JP2018126736A - Particle separation device and particle separation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a particle separation device and a particle separation method capable of accurately separating particles by the simple method since a plurality of elements for accurately aligning the particles on the wall surface of a narrow flow passage such as a sample liquid flow rate, a sheath liquid flow rate, the width and length of the narrow flow passage, the width of an expanded flow passage, a width ratio of the narrow flow passage to the expanded flow passage, the height of each flow passage and the like are mutually related to be complicated in pinched flow fractionation (PFF) and then much time is required for optimization of conditions.SOLUTION: A problem is solved by increasing the accuracy of the position of a flowing particle due to the increase of the hardness of a flow passage material without increasing the accuracy of the generally assumed position of the flowing particle.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a device for separating particles contained in a fluid and a method for manipulating particles contained in a fluid using the device.

  When applying solid materials composed of inorganic materials, organic materials, metals, etc. as materials in various industrial fields, for example, supply as powder as the primary raw material, or form the powder into spherical particles or other shapes And is supplied as a product. These materials (solid materials) are designed by examining their physical properties in order to improve the performance of the final product. In addition, there are many examples where spherical particles are applied as final products, such as grinding balls (alumina, zirconia, silica, etc.), liquid crystal spacers (resin, silica, etc.), chromatographic separation materials, other adsorbents, etc. Is mentioned. In general, such a particle product having a uniform shape, size, and density greatly affects the characteristics of the product as a final form, so that more uniform particles are required.

  There are two methods for producing such particles: a method of developing a technology for producing uniform particles from the beginning, and a method of extracting particles having a required size, density, and shape from particles having a non-uniform distribution. . The former is a relatively new technology and requires a renewal of manufacturing equipment, but the latter can be added to the current equipment and is therefore easily incorporated into the manufacturing process. As the latter technique, there are, for example, a filter separation method (including sieve classification), a gravity classification method, a centrifugal separation method, a cyclone separation method and the like if the particles are several tens of μm or more.

  In the separation and classification methods listed above, it is very difficult to separate particles of several tens of μm or less, particularly particles of 10 μm or less, and it is generally a batch type process and difficult to separate continuously. . In a batch type process, since the amount of treatment per process is determined by the separation yield, relatively large equipment (stock container for raw materials, supply equipment, collection container for unnecessary products) is required. For example, the sieve classification method is suitable for mass processing, but it is necessary to gradually change the size of the sieve mesh, so that it is a batch process. The gravitational classification method is basically a batch process similar to the sieving classification method, and it takes an enormous amount of time to process as the particle size decreases. Although the centrifugal separation method and the cyclone separation method are suitable for high-speed processing, they require a large apparatus and are not suitable for a continuous process.

  However, if classification can be performed continuously, it is possible to reduce the amount of raw materials charged and the amount of production to the limit. Also, in the medical field, there is a process called B / F separation in order to separate components that have not reacted with the fluorescent substance adsorbed on the particles when testing using an antigen-antibody reaction. Particle separation is also used for inspection. However, since many of the particle separations used here use magnetic particles to perform particle separation and washing with a magnet, there is a problem that this process takes time.

  In addition, as a technology for separating finer particles (DNA, vaccines, etc.), the target size can be determined from the mainstream flow trajectory using the Brownian vibrations of the particles, which are regularly arranged with micro-processed bowl-shaped projections called brownia ratchet. There is also a technique of diverting (separating) the DNA or vaccine (Non-patent Document 1). However, since this method is a split flow formation by Brownian vibration as a separation mode, the separation speed is slow, and the main flow formation is performed in the form of electrophoresis, and it takes time for the separation, especially with a diameter of 1 μm or more. For particles, it took a long time to separate, and due to the influence of disturbance during that time, there was a problem that it could not withstand practical use.

  As a method for separating or classifying cells based on their size, a method for measuring and separating the size of cells by measuring forward scattered light using FACS is known. FACS can roughly separate the target from a large amount of fine particles and cells, but it is difficult to accurately separate a small amount due to the influence of the shape and refractive index of the cell, and the target is weak against impact. May be destroyed during separation.

  There are techniques and devices for separating particles of a specific size from particles having various sizes, such as sieve classification, gravity classification, centrifugation, electrophoresis, etc. Each is suitable for batch processing, but unsuitable for continuous separation processing.

  On the other hand, in recent years, research has been reported on the accurate separation of microparticles and cells by using a microchannel structure (microchannel) with a width of several microns to several hundreds of microns, which is manufactured using microfabrication technology. (Non-Patent Documents 2 to 4). In general, a stable laminar flow is formed in a microchannel, and the flow profile can be controlled arbitrarily on the order of micrometers. By using such features, fine particles and cells can be accurately and continuously produced. Can be separated.

  For example, Patent Document 1 reports a technique called “Pinched Flow Fractionation” (hereinafter referred to as “PFF”) that separates cells and microparticles accurately and continuously according to size. The advantage of this method is that it does not require complicated equipment and devices such as optical systems and information processing systems required for the FACS, and enables continuous particulate and cell separation easily. Although it is a separation that is used, it can be separated into multiple fractions, that is, when separating by size, for example, it is separated not only into two stages, large and small, but also into groups of three or more groups according to size In addition, it is possible to separate a large amount and high speed of fine particles and cells by parallelizing the flow paths.

  In PFF, a fluid is introduced from a plurality of branches into a channel whose diameter is partially narrowed (hereinafter referred to as a constricted channel), the position of particles in a direction perpendicular to the flow is controlled, and then the relative diameter When a particle flows through a thick channel (hereinafter referred to as an enlarged channel), the direction of force applied to the particle by the position of the particle in the narrow channel is noticed. It is a technology that can be separated accurately and precisely. In PFF, as described in Patent Document 1, a structure having two inlets can be used. For example, a liquid containing target particles from one inlet and a liquid not containing target particles flow from the other inlet. Can be implemented.

  In PFF, in order to separate more accurately, it is important to align the particles on the wall surface of the constricted flow path. For that purpose, the flow rate of the liquid containing the particles (hereinafter referred to as sample liquid) The ratio of the liquid not included (hereinafter referred to as the sheath liquid) is small, the ratio, the width and length of the narrowed channel, the width of the enlarged channel, the width of the narrowed channel and the enlarged channel It is important to adjust the ratio and the height of each flow path.

JP 2005-205387 A Japanese Patent No. 2845620

Proceedings of the National Academy of Sciences of the United States of America, 96, 23, 13165-13169, 1999. Proceedings of the National Academy of Sciences of the United States of America, 96, 23, 13165-13169, 1999. Analytical Chemistry, 76, 5465-5471, 2004. Lab on a chip, 5, 1233-1239, 2005. Lab on a chip, 6, 974-980, 2006. Lab on a chip, 5,778-784, 2005.

  However, the elements for accurately aligning the particles on the wall of the stenosis channel are the sample liquid flow rate, the sheath liquid flow rate, the width and length of the stenosis channel, the width of the expansion channel, the width of the stenosis channel and the expansion channel. There are a plurality of ratios of the widths of the channels and the heights of the respective channels, and these are intricately related to each other. Therefore, it takes time to optimize the conditions.

  An object of the present invention is to provide a particle separation apparatus capable of separating particles with higher accuracy in a PFF by a simpler method, and a method therefor.

  The present inventors have studied in view of the above problems, and by stiffening the material of the flow path, the expansion of the flow path wall in the outward direction can be suppressed, and thereby the generally assumed position of the flowing particles We found that the accuracy of the flowing particle position (Precision) is increased instead of increasing the accuracy of the particle. Based on this discovery, the inventors have come to the invention of a particle separation apparatus and method for separating particles more accurately in PFF. Here, “accuracy” refers to the degree of deviation between the theoretical value and the measured value of the particle position from the enlarged region wall surface, and “accuracy” refers to the particle position measured from the enlarged region wall surface multiple times. Show how reproducible it is.

  Here, the hardness in this specification refers to durometer hardness (use of type A durometer), and is defined by the amount of deformation of a substance when a constant load is applied. The larger the hardness, the smaller the amount of deformation.

  In the PFF, under the liquid feeding condition in which the particles are aligned with the narrow channel wall, the particle position in the enlarged channel can be predicted from the particle diameter, the narrow channel width, and the enlarged channel width. However, in general, the inner wall surface of the micro flow channel through which the fluid flows is always exposed to the shearing force of the fluid, and thus a force is applied in the direction in which the micro flow channel expands during liquid feeding. Therefore, the prediction of the particle position in the enlarged flow path needs to take into account the expansion of each flow path due to liquid feeding.

  In the case of a flow path made of an elastic silicone resin containing polydimethylsiloxane (hereinafter referred to as PDMS), it is expected that the flow path is susceptible to the expansion of each flow path, particularly the narrow flow path width. Therefore, it can be said that the position in the enlarged flow path can be predicted more accurately by using a material that does not easily expand, that is, by changing to a resin having higher hardness. The depth direction is not particularly limited and may be a material that expands. The width of the stenosis channel referred to here is the direction perpendicular to the wall surface of the inner wall 16a of the stenosis channel (hereinafter referred to as the width direction), and the depth is the direction perpendicular to the width direction (hereinafter referred to as the depth direction). The length of the flow path. However, this method is intended to increase the “accuracy” of the particle position, and is not considered to increase the “accuracy” of flowing all the flowed particles to a certain position every time.

However, in the present invention, it has been found that the accuracy of the position where particles flow increases by increasing the hardness of the flow path. This is considered to be because the unevenness of the flow velocity due to the expansion of the flow channel can be prevented by suppressing the expansion of each flow channel, and the particles are accurately aligned on the wall surface of the narrowed flow channel.
In view of the above, the present invention therefore relates to:
[1] including at least one branch flow path having a fluid introduction port at one end, and a flow path formed by joining the branch flow paths, and being separated from the fluid introduction port of at least one branch flow path A particle separation device into which a fluid containing particles is introduced,
(1) The downstream side expands from the expansion start point on the end side of the flow path formed by merging, and at least one recovery port is provided at the end of the expanded flow path,
(2) The particle separation apparatus, wherein a wall of the flow path from the junction point of the branch flow path to the expansion start point is made of a material that hardly expands outward.
[2] The particle separator according to item 1, wherein the material has a durometer hardness of 40 or more.
[3] The particle separator according to item 1 or 2, wherein the flow rate of the fluid containing the particles to be separated is smaller than the flow rate of the other fluids.
[4] Item 1 to item 3, characterized in that a laminar flow is formed in a flow path formed by a fluid containing particles to be separated and a fluid introduced into another branch flow path. The particle separator according to any one of the above.
[5] Two or more branch channels having a fluid inlet at one end, and a channel formed by joining the branch channels,
(1) The downstream side expands from the expansion start point on the end side of the flow path formed by joining, and at least one recovery port is provided at the end of the expanded flow path,
(2) A method of separating particles using a flow path characterized in that the wall of the flow path from the junction of the branch flow path to the expansion start point is made of a material that does not easily expand outward. And
Introducing a fluid containing particles to be separated from a fluid inlet of at least one branch channel;
Introducing a fluid that does not contain particles to be separated from a fluid inlet of at least one branch channel;
The method comprising the step of separating or recovering particles from at least one recovery port.
[6] The method according to item 5, wherein the material has a durometer hardness of 40 or more.
[7] The method according to item 5 or 6, wherein the flow rate of the fluid containing the particles to be separated is smaller than the flow rate of the other fluid.
[8] Items 5 to 7, characterized in that a fluid containing particles to be separated and a fluid introduced into another branch channel form a laminar flow in a channel formed by joining. The method as described in any one of.
[9] Before introducing the fluid containing the particles to be separated from the fluid introduction port of the at least one branch flow path, a different fluid is introduced only in that the particles are not included in advance, thereby The method according to any one of items 5 to 8, wherein the method is formed.

  According to the present invention from the above viewpoint, the target particles can be separated more accurately by a simple improvement of changing the material of the flow path. Further, the target particles separated by branching the channel end can be recovered. In addition, since the present invention is configured as described above, it exhibits an excellent effect that no complicated operation or equipment is required. In addition, since the present invention is configured as described above, it is possible to continuously separate and collect particles, and to process a large amount of microparticles and cells compared to separation technology of batch processing. Exhibits the excellent effect of being able to.

1 shows a microchip 10 equipped with an embodiment of a continuous particle classification method according to the present invention, FIG. 1 (a) is a view taken in the direction of arrow A in FIG. 1 (b), and FIG. 1 (b) is in FIG. 1 (a). It is sectional drawing by a BB line. Fig. 2 shows a microchip 10 equipped with an embodiment of a continuous particle classification method according to the present invention, Fig. 2 (a) is a view taken in the direction of arrow A in Fig. 2 (b), and Fig. 2 (b) is shown in Fig. 2 (a). It is sectional drawing by a BB line, FIG.2 (c) is an enlarged view of the part 21 in Fig.2 (a). (A) Separation evaluation of 2 μm particles using a microchip 10 made of a material having hardness 70 (Example 1) (b) Separation evaluation of 2 μm particles using a microchip 10 made of a material having hardness 60 ( Example 2) (c) Separation evaluation of 2 μm particles using a microchip 10 made of a material having a hardness of 44 (Example 3) (d) 2 μm particles using a microchip 10 made of a material having a hardness of 30 Of separation (Comparative Example 1) FIG. 4 is an explanatory schematic view showing the behavior of fluid introduced from two branches and the state of particle separation in the microchip 10 shown in FIG. 2A, and FIG. 4 is an enlarged view of a portion 21 in FIG. is there.

  Hereinafter, the best mode for carrying out the present invention will be described in detail with reference to the drawings. However, the present invention can be implemented in many different forms, and is not limited to the following embodiments and examples.

  First, the details of the principle of pinched flow fractionation (PFF) will be described with reference to FIG.

  FIG. 4 shows a narrowed channel and an enlarged channel formed by joining the channels in the microchip of FIGS. 1 and 2. Specifically, it is an enlarged view of a portion 21 in FIG. Further, the fluid 100P and the fluid 100N shown by hatching are a fluid containing particles and a fluid not containing particles, respectively, and the particles 300a and 300b are relatively large particles and relatively small particles, respectively. Yes.

  In FIG. 4, an arrow 200 indicates a streamline profile at the boundary between the constricted flow channel 16 and the enlarged flow channel 17, and arrows 210a and 210b indicate motion vectors of the large particles 300a and the small particles 300b, respectively. ing.

  First, the fluid 100P including particles and the fluid 100N not including particles are continuously supplied from the two inlets 14a and 14b using a syringe pump or the like. At this time, each fluid flows in the flow path 13 while maintaining a stable laminar flow.

  Then, by adjusting the flow rates of the fluid 100P containing particles and the fluid 100N not containing particles, the width of the fluid 100P in the constricted channel 16 (the distance from the channel wall surface to the interface between the fluid 100P and the fluid 100N) The particle size should be smaller than the minimum particle size to be separated. By this operation, all the particles to be separated flow along the inner wall 16a of the constricted flow path, and the position of the particles in the direction perpendicular to the wall surface of the inner wall 16a of the constricted flow path is determined as the size of the particle. Can be made constant.

  Since the streamline spreads as shown in the profile 200 at the boundary between the narrowed channel 16 and the enlarged channel 17, the distance between any streamlines in the narrowed channel 16 is further enlarged in the enlarged channel 17. Is done.

  Therefore, since the position of the particles in the direction perpendicular to the flow in the narrowed flow path 16 differs depending on the size of the particles, the motion vector 210a of the large particles 300a and the small particles at the boundary between the narrowed flow path 16 and the enlarged flow path 17. A difference occurs in the direction of the motion vector 210b of 300b, and the positional difference for each particle size is enlarged in the enlarged flow path 17 to enable classification.

  By observing the separated particles along the detection line 20 in the enlarged flow path 17 using an appropriate detection system, the particle size of the particles and the particle size distribution of the particle group can be examined.

  1 (a) and 1 (b) show a microchip 10 equipped with an embodiment of a continuous classification method according to the present invention, and FIG. 1 (a) is a view taken along arrow A in FIG. 1 (b). FIG.1 (b) is sectional drawing by the BB line in Fig.1 (a).

  The microchip 10 in FIG. 1 has a rectangular cross-sectional shape at any point, and the flow path depth is uniform. The cross-sectional shape is preferably rectangular in order to separate the particles by two factors in the cross-section and from the viewpoint of ease of production of the flow channel structure, but partially has a shape other than rectangular, for example, a circular shape. It may have a cross section, and the depth may be partially different.

  The microchip 10 is a microchip for measuring the particle size and particle size distribution by separating particles according to their sizes and using an appropriate detection system. For example, the microchip 10 is a polymer such as PDMS. It has a flat structure formed of two flat substrates 11 and 12 formed of a material.

  In addition, as a technique used when manufacturing the microchip 10, for example, a manufacturing technique using a mold such as molding or embossing is preferable in that the flow channel structure can be accurately and easily manufactured. Manufacturing techniques such as wet etching, dry etching, nanoimprinting, laser processing, direct electron beam drawing, and machining can also be used.

  In addition, as a material for manufacturing the microchip 10, it is necessary to configure the wall surface of the flow path with a material that does not easily expand, but at least the wall surface of the flow path is configured with a material that does not easily expand in the width direction of the flow path. It is necessary to. More specifically, it is preferably made of a material having a durometer hardness of 40 or more. Examples of usable materials include various polymer materials such as PDMS and acrylic, various metals such as glass, silicone, ceramics, and stainless steel. More preferably, various polymer materials such as PDMS having a durometer hardness of 40 or more, acrylic resin, glass, silicone, ceramics, stainless steel, and the like can be used. Of these materials, any two kinds of materials can be used in combination. However, in order to produce the flow path itself at a low cost and provide a disposable apparatus, it is preferable to use a polymer material at least partially, and it is more preferable that the whole is made of a polymer material. As such a material, PDMS having a durometer hardness of 40 or more, an acrylic resin, and the like are particularly preferable.

  And the flow path 13 is formed in the lower surface 11a of the board | substrate 11, and the depth of a flow path can be set to arbitrary values from 10 nm to 1 cm. It is preferable to set the value from micron to several tens of μm.

  The flow path 13 has inlets 14a and 14b and an outlet 15. The inlets 14a and 14b are fluid inlets for a fluid containing particles and a fluid not containing particles, respectively, and the outlet 15 is an outlet for the fluid.

  In addition, one end of the flow path 13 is formed by two branch flow paths (18a, 18b), and the flow path 13 is composed of a narrowed flow path 16 and an enlarged flow path 17 having two different shapes. Yes. The connection portion between the narrowed channel 16 and the enlarged channel 17 can be referred to as an enlargement start point, and the channel width changes at the enlargement start point as shown in FIG. The enlarged flow path 17 may have an arbitrary shape as long as the flow path width is larger than that of the narrowed flow path 16. Here, the branch channels 18 a and 18 b are connected to the inlets 14 a and 14 b, respectively, and the enlarged channel 17 is connected to the outlet 15.

  The entire length of the flow path 13, that is, the length from one end where the inlets 14 a and 14 b are located to the other end where the outlet 15 is located can be set to an arbitrary value of 1 μm or more. However, it is preferable to set the thickness to about several μm to several tens of mm from the viewpoint of easy flow path fabrication and pressure loss.

  The lengths of the constricted flow channel 16 and the enlarged flow channel 17 can be set to arbitrary values of 10 nm or more, but from the viewpoint of ease of flow channel fabrication and pressure loss, it is about submicron to several tens of mm. It is preferable to set. Further, from the viewpoint of achieving particle alignment, the lower limit of the length of the constricted flow path 16 is preferably 1 μm or more, more preferably 10 μm or more, and the upper limit of the length of the constricted flow path 16 is 500 μm or less. Is preferably 100 μm or less.

  The width of the narrowed channel 16 refers to the length of the channel in the direction perpendicular to the wall surface of the inner wall 16a of the narrowed channel. The width of the narrowed channel 16 can be set to an arbitrary value of 10 nm or more as long as the condition that it is less than the width of the enlarged channel 17 is satisfied. Further, from the viewpoint of achieving particle alignment, the lower limit of the width of the constricted flow channel 16 is preferably 1 μm or more, more preferably 10 μm or more, and the upper limit of the width is preferably 100 μm or less, and further is 20 μm or less. preferable.

  Note that a plurality of outlet ports 15 of the enlarged flow path 17 may be provided. Based on the principle of “Asymmetric Pinched Flow Fractionation (AsPFF)” (Non-Patent Document 5), Separation can also be improved by flowing a large amount of fluid through the outlet port. More specifically, the resistance value of the flow path is appropriately designed, and a particle having a specific size can be introduced only into a specific outlet port from the relationship between the flow path width and the particle diameter of the narrowed flow path 16. This is the principle.

  In the case of a continuous particle separation device having a plurality of outlets and the method thereof, the entire flow channel network is regarded as an analogy of the resistance circuit, and an appropriate flow distribution ratio can be achieved by performing an appropriate design. It is desirable to design based on such an idea.

  And, as a method of adjusting the introduction amount for achieving these flow rate conditions inside the flow path, it is convenient and preferable to introduce the solution from the introduction port using a syringe pump or the like. It is also possible to use a method using a pump, a constant pressure liquid feeding method using a cylinder, a pressure device, or the like, a liquid feeding method using an electroosmotic flow, centrifugal force, or the like. A pressure device that applies negative pressure from the outlet can also be used. In addition, it is desirable that the liquid feeding pressure is in a range where liquid feeding can be stably performed, and is preferably 20 MPa or less from the viewpoint of durability of the flow path and the liquid feeding pump itself.

  In order to achieve stable and efficient separation of particles, it is preferable to maintain a stable laminar flow in the flow path. Specifically, the particles are fed under conditions where the Reynolds number is 1000 or less. It is preferable to perform a liquid operation. However, when a channel structure having a diameter of 1 mm or less is used, it is relatively difficult to form a turbulent flow, and it is easy to achieve such a condition.

  Moreover, although the width | variety of the branch flow paths 18a and 18b used the thing of 100 micrometers, these values can each be set to arbitrary values 10 nm or more.

  As the particles to be separated, fluorescent polystyrene-divinylbenzene beads having a diameter of 2 μm were used, but depending on the purpose, polymer particles such as polystyrene, metal fine particles, ceramic particles, or their surfaces were physically or chemically treated. In addition to the above, particles such as animal and plant cells, bacteria, bacteria, viruses and organelles such as organelles, proteins, protein aggregates, and the like can be used. As long as the maximum diameter of the particles to be classified is equal to or smaller than the width of the constricted flow path 16 in the flow path 13, particles of any size can be separated. For example, when a channel having a width of the narrow channel 16 of 20 μm is used, the diameter of the separated particles is 0.1 μm to 20 μm, and more preferably 1 μm to 10 μm.

  As the fluid in which the particles are suspended, the 0.5 wt% Tween 80 aqueous solution is used, but any liquid or gas can be used, and various solutions can be used depending on the purpose. For example, when polymer particles or metal particles are used as the particles, an organic solution, an ionic fluid, or the like can be used in addition to an aqueous solution containing various chemical substances. Furthermore, when biological particles such as cells are used as the particles, it is preferable to use an aqueous solution that is isotonic with cells, such as a cell culture solution or a buffer solution. However, in the case of cells that are resistant to relatively hypotonic or hypertonic solutions, such as bacteria and plant cells, it is not always necessary to be isotonic. In view of operation, a system in which the difference between the density of the solution and the density of the particles is not large is more preferable. Regarding the viscosities of the sample liquid and the sheath liquid, a system having a small difference in viscosity is more preferable.

  Both the fluid containing particles and the fluid not containing particles may be composed of the same fluid, or may be composed of two or more different fluids.

  The particles introduced into the flow path 13 move in the downstream direction along the flow. At this time, by appropriately adjusting the flow rate of the fluid introduced from the two inlets 14a and 14b, In the narrowed channel 16, the position of the particles in the direction perpendicular to the wall surface of the inner wall 16a of the narrowed channel can be controlled by the size of the particles. Here, in order to align the particles in the sample liquid to the wall surface of the narrow channel, the ratio of the flow rate of the sheath liquid to the sample liquid is preferably 1 or more, more preferably 10 or more, and more preferably 50 or more. Most preferred. The sum of the flow rates of the sample liquid and the sheath liquid is preferably set so that the liquid feeding pressure does not become too large, and the sum of the flow rates of the sample liquid and the sheath liquid is preferably 10 mL / hour or less. Most preferred is 02 mL / hour.

  In the above configuration, the present invention is characterized in that the width of the constricted flow path 16 is difficult to expand. About the depth direction, you may be comprised with the raw material which expand | swells. Since it is cumbersome to change the degree of expansion in the width direction and depth direction of the flow path when manufacturing the flow path, the above-described microchip 10 is manufactured by PDMS having a hardness of 30, 44, 60, and 70. However, the hardness can be set to an arbitrary value, and preferably has a hardness of 40 or more, more preferably a hardness of 70 or more. Here, as described above, the hardness in the present specification refers to durometer hardness (uses a type A durometer), and is defined by the amount of deformation of a substance when a constant load is applied. small.

  The hardness test is evaluated according to JIS K 6253 5 (durometer hardness test) in accordance with the standard of JIS K 6249. In the test, a type A durometer hardness tester is mounted on an indentation depth measuring device consisting of a length measuring device having a measurement range of indentation depth of 0.000 to 2.500 mm and a displacement device, and the length is measured. It is desirable to use a micrometer for the system. The length measuring instrument (the tip of the spindle of the micrometer) is fixed so that it is in vertical coaxial contact with the push needle, and the spindle is moved to give displacement to the push needle. The pusher of the hardness tester is displaced from 100 to 0 according to each display, or the indicated value of the hardness tester for a known indentation depth value is verified. The indentation depth is preferably verified at least at four locations including the instruction values 100 and 0.

  The present invention can be applied not only to the above-described PFF but also to other separation methods. For example, the application to capillary hydrodynamic fractionation (hereinafter referred to as CHDF) using a capillary is described in detail below. Describe.

  As described in Patent Document 2, CHDF is a technique for separating particles of submicron size using hydrodynamics in a capillary. DiMarzio and Guttman (Polymer Letters 7 267 (1969)) It is shown that it depends on factors. That is, (1) large particles cannot approach the capillary wall as much as small particles, so that large particles are distributed near the center in the capillary radial direction and small particles are unevenly distributed outside the cross section. (2) The particles can move at different velocities because of the radial velocity characteristics caused by the fluid passing through the capillary tube. As such, large particles generally move at a faster rate than the average eluent velocity, and sample faster flow streamlines.

  However, here, the fraction of small particles spreads to the fraction of large particles, and there may be a problem in fractionation with large particles. At this time, (1) a step of aligning the particles in the capillary radial direction by fluidly pressing the liquid containing particles on the capillary wall surface with a liquid not containing particles is added upstream of the capillary separation method. (2) It is reported that large particles and small particles can be fractionated by separating large particles and small particles according to the flow velocity distribution in the downstream capillary tube. Application No. 2016-235056).

  The method has been described using a capillary, but the material can be implemented not only using a hard material such as glass or plastic, but also using a silicone resin such as PDMS. At this time, since the particle fraction is greatly affected by the position of the particles in the direction perpendicular to the wall surface of the inner wall 16a of the constricted flow path (particle position in the direction perpendicular to the flow), the silicone resin When the inner diameter of the conductive capillary expands, there is a concern that the separation performance is degraded. Therefore, as the present invention has found, it can be said that the separation performance is improved by using a material having high hardness for the capillary.

Example 1
Hereinafter, an embodiment of a continuous particle classification method according to the present invention will be described in detail with reference to the attached document in accordance with the drawing of FIG.
The microchip 10 was produced using LSR7070 (Momentive, hardness 70). The microchip 10 has a flat structure formed by two flat substrates 11 and 12.
A flow path 13 is formed on the lower surface 11a of the substrate 11. The inlet-side ports 14a and 14b, the outlet 15 (each having a hole diameter of 2 mm), the entire length of the flow path 13, that is, the inlets 14a and 14b The length from one end to the other end with the outlet 15 is 19 mm.
The depths from the flow path 18 to the flow path 17 are all 20 μm, the branch flow paths 18a and 18b (width 100 μm, length 800 μm), the narrow flow path 16 (width 20 μm, length 50 μm), the enlarged flow path 17 (width 500 μm, A flow path having a length of 10 mm) was produced.
The fluid 100P containing the particles was diluted with 1 μg / mL of fluorescent polystyrene-divinylbenzene particles having a diameter of 2 μm (Fluoro-Max; manufactured by Thermo Scientific) using a 0.5 wt% Tween 80 aqueous solution. As the fluid 100N containing no particles, a 0.5 wt% Tween 80 aqueous solution was used.
The flow rate of the fluid 100P was adjusted from the inlet 14a and the flow rate of the fluid 100N from 14b by a syringe pump with a flow rate of 20, 1000 μL / h, respectively. The trajectory of the fluorescent particles passing through the detection line 20 was photographed with a fluorescent microscope as a moving image, and the resolution was evaluated by measuring the passing coordinates from the lower wall surface of FIG. The detection line 20 that is a measurement position was set to a position 150 μm from the right end (enlargement start point) of the constriction flow path 16.
The graph which showed the measurement result in Fig.3 (a) is shown. The horizontal axis is the passing coordinates on the detection line 20 (X μm), and the vertical axis is the count number for each coordinate. It can be seen that the particle distribution is narrower and the separation accuracy is higher than in Comparative Example 1.

Example 2
Particle separation evaluation was performed in the same manner as in Example 1 except that the material used for the production of the microchip 10 was changed to LSR7060 (manufactured by MOMENTIVE, hardness 60). The graph which showed the measurement result in FIG.3 (b) is shown. It can be seen that the particle distribution is narrower and the separation accuracy is higher than in Comparative Example 1.

Example 3
Particle separation evaluation was performed in the same manner as in Example 1 except that the material used for the production of the microchip 10 was changed to SILPOT184 (manufactured by TORAY, hardness 44). The graph which showed the measurement result in FIG.3 (c) is shown. It can be seen that the particle distribution is narrower and the separation accuracy is higher than in Comparative Example 1.

Comparative Example 1
Particle separation was evaluated in the same manner as in Example 1 except that the material used to produce the microchip 10 was changed to LSR7030 (MOMENTIVE, hardness 30). FIG. 3D shows a graph showing the measurement results. Compared with Example 1, it can be seen that the particle distribution is wide and the separation accuracy is low. Using the material having a hardness of 40 or more with the above results, the separation accuracy can be increased.

10 Microchip 11 Substrate 11a Substrate lower surface 12 Substrate 13 Channel 14a, 14b Inlet 15 Outlet 16 Narrow channel 16a Inner wall 17 of narrow channel 16 Expanded channel 18a, 18b Branch channel 20 Detection line 21 of FIG. Partial 100P Fluid containing particles 100N Fluid not containing particles 200 Streamline profiles 210a, b Vectors 300a, b Particles

Claims (9)

Including at least one branch channel having a fluid inlet at one end, and a channel formed by joining the branch channels, and containing particles to be separated from the fluid inlet of at least one branch channel A particle separation device into which a fluid is introduced,
(1) The downstream side expands from the expansion start point on the end side of the flow path formed by merging, and at least one recovery port is provided at the end of the expanded flow path,
(2) The particle separation apparatus, wherein a wall of the flow path from the junction point of the branch flow path to the expansion start point is made of a material that hardly expands outward.   The particle separator according to claim 1, wherein the material has a durometer hardness of 40 or more.   The particle separation device according to claim 1 or 2, wherein a flow rate of a fluid containing particles to be separated is smaller than a flow rate of another fluid.   The laminar flow is formed in a flow path formed by a fluid containing the particles to be separated and a fluid introduced into another branch flow path. The particle separator according to one item. Two or more branch channels having a fluid inlet at one end, and a channel formed by joining the branch channels,
(1) The downstream side expands from the expansion start point on the end side of the flow path formed by joining, and at least one recovery port is provided at the end of the expanded flow path,
(2) A method of separating particles using a flow path characterized in that the wall of the flow path from the junction of the branch flow path to the expansion start point is made of a material that does not easily expand outward. And
Introducing a fluid containing particles to be separated from a fluid inlet of at least one branch channel;
Introducing a fluid that does not contain particles to be separated from a fluid inlet of at least one branch channel;
The method comprising the step of separating or recovering particles from at least one recovery port.   The method according to claim 5, wherein the material has a durometer hardness of 40 or more.   The method according to claim 5 or 6, wherein the flow rate of a fluid containing particles to be separated is smaller than the flow rate of other fluids.   The fluid containing the particles to be separated and the fluid introduced into another branch flow path form a laminar flow in the flow path formed by merging. The method according to claim 1.   Before the step of introducing the fluid containing the particles to be separated from the fluid introduction port of at least one branch flow path, a fluid that is different only in that the particles are not included in advance is introduced to form a laminar flow. A method according to any one of claims 5 to 8, characterized in that