JP6615499B2 - Separation and capture device for cells or liposome particles - Google Patents

Description

  The present invention relates to a particle separation and capture device having a small bending elastic modulus.

  Conventionally, research using particles having a small bending elastic modulus such as cells or liposomes has been conducted. In conducting such research, it may be necessary to sort the particles according to particle size, or to capture the particles and arrange them in a plane to form an array.

  For this purpose, for example, Non-Patent Documents 1 and 2 listed below disclose a method for separating PS beads by DLD (Deterministic Lateral Displacement Deterministic Lateral Replacement Method). Non-Patent Documents 3 and 4 below disclose a method for arranging cells and liposomes in parallel space using a microfluidic device.

L. R. Huang et al., Science, 304, 987 (2004). D. W. Inglis et al., Lab Chip, 6, 655 (2006). A. M. Skelley et al., Nat. Methods, 6, 147 (2009). T. Robinson et al., Lab Chip, 14, 2852 (2014).

  However, in the above-described conventional technology, it has not been possible to continuously select particles such as cells and liposomes having a small bending elastic modulus based on the particle diameter or the bending elastic modulus and arrange them in a plane.

  SUMMARY OF THE INVENTION An object of the present invention is to provide a separation and capture device for particles having a small bending elastic modulus, which can easily sort particles having a small bending elastic modulus according to particle size or bending elastic modulus and arrange them in a plane. It is in.

  In order to achieve the above object, one embodiment of the present invention is a particle separation / capture device having a small bending elastic modulus, and is a specific particle size among particles having a small bending elastic modulus flowing dispersedly in a fluid. Alternatively, the particle capturing unit includes a particle capturing unit that captures particles having a specific bending elastic modulus at the center of distribution and arranges the particles in a plane, and the particle capturing unit includes the trap for capturing the particles and the particle capturing unit for the fluid. And a flow state adjusting unit that maintains a laminar flow state on the upstream side.

  Further, another embodiment of the present invention is a device for separating and capturing particles having a small bending elastic modulus, wherein the particle trajectory of the particles having a small bending elastic coefficient dispersed and flowing in a fluid is changed to the particle size or bending elasticity of the particles. The particle separation part that is separated according to the coefficient and separated for each particle diameter or each bending elastic modulus, and among the particles separated by the particle separation part, a specific particle diameter or a specific bending elastic coefficient is at the center of the distribution. A particle trap that traps certain particles and arranges the particles in a plane, and the particle trap maintains the laminar flow of the trap for trapping the particles and the fluid in the particle separator. And a flow state adjusting unit.

  The particle separation portion is formed in a tunnel-shaped flow path having a rectangular cross section, and is perpendicular to the fluid flow direction, and connects a columnar shape between one and the other surfaces of the tunnel-shaped flow path. It is preferable that the obstacles are arranged at a constant angle with respect to the fluid flow direction.

  The particle trapping portion is formed in a tunnel-shaped channel having a rectangular cross section, and the trap connects between one and the other of the mutually facing surfaces of the tunnel-shaped channel, and It is preferable to have an opening for receiving particles on the upstream side in the fluid flow direction and a drainage port for securing the fluid flow on the downstream side.

  The trap is disposed upstream of the opening in the fluid flow direction, at a position extending a wall-like protrusion that forms the opening, and between the wall-like protrusion and a drainage channel. It is preferable to provide a pair of gate pillar structures that form

  Moreover, it is preferable that the flow state adjusting unit is formed with a dummy trap having an opening for receiving particles on the upstream side in the fluid flow direction. In that case, a dummy trap having an opening for receiving particles on the upstream side and a drainage port for securing a fluid flow on the downstream side is more preferable.

  Further, it is preferable that a drainage port is formed in the dummy trap, and the drainage port is formed in a gap larger than the drainage port of the trap.

  According to the present invention, it is possible to easily select particles having a small bending elastic modulus based on the particle diameter, and arrange them in a plane to form an array.

It is a figure which shows the structural example of the isolation | separation capture device of the particle | grains with a small bending elastic modulus concerning embodiment. It is a figure which shows the example of the arrangement | sequence method of the micro pillar concerning embodiment. It is a top view of the example of the geometrical structure of the trap A concerning embodiment, dummy trap A ', B, B', B ". It is a top view of the modification of trap A concerning an embodiment. It is a figure which shows the particle size distribution obtained by the particle size analysis after the separation capture operation by the separation capture device concerning an Example.

  Hereinafter, modes for carrying out the present invention (hereinafter referred to as embodiments) will be described with reference to the drawings.

  FIGS. 1A and 1B show a configuration example of a particle separation and capture device having a small bending elastic modulus according to the embodiment. FIG. 1A is a plan view, and FIG. 1B is a cross-sectional view taken along line bb in FIG.

  In FIG. 1 (a), the separation / capture device includes a particle separation unit 10 and a particle capture unit 12. The particle separation unit 10 uses the flow direction (orbits) of particles having a small bending elastic modulus dispersed in the fluid (the particle size is not constant and includes various particle sizes) as the particle size of the particles. Different according to the particle size. The particle separation unit 10 is formed in a channel formed in a tunnel shape having a rectangular cross section, and is perpendicular to the fluid flow direction and between one side and the other side (bottom surface and ceiling surface) of the tunnel facing each other. A columnar obstacle to be connected (hereinafter sometimes referred to as a micro pillar) is formed at a certain angle with respect to the flow direction of the fluid, and a DLD is formed. The fluid flowing in the tunnel of the particle separation unit 10 is designed to be in a laminar flow state, and the particles are moved by the fluid to move.

  In FIG. 1B, the flow path 14 of the particle separation unit 10 has a tunnel shape with a rectangular cross section, and the micro pillar 16 has a bottom surface U and a ceiling surface C of the tunnel-shaped flow path 14 facing each other. It is formed as a columnar obstacle that connects them. In FIG. 1B, three micro pillars 16 are described. This is a schematic representation of the micro pillars 16 appearing in the cross-sectional view, and the accurate arrangement of the micro pillars 16 is shown. It is not a representation.

  The micro pillars 16 are arranged at a constant angle with respect to the fluid flow direction. The particle separation unit 10 in the example of FIG. 1A includes two separation regions 10a and 10b having different angles of the arrangement of the micro pillars 16. Although the separation regions 10a and 10b will be described later, the number of separation regions is not limited to two, and an arbitrary number of separation regions can be provided according to the properties, particle sizes, and the like of the particles to be separated.

  FIG. 1A also shows an example of a method for supplying the particles to the separation and capture device for particles having a small bending elastic modulus and a fluid for flowing the particles according to the embodiment. In FIG. 1A, the particles dispersed in an appropriate fluid are introduced from the flow path center of the upstream separation region 10a in the particle separation unit 10 by the flow path I, and two flow paths II and III from both sides thereof. The same fluid as the fluid in which the particles are dispersed using is introduced into the separation region 10a. Thereafter, the fluid flows in the order of the separation region 10b on the downstream side, the particle capturing unit 12 disposed further downstream, and the drain port (not shown) disposed on the downstream side of the particle capturing unit 12. The fluid in which the particles are dispersed is introduced into the separation region 10a by being sandwiched between the fluids supplied to both sides thereof, so that the particles are placed in a laminar flow in the flow path of the particle separation unit 10 (separation region 10a, 10b) can be moved.

  As the fluid, distilled water, ultrapure water, deionized water, an aqueous solution containing sugar or inorganic salts, and a buffer solution are suitable. When the particles are cells, a cell culture solution or a buffer solution having a high salt concentration is preferable.

  FIG. 2 shows an example of a method for arranging the micro pillars 16. In FIG. 2, the micro pillar 16 has a cylindrical shape and is indicated by a circle (◯). The fluid flow direction is indicated by an arrow F, and a line segment parallel to the line connecting the centers of the micro pillars 16 is indicated by Lc. The shape of the micro pillar 16 is not limited to a cylindrical shape, and may be a triangular prism shape, for example.

  As shown in FIG. 2, the micro pillars 16 are arranged such that a line segment Lc connecting the centers forms a certain angle θ with respect to the fluid flow direction F. The angle θ is determined by the shift rate ε of the micro pillar 16. Here, the shift rate ε is the ratio of the center shift with respect to the center-to-center distance of the micropillars in the direction perpendicular to the flow direction F between the micropillars adjacent in the fluid flow direction F (direction parallel to the line segment Lc). It is. That is, as shown in FIG. 2, when the distance between the centers of the micro pillars (the distance between the centers in the fluid flow direction F and the direction perpendicular thereto) is λ, ε × λ is the distance between the adjacent micro pillars. Of the center in the direction orthogonal to the flow direction F. In FIG. 2, g is the distance between the walls of the micro pillar (the shortest distance between the surfaces, that is, the distance between the surfaces on the line segment connecting the centers in the direction orthogonal to the fluid flow direction F). According to Non-Patent Document 2, a particle having a small bending elastic modulus moves along the flow direction F while the flow is blocked by the micropillar, and the flow in the flow direction F while the flow is blocked by the micropillar. The boundary particle size with the particle size moving with the angle component in the orthogonal direction is determined by the values of the inter-wall distance g and the shift rate ε.

  Further, when particles having a small bending elastic modulus are caused to flow through the particle separation unit 10 (DLD), the flow direction (orbit) can be varied depending on the “bending elastic modulus” not only in the particle diameter but also in the same particle diameter. . For this reason, the separation and selection of particles based on the bending elastic modulus are possible. In this case, if the particle size is not uniform, the DLD principle works simultaneously on the difference between the particle size and the flexural modulus. Can be sorted. If particles having a substantially constant particle diameter are selected in advance using the principle of sieving and then flowed to the particle separator 10, the particles can be selected only by the difference in bending elastic modulus.

  As described above, the particle separation unit 10 is formed with two separation regions 10a and 10b. In these separation regions 10a and 10b, the shift rate ε in the separation region 10a is greater than the shift rate ε in the separation region 10b. It is set small. In the separation regions 10a and 10b shown in FIG. 1A, the particle size or bending of the particles separated by changing the value of the shift rate ε between the separation regions 10a and 10b while keeping the distance g between the walls constant. The elastic modulus is controlled.

  The separation regions 10a and 10b of the particle separation unit 10 described above are configured by DLD. However, the present invention is not limited to this, and for example, a pinch flow type particle separation device may be used.

  The particle capturing unit 12 captures particles having a specific particle size (hereinafter referred to as a target particle size) at the center of the particle size distribution among the particles separated by the particle separating unit 10 in a planar manner. Arrange. As described above, among the particles separated by the bending elastic modulus, particles having a specific bending elastic modulus being the center of distribution can be captured and arranged in a plane.

  The particle capturing unit 12 includes a trap that captures the particles and a flow state adjusting unit that maintains the flow state of the fluid in the particle separating unit 10 in a laminar flow. In the example of FIG. 1A, the trap A for trapping particles having a target particle diameter and the dummy traps A ′, B, B ′, B ″ constituting the flow state adjusting unit are arranged in the particle trap 12. Trap A, dummy trap A ′, and dummy traps B, B ′, B ″ have the same geometric structure. These may be similar geometric structures.

  3A and 3B are plan views showing examples of the geometric structure of the trap A and the dummy traps A ′, B, B ′, and B ″. FIG. 3A shows the trap A and the dummy trap. This is an example of A ′, and FIG. 3B is an example of dummy traps B, B ′, B ″. 3A and 3B show a part of the arrangement of the trap A and the dummy traps A ′, B, B ′, B ″. Further, the fluid flows from the left side of the figure. Shall be included.

  In FIG. 3A, the trap A has an opening 18 for receiving particles on the upstream side in the fluid flow direction, and a drain port 20 for securing the fluid flow on the downstream side (bottom side). The opening 18 is formed as a region between two wall-shaped protrusions P protruding upstream in the fluid flow direction. Here, it is necessary to make the gap G of the drainage port 20 as small as possible in the microfabrication so that the trapped particles having a small bending elastic modulus do not slip through with deformation. Therefore, it is preferable that G = x / 5 to x / 4 with respect to the target particle size x of the particles to be captured. Next, it is possible to prevent the trap A from being clogged with particles having a large particle diameter, and the probability that the center of gravity of the particle that bypasses the trap (A) on the front stage (upstream side) will ride on the laminar flow that flows into the trap A on the rear stage (downstream side). Need to be high. For this reason, it is inconvenient if the arrangement interval L of the trap A is too narrow or too wide, so it is preferable to set L = x + 1 to x + 3. Here, the arrangement interval is the distance between the walls of the trap A in the fluid flow direction and the direction orthogonal to the flow direction. Further, it is preferable that the width of the opening 18 formed on the upstream side of the trap A and the width of the wall-like protrusions P on both sides of the opening 18 are the same as L. In the example of FIG. 3, the length of the wall-shaped protrusion P in the fluid flow direction is 3L / 2. However, the length is not limited to this, and the center of gravity of the particles bypassing the trap A in the previous stage is It is only necessary to increase the probability of getting into the laminar flow flowing into the trap A at the subsequent stage. Further, the dummy trap A ′ has the same shape as the trap A.

  As shown in FIG. 3 (a), since traps A are arranged in a plane, if traps A capture particles such as cells and liposomes having a small bending elastic modulus, they can be arranged in a plane at the same time. Therefore, it can be greatly useful for various tests and research on the particles.

  In FIG. 3B, the dummy traps B, B ′, B ″ are formed in a shape similar to that of the trap A. In particular, in the trap B ′, these shapes are larger than the target particle size. It is preferable that the arrangement interval D ′ is designed to be larger than L of the trap A. Further, the width L ′ of the opening 18 and the gap G ′ of the drainage port 20 are determined by the particle size. The flow state in the separation unit 10 is appropriately determined according to the purpose of the flow state adjusting unit for maintaining a laminar flow, and the gap G ′ of the drain port 20 is set so that the laminar flow in the trap region does not change. In addition, it is preferable that the gap G is 2 to 4 times the gap G of the drain port 20 of the trap A. As described above, particles having a larger particle size than the target particle size flow into the trap B ′. Since there is a possibility, the gap G ′ of the drainage port 20 is determined. This point is also taken into consideration when the flow is maintained in the particle separation unit 10 in a laminar flow, even if the dummy traps B, B ′, B ″ are not provided with the drain port 20. Good.

  The trap A and the dummy traps A ′, B, B ′, B ″ are also formed in a tunnel-shaped channel having a rectangular cross section shown in FIG. It is the height which connects between the bottom face U and the ceiling surface C which oppose.

  The dummy traps A ′, B, B ′, B ″ having the above-described configuration are formed in the particle capturing unit 12 together with the trap A, so that the fluid resistance balance in the entire flow path of the particle separating unit 10 and the particle capturing unit 12 is achieved. As a result, a fluid flow field that is symmetric with respect to the central axis of the flow path can be constructed, and as a result, the fluid flows in a laminar flow through the entire flow path, and the particles are separated accurately for each particle size or bending elastic modulus. Also, if any or all of the dummy traps A ′, B, B ′, B ″ are missing, the target particle size particles that should flow into the trap A will be a dummy with a lower fluid resistance. The probability of detouring through an area where there is no trap is increased, and there is a disadvantage that trap A is not captured.

  FIG. 1 (a) also shows the trajectories of particles traveling in the separation regions 10a and 10b. In FIG. 1A, particles having a target particle size determined by the values of the inter-wall distance g and the shift rate ε in the separation regions 10a and 10b are separated from particles having a smaller particle size or larger particles. And guided to the particle capturing unit 12. In FIG. 1 (a), the orbit of the target particle size is indicated by Lt, the orbit of the particle size smaller than the target particle size is indicated by Ls, and the orbit of the particle size larger than the target particle size is indicated by Lb. ing.

  In DLD, particles having a particle size smaller than the target particle size or particles having a small bending elastic modulus move parallel to the direction of introduction into the flow path, and particles having a particle size larger than the target particle size or a particle having a large bending elastic modulus. Moves along the arrangement of the micro pillars with an angular component in a direction perpendicular to the flow direction F. In the example of FIG. 1A, as described above, the separation regions 10a and 10b in which the micropillars having different shift rates ε are formed are arranged adjacent to each other, so that the particles having the target particle diameter or the target bending can be obtained. It is designed so that only particles having an elastic coefficient are moved along the trajectory Lt and guided to the trap A. Thereby, only particles having a target particle diameter or particles having a target bending elastic modulus can be accurately separated. Note that particles having a particle size smaller than the target particle size or particles having a bending elastic modulus smaller than the target bending elastic modulus flow in the direction of the dummy trap B along the trajectory Ls, and have a particle size larger than the target particle size or the target. Particles having a flexural modulus greater than the flexural modulus are expressed in the direction of the dummy trap B ′ along the trajectory Lb.

  In order to accurately separate only particles having a target particle size, the center distance m between the trap A and the dummy trap B and the center between the trap A and the dummy trap B ′ shown in FIG. It is necessary to increase the distance n as much as possible. This is because the particle size of the particles dispersed in the fluid is continuously distributed, and the bending elastic modulus of the particle film is small and soft, and the trajectory can be greatly dispersed in the DLD. However, if the values of m and n are too large, the separation / capture device becomes too large, making it difficult to manufacture. For this reason, the values of m and n are appropriately determined in consideration of the separation accuracy of the particles and the size of the separation capturing device.

  FIG. 4 shows a plan view of a modified example of the trap A. In FIG. 4, the trap A is arranged at a position extending the wall-shaped protrusion P forming the opening 18 on the upstream side of the opening 18 in the fluid flow direction, and the wall-shaped protrusion P. A pair of gate pillar structures P ′ forming a drainage channel 22 is provided. Due to the presence of the gate pillar structure P ′, the space in which the particles are accommodated extends to the upstream side in the fluid flow direction, so that a plurality of particles can be captured and accommodated in the fluid flow direction.

  The dummy traps A ′, B, B ′, B ″ are not for capturing particles, but the flow state is balanced by the fluid resistance in the entire flow path of the particle separating unit 10 and the particle capturing unit 12. Therefore, the gate pole structure P ′ may be formed also in the dummy traps A ′, B, B ′, and B ″.

  The particle separation / capturing device having a small bending elastic modulus according to the above-described embodiment can be manufactured using a conventionally known photolithography technique. As the material, polydimethylsiloxane, glass, acrylic, polycarbonate, or the like can be used for the micro pillar, trap, and dummy trap. Alternatively, the silicon wafer may be directly etched by reactive ion etching to form an uneven pattern such as a micro pillar, trap, dummy trap, etc., and then bonded to glass to form a separation / capture device.

In the embodiment described above, the particles having a small bending elastic modulus (energy required for bending the membrane) are exemplified by cells, liposomes and the like which are easily deformable particles, but are not limited thereto. Here, when the bending elastic modulus of a particle having a small bending elastic modulus is exemplified, the liposome is 8 × 10 ⁻¹⁴ μJ, but the soft liposome is 3.4 to 4.2 × 10 ⁻¹⁴ μJ, and the red blood cell is 0.25 to 0.25. 2.05 × 10 ⁻¹³ μJ and bacteria (cellular slime mold) 0.940 to 2.188 × 10 ⁻¹² μJ. Therefore, the particle | grain separation / capture apparatus with a small bending elastic modulus concerning embodiment can be applied to the particle | grains which have a bending elastic modulus close | similar to the said bending elastic modulus.

  Examples of the present invention will be specifically described below. In addition, the following examples are for facilitating understanding of the present invention, and the present invention is not limited to these examples.

<Production of separation and capture device>
A micropillar, a trap, and a dummy trap were formed on a silicon wafer using a known photolithography technique according to the following procedure, and a separation / capturing device including a particle separation unit 10 and a particle trapping unit 12 was manufactured.

  First, using a maskless exposure apparatus (Nano System Solutions Co., Ltd., D-light DLS-50), a photomask having a transmission part was produced according to the flow path pattern (micropillars, traps, and dummy traps).

  Next, an epoxy resin-based negative photoresist SU8-25 (manufactured by MicroChem) was spin-coated on a silicon wafer, thermally cured, and then the flow path pattern was transferred from the photomask.

  After development, a silicon / SU8-25 mold having a fine uneven shape according to the flow path pattern is obtained. The mold thickness was set according to the target particle size. The thickness of the casting mold (corresponding to the distance between the bottom surface U of the tunnel-shaped flow path 14 and the ceiling surface C shown in FIG. 1B) is a contact surface shape measuring instrument (ULVAC, Inc., Dektak 6M). ). The measured values were 14.4, 18.2, and 23.0 μm for the target particle sizes of 12, 16, and 20 μm, respectively.

  A mold was formed by pouring polydimethylsiloxane (PDMS, manufactured by Toray Dow Corning Co., Ltd.) into the produced mold and curing it. In this mold, 96 to 240 traps A shown in FIGS. 1 (a), 3 (a), and (b) (corresponding to target particle sizes 12, 16, and 20 μm, 96, 160, 240. The number of dummy traps A ′ is also the same.), 56 dummy traps B and 28 to 35 dummy traps B ′ and B ″ are arranged.

  The arrangement interval in the trap A and the width L of the opening 18 are respectively 14, 18, and 22 μm (target particle size x + 2 μm) corresponding to the target particle sizes 12, 16, and 20 μm, and the gap G of the drain port 20 is They were 3, 4, and 4 μm, respectively. The arrangement interval D ′ in the dummy traps B, B ′, B ″ is 40 μm, the width L ′ of the opening 18 is 32 μm, and the gap G ′ of the drainage port 20 is 8 μm.

  Further, the shift rate ε shown in FIG. 2 was set for each target particle size. For a target particle size of 12 μm, the upstream separation region 10a is 1/17, for the downstream separation region 10b, 1/16, and for a target particle size of 16 μm, the upstream separation region 10a is 1/10 for the separation region 10b on the downstream side, and 1/7 for the separation region 10a on the upstream side and 1/6 for the separation region 10b on the downstream side for a target particle size of 20 μm. Further, the distance λ between the centers of the micro pillars was set to 60 μm, and the distance g between the walls of the micro pillars was set to 40 μm. Further, m = n = 8λ = 480 μm shown in FIG. 1A, and the length of the particle separation unit 10 in the flow direction (the total length of the separation regions 10a and 10b) is set to a target particle size of 12 μm. 15.84 mm, 9.12 mm for a target particle size of 16 μm, and 6.24 mm for a target particle size of 20 μm.

  The obtained PDMS flakes are drilled to provide an injection port and a drain port, and then bonded to a cover glass for microscope observation (manufactured by Matsunami Glass Industrial Co., Ltd.) to obtain the target device (separation and capture device). Obtained.

  The bonding was performed using a technique of directly bonding glass and PDMS by irradiating the activated surface with oxygen plasma using an etching apparatus (Samco Corporation, FA-1) and activating it.

<Preparation of liposome>
Liposomes were prepared using the Tsumoto method. The Tsumoto method is known as a method capable of preparing a single membrane liposome with high efficiency.

  The most common method for preparing liposomes is the thin film swelling method. The thin film of lipid bilayer deposited on the container wall is swollen by adding water and then sheared by applying mechanical vibration. To produce liposomes. In the Tsumoto method, the repulsive force between lipid molecules is increased by doping sugar into the above-described lipid thin film, and single-film liposomes are easily formed.

  In this example, liposomes were prepared by the following procedure.

First, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-sn-glycerol, cholesterol, β-BODIPY (registered trademark) FL From a stock solution obtained by dissolving C ₁₂ -HPC (Life Technologies) in chloroform and a stock solution obtained by dissolving fructose in methanol, 500 μL of a mixed solution was prepared in a 6 mL vial (chloroform / methanol = 9: 1 (volume fraction). Rate v / v)).

  Next, the solvent was distilled off under reduced pressure using a rotary evaporator (Tokyo Rika Kikai Co., Ltd., N-1110V) to form a thin film of lipid bilayer at the bottom of the vial. Then, it dried under reduced pressure at normal temperature for 2 to 3 hours or more.

  2 mL of ultrapure water (manufactured by Milli-Q manufactured by Merck & Co., Inc.) was gently poured and allowed to stand for 20 minutes in an incubator (Waken Pharmaceutical Co., Ltd., MODEL 2290) maintained at 40 ° C. Thereafter, a weak mechanical vibration was applied for 1 hour to promote liposome formation.

  The obtained liposome has a non-uniform particle size, and there are particles having a large particle size. Therefore, the liposome suspension was filtered using a nylon pre-filter (Merck Co., Ltd.) and then used for introducing the flow path of the separation and capture device. For devices with a target particle size of 12 and 16 μm, a nylon prefilter with a pore size of 30 μm is used for a device with a target particle size of 20 μm. It was prevented. The purpose of the filtration is to remove liposomes having a large particle size, and the liposomes in the suspension have a nonuniform particle size even after filtration.

<Separation capture operation>
Using a manual injection valve for HPLC (High-Performance Liquid Chromatography) (Rheodyne, Model 7000), the same concentration as the concentration of the fructose contained in the liposome suspension in the flow path of the separation / capture device The aqueous fructose solution prepared in (1) was rapidly replaced.

  Next, the liposome suspension is shown in FIG. 1 (a) while independently controlling the flow rates of the liposome suspension and the buffer solution using two syringe pumps (Model 11 elite manufactured by Harvard Apparatus). From the channel I, the buffer solution was supplied to the separation / capture device from the channels II and III, respectively. Since introduction at a high flow rate causes rupture of the liposome after trapping in the trap, the flow rates of the buffer solution and the liposome suspension are set to 1300 μL / h and 300 μL / h, respectively, in the flow path of the separation / capture device. After the supply, the flow rate of the buffer solution was gradually decreased to 330 to 350 μL / h, and the flow rate of the liposome suspension was decreased to 30 μL / h to stabilize the liposome after trapping in the trap. If the flow rate of the buffer solution is adjusted in the range of 100 to 2000 μL / h, the liposome can be prevented from bursting and the liposome can be stably held in the trap.

<Evaluation>
Separation and capture performance for three types of target particle sizes (12, 16, and 20 μm) were confirmed using the above prepared liposomes with non-uniform particle sizes. The setting of the separation and capture device for each target particle size is as described above.

  The time when the stable laminar flow was formed when the flow rate of the buffer solution was 330 to 350 μL / h and the flow rate of the liposome suspension was 30 μL / h was analyzed as 0 minutes. Liposomes that are trapped in a one-to-one relationship with traps (one liposome is trapped per trap) after 20, 70, and 40 minutes from the start of observation for target particle sizes of 12, 16, and 20 μm, respectively. The number of became the largest.

  The particle size analysis was performed at the time when the number of liposomes captured one-to-one with the trap was the largest. 5A, 5B, and 5C show particle size distributions obtained by particle size analysis. 5A shows a case where the target particle size is 12 μm, FIG. 5B shows a case where the target particle size is 16 μm, and FIG. 5C shows a case where the target particle size is 20 μm. In FIGS. 5A, 5B, and 5C, the horizontal axis represents the particle size and the vertical axis represents the number. As shown in FIGS. 5 (a), (b), and (c), the average particle diameters of 13.8, 17.7, 22.1 μm, and CV (coefficient of variation) are obtained with respect to the target particle diameters of 12, 16, and 20 μm, respectively. ) Obtained 5.3% (n = 154), 9.7% (n = 105) and 11.8% (n = 67), respectively. Thereby, it turns out that the particle | grains in which each target particle size is the center of particle size distribution are captured.

DESCRIPTION OF SYMBOLS 10 Particle separation part, 10a, 10b Separation area | region, 12 particle | grain capture | acquisition part, 14 flow path, 16 micropillar, 18 opening, 20 Drainage port, 22 Drainage channel.

Claims (6)

A particle separation unit that separates the orbit of cells or liposome particles dispersed and flowing in a fluid according to the particle size or bending elastic modulus of the particles, and separates the particle or bending elastic modulus for each particle size;
Among the particles separated by the particle separation unit, particles having a specific particle diameter or a specific bending elastic modulus are captured at the center of the distribution, and the particle capturing unit is arranged in a plane.
With
The particle trapping unit includes a region in which a plurality of traps A that trap the cells or liposome particles are arranged in a plane, and a flow state adjusting unit that maintains a laminar flow state of the fluid in the particle separation unit, , only contains the flow conditioning unit, dummy traps a trap a the same shape 'dummy trap B of shape similar to the and trap a, B', a region B "is planarly arrayed respectively A device for separating and capturing cells or liposome particles , wherein the trap A is formed side by side in a direction orthogonal to the fluid flow direction together with a region in which a plurality of the traps A are arranged in a plane . The trap A, the dummy traps A ′, B, B ′, and B ″ are formed by arranging the dummy trap B ′, the trap A, the dummy traps B, A ′, and B ″ in the direction orthogonal to the fluid flow direction. The apparatus for separating and capturing cells or liposome particles according to claim 1. The particle separation portion is formed in a tunnel-shaped flow path having a rectangular cross section, is perpendicular to the fluid flow direction, and is connected to one side of the tunnel-shaped flow path facing each other. The apparatus for separating and capturing cells or liposome particles according to claim 1 or 2 , wherein the obstacles are arranged at a constant angle with respect to the flow direction of the fluid. The particle trapping portion is formed in a tunnel-shaped channel having a rectangular cross section, and the trap A connects between one and the other surfaces of the tunnel-shaped channel facing each other, and The apparatus for separating and capturing cells or liposome particles according to any one of claims 1 to 3, further comprising an opening for receiving particles on the upstream side in the flow direction and a drainage port for securing a fluid flow on the downstream side. . The trap A is disposed on the upstream side in the fluid flow direction with respect to the opening at a position extending a wall-like protrusion that forms the opening, and a drainage channel is provided between the trap A and the wall-like protrusion. The apparatus for separating and capturing cells or liposome particles according to claim 4 , comprising a pair of gate pillar structures to be formed. The cell according to any one of claims 1 to 5 , wherein a drainage port is formed in the dummy trap B ', and the drainage port is formed in a gap larger than the drainage port of the trap A. Or a device for separating and capturing liposome particles.

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