JP2015051430A - Micro-flow passage structure, and separation method of particle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a micro-flow passage structure in which particles can be separated precisely according to the size thereof, and to provide a separation method using the micro-flow passage structure.SOLUTION: A micro-flow passage structure 1 for separating particles in a fluid according to the size thereof includes: a body part 2 for circulating the fluid from one side in a predetermined direction A to the other side; an inlet part 3 of the fluid; and an outlet part 4 of the fluid. The main part 2 has: a plurality of main flow passages 5 each of which is extended along a first inclination direction B inclined toward the predetermined direction A; and a plurality of branch flow passages 6 each of which is extended along a second inclination direction C inclined toward the opposed side of the predetermined direction A to the first inclination direction B and has crossings with the main flow passages 5. The main flow passages 5 and the plurality of branch flow passages 6 are disposed on the same plane of the main part 2 and a tilt angle formed by the predetermined direction A and the first inclination direction B is larger than 0° and smaller than 45°.

Description

  The present invention relates to a microchannel structure and a particle separation method using the microchannel structure.

  In various fields such as diagnostic medicine, biochemical research, precision machinery industry and food / cosmetics manufacturing, it is essential to separate particles according to size. According to this technology, for example, when blood analysis is performed. It becomes possible to separate leukocytes and erythrocytes, or to separate and select cancer cells present in blood to detect cancer early. Furthermore, in stem cell biology research and regenerative medicine using iPS cells and the like, it is necessary to select cells in a specific differentiation stage. Also, in the synthesis of functional materials such as monodisperse fine particles, fine particle separation by size Therefore, this technology is indispensable.

  Here, conventionally, as a technique for separating particles, a technique using a microchannel has been proposed, and it is expected that particles of a micrometer size can be separated. The principle of such separation includes a method using a magnetic field or an electric field, a method using gravity or centrifugal force, or a method using a laminar flow in a flow channel. Among these, in particular, a laminar flow in a flow channel is used. This approach is expected to be useful because the particles are separated based on size by simply introducing a suspension of particles.

  In recent years, as such a microchannel structure, for example, those described in Non-Patent Documents 1 to 3 below are known. In the structure described in Non-Patent Document 1 below, it is intended to separate particles based on size by utilizing the difference in particle position in a flow path having a narrow portion (pinch portion). In the structure described in Non-Patent Document 2 below, the particle suspension is introduced into one main channel and a plurality of branch channels (branch channels), and the amount of fluid introduced into the branch channels is controlled. It is intended to separate particles based on size. In the structure described in Non-Patent Document 3 below, a plurality of pillars are installed in a flow path, and particles are separated based on size by controlling the distance and phase difference between the pillars.

  Further, in Patent Document 1 below, a device and a substance, in which the first and second separation channels communicate with each other only at the intersection, and the target substance is separated from a fluid containing a plurality of substances by applying a potential or the like to the channel. A separation method is described. Patent Document 2 below describes a technique for separating target cells by rolling the target cells in a certain direction by coating a 3D structure surface arranged at a predetermined location with a cytophilic substance.

JP 2008-119678 A International Publication No. 2013/049860 Pamphlet

“Anal. Chem.”, 2004, 76 (18), 5465-5471. “Lab Chip”, 2005, 5, 1233-1239. “Science”, 2004, Vol. 303 No. 5673, 987-990

  By the way, as described above (Non-Patent Documents 1 to 3), the microchannel structure as described above is expected to be able to freely separate particles of, for example, a micrometer size in various fields. In particular, those having high separation accuracy (separation performance) are desired.

  Note that the device described in Patent Document 1 is not a separation method based on size, but merely concentrates a substance using a difference in mobility with respect to application of a driving force such as an electric field. Furthermore, the first and second separation flow paths are arranged not in the same plane but shifted in the direction perpendicular to the extending direction so as to easily apply an electric field or the like and communicate with each other at the intersection. In the case of separation based on this, it is a completely different technique because it is essential that the flow paths are arranged on the same plane.

  The separation method described in Patent Document 2 is completely different from the separation based on size, and there is a description about separation using a difference in size, but there is no description about the principle or mechanism, There is no description about the concept which can set separation size freely.

  This invention is made | formed in view of the said situation, and makes it a subject to provide the microchannel structure which can isolate | separate particle | grains accurately based on size, and the separation method using the same.

  In order to solve the above problems, a microchannel structure according to the present invention is a microchannel structure for separating particles in a fluid based on a size, and the fluid flows from one side to the other side in a predetermined direction. A main body part that circulates fluid, an inlet part that allows fluid to flow into the main body part, and an outlet part that causes fluid to flow out from the main body part, and the main body part extends along a first inclined direction that is inclined with respect to a predetermined direction. Along with the plurality of main flow paths arranged side by side in the main body and a second inclined direction inclined to the opposite side of the first inclined direction with respect to the predetermined direction, the main flow paths intersect with the main flow path in the main body. A plurality of branch flow paths arranged side by side, and the main flow path and the plurality of branch flow paths exist on the same plane of the main body, and the inclination angle of the first inclination direction with respect to the predetermined direction is: Has an angle greater than 0 ° and less than 45 ° It is characterized by that.

  In this micro-channel structure, asymmetrical flow distribution occurs at the intersection of the main channel and the branch channel while the fluid flows in a predetermined direction as a whole in the main body, and small particles enter the branch channel. On the other hand, large particles flow through the main channel as they are. This is repeated in the plurality of main channels and the plurality of branch channels. As a result, the large particles are circulated to the side where the main channel is inclined, while the small particles are moved to the side where the branch channel is inclined. It becomes possible to distribute. That is, it is possible to separate the particles continuously with a plurality of main flow paths and a plurality of branch flow paths, and to separate the particles with high accuracy.

  Further, in this microchannel structure, the number of branch channels provided on the second inclined direction side with respect to the width of an arbitrary main channel in the plurality of main channels and the arbitrary length of the arbitrary main channel Thus, it is preferable that the size of particles to be introduced from any main channel into the branch channel on the second inclined direction side is set. In other words, by controlling the width of the main channel and the number of branch channels provided on the second inclined direction side with respect to an arbitrary length of the main channel, the intersection of the main channel and the branch channel is controlled. Thus, a desired flow rate distribution can be generated, and particles smaller than a certain set value enter the branch channel, while particles larger than the set value flow through the main channel as they are. This is repeated in the plurality of main channels and the plurality of branch channels, and as a result, particles larger than the set value are circulated to the inclined side of the main channel, while particles smaller than the set value are branched. It becomes possible to distribute to the side where the road is inclined. That is, the particles can be continuously separated with high accuracy based on the desired size in the plurality of main channels and the plurality of branch channels.

  Further, in this microchannel structure, an intersection Ap that is the center of an intersection between an arbitrary main channel in the plurality of main channels and one branch channel that intersects the arbitrary main channel, and a predetermined direction from the intersection Ap. An intersection Bp that is an intersection of the extension line extending to the other side and the center line of another main channel that is adjacent to and intersects the arbitrary main channel, and a branch channel that passes through the intersection Ap and the intersection Bp In the triangle connecting the intersection Cp, which is the center of the intersection with the other main channel, the number n of branch channels on the second inclined direction side between the intersection Bp and the intersection Cp in the other main channel is 2 It can be set as the aspect which satisfy | fills <= n <3356.

In this microchannel structure, the main channel and the branch channel are orthogonal to each other, the width W _{0 of} the main channel, the number n of branch channels provided on the second inclined direction side of the main channel, and the branches The width W _{2 of} the channel, the distance d between the branch channels adjacent to each other on the second tilt direction side of the main channel, the tilt angle θ in the first tilt direction with respect to the predetermined direction, and the length L of the branch channel fulfills the following formula 1, it may be embodiments in which L> W _0.

  In this microchannel structure, the width of the main channel may be set to a width that is not blocked by particles in the fluid.

  Further, as one aspect of the microchannel structure, the main body portion is provided on one side in the predetermined direction and is connected to the main channel and the branch channel, and the parallel direction of the main channel And a second boundary channel that extends along a predetermined direction, and the inlet portion is connected to the first boundary channel and introduces a suspension of particles. A first inlet port that is connected to the first boundary channel and a second inlet port that introduces the buffer solution, the outlet portion has a plurality of outlet ports that lead out the fluid, and the outlet port includes a plurality of outlet ports. Are connected to a part of the main flow path and a part of the plurality of branch flow paths, and are arranged adjacent to each other in a direction along the parallel arrangement direction of the main flow paths. Opening closest to the outlet port located at the end of the first inclined direction side of the outlet ports The other end of the main channel having one end that can be manner which opens the first interface passage.

  As a specific aspect of the microchannel structure, the main body portion has a first boundary channel provided on one side in a predetermined direction and connected to the main channel and the branch channel, It is preferable to have a first inlet port connected to one boundary channel and introducing a suspension of particles.

  At this time, it is preferable that the inlet portion further includes one or a plurality of second inlet ports that are connected to the first boundary channel and introduce the buffer solution. In this case, the introduced buffer solution makes it possible to control the suspension and thus the flow of particles. In order to further improve the particle separation accuracy, it is preferable that at least one of the second inlet ports of the inlet portion is disposed on the first inclined direction side of the first inlet port.

  In addition, the outlet portion has a plurality of outlet ports that lead out the fluid, and the outlet port is connected to a part of the plurality of main flow paths and a part of the plurality of branch flow paths, and the main flow It is preferable that they are provided adjacent to each other in a direction along the direction in which the roads are arranged side by side. In this case, for example, it is possible to derive and collect larger particles from some of the plurality of outlet ports and collect and collect smaller particles from the other outlet ports. .

  In addition, as a preference, at least one of the main channel and the branch channel may have a rectangular channel cross section. Furthermore, as a preference, the main body may have a second boundary channel that is provided on at least one of the one side and the other side in the direction along the parallel direction of the main channels and extends along a predetermined direction.

  Moreover, this invention is a particle | grain separation method which isolate | separates the particle | grains in a fluid based on size using any one of said microchannel structures.

  In this separation method, the inlet portion has a first inlet port for introducing a suspension of particles and a second inlet port for introducing a buffer solution, and the suspension of particles introduced from the first inlet port. The flow rate of the buffer liquid introduced from the second inlet port can be 0.5 or more with respect to the flow rate of 1.0.

  In this particle separation method, the inlet portion has a first inlet port for introducing a suspension of particles and a second inlet port for introducing a buffer solution, and the suspension of particles is supplied from the first inlet port. A plurality of second inlet ports may be introduced and provided on one and / or both sides of the first inlet port.

  In addition, it is possible to adopt an aspect in which the particles are cells and the cells in the fluid are separated based on the size using the microchannel structure.

  Further, in the case where the particles are cells, a step of introducing blood or a cell-containing liquid into the inlet portion of the microchannel structure and a cell separated from a part of the outlet portion of the microchannel structure are collected. And a process.

  According to the present invention, it is possible to accurately separate particles based on size.

It is the perspective view which showed the microchannel structure which concerns on one Embodiment, and was seen from the general front side. It is an enlarged view explaining the motion of the particle | grains in the microchannel structure of FIG. It is a front view showing roughly the modification of the micro channel structure concerning one embodiment. It is the schematic explaining the size of the particle | grains isolate | separated in the microchannel structure of FIG. It is a schematic diagram for explaining the size of the particles to be separated by the main channel width W ₀ and Edaryuro number n can be controlled. It is a graph which shows the result of the particle-separation experiment by the microchannel structure which has the main channel of 1st inclination | tilt angle of 15 degrees, and changed the branch channel number n of the 2nd inclination direction side of the main channel. It is a graph which shows the result of the particle-separation experiment by the microchannel structure which has the main channel of the 1st inclination | tilt angle of 30 degrees, and changed the branch channel number n of the 2nd inclination direction side of the main channel. It is a graph which shows the result of the blood cell isolation | separation experiment by the microchannel structure which has the main flow path of 1st inclination | tilt angle of 15 degrees, and the branch flow path number n of the 2nd inclination direction side of the main flow path is constant. It is a graph which shows the result of the blood cell isolation | separation experiment by a microchannel structure when it has the main channel of 1st inclination | tilt angle 30 degrees, and the branch channel number n of the 2nd inclination direction side of a main channel is constant. It is a graph which has the main flow path of 1st inclination-angle 45 degrees, and shows the result of the particle separation experiment by a micro flow path structure. It is a graph which has the main flow path of 1st inclination | tilt angle of 45 degrees, and shows the result of the blood cell separation experiment by a micro flow path structure.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, the same or equivalent elements will be denoted by the same reference numerals, and redundant description will be omitted. Further, the terms “left”, “right”, “upper”, and “lower” are based on the illustrated directions and are for convenience.

  FIG. 1 is a schematic front view showing a microchannel structure according to an embodiment, and FIG. 2 is an enlarged view for explaining the movement of particles in the microchannel structure of FIG. FIG. 3 is a front view showing an outline of a modified example of the microchannel structure according to the present embodiment. FIG. 4 is a schematic view for setting the size of particles separated in the microchannel structure of FIG. FIG. 5 is a schematic diagram for explaining that the size of particles separated by the main channel width and the number of branch channels n can be controlled. As shown in FIGS. 1 and 2, the microchannel structure 1 of the present embodiment separates particles in a suspension with accuracy based on size, and is a so-called hydrodynamic array for fine particle classification. Configure filtration.

  In the present specification, the fluid is not particularly limited to viscosity, density, temperature, pH, osmotic pressure ratio, ionicity, etc., for example, water, polymer-containing liquid, plasma, blood, body fluid, cell-containing liquid, Examples thereof include a culture solution, a protein solution, and a physiological saline solution. From the viewpoint of development in regenerative medicine, blood, a cell-containing solution and a culture solution are more preferable, and blood and a cell-containing solution are most preferable.

  The particles applicable to the microchannel structure 1 are not particularly limited. For example, synthetic fine particles, magnetic nanoparticles, fluorescent particles, blood cells, cells (animal and plant cells, live cells, dead cells, Antigen-specific T cells, iPS cells, ES cells, stem cells, hepatocytes, cancer cells, tumor cells, rare cells, glial cells, nerve cells, etc.), DNA, proteins, viruses, cell nuclei, mitochondria, chloroplasts , Lysosomes, microorganisms and the like.

  The microchannel structure 1 includes a main body portion 2, an inlet portion 3, and an outlet portion 4. The main body 2 circulates fluid from one side (upper side in the drawing) in the predetermined direction A to the other side (lower side in the drawing) as an overall flow. The main body 2 has a long plate-like outer shape in the predetermined direction A. For example, the length in the longitudinal direction is 4 cm and the length in the short direction is 1.5 cm.

  The main body 2 has a main flow path 5, a branch flow path 6, and a side boundary flow path (second boundary flow path) as flow paths provided so as to be configured in a plane in the main body 2. 7 and an inlet side boundary flow path (first boundary flow path) 8. The main flow path 5 extends linearly on the same plane as the branch flow path 6 along the first inclined direction B inclined with respect to the predetermined direction A. In other words, the main channel 5 extends in the predetermined direction A and inclines toward one side (the right side in the drawing) in the short direction of the main body 2 as it goes downstream.

The inclination angle θ of the first inclination direction B with respect to the predetermined direction A is an angle larger than 0 ° and smaller than 45 ° as preferable for separating particles with high accuracy. The reason for this will be described later. The main flow path 5 is juxtaposed at regular intervals as an example in a region other than the upstream end (one side in the predetermined direction A) of the main body 2 and both sides in the short direction. As an example, the main channel 5 here has a rectangular channel cross section. Further, the width W ₀ of the main channel 5 is a width that is not blocked by particles in the fluid.

The number of the main flow paths 5 can be arbitrarily set. For example, as in a micro flow path structure 1A (see FIG. 4) according to a modified example described later, the intervals between the main flow paths 5 are constant and separated. from the viewpoint of precision, the other end 5b of the main channel 5 having closest to open end to the outlet port 12 ₆ (one end) 5a on the edge of the first inclined direction B side, the inlet-side interface passage 8 It is preferable that there are four or more main flow paths 5 opened in the inlet side boundary flow path (first boundary flow path) 8, and in order to further improve the separation accuracy, 6 or more Preferably it is 10 or more.

  The branch channel 6 extends linearly along a second inclined direction C that is inclined to the opposite side of the first inclined direction B with respect to the predetermined direction A. In other words, the branch flow path 6 extends in the predetermined direction A and is inclined toward the other side (the left side in the drawing) in the short direction of the main body 2 as it goes downstream.

  The branch channel 6 constitutes a branch channel that branches from the main channel 5 and extends so as to connect the adjacent main channels 5. Specifically, the branch channel 6 intersects with the main channel 5 in a region other than the upstream end portion and the lateral sides of the main body 2, and all the branch channels 6 are mainstream. It may not be completely intersected with the path 5, and a part of the branch channel 6 may intersect with the main channel 5. However, from the viewpoint of separation accuracy and efficiency, it is more preferable that the main channel 5 and the branch channel 6 completely intersect.

  The presence of the main flow path 5 and the plurality of branch flow paths 6 on the same plane of the main body 2 means that the main flow path 5 and the branch flow paths 6 are provided on one surface of a substantially planar member. Therefore, it means a state in which each flow path is provided from one surface side to the other surface side of one solid member, or a state in which each flow path is provided in a plurality of members and the flow paths face each other. do not do. It is preferable that the depth of the main channel 5 and the depth of the branch channel 6 directly connected to the main channel 5 substantially coincide with each other. The depth is not constant throughout the main body 2 but may vary stepwise, but it is more preferable that the depth is constant from the viewpoint of manufacturing.

  In addition, the shape of the channel cross section of the main channel 5 and the branch channel 6 is not particularly limited, and may be a semicircular arc shape, for example. However, in order to accurately control the flow rate introduced from the main flow path 5 to the branch flow path 6, at least one of the main flow path 5 and the branch flow path 6 preferably has a rectangular cross section. Preferably, both the main channel 5 and the branch channel 6 have a rectangular shape.

  Further, the branch channel 6 does not necessarily have to be orthogonal to the main channel 5, but it is more preferable that the branch channel 6 is orthogonal because separation design based on size is easy. In addition, the branch flow paths 6 do not always have to be arranged at regular intervals in the main body 2. For example, the intervals may vary stepwise, but the regular intervals are more preferable from the viewpoint of easy manufacture. .

  The side boundary flow path 7 constitutes a flow path in the boundary region of the main body 2 and is provided on both one side and the other side along the parallel direction of the main flow path 5, along the predetermined direction A. It is an extending flow path. Specifically, it extends linearly along a predetermined direction A at both lateral sides of the main body 2. The side boundary channel 7 is connected to and communicates with the main channel 5 and the branch channel 6. As with the main channel 5 and the branch channel 6, the side boundary channel 7 here has a rectangular channel cross section. The side boundary channel 7 may not be communicated with at least one of the main channel 5 and the branch channel 6. Moreover, although the side boundary flow path 7 which concerns on this embodiment is provided in both the one side and the other side along the parallel arrangement direction of the main flow path 5, you may make it provide only in either one side. .

  The inlet-side boundary flow path 8 is a flow path provided on one side in the predetermined direction A and connected to the main flow path 5 and the branch flow path 6, specifically, provided at the upstream end of the main body 2. The main flow path 5, the branch flow path 6, and the side boundary flow path 7 are connected to and communicated with each other.

  The inlet portion 3 flows fluid into the main body portion 2 and is provided on one side of the main body portion 2 in the predetermined direction A. The inlet portion 3 has a first inlet port 9 and a plurality (two in this case) of second inlet ports 10 and 11. The first inlet port 9 is an inlet for introducing a suspension of particles. The first inlet port 9 is connected to and communicated with a position closer to the other side portion in the short side direction in the inlet side boundary flow path 8. The first inlet port 9 includes an introduction flow path 9x extending linearly.

  The second inlet ports 10 and 11 are inlets for introducing a buffer solution. These second inlet ports 10 and 11 are arranged so as to sandwich the first inlet port 9 in the short direction. Specifically, the second inlet port 10 is connected to and communicated with the position on the other side of the first inlet port 9 in the inlet side boundary flow path 8, and the second inlet port 11 The side boundary channel 8 is connected to and communicated with a position on one side of the first inlet port 9. The second inlet port 10 includes an introduction flow path 10x that extends in a straight line, while the second inlet port 11 includes an introduction flow path 11x that branches toward the downstream side (four branches in this case). It is out. In the present embodiment, the second inlet ports 10 and 11 are provided on both sides of the first inlet port 9. However, the second inlet port 10 or the second inlet is provided only on one side of the first inlet port 9. It is also possible to provide the port 11.

Further, the positions of the first inlet port 9 and the second inlet ports 10 and 11 connected to the inlet-side boundary flow path 8 are not particularly limited. For example, the second inlet port 10x includes the outlet port 12 ₁ , the first inlet port 10x, and the first inlet port 10x. The inlet port 9x has four outlet ports 12 ₂ and a second inlet port 11x (four branches in this case) located opposite to each other in the predetermined direction A from the outlet ports 12 ₃ , 12 ₄ , 12 ₅ , 12 ₆ . Thus, it is preferable because the fluid flowing through the main body portion 2 easily flows from the inlet portion 3 toward the outlet portion 4 evenly.

The outlet portion 4 allows the fluid as a suspension containing the buffer liquid to flow out from the main body portion 2, and is provided on the other end side of the main body portion 2 in the predetermined direction A. The outlet portion 4 has a plurality of (here, six) outlet ports 12 _{1 to} 12 ₆ . The plurality of outlet ports 12 _{1 to} 12 ₆ are outlets through which the fluid is divided and led out, and the particles dispersed in the fluid can be separated and led out for each size.

Further, a plurality of outlet ports 12 ₁ to 12 _6, in some cases to integrate a plurality of outlet ports 12 ₁ to 12 ₆ derivation destination, by reducing the number of holes of collection outlets, easy to manufacture and efficient recovery You can also In addition, the flow rate ratio from each outlet is controlled by adjusting the flow path width, depth and length of the outlets of the plurality of outlet ports 12 _{1 to} 12 ₆ ( _six here), It is also possible to set the efficiency.

These outlet ports 12 _{1 to} 12 ₆ are arranged adjacent to each other in the short direction of the main body 2 (the direction along the parallel direction of the main flow paths 5) without gaps, and the plurality of main flow paths 5 are arranged. And a part of the plurality of branch channels 6 are respectively connected.

  Next, the correlation between the predetermined direction A, the first inclined direction B of the main channel 5, the second inclined direction C of the branch channel 6, and the particle size control will be described. In the following description, when the first inclined direction B of the main flow path 5 faces one side (the right side in the drawing) in the short direction with respect to the predetermined direction A, that is, the first inclined direction B is the predetermined direction A. When the direction is opposite to the second inclination direction C with reference to the angle, the inclination angle of the first inclination direction B (hereinafter referred to as “first inclination angle”) θ is positive and the same direction as the predetermined direction A Will be described on the premise that the first inclination angle θ is negative when it is “0 °” and faces the same side as the second inclination direction C with respect to the predetermined direction A.

  When the flow rate introduced into the inlet side boundary flow path 8 of the first inlet port 9 for introducing the particle suspension and the second inlet ports 10 and 11 for introducing the buffer liquid is equal, Since particles with a smaller inclination angle θ (closer to 0 °) are more likely to flow into the main flow path 5, the outlet of the smaller particles is closer to the first inclination direction B than when the first inclination angle θ is large. Shift to exit port.

  However, even in such a case, the flow rate introduced into the inlet-side boundary flow path 8 of the second inlet port 11 is set relatively to the first inlet port 9 and the second inlet port 10 for introducing the buffer solution. By increasing the size, the outlet of small particles can be shifted to the outlet port closer to the second inclined direction C. However, when the first inclination angle θ is 0 ° or less, the main flow path 5 extends in the second inclination direction C side, so that all particles are in the first position regardless of the flow rate of any of the inlet ports 9, 10, 11. 2 Derived from the exit port near the inclined direction C, separation is not possible. Therefore, the first inclination angle θ is preferably larger than 0 °.

  On the other hand, when the first inclination angle θ is larger than 0 °, the particles easily flow into the branch channel 6, so that particles smaller than the width of the branch channel 6 are easily introduced into the branch channel 6, The outlet shifts to an outlet port near the second inclined direction C. However, by increasing the flow rate introduced into the inlet-side boundary flow path 8 of the second inlet port 10 relative to the first inlet port 9 and the second inlet port 11, the outlet of large particles is inclined to the first slope. It is possible to shift to an exit port closer to direction B. However, when the first inclination angle θ is 45 ° or more, the introduction of particles into the branch channel 6 cannot be suppressed even if the flow rate of the second inlet port 10 is increased. Most of the particles are introduced into the branch channel 6 and all particles are led out from the outlet port near the second inclined direction C and cannot be separated.

  As described above, the first inclination angle θ with respect to the predetermined direction A needs to be larger than 0 ° and smaller than 45 °. In addition, since the main body 2 becomes longer in the predetermined direction A as the first inclination angle θ is smaller, it becomes difficult to manufacture, and at the same time, when a plurality of microchannel structures 1 are arranged in parallel for the purpose of mass processing, the space is effectively used. Becomes difficult. Accordingly, the first inclination angle θ is more preferably 5 ° or more and 40 ° or less, further preferably 10 ° or more and 35 ° or less, and most preferably 15 ° or more and 30 ° or less.

Note that the microchannel structure 1A according to the modification shown in FIG. 3 is preferable in order to derive large particles from the outlet port closer to the first inclination direction B and increase the separation accuracy. If the microchannel structure 1A, among the plurality of outlet ports 12 ₁ to 12 _6, the main channel 5 having a closest to one end 5a which is open to the outlet port 12 ₆ on the edge of the first inclined direction B side The other end 5b of the first end is opened to the inlet side boundary flow path (first boundary flow path) 8.

  Next, particle separation based on size will be described with reference to FIGS. 4 and 5. Since the basic principle of the microchannel structure 1 and the microchannel structure 1A is the same, the following description is based on the microchannel structure 1.

The separation of the particles based on the size is performed by dividing the width W ₀ of the main channel 5 (see FIG. 5A) and the number n of the branch channels 6 provided on the second inclined direction C side of the main channel 5 (FIG. 5). (See (b)). Specifically, it is assumed that the plurality of main channels 5 and the plurality of branch channels 6 are formed under the same conditions (width, shape, etc.). Here, among the plurality of main flow paths 5, an arbitrary main flow path 5 is specified as the reference main flow path 5, and an arbitrary length of the reference main flow path 5 is specified as the reference length Db. Further, by setting the number n of the branch flow paths 6 provided on the second inclined direction C side with respect to the reference length Db of the reference main flow path 5, it is possible to separate particles based on the size. Note that the number n of the branch flow paths 6 provided on the second inclination direction C side with respect to the reference length Db of the reference main flow path 5 intersects the reference main flow path 5 with all the branch flow paths 6. In this case, the number of branch channels 6 that intersect the main channel 5 in the range of the reference length Db can be considered as n.

Here, for example, when the fluid flowing through the main channel 5 is a laminar flow, the velocity distribution of the fluid flowing through the main channel 5 shows a parabola as shown in FIG. In this case, when _{W 1} the maximum radius of the particle Ps introduced into Edaryuro 6, the flow through the main passage 5 _{Q 0,} and the introduction flow rate to _{Q 1} to Edaryuro 6, for _{Q 1} for _{Q 0} The ratio can be said to be equal to the ratio of S _{1 to} the parabolic area S ₁ + S ₂ . That is, Q ₀ : Q ₁ = S ₁ + S ₂ : S ₁ . Here, S ₁ is a parabolic integral value in the range from 0 to W ₁ , S ₁ + S ₂ is a parabolic integral value in the range from 0 to W ₀ , and Q ₀ is 100% _{When the} ratio introduced into ₁ is Q ₁ %, the following (formula I) is derived.

For example, in the micro channel structure 1, if the fluid flowing through the main body portion 2 has a flow uniformly toward the inlet portion 3 to outlet portion 4, the flow rate Q ₁ through the branch passage 6 in Figure 4, any particles It can be said that this is inversely proportional to the number n of branch channels 6 that must pass through at least in order to reach the next main channel 5 on the side of the second inclination direction C from the main channel 5 with the above. That is, the following (formula II) is established.

This is because, for example, when the width W ₀ of the main flow path 5 is constant, one branch increases as the number of branch flow paths n provided on the second inclined direction C side of the main flow path 5 increases regardless of the width of the branch flow path 6. Since the amount introduced into the channel 6 is reduced, the size of the particles introduced into the branch channel 6 is reduced. Conversely, the smaller the number n of branch channels connected to the main channel 5 is, the smaller one branch channel 6 is. This indicates that the amount introduced into the flow path increases, and the particles introduced into the branch channel 6 increase.

For example, when the number of branch channels n connected to the main channel 5 is constant, the larger the width W ₀ of the main channel 5, the larger the amount introduced into one branch channel 6. The particles introduced into the channel 6 increase, and conversely, the smaller the width W ₀ of the main channel 5, the smaller the particle introduced into the branch channel 6. Therefore, Formula III is derived from Formula I and Formula II when Q ₀ is 100%.

From this formula III, it can be said that the size of particles introduced from the main channel 5 to the branch channel 6 can be freely set by the main channel width W ₀ and the number of branch channels n connected to the main channel 5. However, the main flow path width W ₀ and the number of branch flow paths n set in the design have a substantial application range in addition to being manufactured with a certain error range in the manufacturing technology, and within that range, the formula III is To establish. For example, with respect to n calculated from Formula III, the value of n has an error range of about 10% due to actual manufacturing accuracy. Therefore, n calculated from Formula III is n × 0.9 or more. , And a natural number range of n × 1.1 or less.

As long as the fluid flowing through the main flow path 5 forms a laminar flow, the velocity distribution in the main flow path 5 draws a parabola and Formula III is established. Generally, since the Reynolds number is a laminar flow having a Reynolds number of about 1.0 or less in the microchannel, the main channel width W ₀ is not particularly limited, but the range of the main channel width W ₀ for forming the laminar flow will be described. .

  The introduction pressure P of the fluid introduced into the main channel 5 is set to 0.1 to 1.0 atm. This reflects the minimum and maximum range of the pressure difference generated between the end points of the main flow path 5 when the fluid is actually flowed.

First, the introduction pressure is 0.1 atm, the fluid is water, the viscosity μ = 1 (mPa · sec), and the specific gravity ρ = 1 × 10 ³ (kgm ⁻³ ). The flow path is a circular pipe for calculation. Since the diameter d ₅ (m) of the circular pipe and the length L ₅ of the main flow path ₅ are approximately 1,000 times the circular pipe diameter d ₅ which is the main flow path width W ₀ (L ₅ = 1,000 d ₅ ), From the Hagen Poiseuille's equation, the flow rate flowing through the circular tube diameter d ₅ and circular tube length L ₅ = 1000d ₅ is ΔP = 128 × μ × L × Q / (π × d ₅ ⁴ ). Therefore, the flow rate Q = 78.125 × π × d ₅ ³ (m ³ / sec).

When this is divided by the cross-sectional area of the tube and converted to an average linear velocity v, v = 312.5d ₅ (m / sec). From Reynolds number Re = ρ × v × d ₅ / μ at this time, Re = 3.125 × 10 ⁸ × d ₅ ² . Assuming that the maximum Reynolds number is 2,000, which is a laminar flow, the main channel width W ₀ is preferably less than 2.5 mm from the circular tube diameter d ₅ = 2.5 mm at this time. In a rectangular channel actually used, the Reynolds number Re = 0.196 when the main channel width is 25 μm and the main channel depth is 20 μm, and the Reynolds number Re = 0195 with a circular tube diameter of 25 μm. Reynolds numbers are not significantly different and both are within the range of laminar flow.

Also, here was the length _L 5 = 1,000 D ₅ of the main flow passage 5, for example, the main flow path length _{(L 5)} when the short 500d _5, the main channel width as a Reynolds number less than 2,000 1. less than 8 mm, less than 3.6mm when the main channel length conversely long 2,000d _5. Furthermore, when the flow rate for introducing the particle suspension is doubled, the main flow path width W _{0 with} a Reynolds number of less than 2,000 when the introduction pressure P = 0.2 atm and L ₅ = 2,000d ₅ is less than 2.5 _mm, less than 1.8mm when L ₅ = 1,000d _5, when L 5 = 500d ₅ is less than 1.3 mm. When the assumed maximum introduction pressure P = 1.0 atm, L ₅ = 2,000d ₅ and the Reynolds number less than 2,000, the main flow path width W ₀ is less than 1.1 mm, and L ₅ = 1,000d ₅ Is less than 0.8 mm, and when L ₅ = 500d ₅ is less than 0.6 mm.

From the practical viewpoint, the lower limit of the main flow path width W ₀ needs to be larger than the maximum diameter of the particles flowing in the fluid. That is, it is preferable that W ₀ ≧ 2W ₁ . From the above, the main flow path width W ₀ is preferably 2 W ₁ or more and less than 3.6 mm because it is a laminar flow, more preferably 2 W ₁ or more and less than 2.5 mm, more preferably 2 W ₁ or more and less than 1.1 mm, and most preferably 2 W ₁ or more. It is less than 0.8 mm. Within this range, the formula III is satisfied.

In addition to the above, when the main channel width W ₀ is remarkably large with respect to the size of the maximum particle radius to be separated, for example, W ₀ = 100 W ₁ , a plurality of micro channel structures are parallelized for the purpose of mass processing. It is not preferable because the space cannot be effectively used. Therefore, it is preferable that 2W ₁ ≦ W ₀ <100W _1, that is, the number of branch channels n connected to an arbitrary main channel 5 is 2 or more and less than 3,356 (2 ≦ n <3356), more preferably 2W ₁ ≦ W ₀ <80 W _1, that is, n is 2 or more and less than 2,151, and most preferably 2W ₁ ≦ W ₀ <50 W _1, that is, n is 2 or more and less than 845.

  Further, the number of branch channels n will be described in detail. First, for example, as shown in FIG. 5B, the approximate center of the intersection of an arbitrary main flow path 5 in the plurality of main flow paths 5 and one branch flow path 6 that intersects the main flow path 5. The point is specified as an intersection Ap (see FIG. 5B). Next, an intersection Bp that is an intersection of an extension line Ar extending from the intersection Ap to the other side in the predetermined direction A and a center line of another main channel 5 adjacent to the arbitrary main channel 5 and intersecting the extension line Ar. Is identified. Next, the intersection Cp that is the approximate center of the intersection between the branch channel 6 passing through the intersection Ap and the other main channel 5 passing through the intersection Bp is specified, and the intersections Ap, Bp, Cp are connected by a straight line. A triangle T is specified. Here, the number n of branch channels provided on the second inclined direction C side between the intersection Bp and the intersection Cp in the other main channels 5 corresponds to the number n of the branch channels described above.

Furthermore, the width W ₂ of Edaryuro 6 can be arbitrarily set, it is preferable to set the maximum diameter greater width than the particles to be introduced into Edaryuro 6, the main channel width smaller than the width W ₀ It is preferable that Also, depending on the specifications of the manufacturing apparatus to be used, for example, when the aspect ratio, which is the ratio of the depth of the flow path to the width of the flow path, needs to be less than 2, the width of the branch flow path 6 is 2 of the depth. It must be at least 1 / min. That is, the width W ₂ of the branch channel 6 is preferably 2W ₀ or more and not less than the value obtained by dividing the depth of the branch channel by the aspect ratio.

When the branch channel 6 and the main channel 5 are orthogonal to each other, as shown in FIG. 5B, the length L of the branch channel 6, the first inclination angle θ, and the second inclination direction C side of the main channel 5. The number n of branch channels 6 provided, the width W _{2 of} the branch channels 6, the distance d between the branch channels 6 adjacent to each other on the second inclined direction C side of the main channel 5, the number n of branch channels, Then, the following formula IV is established from the trigonometric function.

Preferably the L of time is greater than the main channel width W _0. For example, when separating a size of several tens of microns such as a blood cell, L is about 1 cm from the viewpoint of making the entire body 2 compact and making effective use of space even when a plurality of bodies 2 are used together. It is preferable to be less than about.

  As a material of the microchannel structure 1 of the above-described embodiment, PDMS (polydimethylsiloxane) which is a kind of silicone rubber can be preferably used. In this case, for example, activation is performed using plasma. Therefore, it is possible to easily join to another member (glass or the like). The material of the microchannel structure 1 is not particularly limited. For example, in addition to PDMS / glass, various polymer materials such as acrylic, silicon, ceramics, metal (stainless steel, etc.) are used. Alternatively, these materials can be used alone or in combination.

  Further, as an example, the microchannel structure 1 can be manufactured by soft lithography (that is, molding using a microstructure patterned by a photolithography process as a template). The method for producing the microchannel structure 1 is not limited, and for example, other molding, embossing, wet etching, dry etching, laser processing, electron beam direct drawing, machining, or the like may be used. it can.

  In the microchannel structure 1 described above, as exemplified below, cells can be separated accurately, simply and efficiently while using a laminar flow formed in a fine channel. Note that the microchannel structure 1A according to the modification also has substantially the same operations and effects.

As shown in FIG. 1, blood or cell-containing liquid is continuously introduced from the first inlet port 9, and buffer liquid is continuously introduced from the second inlet ports 10 and 11. As a result, the overall flow in the main body 2 is in the predetermined direction A, but asymmetrical flow distribution occurs at each intersection (also referred to as each grid point or each branch point). Therefore, as shown in FIG. 2, the small cells PS in the blood or the cell-containing liquid flow with S-shaped movement. Specifically, after entering the main channel 5 from the branch channel 6 and flowing through the main channel 5, the branch channel 6 is entered again. On the other hand, the large cells P _B in the blood or cell-containing liquid go straight through the main channel 5 without entering the branch channel 6.

More specifically, as shown in FIG. 4, for example, the cell P _B having a cell radius r _p larger than the fluid width W ₁ drawn into the branch channel 6 and flowing in (r _p > W ₁ ) The main flow path 5 goes straight without being introduced into the branch flow path 6 at each branch point. On the other hand, cell radius _{r p} is the fluid width _{W 1} below (the _{r p} ≦ _{W 1)} cell _{P S} becomes possible to flow at S-motion at each branch point.

For example, when the width of the main flow path 5 is increased after the inclination angle and the number density ratio of the main flow path 5 and the branch flow path 6 are set to constant values, the fluid that is drawn into the branch flow path 6 and flows in The width W ₁ can be increased and larger particles can be fractionated into the branch channel 6. Further, for example, if the inclination density and the channel width of the main channel 5 and the branch channel 6 are set to constant values and the number density ratio of the branch channel 6 is increased, the main channel 5 and the branch channel 6 are pulled out to the branch channel 6. Incoming W ₁ can be reduced, and only smaller particles can be fractionated into the branch channel 6. Thus, the size of the particles to be separated is controlled by appropriately setting the channel width of the main channel 5 and the number density ratio of the main channel 5 and the branch channel 6 and increasing / decreasing the fluid width W _1. Can do.

Incidentally, theoretically, for example, when the width of the main flow path 5 is 25 μm and the number density ratio of the main flow path 5 and the branch flow path 6 is 1:40, W1 is about 2.5 μm, and the particles are smaller than 5 μm in diameter. P _B is considered to be separated into the branch channel 6.

This is repeated in the plurality of main flow paths 5 and the plurality of branch flow paths 6. As a result, as shown in FIG. 2, the cells P _B having a certain size are separated from the entire flow, with at road 5 flows into shown lower right direction (the side of the main channel 5 is inclined), it is separated from the smaller cells P _S. Then, it derived from a plurality of outlet ports ₁₂ 1 to 12 ₆ the right side in (outlet port 12 _6, the outlet ports ₁₂ 5, 12 ₆ or outlet port ₁₂ 4-12 _6).

At the same time, the small cell P _S, is separated from the entire flow, it flows into the lower left direction (the side where Edaryuro 6 is inclined) with at Edaryuro 6, is separated from the large cell P _B. Then, it derived from the left side of the plurality of outlet ports ₁₂ 1 to 12 ₆ (outlet port 12 _1, the outlet port ₁₂ 1, 12 _2, or outlet port ₁₂ 1 to 12 _3).

  In the case of separating particles at a higher throughput, in addition to the two-dimensional parallelization, the possibility of realizing a higher throughput can be expected by stacking the microchannel structures 1 three-dimensionally.

  When the microchannel structure 1 is used to separate particles, the flow rate of the particle suspension introduced from the first inlet port 9x into the inlet side boundary channel (first boundary channel) 8 is set to 1. 0, the flow rate of the buffer liquid introduced from the respective inlet ports of the second inlet ports 10x and 11x (here, four) into the inlet side boundary flow path (first boundary flow path) 8 is 0.5 or more. In order to further improve the separation accuracy, it is preferable to set each flow rate of 11x to 1.0 or more, and in order to further improve the separation efficiency in addition to the separation accuracy, all flow rates of 10x and 11x Is preferably 1.0 or more.

  For example, when separating particles in a fluid that contains foreign substances or fluids that easily aggregate such as blood, the boundary flow on the inlet side is prevented so that the foreign substances and aggregates are not introduced into the main flow path 5 and the branch flow paths 6. The path 8 is preferably provided with a function like a coarse filter. Although the shape and structure are not particularly limited, specifically, a mesh structure in which pins such as pillars are erected is used.

  Incidentally, in the above-described embodiment, two types of particles are separated based on the size as an example. However, for example, by appropriately setting the inclined lattice structure of the main channel 5 and the branch channel 6, three or more types of particles can be divided into multiple stages. It is also possible to separate them. Further, for example, by using a tilted grating structure that is symmetrical to the left and right lines, it is possible to efficiently concentrate the particles to be separated. Also in this respect, in the above-described embodiment, it is possible to efficiently separate and collect an amount of cells required for diagnostic medical treatment or the like.

  As described above, the microchannel structures 1 and 1A according to the present embodiment and the modified examples have been described. However, according to the microchannel structures 1 and 1A and the method of separating particles using these, the main body While the fluid flows in the predetermined direction A as a whole in the part 2, an asymmetric flow distribution occurs at the intersection of the main channel 5 and the branch channel 6, and small particles enter the branch channel 6 while large The particles flow through the main channel 5 as they are. This is repeated in the plurality of main channels 5 and the plurality of branch channels 6. As a result, the branch channel 6 is inclined for small particles while the main particles 5 are circulated toward the inclined side. It becomes possible to distribute to the side to do. That is, the particles can be continuously separated by the plurality of main channels 5 and the plurality of branch channels 6, and the particles can be accurately separated.

In the present embodiment, the width W ₀ of the arbitrary main flow path 5 in the plurality of main flow paths 5 and the second inclination direction C side with respect to the arbitrary length (reference length) Db of the arbitrary main flow path 5 The size of the particles to be introduced from the arbitrary main channel 5 to the branch channel 6 on the second inclined direction C side is preferably set according to the number of the branch channels 6 provided in the channel. That is, the width W ₀ of the main flow path 5 and the number n of the branch flow paths 6 provided on the second inclined direction C side with respect to the arbitrary length (reference length) Db of the main flow path W ₀ are controlled. By doing so, a desired flow rate distribution can be generated at the intersection of the main flow path 5 and the branch flow path 6, and particles smaller than a certain set value enter the branch flow path 6 while larger than the set value. The particles flow through the main channel 5 as they are. This is repeated in the plurality of main flow paths 5 and the plurality of branch flow paths 6, and as a result, for particles larger than the set value, the particles smaller than the set value are circulated to the side where the main flow path 5 is inclined. Can be distributed to the side on which the branch channel 6 is inclined. That is, the particles can be continuously separated with high accuracy based on the desired size by the plurality of main channels 5 and the plurality of branch channels 6.

  Further, in the particle separation method according to the present embodiment, the second boundary ports 10 and 11 are connected to the inlet side boundary flow path (the first boundary flow path (the first flow rate) with respect to the flow rate 1.0 of the particle suspension introduced from the first inlet port 9. The flow rate of the buffer liquid introduced into the (one boundary flow path) 8 can be 0.5 or more. In the present embodiment, the second inlet ports 10 and 11 are provided on both sides of the first inlet port 9, but only one of them may be provided.

  Moreover, it can also be set as the method (particle separation method) which isolate | separates a cell by making the particle | grains isolate | separated into a cell. In this method of separating cells, blood or a cell-containing liquid was introduced into the inlet portion 3 of the microchannel structure 1, 1A and separated from a part of the outlet portion 4 of the microchannel structure 1, 1A. And a step of collecting cells.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by a following example.

  In the present invention, the size of the separated particles can be freely set by controlling the main channel width and the number of branch channels. Therefore, the size of the particles can be set by controlling the number of branch flow paths. Therefore, the following example shows the result when the main flow path width is 25 μm as one representative. The specifications of the microchannel structure according to each example and comparative example are shown in Table 1.

[Example 1]
Using the microchannel structure 1 (Example 1) corresponding to the above-described embodiment, a fluorescent particle separation experiment was performed. Here, the first inclination angle θ15 ° of the main channel 5, the channel width of the main channel 5 (main channel width) is 25 μm, the channel width of the branch channel 6 (branch channel width) is 15 μm, and the interval between the branch channels is 20 μm. It was. The channel depths (heights) of the main channel 5 and the branch channel 6 were both 25 μm. The main channel 5 and the branch channel 6 were always formed to be orthogonal to each other, and the number of branch channels n was changed between 35 and 100. As described above, an experiment was performed in which the number of branch channels n intersecting the arbitrary main channel 5, that is, the number of branch channels n provided on the second inclined direction side of the arbitrary main channel 5 was changed between 35 and 100. It was.

  Suspension is continuously introduced at 20 μL / min from the first inlet port 9 into the microchannel structure 1 according to the first embodiment, and 20 μL of buffer solution is supplied from the second inlet ports 10 and 11 on both sides thereof. / Min and 80 μL / min, respectively. As the particles, fluorescent particles having a diameter of 3.0 μm and a diameter of 9.9 μm were used, and fluorescent particles having a diameter of 4.8 μm were also used. A 0.5% Tween 80 aqueous solution (polyoxyethylene sorbitan oleate) was used as the buffer solution. The result is shown in FIG. FIG. 6 is a graph showing the results of the particle separation experiment.

In FIG. 6, the result when the branch channel number n is 35 and the result when the branch channel number n is 100 are shown. Each number on the horizontal axis corresponds to the outlet ports 12 _{1 to} 12 ₆ (the same applies to FIGS. 7 to 11).

  First, in the case of the number of branch flow paths n35, the particle size separated based on the above-mentioned formula III was set to have a diameter of 5.0 μm and verified by this experiment. As a result of this experiment, as shown in FIG. 6, 4.8 μm particles smaller than 5.0 μm are derived from the outlets 2 and 3 by about 80%, and 9.9 μm particles are derived from the outlet 5 by 70% or more. Thus, separation of particles according to the set size was observed.

  Next, under the same conditions, only the branch channel number n was changed to 100, and the experiment was performed by setting the size of the separated particles to be 3.0 μm in diameter. As a result, as shown in FIG. 6, more than 90% of 3.0 μm particles having substantially the same size are derived from the outlets 2 and 3, and about 76% of 4.8 μm particles are derived from the outlets 4 and 5. About 84% of the 9 μm particles were derived from the outlet 6, and three types of particles were separable from different outlets.

[Example 2]
The same experiment as in Example 1 was performed except that the inclination angle θ30 ° of the main channel 5 and the number of branch channels n41 and 100 were used. The result of Example 2 is shown in FIG.

  First, when the number of branch channels n was 41, the experiment was performed by setting the particle size separated based on the above-described formula III to be 4.5 μm in diameter. As a result, as shown in FIG. 7, particles of 3.0 μm smaller than 4.5 μm are derived from the outlet 1 by about 92%, and particles of 9.9 μm are derived from the outlets 5 and 6 by 99% or more, Separation of particles according to the set size was observed. Next, under the same conditions, only the branch channel number n was changed to 100, and the experiment was performed by setting the size of the separated particles to be 3.0 μm in diameter. As a result, as shown in FIG. 7, the particle size to be separated is 3.0 μm in diameter, and 3.0 μm particles of approximately the same size are derived 100% from the outlets 2 and 3, and the 9.9 μm particle is the outlet 5. And 6 to 100%, the two sizes of particles could be separated from different outlets.

[Example 3]
The microchannel structure 1 (Example 3) in which the inclination angle θ of the main channel 5 is 15 °, the number of branch channels n is 193, and the size of the separated particles is 2.1 μm in diameter is used. Then, blood cell separation experiments using prepared blood and peripheral blood were performed. Before describing the details of the experimental results of Example 3, “prepared blood preparation”, “peripheral blood”, and “calculation of blood cell recovery rate” will be described first.

(Preparation of prepared blood)
ACD-A (acid citrate dextrose-A) as an anticoagulant was added to fresh peripheral blood of a healthy person (healthy human) so that the ratio of blood: ACD-A = 8: 1 was added, and the mixture was thoroughly mixed by inversion. 15 mL of Ficoll-Paque PLUS (manufactured by GE Healthcare), which is a density gradient centrifugation medium, is added to a 50 mL centrifuge tube, and then diluted 2-fold with a D-PBS (Dulbecco's Phosphate Buffered Saline) solution (manufactured by Life Technologies). The ACD-A-added peripheral blood 30 mL was carefully layered so that a clear interface was formed. After centrifuging at 800 × g for 15 minutes without an accelerator or brake, about 7 mL of a buffy coat layer rich in mononuclear cells was collected and transferred to another centrifuge tube. The buffy coat layer was further centrifuged at 1,430 × g for 10 minutes with an accelerator and a brake, and then about 6.2 mL of the supernatant was removed. The residual cell suspension was passed through a 30 μm mesh and mixed 1: 1 with peripheral blood added with ACD-A to prepare prepared blood. The required amount of prepared blood was diluted 5-fold with a D-PBS solution containing 0.5% BSA (Bovine Serum Albumin).

(Peripheral blood)
ACD-A (acid citrate dextrose-A) as an anticoagulant was added to fresh peripheral blood of healthy persons (healthy humans) so that blood: ACD-A = 8: 1 was added, and the mixture was thoroughly inverted to obtain peripheral blood. .

(Separation of blood cells)
Prepared blood or peripheral blood diluted 5 times is introduced from the first inlet port 9 at the inlet 3 of the manufactured microchannel structure 1 using a syringe pump, and the second inlet ports 10 and 11 on both sides of the blood are introduced from 0 to 0. A D-PBS solution containing 5% BSA was introduced. The respective introduction speeds were 15 μL / min for the inlet ports 9 and 10 and 60 μL / min for the inlet port 11. The solutions recovered from the outlets 1 and 2, the outlets 3 and 4, and the outlets 5 and 6 were each collected in one container to obtain a recovered cell solution.

(Calculation of blood cell recovery rate)
Using a multi-item automatic blood cell analyzer (manufactured by Sysmex, model XS-800i), the white blood cell concentration, red blood cell concentration, and platelet concentration of the prepared blood, 5 times diluted prepared blood and the collected cell solution were measured. The number of white blood cells, red blood cells, and platelets in each specimen was calculated by multiplying each concentration by the volume of the solution. Next, the lymphocyte ratio, monocyte ratio, and granulocyte ratio in leukocytes were measured. Each specimen was stained with an anti-human CD45-FITC antibody and an anti-human CD14-PE antibody and then subjected to hemolysis treatment, and the CD14 positive cell ratio was measured by flow cytometry (manufactured by Beckman Coulter, model Cytomics FC500). The number of monocytes in each specimen is calculated by the white blood cell count × CD14 positive ratio. The red blood cell recovery rate, platelet recovery rate, white blood cell recovery rate, lymphocyte recovery rate and monocyte recovery rate in the solution recovered from each outlet (exit 1, 2, exit 3, 4, exit 5, 6) are expressed by the following formula ( Calculated from A) to (E).

Red blood cell recovery rate (%) = (Red blood cell concentration of recovered cell solution × Volume of recovered cell solution) / (Red blood cell concentration of 5-fold diluted blood × Volume of 5-fold diluted blood discharged from syringe pump) × 100 (A)
Platelet recovery rate (%) = (platelet concentration of recovered cell solution × capacity of recovered cell solution) / (platelet concentration of 5-fold diluted blood × volume of 5-fold diluted blood discharged from syringe pump) × 100 (B)
・ White blood cell recovery rate (%) = white blood cell count of recovered cell solution / 5 white blood cell count of diluted blood × 100 formula (C)
・ Lymphocyte recovery rate (%) = number of lymphocytes in recovered cell solution / 5 times number of lymphocytes in diluted blood × 100 Formula (D)
Monocyte recovery rate (%) = Monocyte count of recovered cell solution / 5 monocyte count of diluted blood × 100 Expression (E)

  The results of the blood cell separation experiment of Example 3 are shown in FIG. In the graph, the outlets 1 and 2 are grouped into the outlet 1, the outlets 3 and 4 are grouped into the outlet 3, and the outlets 5 and 6 are grouped into the outlet 5.

  As a result of blood cell separation of the prepared blood, leukocytes with a diameter of about 6 to 20 μm are collected from the outlets 5 and 6, and red blood cells with a thickness of 2 to 3 μm and platelets with a diameter of 2 to 3 μm are collected from the outlets 5 and 6. They were 2.6% and 1.2%, respectively. Red blood cells and platelets were almost recovered from outlets 1 to 4, and the recoveries were about 88% and about 96%, respectively. That is, only small erythrocytes and platelets are drawn into the branch channel 6, and white blood cells having a large diameter go straight through the main channel 5, and red blood cells and platelets are collected from the outlets 1 to 4, and high from the outlets 5 and 6. Leukocytes were obtained with high purity and high recovery rate, and separation of leukocytes from erythrocytes and platelets was confirmed.

  Next, as a result of blood cell separation of peripheral blood, leukocytes are recovered from about 93% outlets 5 and 6, and recovery rates of red blood cells and platelets from outlets 5 and 6 are as high as 0.0% and 0.0%, respectively. Leukocyte separation was confirmed. Red blood cells and platelets were almost recovered from outlets 1 to 4, and the recovery rates were about 100% and about 75%, respectively, and separation of white blood cells and red blood cells / platelets could be confirmed.

[Example 4]
Exactly the same microchannel structure as in Example 3, except that the inclination angle θ of the main channel 5 is 30 °, the number of branch channels n is 41, and the size of the separated particles is 4.5 μm in diameter. 1 (Example 4) was used, and the same blood cell separation experiment as in Example 3 was performed. The results are shown in FIG. In the graph, the outlets 1 and 2 are grouped into the outlet 1, the outlets 3 and 4 are grouped into the outlet 3, and the outlets 5 and 6 are grouped into the outlet 5.

  As a result of blood cell separation of the prepared blood, among leukocytes, monocytes having a large size of about 12 to 20 μm are recovered from the outlet 5 to about 73%, and red blood cells having a thickness of 2 to 3 μm and platelets having a diameter of 2 to 3 μm are extracted from the outlet 1. From 98 to 98% and 100% were recovered respectively. Among leukocytes, small lymphocytes having a diameter of 6 to 14 μm were recovered from the outlets 1 to 4 by about 77%. That is, monocytes far larger than the separation size of 4.5 μm were obtained from the outlets 5 to 6 with high purity and high recovery rate, and separation of erythrocytes, platelets, lymphocytes and monocytes could be confirmed.

  Next, as a result of blood cell separation of peripheral blood, monocytes were collected from about 90% outlets 5 to 6, and recovery rates of red blood cells and platelets from outlets 1 to 4 were collected about 95% and about 84%, respectively. Lymphocytes were recovered about 94% from outlets 1-4. That is, monocytes far larger than the separation size of 4.5 μm were obtained with high purity and high recovery rate only from the outlets 5 to 6, and separation of erythrocytes, platelets, lymphocytes and monocytes could be confirmed.

[Comparative Example 1]
The same experiment as in Example 1 was performed except that the inclination angle θ45 ° of the main flow path 5 and the number of branch flow paths n were set to 14. The result of Comparative Example 1 is shown in FIG. As shown in FIG. 10, when the inclination angle θ is 45 °, the diameter set as the particle size to be separated is 8.2 μm, but the particles having a diameter of 9.9 μm larger than 8.2 μm are also discharged from the outlet 1 and About 76% was derived from 2, and no separation of particles according to the set size was observed.

[Comparative Example 2]
An experiment similar to the experiment using the prepared blood of Example 3 was performed except that the inclination angle θ45 ° of the main channel 5 and the number of branch channels n were set to 14. The result of Comparative Example 2 is shown in FIG. As shown in FIG. 11, when the inclination angle is 45 °, monocytes larger than the diameter of 8.2 μm are derived from the outlets 1 and 2 by 57% even though the diameter set as the separated size is 8.2 μm. The cell separation according to the set size was not observed.

DESCRIPTION OF SYMBOLS 1, 1A ... Microchannel structure, 2 ... Main-body part, 3 ... Inlet part, 4 ... Outlet part, 5 ... Main channel, 6 ... Branch channel, 7 ... Side boundary channel (2nd boundary channel) , 8... Entrance side boundary channel (first boundary channel), 9... First inlet port, 10, 11..., Second inlet port, 12 ₁ , 12 ₂ , 12 ₃ , 12 ₄ , 12 ₅ , 12 ₆ . Outlet port, A ... predetermined direction, B ... first tilt direction, C ... second tilt direction.

Claims (11)

A microchannel structure for separating particles in a fluid based on size,
A main body for circulating the fluid from one side to the other side in a predetermined direction;
An inlet for flowing the fluid into the main body;
An outlet for allowing the fluid to flow out of the main body,
The main body is
A plurality of main flow paths that extend along the first inclined direction that is inclined with respect to the predetermined direction, and that are arranged in parallel in the main body,
A plurality of branch channels extending in a second inclined direction inclined to the opposite side of the first inclined direction with respect to the predetermined direction, and arranged side by side so as to intersect the main channel in the main body portion; Have
The main channel and the plurality of branch channels exist on the same plane of the main body,
The microchannel structure, wherein an inclination angle of the first inclination direction with respect to the predetermined direction is greater than 0 ° and less than 45 °.   The arbitrary main flow path in the plurality of main flow paths and the number of the branch flow paths provided on the second inclined direction side with respect to the arbitrary length of the arbitrary main flow path The microchannel structure according to claim 1, wherein a size of the particle introduced from the main channel into the branch channel on the second inclined direction side is set. An intersection Ap that is the center of the intersection of any of the plurality of main channels and one branch channel that intersects the arbitrary main channel;
An intersection Bp that is an intersection of an extension line extending from the intersection Ap to the other side in the predetermined direction and a center line of another main flow path adjacent to the arbitrary main flow path and intersecting the extension line;
In a triangle connecting the intersection Cp, which is the center of the intersection of the branch flow path passing through the intersection Ap and the other main flow path passing through the intersection Bp,
3. The microchannel according to claim 1, wherein the number n of the branch channels on the second inclined direction side between the intersection Bp and the intersection Cp in the other main channel satisfies 2 ≦ n <3356. Structure. The main channel and the branch channel are orthogonal to each other, the width W _{0 of} the main channel, the number n of the branch channels provided on the second inclined direction side of the main channel, the branch channel A width W ₂ , an interval d between the branch channels adjacent to each other on the second tilt direction side of the main channel, an inclination angle θ in the first tilt direction with respect to the predetermined direction, and a length L of the branch channel There fulfills equation 1, L> microchannel structure W ₀ in which any one of claims 1 to 3.
  The microchannel structure according to any one of claims 1 to 4, wherein the width of the main channel is set to a width that is not blocked by particles in the fluid. The main body portion is provided on the one side in the predetermined direction and is connected to the main flow channel and the branch flow channel, and at least one side and the other side along the parallel direction of the main flow channel A second boundary channel that is provided in any one direction and extends along the predetermined direction;
The inlet has a first inlet port connected to the first boundary channel and introducing the suspension of particles, and a second inlet port connected to the first boundary channel and introduced a buffer solution. And
The outlet portion has a plurality of outlet ports for leading the fluid,
The outlet port is connected to a part of the plurality of main flow paths and a part of the plurality of branch flow paths, and is arranged adjacent to the direction along the parallel arrangement direction of the main flow paths. Is provided as
The other end of the main channel having one end opened closest to an outlet port located at an end on the first inclined direction side among the plurality of outlet ports is open to the first boundary channel. The microchannel structure according to any one of 1 to 5.   The particle | grain separation method which isolate | separates the particle | grains in a fluid based on size using the microchannel structure as described in any one of Claims 1-6. The inlet portion has a first inlet port for introducing the suspension of particles and a second inlet port for introducing a buffer solution,
The particle according to claim 7, wherein a flow rate of the buffer liquid introduced from the second inlet port is 0.5 or more with respect to a flow rate of 1.0 of the suspension of the particles introduced from the first inlet port. Separation method. The inlet portion has a first inlet port for introducing the suspension of particles and a second inlet port for introducing a buffer solution,
The particle separation method according to claim 7 or 8, wherein the particle suspension is introduced from the first inlet port, and a plurality of the second inlet ports are provided on one side or both sides of the first inlet port.   The method for separating particles according to any one of claims 7 to 9, wherein the particles are cells.   The method includes: introducing blood or a cell-containing liquid into the inlet portion of the microchannel structure; and collecting cells separated from a part of the outlet portion of the microchannel structure. The method for separating particles as described.