Separation Device

Abstract

To provide a technique for suppressing accumulation of target particles in a sample supply part of a separation device.SOLUTION: A separation device for separating target particles contained in a fluid sample containing the target particles and non-target particles from the fluid sample includes a sample supply hole 11 for supplying the fluid sample to the separation device, a port 12 connected to the sample supply hole 11, and a separation region 30 to which the fluid sample is supplied from a sample outlet 13 of the port 12 and that separates the target particles from the fluid sample, in which the longest distance L between an upstream side inner wall surface 15 on the upstream side of the sample supply hole 11 of the port 12 and the center of gravity of the sample supply hole 11 is 1.5 mm or less.SELECTED DRAWING: Figure 1

Description

[Technical field]

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

[0002] A method using a microfluidic device (separation device) that applies DLD (Deterministic Lateral Displacement) is known as a method for separating target substances and cells from a liquid sample containing organic matter, biopolymers, cells, etc. (See, for example, Non-Patent Document 1.) This method makes it possible to separate target particles according to their size from multiple types of particles contained in a liquid sample.

[0003] The method using the separation device described above uses capture particles with capture molecules fixed to their surface that can bind to target substances or cells in a liquid sample. The capture particles are mixed with the liquid sample to form target particles, and then the target particles can be separated and collected using a separation device (see, for example, Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Inglis DW et al., Critical particle size for fractionation by deterministic lateral displacement, Lab Chip, 2006, vol. 6, 655-658. [Patent documents]

[0005] [Patent Document 1] International Publication No. 2016 / 136273 Summary of the Invention [Problem to be solved by the invention]

[0006] However, when a fluid sample containing target particles is supplied to a sample supply portion of a separation device, the target particles tend to accumulate in the sample supply portion, resulting in a decrease in the recovery rate of the target particles.

[0007] Therefore, an object of the present invention is to provide a technique for suppressing target particles from accumulating in the sample supply portion of a separation device. [Means for solving the problem]

[0008] The present invention includes the following aspects. [1] A separation device for separating target particles contained in a fluid sample containing target particles and non-target particles from the fluid sample, the separation device having a sample supply hole for supplying the fluid sample to the separation device, a port connected to the sample supply hole, and a separation area into which the fluid sample is supplied from the sample outlet of the port and which separates the target particles from the fluid sample, wherein the longest distance L between the upstream inner wall surface of the port upstream of the sample supply hole and the center of gravity of the sample supply hole is 1.5 mm or less. [2] The separation device according to [1], further comprising a liquid delivery path for supplying the fluid sample to the separation device, the liquid delivery path being connected to the sample supply hole. [3] The separation device according to [2], wherein the liquid transfer path is connected approximately perpendicular to the separation device. [4] The separation device according to any one of [1] to [3], wherein the specific gravity of the target particles is 0.85 to 1.15 times the specific gravity of the fluid component of the fluid sample. [5] A separation device according to any one of [1] to [4], wherein the target particle is a cell or a complex of a cell and a carrier on which a capture molecule is immobilized, and the carrier is made of one or more materials selected from the group consisting of resin, metal, glass, and silica. Effect of the Invention

[0009] According to the present invention, it is possible to provide a technique for suppressing target particles from accumulating in a sample supply portion of a separation device. [Brief description of the drawings]

[0010] [Figure 1] FIG. 2 is a schematic diagram showing a separation channel section 6 formed inside a separation device 1A. [Figure 2] FIG. 1 is a partial cross-sectional front view of a separation device 1A. [Figure 3] FIG. 2 is a partially cutaway perspective view of the vicinity of a sample supply section 10 of a separation device 1A. [Figure 4] FIG. 2 is a partial cross-sectional front view of the vicinity of a sample supply section 10 of a separation device 1A. [Figure 5] FIG. 2 is a partial cross-sectional plan view of the vicinity of a sample outlet 13 of a separation device 1A. [Figure 6] FIG. 2 is a plan view of a separation region 30 of the separation device 1A. [Figure 7] FIG. 1 is a schematic diagram illustrating a DLD. [Figure 8] FIG. 10 is a schematic diagram illustrating a captured particle 73. [Figure 9] FIG. 2 is a schematic diagram illustrating a target particle 70. [Figure 10] FIG. 2 is a plan view of a separation device 1B. [Figure 11] This is the result of a simulation analysis of the trajectory of a fluid sample when the port diameter of the sample supply section is 6 mm. [Figure 12] This is the result of a simulation analysis of the trajectory of a fluid sample when the port diameter of the sample supply section is 1.5 mm. [Figure 13] This is the result of a simulation analysis of the flow rate of the fluid sample when the port diameter of the sample supply section is 6 mm. [Figure 14] This is the result of a simulation analysis of the flow rate of the fluid sample when the port diameter of the sample supply section is 1.5 mm. [Figure 15]This is the result of analyzing the position of the fluid sample at each time by simulation when the port diameter of the sample supply unit is 6 mm. [Figure 16] This is the result of analyzing the position of the fluid sample at each time by simulation when the port diameter of the sample supply unit is 1.5 mm. [Figure 17] This is a photograph of particles in the port immediately after the start of liquid feeding of the fluid sample when the port diameter of the sample supply unit is 6 mm. [Figure 18] This is a photograph of particles in the port 15 seconds after the start of liquid feeding of the fluid sample when the port diameter of the sample supply unit is 6 mm. [Figure 19] This is a photograph of particles in the port immediately after the start of liquid feeding of the fluid sample when the port diameter of the sample supply unit is 1.5 mm. [Figure 20] This is a photograph of particles in the port 15 seconds after the start of liquid feeding of the fluid sample when the port diameter of the sample supply unit is 1.5 mm.

Embodiments for Carrying Out the Invention

[0011] [Separation Device] First, a separation device according to one embodiment of the present invention will be described. FIG. 1 is a schematic diagram showing a separation channel portion 6 formed inside the separation device 1A. FIG. 2 is a front cross-sectional view of the separation device 1A. The device of this embodiment is for separating the target particles contained in a fluid sample containing target particles and non-target particles from the fluid sample.

[0012] The separation device 1A is formed by joining a plate-shaped lid member 5 and a base plate 4. The separation channel portion 6 is a space secured between the plate-shaped lid member 5 and the base plate 4 by a recess formed in a groove shape in the plate-shaped lid member 5.

[0013] 1, the separation channel section 6 of the separation device 1A includes a buffer supply section 20 that supplies a buffer, a sample supply section 10 that supplies a fluid sample, a channel 22 extending from the buffer supply section 20, a first collection section 40 that collects target particles, and a second collection section 50 that collects non-target particles. The channel 22 is formed in the shape of a groove in the plate-shaped cover member 5, and is a space secured between the plate-shaped cover member 5 and the base plate 4.

[0014] 1, the flow channel 22 of the separation flow channel section 6 of the separation device 1A has an expansion section 24 downstream of the buffer supply section 20. The expansion section 24 is formed by expanding the flow channel 22 in the direction of the width of the flow channel, which is perpendicular to the extension direction of the flow channel 22 in the surface direction of the plate-shaped cover member 5. The flow channel 22 also has a separation region 30 downstream of the expansion section 24, which separates target particles and non-target particles contained in the sample. The first collection section 40 and the second collection section 50 are connected to the downstream end of the separation region 30. The width of the expansion section 24 is designed to be wider than the width of the portion of the flow channel 22 between the buffer supply section 20 and the sample supply section 10 and the width of the separation region 30 .

[0015] 1, the extension section 24 is provided with a partition wall 25 that protrudes from the plate-shaped cover member 5 to secure the sample supply section 10. The partition wall 25 is formed so as to surround a partial area in the planar direction of the plate-shaped cover member 5 inside the extension section 24. The sample supply section 10 refers to the entire inner space surrounded by the partition wall 25. However, the downstream end of the partition wall 25 has a discontinuous portion, which is open as the sample outlet 13. The partition wall 25 is formed at a position spaced apart from the inner walls on both sides of the channel 22 in the channel width direction. The space between the partition wall 25 and the inner wall of the channel 22 forms part of the channel 22.

[0016] 1 and 2, a buffer supply hole 21 that communicates with the buffer supply unit 20 is formed through the plate-shaped lid member 5. The buffer supply hole 21 is provided in the center of the buffer supply unit 20 in the surface direction of the plate-shaped lid member 5.

[0017] Fig. 3 is a partially cutaway perspective view of the separation device 1A in the vicinity of the sample supply section 10. Fig. 4 is a partial front cross-sectional view of the separation device 1A in the vicinity of the sample supply section 10. 3 and 4, a sample supply hole 11 that communicates with the sample supply part 10 is formed through the plate-shaped cover member 5. The sample supply hole 11 is provided in the center of the sample supply part 10 in the surface direction of the plate-shaped cover member 5. In this specification, the sample supply portion 10 may be referred to as a port.

[0018] 4, the sample supply hole 11 may be connected to a liquid supply path 14 that supplies a fluid sample to the sample supply unit 10. The liquid supply path 14 of the sample supply unit 10 may be connected approximately perpendicular to the separation device 1A.

[0019] 1 and 4, the sample outlet side of the sample supply part 10 is hereinafter referred to as the downstream side, and the side opposite the sample outlet side is referred to as the upstream side (rear side). The inner wall surface of the partition wall 25 opposite the sample outlet side is the upstream inner wall surface 15.

[0020] The longest distance L between the center of gravity of the sample supply hole 11 in the planar direction of the plate-shaped cover member 5 and the upstream inner wall surface 15 is 1.5 mm or less.

[0021] As illustrated in Figure 1, the portion of the flow path 22 between the buffer supply section 20 and the sample supply section 10 is connected to the portion outside the partition 25 in the expansion section 24 of the flow path 22, and the portion of the flow path 22 near the downstream end of the partition 25 forms a buffer outlet 23.

[0022] The fluid sample supplied from the sample supply hole 11 by the liquid supply path 14 flows inside the sample supply unit 10 , flows out from the sample outlet 13 , and enters the separation region 30 .

[0023] The buffer supplied from the buffer supply hole 21 flows inside the buffer supply unit 20, passes through the portion between the buffer supply unit 20 and the sample supply unit 10 in the flow path 22, flows out from the buffer outlet 23, and flows into the separation region 30.

[0024] FIG. 5 is a partial cross-sectional plan view near the sample outlet 13. As illustrated in FIGS. 1 and 5, the sample outlet 13 is disposed close to the buffer outlet 23. The fluid sample 2 discharged from the sample outlet 13 and the buffer 3 discharged from the buffer outlet 23 smoothly merge at the confluence portion 31 on the downstream side of the sample outlet 13 and the buffer outlets 23 on both sides thereof, and flow into the separation region 30.

[0025] As illustrated in FIG. 5, in the separation region 30, the fluid sample 2 discharged from the sample outlet 13 and the buffer 3 discharged from the buffer outlet 23 form a laminar flow.

[0026] FIG. 6 is a plan view of the separation region 30 of the separation device 1A. As illustrated in FIGS. 5 and 6, the separation region 30 continuously has pillars 32 that separate particles by size. Among the particles that flow into the separation region 30, the target particles 70 having a certain size or more move obliquely instead of straight ahead with respect to the flow direction, and the non-target particles 71 smaller than a certain size move straight along the flow direction.

[0027] FIG. 7 is a schematic diagram for explaining DLD. The method of separating particles by the size of the particles in the separation region 30 described above may be the DLD method (Deterministic Lateral Displacement) (for example, see Non-Patent Document 1). As shown in FIG. 7, when the critical diameter (the above-mentioned certain size) of the particles moving obliquely with respect to the flow direction is defined as Dc, the pillar interval in the direction perpendicular to the fluid flow direction is defined as g, the deviation angle of the pillar 32 is defined as θ, and ε = tanθ, Dc = 1.4gε 0.48 It is known that it becomes like this. Here, the above-mentioned certain size corresponds to the critical diameter Dc. However, it is preferably 0.06 < ε < 0.1.

[0028] By using the separation region 30 in which the pillars 32 are arranged with the pillar interval g and the deviation angle θ of the pillars 32 so that the critical diameter Dc becomes a desired size, it is possible to separate the target particles 70 having a diameter larger than the critical diameter and the non-target particles 71 having a diameter smaller than the critical diameter.

[0029] As illustrated in FIG. 1, the first recovery unit 40 has a first recovery flow path 41 into which a fluid containing particles larger than the critical diameter Dc flows, and a first recovery port 42 for recovering the fluid containing particles larger than the critical diameter Dc. The second recovery unit 50 has a second recovery flow path 51 into which a fluid containing particles smaller than the critical diameter Dc flows, and a second recovery port 52 for recovering the fluid containing particles smaller than the critical diameter Dc.

[0030] The fluid sample 2 and the buffer 3 that have merged in the confluence part 31 flow into the separation region 30. Particles larger than the critical diameter Dc move in a direction oblique to the flow direction, flow into the first recovery flow path 41, and are recovered from the first recovery port 42. Particles smaller than the critical diameter Dc travel straight along the flow direction, flow into the second recovery flow path 51, and are recovered from the second recovery port 52.

[0031] FIG. 8 is a schematic diagram for explaining the captured particles 73. As illustrated in FIG. 8, the fluid sample 2 contains the target substance 72 and the non-target substance 76. As illustrated in FIG. 8, the captured particles 73 that bind to the target substance 72 are carriers 75 to which the capture molecules 74 that bind to the target substance 72 are fixed.

[0032] FIG. 9 is a schematic diagram for explaining the target particles 70. As illustrated in FIG. 9, the captured particles 73 bind to the target substance 72 to form the target particles 70 that are a complex, but do not bind to the non-target substance 76.

[0033] By using the separation device 1A according to the embodiment, the target particles 70 can be separated from the fluid sample 2. The target particles 70 are larger than the target substance 72 and also larger than the capture particles 73. Here, the non-target particles 71 (not shown) refer to particles smaller than the target particles 70 contained in the fluid sample 2.

[0034] By using the separation device 1A according to the embodiment, the target particles 70 can be separated from the fluid sample 2 containing the target substance 72 and the capture particles 73 in the fluid sample 2.

[0035] The separation device 1A according to the present embodiment has two recovery channels, i.e., the first recovery channel 41 and the second recovery channel 51. However, the number of recovery channels is not limited to two, and the number of recovery channels of the separation device 1A may be three or more.

[0036] The shapes of the sample supply hole 11 and the buffer supply hole 21 are not particularly limited, and may be circular, or may be polygonal such as triangular, square, rectangular, etc.

[0037] Tubes may be joined to the sample supply hole 11, the buffer supply hole 21, the first recovery port 42, and the second recovery port 52, or joints such as syringes may be provided.

[0038] The fluid sample 2 and the buffer 3 may be supplied at a constant rate to the sample supply hole 11 and the buffer supply hole 21, respectively. For example, the fluid sample 2 and the buffer 3 can be supplied using a syringe pump or the like.

[0039] The fluid sample 2 may include body fluids such as blood, saliva, urine, tears, and lymph; lysates of tissues, cells, etc.; culture solutions of tissues, cells, etc.; excreted suspensions; liquids in contact with cells, tissues, organisms, etc. The fluid sample 2 may have an arbitrary diluent added to the above-mentioned liquids. Examples of the target substance 72 include substances such as cells, proteins, nucleic acids, lipids, and sugar chains.

[0040] The target particle 70 may be a complex of the target substance 72 and the capture particle 73, or may be the target substance 72 itself. The target particle 70 may be, for example, a cell itself.

[0041] Examples of the capture molecule 74 include, but are not limited to, antibodies, lectins, aptamers, cell adhesion molecules, sugar chains, etc., and any molecule may be used as long as it recognizes the target substance 72.

[0042] Examples of materials for the carrier 75 include resin, metal, glass, and silica. The carrier 75 may be made of one type of material or a combination of these materials. Preferred materials for the carrier 75 include polystyrene and latex.

[0043] The specific gravity of the carrier 75 is preferably 0.85 to 1.15 times the specific gravity of the fluid sample 2. The specific gravity of the target particles formed by binding the carrier 75 and the objective substance 72 is preferably 0.85 to 1.15 times the specific gravity of the fluid sample 2.

[0044] In order to separate the non-target particles 71 and the target particles 70 in the separation region 30, it is preferable to set the critical diameter Dc to be equal to or larger than the size of the non-target particles 71 and equal to or smaller than the size of the target particles 70.

[0045] For example, if target substance 72 is a cell having a size of 9 to 15 μm and non-target particle 71 is a cell having a size of 7 to 13 μm, the size of captured particle 73 is preferably 5 μm or more. More specifically, the size of captured particle 73 is preferably 15 μm to 50 μm, and more preferably 20 μm to 40 μm.

[0046] For example, when separating circulating tumor cells (CTCs) from blood as target particles 70, the size of red blood cells is 6 to 8 μm, the size of white blood cells is 9 to 15 μm, and the size of CTCs is 10 to 20 μm, so the critical diameter Dc is preferably 20 μm or more, and more preferably 30 μm or more. The size of capture particles 73 is preferably 7 μm to 60 μm, more preferably 10 μm to 55 μm, even more preferably 15 μm to 50 μm, and even more preferably 20 μm to 40 μm.

[0047] The structure of the pillars 32 is not particularly limited, and examples thereof include polygonal pillar structures such as triangular pillars, quadrangular pillars, hexagonal pillars, and octagonal pillars, and cylindrical structures.

[0048] Materials that can be used for the members that come into contact with the fluid sample 2 and the buffer 3 include glass, silicone, PDMS (polydimethylsiloxane), plastic, etc. The above-mentioned materials are preferably materials that do not adsorb the target substance 72. Materials whose surfaces that come into contact with the fluid sample 2 and the buffer 3 are coated with MPC (2-methacryloyloxyethyl phosphorylcholine), etc., which prevents adhesion of biological materials, may also be used.

[0049] As the buffer flowing in through the buffer supply hole 21, physiological saline, PBS, etc. can be used to prevent adverse effects on cells and denaturation of proteins.

[0050] When a fluid sample 2 containing target particles 70 is introduced through the sample supply hole 11, the fluid sample 2 flows through the sample supply section (port) 10. As will be described later in the examples, the target particles 70 in the fluid sample 2 may remain near the upstream inner wall surface 15 upstream of the sample supply hole 11.

[0051] As described later in the examples, the longer the longest distance L between the center of gravity of the sample supply hole 11 and the upstream inner wall surface 15 upstream of the sample supply hole 11, the smaller the flow rate of the fluid sample 2 flowing near the upstream inner wall surface 15 of the sample supply section 10, and the more likely it is to stagnate near the upstream inner wall surface 15 of the port 12.

[0052] When the target particles 70 accumulate near the upstream inner wall surface 15 of the sample supply section 10, the target particles 70 are less likely to flow into the separation region 30, and as a result, the recovery rate of the target particles 70 by the separation device 1A decreases.

[0053] In order to prevent target particles 70 from accumulating near the upstream inner wall surface 15 of the sample supply section 10, it is preferable that the longest distance L between the center of gravity of the sample supply hole 11 and the upstream inner wall surface 15 of the port 12 be 1.5 mm or less.

[0054] The shape of the upstream inner wall surface 15 of the sample supply section 10 is not particularly limited, but it is preferably arc-shaped to prevent target particles 70 from stagnation near the upstream inner wall surface 15 of the sample supply section 10.

[0055] Fig. 10 is a plan view of a modified separation device 1B. As illustrated in Fig. 10, in the separation device 1B, the sample supply section 10 may have an aggregate removal region 60 upstream of the sample outlet 13. The aggregate removal region 60 may have a plurality of removal sections. The removal sections preferably have a pillar structure.

[0056] As illustrated in FIG. 10, the aggregate removal region 60 includes removal portions 61 and 62, the removal portion 61 having a pillar 61a (not shown), and the removal portion 62 having a pillar 62a (not shown).

[0057] The spacing between the pillars of the removal sections 61, 62 is preferably wider than the spacing between the target particles 70 formed by binding the target substance 72 and the capture particles 73. The spacing between the pillars 61a and 62a is preferably 80 μm to 250 μm, more preferably 100 μm to 230 μm, and even more preferably 120 to 220 μm.

[0058] The structures of the pillars 61a and 62a are not particularly limited, and examples thereof include polygonal pillar structures such as triangular prisms, quadrangular prisms, hexagonal prisms, octagonal prisms, and cylindrical structures.

[0059] The pillar interval of the pillar 61a is preferably equal to or greater than the pillar interval of the pillar 62a. By setting the pillar interval as described above, the minute aggregates contained in the fluid sample 2 are removed in order from the larger ones, thereby increasing the removal efficiency of the minute aggregates, preventing the minute aggregates from clogging in the separation region 30, and enabling more of the fluid sample 2 to be processed.

[0060] By setting each pillar interval in this way, the minute aggregates contained in the fluid sample 2 can be removed more efficiently. By removing the minute aggregates upstream of the separation region 30, it is possible to prevent the minute aggregates from clogging the pillars 32 in the separation region 30 and improve the separation efficiency in the separation region 30.

Example

[0061] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0062] [Experimental Example 1] For the case where the port diameter of the sample supply section (port) is 6 mm and the case where it is 1.5 mm, the trajectory of the fluid supplied to the sample supply section was analyzed by simulation. As the simulation software, FloDEF (manufactured by Mentor Graphics Corporation) was used.

[0063] Twice the longest distance L between the centroid of the sample supply hole and the inner wall surface on the upstream side of the port corresponds to the port diameter. Also, the flow rate was set to (50 μL / min).

[0064] First, for the case where the port diameter is 6 mm, the trajectory of the fluid was analyzed by simulation. The results are shown in Fig. 11. In Fig. 11, the plurality of curves extending from the sample supply holes represent the trajectories of the inflowing fluid.

[0065] As a result, it was revealed that the fluid flowing in from the sample supply hole may flow out directly from the outlet of the sample supply section or may flow from the sample supply hole to the inner wall surface on the upstream side of the port.

[0066] Next, for the case where the port diameter is 1.5 mm, the trajectory of the fluid was also analyzed by simulation. The results are shown in Fig. 12.

[0067] As a result, similar to the case where the port diameter is 6 mm, it was revealed that the fluid flowing in from the sample supply hole may flow out directly from the outlet of the sample supply section or may flow from the sample supply hole to the inner wall surface on the upstream side of the port.

[0068] [Experimental Example 2] Under the same conditions as in Experimental Example 1, the flow velocity and the flow direction of the fluid at each position of the sample supply section were visualized based on the values calculated by simulation.

[0069] First, for the case where the port diameter is 6 mm, the flow velocity and the flow direction of the fluid were analyzed by simulation. The results are shown in Fig. 13.

[0070] As a result, it was revealed that the velocity of the fluid near the inner wall surface on the upstream side of the port is slower compared to the velocity of the fluid near the outlet of the sample supply section.

[0071] Next, for the case where the port diameter is 1.5 mm, the flow velocity and the flow direction of the fluid were analyzed by simulation. The results are shown in Fig. 14.

[0072] As a result, similar to the case where the port diameter is 6 mm, it was revealed that the velocity of the fluid near the inner wall surface on the upstream side of the port is slower compared to the velocity of the fluid near the outlet of the sample supply section.

[0073] It was also found that when the port diameter was 6 mm, the fluid velocity near the upstream inner wall surface of the port was slower than when the port diameter was 1.5 mm.

[0074] [Experimental Example 3] The time (residence time) from when the fluid sample is supplied to the sample supply section until it reaches the sample outlet was analyzed by simulation.

[0075] First, we analyzed the residence time by simulation for a port diameter of 6 mm. The results are shown in Figure 15.

[0076] In Fig. 15, the horizontal axis indicates the elapsed time after the fluid sample flowed in, and the vertical axis indicates the displacement from the center (center of gravity) of the sample supply hole toward the sample outlet. Each curve in Fig. 15 indicates that the fluid sample passes through each trajectory similar to that in Example 2. However, the displacement toward the outlet is considered positive.

[0077] As a result, it was found that when the port diameter was 6 mm, the time from when the fluid sample flowed in until it reached the sample outlet was 2 to 8 seconds. It was also found that of the fluid that flowed in from the sample supply hole, the fluid that flowed near the upstream inner wall surface of the port took a long time to reach the outlet.

[0078] Next, we analyzed the residence time by simulation when the port diameter was 1.5 mm. The results are shown in Figure 16.

[0079] As a result, it was revealed that when the port diameter was 1.5 mm, the time from when the fluid sample flowed in until it reached the sample outlet was 0.2 to 0.7 seconds.

[0080] These results reveal that when the port diameter is 6 mm, the time it takes to reach the sample outlet is significantly longer than when the port diameter is 1.5 mm.

[0081] The fact that it takes a long time to reach the sample outlet means that the velocity of the fluid sample passing through that trajectory is slow. That is, a target particle attempting to pass through a trajectory where the time to reach the sample outlet is significantly long suggests that the force received by the target particle from the flow of the fluid sample is weak, and thus it is more likely to stay in the sample supply section.

[0082] [Experimental Example 4] After a fluid sample containing particles was supplied to the sample supply section, the state of the particles moving within the port was observed.

[0083] First, a fluid containing particles was supplied to a sample supply section with a port diameter of 6 mm, and the state of the particles moving within the port was observed. FIG. 17 is a photograph showing the state within the port immediately after supplying the fluid containing particles, and FIG. 18 is a photograph showing the state within the port 15 seconds after supplying the fluid containing particles.

[0084] As a result, when the port diameter was 6 mm, it was revealed that when comparing immediately after supplying the fluid to 15 seconds later, the particles in the fluid near the inner wall surface on the upstream side of the port had hardly moved.

[0085] Next, a fluid containing particles was supplied to a sample supply section with a port diameter of 3 mm, and the state of the particles moving within the port was observed. FIG. 19 is a photograph showing the state within the port immediately after supplying the fluid containing particles, and FIG. 20 is a photograph showing the state within the port 15 seconds after supplying the fluid containing particles.

[0086] As a result, when the port diameter was 3 mm, it was revealed that when comparing immediately after supplying the fluid to 15 seconds later, the particles in the fluid near the inner wall surface on the upstream side of the port had moved within 15 seconds.

[0087] From these results, it became clear that when the port diameter is large, the particles in the fluid near the inner wall surface on the upstream side of the port stay, and the time to reach the outlet becomes significantly long.

[0088] From the above results, it became clear that particles remain when the longest distance L between the center of the sample supply hole (center of gravity) and the upstream inner wall surface of the port is 3 mm, and that particles do not remain when it is 1.5 mm. [Industrial Applicability]

[0089] According to the present invention, it is possible to provide a technique for suppressing target particles from accumulating in a sample supply portion of a separation device. [Explanation of symbols]

[0090] 1A...Separation device, 1B...Separation device, 2...Fluid sample, 3...Buffer, 4...Base plate, 5...Plate-shaped cover member, 6...Separation flow path portion, 10...Sample supply portion (port), 11...Sample supply hole, 13...Sample outlet, 14...Liquid transfer path, 15...Upstream inner wall surface, 20...Buffer supply portion, 21...Buffer supply hole, 22...Flow path, 23...Buffer outlet, 24...Expansion portion, 25...Bulkhead, 30...Separation Region, 31...confluence portion, 32...pillar, 40...first recovery portion, 41...first recovery channel, 42...first recovery port, 50...second recovery portion, 51...second recovery channel, 52...second recovery port, 60...aggregate removal region, 61...removal portion, 61a...pillar, 62...removal portion, 62a...pillar, 70...target particle, 71...non-target particle, 72...target substance, 73...capture particle, 74...capture molecule, 75...carrier, 76...non-target substance

Claims

1. A separation device for separating target particles contained in a fluid sample including target particles and non-target particles from the fluid sample, a sample supply hole for supplying the fluid sample to the separation device, a port connected to the sample supply hole and being a space into which the fluid sample is supplied, a partition wall surrounding the port, a separation region where the fluid sample is supplied from a sample outlet of the port which is a discontinuous portion of the partition wall, and the target particles are separated from the fluid sample, a liquid feed path for supplying the fluid sample to the separation device, and having, the liquid feed path is connected to the sample supply hole and is connected to the separation device substantially perpendicularly, the target particles are cells or a complex of a cell and a carrier to which a capture molecule is fixed, the carrier being made of one or more materials selected from the group consisting of resin, metal, glass, and silica, the separation region has a plurality of pillars, and the target particles are separated by the deterministic lateral displacement (DLD) method, the longest distance L between an upstream inner wall surface which is an inner wall surface on the opposite side of the sample outlet in the partition wall and located upstream of the sample supply hole of the port and the center of gravity of the sample supply hole is 1.5 mm or less, the upstream inner wall surface has a shape curved in an arc shape toward the upstream side, a separation device.

2. The separation device according to claim 1, wherein the specific gravity of the target particles is 0.85 times or more and 1.15 times or less the specific gravity of the fluid component of the fluid sample.

3. There is a buffer supply unit for supplying a buffer upstream of the partition wall, a flow path extending from the buffer supply unit to the separation region downstream thereof, is configured to be narrower in width than the buffer supply unit at a position upstream of the partition wall, and also, at the position where the partition wall is provided, the width expands along the shape of the partition wall. The separation device according to claim 1 or 2.

Images