WO2020255753A1

WO2020255753A1 - Separation device and separation method

Info

Publication number: WO2020255753A1
Application number: PCT/JP2020/022295
Authority: WO
Inventors: 憲彰新井
Original assignee: 凸版印刷株式会社
Priority date: 2019-06-21
Filing date: 2020-06-05
Publication date: 2020-12-24

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

Separation device and separation method

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 (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.

International Publication No. 2016/136273

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.

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.

According to the present invention, it is possible to provide a technique for accurately separating target particles in a fluid sample.

It is a top view explaining the structure of the separation device in one aspect of this invention. 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.

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.

[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)

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.

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.

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)

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.

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.

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)

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.

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.

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)

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.

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%.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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). ]

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)

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). 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.

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.

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.

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. Further, as the material of the separation device, for example, glass, silicone, dimethylpolysiloxane, plastic or the like can be used.

[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.

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.

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.

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.

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.

[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.

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.

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.

(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.

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.

[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.

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.

(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.

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.

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.

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)

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

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)
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)
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)
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.
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.
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.