式（３）を満たすということは、合流部１２６における第２のバイパス流路１２２２の幅に対する第１のバイパス流路１２２１の幅の比よりも、合流部１３３における第２の回収流路１３２の幅に対する第１の回収流路１３１の幅の比のほうが大きいことを意味する。 The cross-sectional areas α and β refer to the cross-sectional areas at the positions where the separation flow path 110 first branches into the first recovery flow path 131 and the second recovery flow path 132 in the branch portion 133. The fact that the cross-sectional areas a, b, α, and β satisfy the above equation (3) is more than the ratio of the width of the first bypass flow path 1221 to the width of the second bypass flow path 1222 at the merging portion 126. It means that the ratio of the width of the first recovery flow path 131 to the width of the second recovery flow path 132 in the merging portion 133 is larger.
　その結果、流体試料が、回収部１３０において第１の回収流路１３１に回収されやすくなる。このため、流体試料が、標的粒子が回収される第２の回収流路１３２に混入しにくくなり、標的粒子の回収を精度よく行うことができる。 As a result, the fluid sample is easily collected in the first collection flow path 131 in the collection unit 130. Therefore, the fluid sample is less likely to be mixed into the second recovery flow path 132 in which the target particles are recovered, and the target particles can be recovered with high accuracy.
　あるいは、断面積ａ及びｂの大きさとは関係なく、αがβ以上の大きさを有していてもよい。すなわち、α及びβは、下記式（４）を満たすことが好ましい。
　α≧β…（４） Alternatively, α may have a size of β or more regardless of the sizes of the cross-sectional areas a and b. That is, it is preferable that α and β satisfy the following formula (4).
α ≧ β… (4)
　断面積α及びβが上記式（４）を満たすということは、合流部１３３における回収流路１３１の幅が合流部１３３における回収流路１３２の幅よりも大きいことを意味する。その結果、流体試料が、回収部１３０において第１の回収流路１３１に回収されやすくなる。このため、流体試料が、標的粒子が回収される第２の回収流路１３２に混入しにくくなり、標的粒子の回収を精度よく行うことができる。 The fact that the cross-sectional areas α and β satisfy the above equation (4) means that the width of the recovery flow path 131 at the confluence portion 133 is larger than the width of the recovery flow path 132 at the confluence portion 133. As a result, the fluid sample is easily collected in the first collection flow path 131 in the collection unit 130. Therefore, the fluid sample is less likely to be mixed into the second recovery flow path 132 in which the target particles are recovered, and the target particles can be recovered with high accuracy.
　本実施形態の分離デバイスにおいて、合流部１２６における第２の流路１２５の流れ方向に垂直な面における断面積Ｃが、分離流路１１０の流れ方向に垂直な面における断面積Ｄに対して５～３０％であることが好ましい。 In the separation device of the present embodiment, the cross-sectional area C of the merging portion 126 on the plane perpendicular to the flow direction of the second flow path 125 is 5 with respect to the cross-sectional area D on the plane perpendicular to the flow direction of the separation flow path 110. It is preferably about 30%.
　実施例において後述するように、上記の断面積Ｃ及びＤが上記の範囲にある分離デバイスによれば、回収部１３０において、流体試料を第１の回収流路１３１に確実に回収することができる傾向にある。断面積Ｃが大きいほど、単位時間あたりに分離デバイスに導入することができる流体試料の体積が大きくなり、標的粒子を分離する処理速度を向上させることができる。分離流路１１０において、断面積Ｄが一定でない場合には、分離流路１１０の流れ方向に垂直な面における断面積の平均値を断面積Ｄとしてもよい。 As will be described later in the examples, according to the separation device in which the cross-sectional areas C and D are in the above range, the fluid sample can be reliably recovered in the first recovery flow path 131 in the recovery unit 130. There is a tendency. The larger the cross-sectional area C, the larger the volume of the fluid sample that can be introduced into the separation device per unit time, and the faster the processing speed for separating the target particles can be improved. When the cross-sectional area D of the separation flow path 110 is not constant, the average value of the cross-sectional areas on the plane perpendicular to the flow direction of the separation flow path 110 may be set as the cross-sectional area D.
　本明細書における分離流路１１０の流れ方向に垂直な面における断面積の平均値は、以下の方法で算出される。分離流路１１０の流れ方向に垂直な面における断面積における最も小さい断面積の部分と最も大きい断面積の部分との間の１０点を等間隔に選択し、それぞれの断面積を測定する。１０点の断面積の値の総和を測定点、即ち１０で割った値を平均値とする。 The average value of the cross-sectional areas on the plane perpendicular to the flow direction of the separation flow path 110 in the present specification is calculated by the following method. Ten points between the smallest cross-sectional area and the largest cross-sectional area in the plane perpendicular to the flow direction of the separation flow path 110 are selected at equal intervals, and the respective cross-sectional areas are measured. The sum of the cross-sectional area values of 10 points is taken as the measurement point, that is, the value divided by 10 is taken as the average value.
　本明細書において、分離対象となる粒子としては、例えば、ビーズ、又は細胞等が挙げられる。ビーズは、その表面に、標的タンパク質等を特異的に認識する特異的結合物質を結合したものであってもよい。特異的結合物質としては、抗体、抗体断片、アプタマー等が挙げられる。ビーズの材質は、特に限定されず、シリカ、ポリスチレン、ラテックス、又は金属等が挙げられる。ビーズは、磁気粒子であってもよい。 In the present specification, examples of particles to be separated include beads, cells, and the like. The beads may be those in which a specific binding substance that specifically recognizes a target protein or the like is bound to the surface thereof. Specific binding substances include antibodies, antibody fragments, aptamers and the like. The material of the beads is not particularly limited, and examples thereof include silica, polystyrene, latex, and metal. The beads may be magnetic particles.
　このようなビーズを流体試料に混合することにより、流体試料中の標的タンパク質等をビーズに結合させることができる。更に、当該ビーズを上述した分離デバイスで回収することにより、流体試料中の標的タンパク質等を回収することができる。 By mixing such beads with the fluid sample, the target protein or the like in the fluid sample can be bound to the beads. Further, by recovering the beads with the separation device described above, the target protein or the like in the fluid sample can be recovered.
　また、特異的結合物質として、特定の細胞又はエクソソーム等の粒子の表面に存在するタンパク質を特異的に認識する抗体等を結合したビーズを用いることにより、当該細胞又はエクソソーム等をビーズに結合させることができる。そして、このようなビーズを上述した分離デバイスで回収することにより、流体試料中の特定の細胞又はエクソソーム等を回収することができる。 In addition, by using beads to which an antibody or the like that specifically recognizes a protein existing on the surface of a particle such as a specific cell or an exosome is bound as a specific binding substance, the cell or the exosome or the like is bound to the bead. Can be done. Then, by recovering such beads with the separation device described above, specific cells, exosomes, etc. in the fluid sample can be recovered.
　細胞としては、特に限定されず、特異的なマーカータンパク質を細胞表面に発現したあらゆる細胞が挙げられる。細胞は、例えば血液中の循環腫瘍細胞（以降、ＣＴＣと称することがある）であってもよい。 The cell is not particularly limited, and examples thereof include any cell expressing a specific marker protein on the cell surface. The cell may be, for example, a circulating tumor cell in blood (hereinafter, may be referred to as CTC).
　また、例えば、目的細胞と目的外細胞の大きさが近似している場合、両者を分離することが困難であることがある。このような場合、適切な大きさのビーズを目的細胞のみに特異的に結合させて、目的細胞を見かけ上大きな粒子にすることにより、目的外細胞との大きさの差を大きくし、分離デバイスで分離しやすくすることができる。 Also, for example, when the size of the target cell and the size of the non-target cell are similar, it may be difficult to separate the two. In such a case, beads of an appropriate size are specifically bound only to the target cells to make the target cells apparently large particles, thereby increasing the size difference from the non-target cells and separating the device. Can be easily separated with.
　本明細書において、粒子が球体である場合には、粒子の大きさとは、当該球体の直径であってよい。また、粒子が球体でない場合には、当該粒子と同じ体積の球体を想定し、当該球体の直径を粒子の大きさとしてもよい。 In the present specification, when the particle is a sphere, the size of the particle may be the diameter of the sphere. When the particle is not a sphere, a sphere having the same volume as the particle may be assumed, and the diameter of the sphere may be the size of the particle.
　以下、ＤＬＤについて説明する。ＤＬＤとは、わずかにずらしながら配置された微小なピラーのアレイに粒子を含む流体の層流を通過させた際に、大きい粒子はピラー周囲に生じる流れの変化により斜めに流れるのに対し、小さい粒子は層流に乗って直線的に進む性質を利用して、粒子を大きさにより分離する方法である。 The DLD will be described below. DLD is small when a laminar flow of fluid containing particles is passed through an array of minute pillars arranged with a slight shift, while large particles flow diagonally due to changes in the flow that occur around the pillars. Particles are a method of separating particles according to their size by utilizing the property of traveling linearly on a laminar flow.
　非特許文献１に記載のＤＬＤ原理に基づいて、分離したい粒子、つまり標的粒子の粒径により、斜方向に変位する粒子直径の閾値（Ｄｃ）を設定することができる。より具体的には、閾値Ｄｃの設定は、下記式（５）から求めることができる。
　Ｄｃ＝２ηＧε　…（５）
［式（５）中、Ｄｃは斜方向に変位する粒子の直径の閾値であり、ηは変数であり、Ｇはピラー間ギャップであり、ε：ピラーのずれ角度（ｔａｎθ）である。］ Based on the DLD principle described in Non-Patent Document 1, the threshold value (Dc) of the particle diameter displaced in the oblique direction can be set according to the particle size of the particles to be separated, that is, the target particles. More specifically, the setting of the threshold value Dc can be obtained from the following equation (5).
Dc = 2ηGε… (5)
[In equation (5), Dc is the threshold value of the diameter of the particles displaced in the oblique direction, η is a variable, G is the gap between pillars, and ε: the deviation angle of pillars (tan θ). ]
　そして、上記式（５）を解くと、下記式（６）に示す近似式が得られる。
　Ｄｃ＝１．４Ｇε０．４８　…（６）
　そして、経験則から、０．０６＜ε＜０．１程度の場合に標的粒子の良好な分離を行うことができることから、ε＝ｔａｎθ＝１／１５＝０．０６７を用いて、下記式（７）を導くことができる。
　Ｇ≒２．６２０５７Ｄｃ　…（７） Then, by solving the above equation (5), an approximate equation shown in the following equation (6) can be obtained.
Dc = 1.4Gε 0.48 ... (6)
Then, from the rule of thumb, since good separation of the target particles can be performed when 0.06 <ε <0.1, the following equation (ε = tanθ = 1/15 = 0.067) is used. 7) can be derived.
G≈2.62057Dc ... (7)
　そして、上記の式（７）から、斜方向に変位する粒子直径の閾値Ｄｃに基づいて、ピラー間ギャップＧを算出する。そして、ピラー間ギャップGのピラー群（以降、障害物構造と称することがある）が設置された基本構造部分を有する細胞分離デバイスを作製することができる。このようにして、目的の閾値Ｄｃを有するＤＬＤマイクロ流路を有する分離デバイスを作製することができる。 Then, from the above equation (7), the inter-pillar gap G is calculated based on the threshold value Dc of the particle diameter displaced in the oblique direction. Then, a cell separation device having a basic structural portion in which the pillar group of the inter-pillar gap G (hereinafter, may be referred to as an obstacle structure) is installed can be produced. In this way, a separation device having a DLD microchannel having a desired threshold Dc can be made.
　図２は、決定論的横置換法（ＤＬＤ）の基本原理を説明する図である。図２は、ＤＬＤマイクロ流路の基本構造２０（以降、分離エリアと称することがある）を示す。図２中、矢印方向に向かう流体の流れ方向に対し、一定の規則にしたがって斜めに配列した障害物構造（以降、ピラーと称することがある）２１が設けられている。矢印方向に向かう流体中において、障害物構造２１周辺部においては流れ速度の変化が生じる。この連続して配列された障害物構造２１による流れ速度の変化を利用し、流体中に含まれる粒子の直径が閾値以上である場合、その粒子の進行方向を変化させることができる。 FIG. 2 is a diagram illustrating the basic principle of the deterministic transverse substitution method (DLD). FIG. 2 shows the basic structure 20 of the DLD microchannel (hereinafter, may be referred to as a separation area). In FIG. 2, an obstacle structure (hereinafter, may be referred to as a pillar) 21 arranged diagonally according to a certain rule with respect to the flow direction of the fluid in the direction of the arrow is provided. In the fluid directed in the direction of the arrow, the flow velocity changes around the obstacle structure 21. By utilizing the change in flow velocity due to the continuously arranged obstacle structures 21, when the diameter of the particles contained in the fluid is equal to or larger than the threshold value, the traveling direction of the particles can be changed.
　進行方向の変化について、閾値以上の大きさを有する粒子２２は、連続して配列された障害物構造２１の配置にしたがい斜め方向に変位していく。一方、閾値未満の大きさを有する粒子２３は、流れ方向に沿って障害物構造２１を迂回しながら概ね直進する。 Regarding the change in the traveling direction, the particles 22 having a size equal to or larger than the threshold value are displaced in the oblique direction according to the arrangement of the obstacle structures 21 arranged continuously. On the other hand, the particles 23 having a size less than the threshold value generally travel straight along the flow direction while bypassing the obstacle structure 21.
　このように、目的とする大きさの粒子を分離可能な閾値設定を行った障害物構造２１の配列パターンを公知の方法（非特許文献１）にしたがって設計することにより、流体中に含まれる標的粒子の大きさに依存して分離することができる。また、流れ方向下流部にそれぞれ異なる回収流路を設けることにより、分離した粒子をそれぞれ回収することができる。 In this way, by designing the arrangement pattern of the obstacle structure 21 in which the threshold value is set so that particles of the target size can be separated according to a known method (Non-Patent Document 1), the target contained in the fluid It can be separated depending on the size of the particles. Further, by providing different recovery channels in the downstream portion in the flow direction, the separated particles can be recovered respectively.
　なお、障害物構造２１の形状は、図２に示すような円柱構造に限定されない。障害物構造２１は、目的とする流れ速度変化を生じさせることのできる形状であれば、例えば三角柱、四角柱等の多角柱構造とすることも可能である。 The shape of the obstacle structure 21 is not limited to the cylindrical structure as shown in FIG. The obstacle structure 21 may have a polygonal prism structure such as a triangular prism or a quadrangular prism as long as it has a shape capable of causing a desired change in flow velocity.
　本実施形態の分離デバイスの各流路及びＤＬＤの微小なピラーのアレイは、公知の方法を適宜選択して作製することができる。また、分離デバイスの材質としては、例えば、ガラス、シリコーン、ジメチルポリシロキサン、又はプラスチック等を用いることができる。 Each flow path of the separation device of the present embodiment and an array of minute pillars of the DLD can be produced by appropriately selecting a known method. Further, as the material of the separation device, for example, glass, silicone, dimethylpolysiloxane, plastic or the like can be used.
［分離方法］
　一実施形態において、本発明は、閾値以上の大きさを有する標的粒子及び閾値未満の大きさを有する非標的粒子を含む流体試料から、前記標的粒子を分離する方法であって、上述したいずれかの分離デバイスの前記第１の導入口から前記バッファーを導入し、前記第２の導入口から前記流体試料を導入することと、前記回収部から前記標的粒子を含む流体又は前記非標的粒子を含む流体を回収することとを含む方法を提供する。 [Separation method]
In one embodiment, the present invention is a method for separating the target particles from a fluid sample containing target particles having a size equal to or larger than a threshold and non-target particles having a size smaller than the threshold, and any of the above-mentioned ones. The buffer is introduced from the first inlet of the separation device, the fluid sample is introduced from the second inlet, and the fluid containing the target particles or the non-target particles is contained from the recovery unit. A method including recovering a fluid is provided.
　上述した分離デバイスにおいて、回収部は、第１の回収流路と、第２の回収流路とを有している。第１の回収流路は、分離流路の第１のバイパス流路側に接続している。第２の回収流路は、分離流路の前記第２のバイパス流路側に接続している。非標的粒子は、第１の回収流路が接続する排出口から回収される。標的粒子は、第２の回収流路が接続する排出口から回収される。 In the separation device described above, the recovery unit has a first recovery flow path and a second recovery flow path. The first recovery flow path is connected to the first bypass flow path side of the separation flow path. The second recovery flow path is connected to the second bypass flow path side of the separation flow path. The non-target particles are recovered from the outlet to which the first recovery channel is connected. The target particles are collected from the discharge port to which the second collection channel is connected.
　本実施形態の方法により、閾値以上の大きさを有する標的粒子及び閾値未満の大きさを有する非標的粒子を含む流体試料から、標的粒子を精度よく分離することができる。本実施形態の方法において、粒子は上述したものと同様である。 According to the method of the present embodiment, the target particles can be accurately separated from the fluid sample containing the target particles having a size equal to or larger than the threshold value and the non-target particles having a size smaller than the threshold value. In the method of this embodiment, the particles are similar to those described above.
　本発明は、別の側面として以下の態様を含む。
［７］閾値以上の大きさを有する標的粒子及び閾値未満の大きさを有する非標的粒子を含む流体試料から、前記標的粒子を分離する分離デバイスであって、流体中の粒子を大きさにより分離する分離流路と、前記分離流路の上流側に接続され、前記分離流路に前記流体試料及びバッファーを導入する流体導入部と、前記分離流路の下流側に接続され、前記標的粒子を含む流体及び前記非標的粒子を含む流体をそれぞれ回収する回収部と、を有し、前記流体導入部は、前記バッファーを導入する第１の導入口と、前記第１の導入口に接続され、前記バッファーが流れる第１の流路と、前記第１の流路を第１のバイパス流路及び第２のバイパス流路に分岐する第１の分岐部と、前記第１の分岐部の下流側に位置し、前記流体試料を導入する第２の導入口と、前記第２の導入口に接続され、前記流体試料が流れる第２の流路と、前記第１のバイパス流路、前記第２の流路及び前記第２のバイパス流路が合流する合流部と、を有し、前記第１の分岐部における前記第１のバイパス流路の流れ方向に垂直な面における断面積Ａと、前記第１の分岐部における前記第２のバイパス流路の流れ方向に垂直な面における断面積Ｂと、前記合流部における前記第１のバイパス流路の流れ方向に垂直な面における断面積ａと、前記合流部における前記第２のバイパス流路の流れ方向に垂直な面における断面積ｂとが、下記式（１）を満たし、前記断面積ｂに対する前記断面積ａの比であるａ／ｂが、０．０５以上０．８以下であり、前記回収部は、前記非標的粒子を含む流体が流れる第１の回収流路と、前記標的粒子を含む流体が流れる第２の回収流路と、を有し、前記第１の回収流路は前記分離流路の前記第１のバイパス流路側に接続しており、前記第２の回収流路は、前記分離流路の前記第２のバイパス流路側に接続している、分離デバイス。
　（Ａ／Ｂ）≦（ａ／ｂ）…（１）
［８］前記断面積ｂに対する前記断面積ａの比であるａ／ｂが、０．０８以上０．７５以下である、［７］に記載の分離デバイス。
［９］前記断面積ｂに対する前記断面積ａの比であるａ／ｂが、０．１以上０．７以下である、［７］に記載の分離デバイス。
［１０］前記断面積Ａ、前記断面積Ｂ、前記断面積ａ、及び前記断面積ｂ、が（Ａ／Ｂ）＝（ａ／ｂ）を満たす、［７］～［９］のいずれか一つに記載の分離デバイス。
［１１］前記回収部は、前記分離流路を第１の回収流路及び第２の回収流路に分岐する第２の分岐部を有し、前記第２の分岐部における前記第１の回収流路の流れ方向に垂直な面における断面積αと、前記第２の分岐部における前記第２の回収流路の流れ方向に垂直な面における断面積βが、下記式（３）を満たす、［７］～［１０］のいずれか一つに記載の分離デバイス。
　（ａ／ｂ）＜（α／β）…（３）
［１２］前記合流部における前記第２の流路の流れ方向に垂直な面における断面積が、前記分離流路の流れ方向に垂直な面における断面積の５～３０％である、［７］～［１１］のいずれか一つに記載の分離デバイス。
［１３］前記分離流路は、決定論的横置換法により前記標的粒子を分離する、［７］～［１２］のいずれか一つに記載の分離デバイス。
［１４］前記閾値以上の大きさを有する標的粒子及び前記閾値未満の大きさを有する非標的粒子を含む前記流体試料から、前記標的粒子を分離する方法であって、［７］～［１３］のいずれか一つのいずれかに記載の分離デバイスの前記第１の導入口から前記バッファーを導入し、前記第２の導入口から前記流体試料を導入することと、前記回収部から前記標的粒子を含む流体又は前記非標的粒子を含む流体を回収することと、を含む、方法。 The present invention includes the following aspects as another aspect.
[7] A separation device that separates the target particles from a fluid sample containing target particles having a size equal to or larger than the threshold and non-target particles having a size smaller than the threshold, and separates the particles in the fluid according to the size. The separation flow path is connected to the upstream side of the separation flow path, the fluid introduction section for introducing the fluid sample and the buffer into the separation flow path, and the target particle connected to the downstream side of the separation flow path. It has a recovery unit for recovering the containing fluid and the fluid containing the non-target particles, respectively, and the fluid introduction unit is connected to a first introduction port for introducing the buffer and the first introduction port. A first flow path through which the buffer flows, a first branch portion that branches the first flow path into a first bypass flow path and a second bypass flow path, and a downstream side of the first branch portion. A second introduction port for introducing the fluid sample, a second flow path connected to the second introduction port and through which the fluid sample flows, the first bypass flow path, and the second flow path. A cross-sectional area A in a plane perpendicular to the flow direction of the first bypass flow path in the first branch portion, and the cross-sectional area A, which has a flow path and a confluence portion where the second bypass flow path merges. A cross-sectional area B on a plane perpendicular to the flow direction of the second bypass flow path at the first branch portion, and a cross-sectional area a on a plane perpendicular to the flow direction of the first bypass flow path at the confluence portion. The cross-sectional area b on the plane perpendicular to the flow direction of the second bypass flow path at the confluence satisfies the following formula (1), and a / b which is the ratio of the cross-sectional area a to the cross-sectional area b is , 0.05 or more and 0.8 or less, and the recovery unit includes a first recovery flow path through which the fluid containing the non-target particles flows, and a second recovery flow path through which the fluid containing the target particles flows. The first recovery flow path is connected to the first bypass flow path side of the separation flow path, and the second recovery flow path is the second bypass flow of the separation flow path. A separate device connected to the roadside.
(A / B) ≤ (a / b) ... (1)
[8] The separation device according to [7], wherein a / b, which is the ratio of the cross-sectional area a to the cross-sectional area b, is 0.08 or more and 0.75 or less.
[9] The separation device according to [7], wherein a / b, which is the ratio of the cross-sectional area a to the cross-sectional area b, is 0.1 or more and 0.7 or less.
[10] Any one of [7] to [9], wherein the cross-section A, the cross-section B, the cross-section a, and the cross-section b satisfy (A / B) = (a / b). Separation device described in 1.
[11] The recovery unit has a second branching portion that branches the separation flow path into a first recovery flow path and a second recovery flow path, and the first recovery section in the second branching section. The cross-sectional area α on the plane perpendicular to the flow direction of the flow path and the cross-sectional area β on the plane perpendicular to the flow direction of the second recovery flow path at the second branch portion satisfy the following equation (3). The separation device according to any one of [7] to [10].
(A / b) <(α / β) ... (3)
[12] The cross-sectional area of the confluence portion on the plane perpendicular to the flow direction of the second flow path is 5 to 30% of the cross-sectional area of the plane perpendicular to the flow direction of the separation flow path [7]. The separation device according to any one of [11].
[13] The separation device according to any one of [7] to [12], wherein the separation channel separates the target particles by a deterministic transverse substitution method.
[14] A method for separating the target particles from the fluid sample containing the target particles having a size equal to or larger than the threshold value and the non-target particles having a size smaller than the threshold value, [7] to [13]. The buffer is introduced from the first introduction port of the separation device according to any one of the above, the fluid sample is introduced from the second introduction port, and the target particles are introduced from the recovery unit. A method comprising recovering a fluid comprising or a fluid comprising said non-target particles.
　次に実施例を示して本発明を更に詳細に説明するが、本発明は以下の実施例に限定されるものではない。 Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.
［実験例１］
　図１Ａ及び図１Ｂに示す構造を有し、下記表１に記載の各断面積を有する試験例１～８の分離デバイスに流体試料及びバッファーを導入した場合の、流体試料の層流の流れをシミュレーションした。 [Experimental Example 1]
The laminar flow flow of the fluid sample when the fluid sample and the buffer are introduced into the separation devices of Test Examples 1 to 8 having the structures shown in FIGS. 1A and 1B and having the respective cross-sectional areas shown in Table 1 below. I simulated it.
　表１中、Ａは、図１Ａ及び図１Ｂに示す第１の分岐部１２３における第１のバイパス流路１２２１の流れ方向に垂直な面における断面積（μｍ２）である。また、Ｂは、図１Ａ及び図１Ｂに示す第１の分岐部１２３における第２のバイパス流路１２２２の流れ方向に垂直な面における断面積（μｍ２）である。また、ａは、図１Ａ及び図１Ｂに示す合流部１２６における第１のバイパス流路１２２１の流れ方向に垂直な面における断面積（μｍ２）である。また、ｂは、図１Ａ及び図１Ｂに示す合流部１２６における第２のバイパス流路１２２２の流れ方向に垂直な面における断面積（μｍ２）である。また、Ｃは、図１Ａ及び図１Ｂに示す合流部１２６における第２の流路１２２２の流れ方向に垂直な面における断面積（μｍ２）である。また、Ｄは、図１Ａ及び図１Ｂに示す分離流路１１０の流れ方向に垂直な面における断面積（μｍ２）である。表１には、ａ／ｂ、Ａ／Ｂ、及びＣ／Ｄの値も示す。シミュレーションにおいて、Ａ及びＢの値は考慮しなかったが、Ａ／Ｂ＝ａ／ｂとした。 In Table 1, A is a cross-sectional area (μm 2 ) in a plane perpendicular to the flow direction of the first bypass flow path 1221 in the first branch portion 123 shown in FIGS. 1A and 1B. Further, B is a cross-sectional area (μm 2 ) in a plane perpendicular to the flow direction of the second bypass flow path 1222 in the first branch portion 123 shown in FIGS. 1A and 1B. Further, a is a cross-sectional area (μm 2 ) in a plane perpendicular to the flow direction of the first bypass flow path 1221 in the confluence portion 126 shown in FIGS. 1A and 1B. Further, b is a cross-sectional area (μm 2 ) in a plane perpendicular to the flow direction of the second bypass flow path 1222 at the confluence portion 126 shown in FIGS. 1A and 1B. Further, C is a cross-sectional area (μm 2 ) in a plane perpendicular to the flow direction of the second flow path 1222 in the confluence portion 126 shown in FIGS. 1A and 1B. Further, D is a cross-sectional area (μm 2 ) in a plane perpendicular to the flow direction of the separation flow path 110 shown in FIGS. 1A and 1B. Table 1 also shows the values for a / b, A / B, and C / D. In the simulation, the values of A and B were not considered, but A / B = a / b.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　シミュレーションにはＦｌｏＤＥＦ（メンター・グラフィックス・コーポレーション製）を使用した。流体試料として比重１の流体を下記表２に示す流速で送液し、バッファーとして比重１の流体を下記表２に示す流速で送液した場合をそれぞれシミュレーションした。また、分離流路の長さは、無限遠とした。シミュレーションにより、分離流路における合流部から０．１ｍｍの領域における層流の流跡線、及び、合流部から１．８～１．９ｍｍの領域における流跡線を描画した。 FloDEF (manufactured by Mentor Graphics Corporation) was used for the simulation. A simulation was performed in which a fluid having a specific gravity of 1 was sent as a fluid sample at the flow velocity shown in Table 2 below, and a fluid having a specific gravity of 1 was sent as a buffer at the flow velocity shown in Table 2 below. The length of the separation flow path was set to infinity. By simulation, the laminar flow trace line in the region 0.1 mm from the confluence in the separation flow path and the laminar flow line in the region 1.8 to 1.9 mm from the confluence were drawn.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　図３の（ａ）～（ｇ）は、それぞれ試験例１～７の分離デバイスのシミュレーション結果を示す図である。また、図４は、シミュレーションにより算出された、分離流路における合流部から１．８ｍｍの位置の流跡線のうち、最も上の位置（流れ方向に垂直な面における位置）の流跡線と、合流部における流跡線のうち、最も上の位置（流れ方向に垂直な面における位置）の流跡線の差、すなわち、流体試料の層流が、分離流路においてどれだけ移動したかを示すグラフである。 (A) to (g) of FIG. 3 are diagrams showing simulation results of the separation devices of Test Examples 1 to 7, respectively. Further, FIG. 4 shows the trace line at the highest position (position on the plane perpendicular to the flow direction) among the trace lines at the position 1.8 mm from the confluence in the separation flow path calculated by the simulation. , The difference between the trace lines at the highest position (the position on the plane perpendicular to the flow direction) among the trace lines at the confluence, that is, how much the laminar flow of the fluid sample moved in the separation flow path. It is a graph which shows.
　その結果、試験例１～７のいずれの流路においても、流体試料の層流が第１のバイパス流路側に移動することが明らかとなった。移動量（ずれ量）は最大で約３０μｍであった。流体試料の層流が第１のバイパス流路側に移動すると、流体試料は、回収部において第１の回収流路に回収されやすくなる。このため、流体試料が、標的粒子が回収される第２の回収流路に混入しにくくなり、標的粒子の回収を精度よく行うことができる。 As a result, it was clarified that the laminar flow of the fluid sample moved to the first bypass flow path side in any of the flow paths of Test Examples 1 to 7. The maximum amount of movement (displacement) was about 30 μm. When the laminar flow of the fluid sample moves to the first bypass flow path side, the fluid sample is easily collected in the first recovery flow path in the recovery unit. Therefore, the fluid sample is less likely to be mixed into the second recovery flow path in which the target particles are recovered, and the target particles can be recovered with high accuracy.
［実験例２］
　実験例１でシミュレーションした試験例１、４、５、７の分離デバイスを実際に製造した。また、比較のために試験例８の分離デバイスを製造した。製造した各試験例の分離デバイスの各断面積を下記表３に示す。表３中、Ａ、Ｂ、a、ｂ、α、β、Ｃ、Ｄは、分離デバイスにおいて、それぞれ表１におけるものと同様の位置の断面積（μｍ２）を表す。また、表３には、ａ／ｂ、Ａ／Ｂ、Ｃ／Ｄの値も示す。全ての分離デバイスにおいて、分離流路の長さは１２ｍｍであった。 [Experimental Example 2]
The separation devices of Test Examples 1, 4, 5, and 7 simulated in Experimental Example 1 were actually manufactured. In addition, the separation device of Test Example 8 was manufactured for comparison. Table 3 below shows the cross-sectional areas of the separated devices of each of the manufactured test examples. In Table 3, A, B, a, b, α, β, C, and D represent the cross-sectional areas (μm 2 ) of the positions similar to those in Table 1 in the separation device, respectively. Table 3 also shows the values of a / b, A / B, and C / D. In all separation devices, the length of the separation channel was 12 mm.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　続いて、これらの分離デバイスに流体試料及びバッファーを導入し、流体試料の層流の流れを観察した。流体試料として、ヒトから採取した血液を、１％ＢＳＡ、５ｍＭ　ＥＤＴＡ含有ＰＢＳバッファーで２倍希釈したサンプルを送液した。また、バッファーとして、１％ＢＳＡ、５ｍＭ　ＥＤＴＡ含有ＰＢＳバッファーを送液した。試験例１、４、５、７の分離デバイスにおいては、流体試料及びバッファーをそれぞれ上記表２と同じ流速で送液した。試験例８の分離デバイスにおいては、流体試料を流速１００μＬ／分で送液し、バッファーを流速１，０００μＬ／分で送液した。 Subsequently, a fluid sample and a buffer were introduced into these separation devices, and the laminar flow of the fluid sample was observed. As a fluid sample, a sample obtained by diluting blood collected from a human with a PBS buffer containing 1% BSA and 5 mM EDTA 2-fold was sent. In addition, as a buffer, a PBS buffer containing 1% BSA and 5 mM EDTA was sent. In the separation devices of Test Examples 1, 4, 5, and 7, the fluid sample and the buffer were fed at the same flow rates as in Table 2 above. In the separation device of Test Example 8, the fluid sample was fed at a flow rate of 100 μL / min, and the buffer was fed at a flow rate of 1,000 μL / min.
　図５の（ａ）～（ｅ）は、それぞれ試験例１、４、５、７、及び８の各分離デバイスの分離流路の先頭部分、中間部分、及び尾部部分を撮影した写真である。 (A) to (e) of FIG. 5 are photographs of the head portion, the middle portion, and the tail portion of the separation flow path of each of the separation devices of Test Examples 1, 4, 5, 7, and 8, respectively.
　その結果、図５の（ａ）～（ｄ）に示すように、試験例１、４、５、及び７の分離デバイスでは、いずれも流体試料の層流が流体試料の層流が第１のバイパス流路側に移動することが明らかとなった。上述したように、流体試料の層流が第１のバイパス流路側に移動すると、流体試料は、回収部において第１の回収流路に回収されやすくなる。このため、流体試料が、標的粒子が回収される第２の回収流路に混入しにくくなり、標的粒子の回収を精度よく行うことができる。 As a result, as shown in FIGS. 5A to 5D, in each of the separation devices of Test Examples 1, 4, 5, and 7, the laminar flow of the fluid sample is the first laminar flow of the fluid sample. It became clear that it moved to the bypass flow path side. As described above, when the laminar flow of the fluid sample moves to the first bypass flow path side, the fluid sample is easily collected in the first recovery flow path in the recovery unit. Therefore, the fluid sample is less likely to be mixed into the second recovery flow path in which the target particles are recovered, and the target particles can be recovered with high accuracy.
　一方、図５の（ｅ）に示すように、試験例８の分離デバイスでは、流体試料の層流が第２のバイパス流路側に移動する様子が観察された。この状態では、流体試料が第２の回収流路に混入し、標的粒子の分離能が低下してしまう場合がある。 On the other hand, as shown in FIG. 5 (e), in the separation device of Test Example 8, it was observed that the laminar flow of the fluid sample moved to the second bypass flow path side. In this state, the fluid sample may be mixed into the second recovery flow path, and the separation ability of the target particles may be reduced.
　以上の結果から、上記断面積Ａ、Ｂ、ａ、ｂが下記式（１）を満たす分離デバイスでは、流体試料中の標的粒子を精度よく分離することができることが明らかとなった。
　（Ａ／Ｂ）≦（ａ／ｂ）…（１） From the above results, it was clarified that the separation device in which the cross-sectional areas A, B, a, and b satisfy the following formula (1) can accurately separate the target particles in the fluid sample.
(A / B) ≤ (a / b) ... (1)
　本発明によれば、流体試料中の標的粒子を精度よく分離する技術を提供することができる。 According to the present invention, it is possible to provide a technique for accurately separating target particles in a fluid sample.
　２０…ＤＬＤマイクロ流路の基本構造（分離エリア）、２１…障害物構造（ピラー）、２２…閾値以上の大きさを有する粒子、２３…閾値未満の大きさを有する粒子、１００…分離デバイス、１１０…分離流路、１１１…ピラー、１２０…流体導入部、１２１…第１の導入口、１２２…第１の流路、１２２１…第１のバイパス流路、１２２２…第２のバイパス流路、１２２３…隔壁、１２３…第１の分岐部、１２４…第２の導入口、１２５…第２の流路、１２６…合流部、１３０…回収部、１３１…第１の回収流路、１３２…第２の回収流路、１３４，１３５…排出口、Ａ，Ｂ，ａ，ｂ，α，β，Ｃ，Ｄ…断面積。 20 ... Basic structure (separation area) of DLD microchannel, 21 ... Obstacle structure (pillar), 22 ... Particles having a size equal to or more than a threshold value, 23 ... Particles having a size less than a threshold value, 100 ... Separation device, 110 ... Separation flow path, 111 ... Pillar, 120 ... Fluid introduction part, 121 ... First introduction port, 122 ... First flow path, 1221 ... First bypass flow path, 1222 ... Second bypass flow path, 1223 ... partition, 123 ... first branch, 124 ... second inlet, 125 ... second flow path, 126 ... confluence, 130 ... recovery section, 131 ... first recovery flow path, 132 ... first 2 recovery channels, 134, 135 ... Discharge ports, A, B, a, b, α, β, C, D ... Cross-sectional area.

Claims (6)

  1. 　閾値以上の大きさを有する標的粒子及び閾値未満の大きさを有する非標的粒子を含む流体試料から、前記標的粒子を分離する分離デバイスであって、
    　流体中の粒子を大きさにより分離する分離流路と、
    　前記分離流路の上流側に接続され、前記分離流路に前記流体試料及びバッファーを導入する流体導入部と、
    　前記分離流路の下流側に接続され、前記標的粒子を含む流体及び前記非標的粒子を含む流体をそれぞれ回収する回収部と、を有し、
    　前記流体導入部は、
    　前記バッファーを導入する第１の導入口と、前記第１の導入口に接続され、前記バッファーが流れる第１の流路と、
    　前記第１の流路を第１のバイパス流路及び第２のバイパス流路に分岐する第１の分岐部と、
    　前記第１の分岐部の下流側に位置し、前記流体試料を導入する第２の導入口と、前記第２の導入口に接続され、前記流体試料が流れる第２の流路と、
    　前記第１のバイパス流路、前記第２の流路及び前記第２のバイパス流路が合流する合流部と、を有し、
    　前記第１の分岐部における前記第１のバイパス流路の流れ方向に垂直な面における断面積Ａと、
    　前記第１の分岐部における前記第２のバイパス流路の流れ方向に垂直な面における断面積Ｂと、
    　前記合流部における前記第１のバイパス流路の流れ方向に垂直な面における断面積ａと、
    　前記合流部における前記第２のバイパス流路の流れ方向に垂直な面における断面積ｂとが、下記式（１）を満たし、
    　前記回収部は、
    　前記非標的粒子を含む流体が流れる第１の回収流路と、
    　前記標的粒子を含む流体が流れる第２の回収流路と、を有し、
    　前記第１の回収流路は前記分離流路の前記第１のバイパス流路側に接続しており、前記第２の回収流路は、前記分離流路の前記第２のバイパス流路側に接続している、
    　分離デバイス。
    　（Ａ／Ｂ）≦（ａ／ｂ）…（１）     A separation device that separates the target particles from a fluid sample containing target particles having a size equal to or larger than a threshold value and non-target particles having a size smaller than the threshold value.
    A separation flow path that separates particles in a fluid according to size,
    A fluid introduction section connected to the upstream side of the separation flow path and introducing the fluid sample and buffer into the separation flow path,
    It is connected to the downstream side of the separation flow path and has a recovery unit for collecting the fluid containing the target particles and the fluid containing the non-target particles, respectively.
    The fluid introduction section
    A first inlet for introducing the buffer, a first flow path connected to the first inlet, and a flow path through which the buffer flows.
    A first branch portion that branches the first flow path into a first bypass flow path and a second bypass flow path, and
    A second introduction port for introducing the fluid sample, a second flow path connected to the second introduction port, and a second flow path through which the fluid sample flows, located on the downstream side of the first branch portion.
    It has a first bypass flow path, a second flow path, and a confluence portion where the second bypass flow path merges.
    The cross-sectional area A on the plane perpendicular to the flow direction of the first bypass flow path in the first branch portion,
    The cross-sectional area B on the plane perpendicular to the flow direction of the second bypass flow path in the first branch portion,
    The cross-sectional area a on the plane perpendicular to the flow direction of the first bypass flow path at the confluence,
    The cross-sectional area b on the plane perpendicular to the flow direction of the second bypass flow path at the confluence satisfies the following equation (1).
    The collection unit
    The first recovery flow path through which the fluid containing the non-target particles flows, and
    It has a second recovery channel through which the fluid containing the target particles flows, and has.
    The first recovery flow path is connected to the first bypass flow path side of the separation flow path, and the second recovery flow path is connected to the second bypass flow path side of the separation flow path. ing,
    Separation device.
    (A / B) ≤ (a / b) ... (1)
  2. 　前記断面積ａ及び前記断面積ｂが、下記式（２）を満たす、請求項１に記載の分離デバイス。
    　ａ≦ｂ…（２）     The separation device according to claim 1, wherein the cross-sectional area a and the cross-sectional area b satisfy the following formula (2).
    a ≦ b ... (2)
  3. 　前記回収部は、前記分離流路を第１の回収流路及び第２の回収流路に分岐する第２の分岐部を有し、
    　前記第２の分岐部における前記第１の回収流路の流れ方向に垂直な面における断面積αと、
    　前記第２の分岐部における前記第２の回収流路の流れ方向に垂直な面における断面積βが、下記式（３）を満たす、請求項１又は２に記載の分離デバイス。
    　（ａ／ｂ）＜（α／β）…（３）     The recovery section has a second branch section that branches the separation flow path into a first recovery flow path and a second recovery flow path.
    The cross-sectional area α in the plane perpendicular to the flow direction of the first recovery flow path in the second branch portion,
    The separation device according to claim 1 or 2, wherein the cross-sectional area β in the plane perpendicular to the flow direction of the second recovery flow path in the second branch portion satisfies the following formula (3).
    (A / b) <(α / β) ... (3)
  4. 　前記合流部における前記第２の流路の流れ方向に垂直な面における断面積が、前記分離流路の流れ方向に垂直な面における断面積の５～３０％である、請求項１～３のいずれか一項に記載の分離デバイス。 Claims 1 to 3, wherein the cross-sectional area of the merging portion on the plane perpendicular to the flow direction of the second flow path is 5 to 30% of the cross-sectional area of the plane perpendicular to the flow direction of the separation flow path. The separation device according to any one item.
  5. 　前記分離流路は、決定論的横置換法により前記標的粒子を分離する、請求項１～４のいずれか一項に記載の分離デバイス。 The separation device according to any one of claims 1 to 4, wherein the separation flow path separates the target particles by a deterministic transverse substitution method.
  6. 　前記閾値以上の大きさを有する標的粒子及び前記閾値未満の大きさを有する非標的粒子を含む前記流体試料から、前記標的粒子を分離する方法であって、
    　請求項１～５のいずれか一項に記載の分離デバイスの前記第１の導入口から前記バッファーを導入し、前記第２の導入口から前記流体試料を導入することと、
    　前記回収部から前記標的粒子を含む流体又は前記非標的粒子を含む流体を回収することと、を含む、方法。     A method for separating the target particles from the fluid sample containing the target particles having a size equal to or larger than the threshold value and the non-target particles having a size smaller than the threshold value.
    The buffer is introduced from the first inlet of the separation device according to any one of claims 1 to 5, and the fluid sample is introduced from the second inlet.
    A method comprising recovering a fluid containing the target particles or a fluid containing the non-target particles from the recovery unit.
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* Cited by examiner, † Cited by third party
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