Fine Particle Sampling Device and Fine Particle Sampling Method

Abstract

The purpose of the present invention is to provide a technique that makes it possible to form stable droplets.　Provided is a microparticle batching device that includes a microchip comprising: a main channel through which a liquid that includes microparticles flows; a sheath fluid channel which communicates with the main channel and through which a sheath fluid flows; and a sheath fluid introduction section into which the sheath fluid is introduced. The microparticle batching device vibrates the sheath fluid flowing through the sheath fluid introduction section. Also provided is a microparticle batching method in which, in a microchip, sheath fluid flowing through a sheath fluid introduction section is vibrated, the microchip comprising: at least a main channel through which a liquid that includes microparticles flows; a sheath fluid channel which communicates with the main channel and which supplies a sheath fluid; and a sheath fluid introduction section into which the sheath fluid is introduced.

Description

Technical Field

[0001] The present technology relates to a microparticle separation device and a microparticle separation method.

Background Art

[0002] To separate microparticles, various devices have been developed so far. In particular, a device for separating cells is called a "cell sorter". In a cell sorter, generally, vibration is applied to a flow cell or a microchip by a vibration element or the like to atomize the fluid discharged from the flow path into droplets. The droplets separated from the fluid are given a positive (+) or negative (-) charge, and then the traveling direction thereof is changed by a deflection plate or the like and collected in a predetermined container or the like.

[0003] For example, Patent Document 1 discloses separation using a microchip, in which droplets are formed by applying vibration to an orifice of the microchip by a vibration element.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In a flow cytometer, a control technique for stably forming droplets is one of the important factors for improving the accuracy of separation. Here, if the formation of droplets is unstable, such as the break-off point (BOP) at which the fluid discharged from the discharge port of the flow path becomes droplets is unstable, the time for charging the droplets with an electric charge also becomes unstable. As a result, it is known that the separation of microparticles also becomes unstable.

[0006] On the other hand, at frequencies on the order of the vibration frequency used by the flow cytometer for droplet formation (on the order of several 10 kHz to 100 kHz), since the microchip has a large number of higher-order eigenvalues and corresponding mode shapes, there can be multiple frequencies at which the microchip is affected according to its eigenvalues. Therefore, when vibration is applied to the orifice of the microchip, the vibration intensity of the microchip changes complexly depending on the frequency, which may lead to destabilization of the break-off point.

[0007] Therefore, the main object of this technology is to provide a technology capable of forming stable droplets.

Means for Solving the Problem

[0008] In this technology, first, a microchip is provided that includes a main flow path through which a liquid containing microparticles flows, a sheath liquid flow path that communicates with the main flow path and through which a sheath liquid flows, and a sheath liquid introduction part for introducing the sheath liquid, and a microparticle sorting device that vibrates the sheath liquid flowing through the sheath liquid introduction part is provided. In this technology, a connection member that can be attached to the microchip and has a sheath liquid introduction connection part that connects to the sheath liquid introduction part may be further provided. In this technology, a vibration element may be attached to the connection member. In this technology, the driving frequency of the vibration element may be different from the resonance frequency of the flow path in the microchip. In this technology, the driving frequency of the vibration element may be within a range of ±10% from the resonance frequency of the flow path in the microchip. In this technology, the sheath liquid introduction connection part may have a sheath liquid converging part whose width gradually or partially narrows from the vibration element side toward the sheath liquid introduction part side. In this technology, the sheath liquid converging part may gradually or partially decrease in height from the vibration element side toward the sheath liquid introduction part side. In this technology, a connection part having a tubular part that communicates with the tip of the sheath liquid converging part may be provided between the sheath liquid converging part and the sheath liquid introduction part. In the present technology, a tubular member may be inserted inside the tubular portion. In the present technology, at least a part of the tubular portion and / or the tubular member may be made of any one or more selected from the group consisting of an elastomer, a resin, and a metal. In the present technology, the sheath liquid converging portion may be substantially conical, substantially polygonal pyramidal, or a solid of revolution of an exponential function or a parabola. In the present technology, the sheath liquid converging portion may be made of a resin, a metal, or a transparent member. In the present technology, an electrode may be inserted into the sheath liquid converging portion. In the present technology, a swirling flow for swirling the sheath liquid may be generated in the sheath liquid converging portion. In the present technology, the sheath liquid inlet for introducing the sheath liquid into the sheath liquid converging portion may be located at a position away from the center of the sheath liquid converging portion.

[0009] Further, in the present technology, there is provided a microparticle sorting device having a microchip including a main flow path through which a liquid containing microparticles flows, a sheath liquid flow path communicating with the main flow path and supplying a sheath liquid, and a sheath liquid introduction portion for introducing the sheath liquid, a light irradiation portion for irradiating light onto the microparticles, a light detection portion for detecting light from the microparticles, and a processing portion for processing a signal obtained from the light detection portion, and vibrating the sheath liquid flowing through the sheath liquid introduction portion.

[0010] Furthermore, in the present technology, there is also provided a microparticle sorting method for vibrating the sheath liquid flowing through the sheath liquid introduction portion in a microchip including at least a main flow path through which a liquid containing microparticles flows, a sheath liquid flow path communicating with the main flow path and supplying a sheath liquid, and a sheath liquid introduction portion for introducing the sheath liquid.

Brief Description of the Drawings

[0011]

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Embodiments for Carrying Out the Invention

[0012] Hereinafter, preferred embodiments for carrying out this technology will be described with reference to the drawings. The embodiments described below show an example of a typical embodiment of this technology, and thus the scope of this technology should not be construed narrowly. The description will be made in the following order. 1. First Embodiment (Micro Particle Separation Device 100) (1) Microchip M (2) Connection member C 2. Second Embodiment (Micro Particle Separation Device 100) 3. Third Embodiment (Micro Particle Separation Device 100) [Example Form 1 of Connection Port] [Example Form 2 of Connection Port] [Example Form 3 of Connection Port] [Example Form 4 of Connection Port] 4. Fourth Embodiment (Micro Particle Separation Device 100) (1) Light irradiation unit 103 (2) Light detection unit 104 (3) Processing unit 105 (4) Separation unit 106 (including charging unit 106c) (5) Storage unit 107 (6) Display unit 108 (7) Input unit 109 (8) Control unit 110 5. Fifth Embodiment (Micro Particle Separation Method)

[0013] 1. First Embodiment (Micro Particle Separation Device 100)

[0014] FIG. 1 is a diagram showing a configuration example of the micro particle separation device 100 according to the first embodiment. The micro particle separation device 100 according to the present embodiment includes a main flow path M2 through which a liquid containing micro particles flows, a sheath liquid flow path M41 that communicates with the main flow path and supplies a sheath liquid, and a sheath liquid introduction unit M4 that introduces the sheath liquid, and has a microchip M that vibrates the sheath liquid flowing through the sheath liquid introduction unit M4. Hereinafter, each unit will be described in detail.

[0015] (1) Microchip M

[0016] FIG. 2 is a diagram showing a configuration example of the microchip M, and FIG. 3 is a diagram showing a configuration example of the orifice M1 of the microchip M. A in FIG. 2 is a top view, and B in FIG. 2 is a cross-sectional view corresponding to the P-P cross-section in A. Also, A in FIG. 3 is a top view, B in FIG. 3 is a cross-sectional view, and C in FIG. 3 is a front view.

[0017] As shown in A of FIG. 2, the microchip M includes a sheath liquid flow path M41 that communicates with the main flow path M2 and through which the sheath liquid flows, a sheath liquid introduction portion M4 that introduces the sheath liquid, a sample liquid flow path M31 that communicates with the main flow path M2 and through which a sample liquid containing fine particles flows, a sample liquid introduction portion M3 that introduces the sample liquid, and a confluence portion where the sample flow is introduced and merges with the sheath liquid. The sheath liquid introduced from the sheath liquid introduction portion M4 is divided and fed in two directions, and then merges with the sample liquid at the confluence portion with the sample liquid introduced from the sample liquid introduction portion M3, sandwiching the sample liquid from two directions. As a result, a three-dimensional laminar flow is formed in which the sample liquid laminar flow is positioned at the center of the sheath liquid laminar flow at the confluence portion.

[0018] M51 shown in A of FIG. 2 indicates a suction flow path for applying a negative pressure to the main flow path M2 to temporarily reverse the flow and eliminate clogging or bubbles when clogging or bubbles occur in the main flow path M2. A suction opening M5 connected to a negative pressure source such as a vacuum pump is formed at one end of the suction flow path M51. The other end of the suction flow path M51 is connected to the main flow path M2 at the communication port M52.

[0019] The three-dimensional laminar flow is narrowed in the narrowing portions M61 (see A of FIG. 2) and M62 (see A and B of FIG. 3) formed such that the area of the cross-section perpendicular to the liquid feeding direction gradually or stepwise decreases from the upstream to the downstream in the liquid feeding direction. Thereafter, the three-dimensional laminar flow is discharged as a fluid stream from the orifice M1 provided at one end of the flow path.

[0020] Here, in the prior art, for the fluid stream ejected from the orifice M1, the vibration element applied vibration to the orifice M1 to atomize it into droplets. However, in the present technology, the fluid stream ejected from the orifice M1 is atomized by applying vibration to the sheath fluid flowing through the sheath fluid introduction part M4, as will be described later.

[0021] The orifice M1 is open in the end face direction of the substrate layers Ma and Mb, and a notch M11 is provided between the opening position thereof and the end face of the substrate layer. The notch M11 is formed by cutting out the substrate layers Ma and Mb between the opening position of the orifice M1 and the substrate end face such that the diameter L1 of the notch M11 is larger than the opening diameter L2 of the orifice M1 (see C in FIG. 3). The diameter L1 of the notch M11 is preferably formed to be more than twice as large as the opening diameter L2 of the orifice M1 so as not to impede the movement of the droplets ejected from the orifice M1.

[0022] In the present technology, "micro" means that at least a part of the flow path included in the microchip M has dimensions on the order of μm, particularly a cross-sectional dimension on the order of μm. That is, in the present technology, a "microchip" refers to a chip including a flow path on the order of μm, particularly a chip including a flow path having a cross-sectional dimension on the order of μm. For example, a chip including a particle separation part composed of a flow path having a cross-sectional dimension on the order of μm can be called a microchip according to the present technology.

[0023] The microchip M can be manufactured by a method known in the art. For example, the microchip M is formed by bonding substrate layers Ma and Mb in which a main flow path M2 is formed. The formation of the main flow path M2 in the substrate layers Ma and Mb can be performed, for example, by injection molding of a thermoplastic resin using a mold. The flow path may be formed in all of two or more substrates, or may be formed only in a part of two or more substrates. Further, the microchip M may be formed by bonding substrates from above, below, or both directions with respect to the plane of the substrate in which each flow path is formed, and may be formed by three or more substrates.

[0024] As the material for forming the microchip M, materials known in the art can be used. For example, polycarbonate (PC), cycloolefin polymer, polypropylene, PDMS (polydimethylsiloxane), polymethyl methacrylate (PMMA), polyethylene, polystyrene, glass, silicon, etc. can be mentioned, but it is not limited thereto. Among them, in particular, since it has excellent processability and a microchip can be manufactured at low cost using a molding device, for example, polymer materials such as polycarbonate, cycloolefin polymer, and polypropylene are particularly preferable.

[0025] The microchip M is preferably transparent. For example, at least a portion through which light (laser light and scattered light) passes in the microchip M may be transparent, or the entire microchip M may be transparent.

[0026] In the present technology, the "sample" contained in the sample liquid is particularly fine particles, and the fine particles may be particles having a size capable of flowing in the flow path in the microchip M. In the present technology, the fine particles may be appropriately selected by those skilled in the art. In the present technology, examples of the fine particles may include biological fine particles such as cells, cell aggregates, microorganisms, ribosomes, and synthetic fine particles such as gel particles, beads, latex particles, polymer particles, and industrial particles. Biological microparticles (also referred to as "bioparticles") can include chromosomes, ribosomes, mitochondria, and organelles (cellular organs) that make up various cells. Cells can include animal cells (e.g., blood cell line cells, etc.) and plant cells. The cells can particularly be blood system cells or tissue system cells. The blood system cells can be, for example, floating cells such as T cells and B cells. The tissue system cells can be, for example, adherent cultured cells or adherent cells separated from tissues. Cell aggregates can include, for example, spheroids, organoids, etc. Microorganisms can include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Furthermore, the biological microparticles can also include biological macromolecules such as nucleic acids, proteins, and their complexes. These biological macromolecules can be, for example, those extracted from cells, or those contained in a blood sample or other liquid sample. Synthetic microparticles can be, for example, microparticles composed of organic or inorganic polymer materials or metals. Organic polymer materials can include polystyrene, styrene divinylbenzene, polymethyl methacrylate, etc. Inorganic polymer materials can include glass, silica, magnetic materials, etc. Metals can include gold colloids, aluminum, etc. The synthetic microparticles can be, for example, gel particles, beads, etc., and particularly can be gel particles or beads to which one or more combinations selected from oligonucleotides, peptides, proteins, and enzymes are bound.

[0027] The shape of the microparticles may be spherical or substantially spherical, or may be non-spherical. The size and mass of the microparticles can be appropriately selected by those skilled in the art according to the size of the flow path of the microchip M. On the other hand, the size of the flow path of the microchip M can also be appropriately selected according to the size and mass of the microparticles. In this technology, chemical or biological labels, such as fluorescent dyes, fluorescent proteins, etc., can be attached to the microparticles as necessary. Such labels can make the detection of the microparticles easier. The labels to be attached can be appropriately selected by those skilled in the art. Molecules (such as antibodies, aptamers, DNA, RNA, etc.) that specifically react with the microparticles can bind to the labels. In this technology, it is preferable that the microparticles are biological particles, and in particular, they can be cells.

[0028] (2) Connection member C

[0029] The microparticle sorting device 100 according to this embodiment is attachable to the microchip M and further includes a connection member having a sheath liquid introduction connection part C2 that connects to the sheath liquid introduction part M4.

[0030] In the connection member C shown in FIG. 1, it has at least a sample liquid introduction connection part C1 that connects to the sample liquid introduction part M3 and a sheath liquid introduction connection part C2 that connects to the sheath liquid introduction part M4.

[0031] By using a connection member C that is detachable from the microchip M, when continuously sorting a large number of different microparticles using one device, a part of the components of the device can be removed. Therefore, even if the microparticles contained in the previously sorted fluid flow remain in the component, the entire component can be removed, reducing the risk of contamination. Also, by using the microchip M and the connection member C disposable for each sample, the labor of the cleaning operation performed when changing the sample can be saved, reducing the burden on the operator.

[0032] The sheath liquid introduction connection part C2 may have a liquid delivery tube capable of delivering liquid from the sheath liquid delivery part 101. Further, the liquid delivery tube may have a tube connection part that directly connects to the sheath liquid delivery part 101. In this case, it is preferable that the tube connection part is configured so that the liquid in the liquid delivery tube does not come into contact with the outside air. Thereby, the cleanliness of the sheath liquid can be ensured.

[0033] The sample liquid introduction connection part C1 may have a tube fixing part for fixing a liquid delivery tube capable of delivering liquid from the sample liquid delivery part 102. Thereby, the labor for attaching and fixing each tube can be saved, the complication of the operation during measurement can be prevented, and the burden on the operator can be reduced. Also, by making these members disposable for each sample, contamination can be prevented.

[0034] The liquid delivery tube can be formed integrally with the connection member C, but it can also be formed separately. For example, the liquid delivery tube and the tube fixing part capable of delivering liquid from the sample liquid delivery part 102 are formed so as to be detachable from the connection member C, and it is possible to facilitate the connection with the sample liquid delivery part 102 arranged at a location different from the sheath liquid delivery part 101.

[0035] In the present embodiment, a vibration element C3 is attached to the connection member C. Thereby, vibration can be propagated to the sheath liquid flowing through the sheath liquid introduction part M4 of the microchip M, and after jetting from the nozzle, droplet formation can be induced.

[0036] The driving frequency of the vibration element C3 is preferably different from the resonance frequency of the flow path in the microchip M. The reason will be described in detail below.

[0037] As shown in Fig. 4, a standing wave occurs between the vibration element C3 and the nozzle, and the fluctuating pressure near the nozzle changes depending on the shape, dimensions, material, and driving frequency of the flow path. The state in which the fluctuating pressure is maximized is called resonance (see Fig. 5). For example, the resonance frequency can be lowered by increasing the length of the flow path, and the resonance frequency can be adjusted by combining such properties. The detailed frequency characteristics of the fluctuating pressure can be estimated by using commercially available acoustic analysis software or the like. If resonance occurs at the driving frequency to be used, droplets can be formed with a small amount of vibration energy. However, since the way the fluctuating pressure changes reverses before and after the resonance frequency of the flow path in the microchip M and the change is also steep, droplet formation at the resonance frequency is inferior in terms of stability. Therefore, as shown in Fig. 5, by shifting the driving frequency slightly from the resonance frequency, both an improvement in efficiency due to resonance and stability can be achieved.

[0038] In this technology, it does not matter whether the driving frequency is shifted to the higher side or the lower side from the resonance frequency. Usually, the driving frequency is set as a frequency band with a certain width for adjustment. The amount of shift is not particularly limited as long as the driving frequency band does not cross the resonance frequency even in the presence of disturbances such as temperature changes, and it may be set in consideration of the desired efficiency improvement effect and the balance of stability within the frequency band. In this technology, the driving frequency of the vibration element can be, for example, within the range of ±10% from the resonance frequency of the flow path in the microchip M.

[0039] In this embodiment, it is preferable that the sheath liquid introduction connection part C2 has a sheath liquid convergence part C21 whose width gradually or partially narrows from the side where the vibration element C3 is attached toward the sheath liquid introduction part M4 side. Thereby, the thickness of the flow path in the sheath liquid introduction connection part C2 is gradually narrowed from the thickness of about the vibration element C3 to the thickness of about the sheath liquid introduction part M4, connecting the scale of the size of about the vibration element C3 and the scale of the size of about the flow path, and concentrating the vibration energy of the vibration element C3 near the sheath liquid introduction part M4, so that the vibration energy can be efficiently sent into the flow path in the microchip M with a small driving voltage. Hereinafter, how the vibration is propagated to the sheath liquid near the sheath liquid introduction part M4 of the microchip M will be described in detail.

[0040] The sheath liquid is supplied from the sheath liquid feeding part 101 to the sheath liquid convergence part C21, and the sheath liquid is vibrated by the vibration element C3 arranged upstream of the convergence part C21. The vibration element C3 is composed of, for example, a piezoelectric element part and a piston part, and each is firmly bonded by an adhesive or the like. The structure of the piezoelectric element part is not limited as long as the finally extracted vibration can be vibrated in the X direction (see B in FIG. 1) with the required amplitude at the target vibration frequency. For example, structures such as a laminated type, a square plate type, a disk type, and a tube type are conceivable (see A to C in FIG. 6). Further, as the vibration element C3, a magnetic force such as a permanent magnet and a solenoid may be used. Furthermore, instead of the structure in which a piston adhered to such a piezoelectric element is inserted into the convergence part C21, a structure in which a bent piezoelectric element is attached to the top surface of the convergence part C21 as shown in FIG. 7 may be used. The sheath liquid is sent into the chip from the sheath liquid introduction part M4 of the microchip M, and the vibration of the vibration element C3 propagates through the sheath liquid to induce droplet formation after ejection from the nozzle.

[0041] As the vibration element C3, for example, a piezoelectric element such as a piezo element can be used, but as described above, a vibration element C3 that converts electrical energy into vibration through magnetic force such as a permanent magnet and a solenoid can be used. Also, the vibration frequency is not limited to the ultrasonic region of 20 kHz or more, and can be appropriately set according to the size of the droplets to be formed.

[0042] The shape of the sheath liquid converging portion C21 preferably gradually or partially decreases in height from the vibration element C3 side toward the sheath liquid introduction portion M4 side. Specifically, for example, it can be a substantially conical shape, a substantially polygonal pyramid shape, or a solid of revolution of an exponential function or a parabola. By designing it into these shapes, it is possible to efficiently concentrate the vibration energy. If the vibration element C3 can generate sufficient vibration energy and there are no problems with assemblability, it is not limited to these shapes, but generally, the form of gradually narrowing the flow path provides greater benefits (for example, since the amplitude of the vibration element C3 may be small, droplets can be formed with a low driving voltage of the piezoelectric element, increasing the degree of freedom in the selection and design of the piezoelectric element).

[0043] When selecting a substantially polygonal pyramid shape as the shape of the sheath liquid converging portion C21, a flat surface portion can be provided. Therefore, particularly when the sheath liquid converging portion C21 is formed of a transparent member, there is an advantage that the inside of the converging portion C21 is easy to see and it is easy to confirm bubbles and the like. Note that a substantially polygonal pyramid with small inner angles like a substantially triangular pyramid is not desirable because bubbles are likely to be trapped at each corner and it is difficult to remove the bubbles. For example, a substantially polygonal pyramid with large inner angles like a substantially polygonal pyramid with six or more sides is desirable. Also, from the viewpoint of not inhibiting the swirling flow described later and enhancing the effect, a polygonal pyramid with large inner angles is more desirable. Furthermore, from the viewpoint of bubble removal or not inhibiting the swirling flow, it is desirable not to make each corner sharp and to provide an appropriate radius R. Providing an appropriate radius R includes, for example, providing an R of about 1 / 10 or more of the radius of the inscribed circle of the polygon.

[0044] Note that the detailed shape and dimensions of the sheath liquid converging portion C21 are preferably determined while checking its acoustic characteristics. Thereby, the utilization efficiency of the vibration energy can be further increased.

[0045] As the material for forming the sheath liquid converging portion C21, materials known in the art can be used. In the present technology, however, it is preferable to form the sheath liquid converging portion C21 with a resin, a metal, or a transparent member. As the resin, for example, polyetheretherketone (PEEK) or the like can be used. As the transparent member, for example, polymethyl methacrylate (PMMA), polycarbonate (PC), or the like can be used. By forming the sheath liquid converging portion C21 with a transparent member, the inside of the sheath liquid converging portion C21 can be observed. As the metal, for example, stainless steel, aluminum alloy, titanium alloy, or the like can be used. By forming the sheath liquid converging portion C21 with a metal, it is possible to omit the electrode for droplet charging.

[0046] FIG. 8 is a diagram showing the electrode C4 disposed near the sheath liquid converging portion C21. When charging the formed droplets, when the sheath liquid converging portion C21 is formed of an insulator such as resin, as shown in FIG. 8, by inserting the electrode C4 into the sheath liquid converging portion C21, the droplets can be charged through the sheath liquid. The purpose is to make the distance between the droplet splitting point and the electrode C4 as close as possible and perform charging at a more ideal timing.

[0047] In the case of this configuration, fine bubbles may get caught at the boundary between the hole of the sheath liquid converging portion C21 and the electrode C4, and it is particularly difficult to remove the bubbles when the flow stagnates. Therefore, in the present technology, it is preferable to generate a swirling flow that swirls the sheath liquid in the sheath liquid converging portion C21. As a result, the swirling flow generated eliminates the stagnant portions, making it possible to easily remove the bubbles.

[0048] In addition, the sheath liquid converging portion C21 corresponds to a portion where the flow path becomes wider in the entire flow path system, and air bubbles tend to stay inside it. When air bubbles stay, the vibration energy is absorbed by the air bubbles, which affects the break-off point. Therefore, it is conceivable to set the sheath liquid inlet C20 for introducing the sheath liquid into the sheath liquid converging portion C21 at a position away from the center of the sheath liquid converging portion C21. Specifically, for example, the position of the sheath liquid inlet C20 is offset upward from the center of the sheath liquid converging portion C21 and further arranged in the upstream portion. Thereby, a swirling flow is generated in the sheath liquid converging portion C21, and by preventing the formation of stagnant portions, air bubbles do not stay. Also, since the specific gravity of air bubbles is smaller than that of the sheath liquid, they tend to gather at the center of the flow path and flow down easily.

[0049] Note that if the intensity of the swirling flow generated is sufficient to cause the air bubbles to flow down, the offset may be in the lower direction instead of the upper direction of the sheath liquid converging portion C21. Also, there is a configuration (see Fig. 8) in which an electrode C4 is inserted to charge the droplets in this portion. However, since fine air bubbles may get caught at the boundary between the hole opened in the sheath liquid converging portion C21 and the electrode C4, the effect of peeling off and removing the air bubbles adhering around the electrode C4 by the swirling flow can also be expected.

[0050] In this embodiment, the microchip M and the connection member C can be appropriately removed as needed and may be disposable. Also, the vibration element C3 attached to the connection member C may be circulated while remaining attached to the connection member C in advance. In this case, the vibration element C3 may be disposable.

[0051] 2. Second Embodiment (Fine Particle Separation Device 100)

[0052] A and B in Fig. 9 are diagrams showing a configuration example of the fine particle separation device according to the second embodiment. A in Fig. 9 is a schematic top view, and B in Fig. 9 is a schematic cross-sectional view. In the above-described first embodiment, a confluence portion is formed in the microchip M where the sample stream is introduced and merged with the sheath liquid, and the introduction of the sample liquid into the sheath liquid is performed within the microchip M. However, in the present embodiment, the sample liquid is introduced into the sheath liquid convergence portion C21, and the configuration has a flow path in a form that converges the core stream containing the sample liquid by hydrodynamic focusing. Note that the present embodiment is the same as the above-described first embodiment except that the method of introducing the sample liquid into the sheath liquid is different.

[0053] In the present embodiment, as shown in FIG. 9, upstream of the position of the sample liquid inlet for introducing the sample liquid into the sheath liquid convergence portion C21, after removing the swirling component of the flow by the rectifying plate formed inside the sheath liquid convergence portion C21, the sample liquid is introduced. Thereby, it is possible to prevent the core stream from being disturbed by the swirling flow. The rectifying plate can be formed of, for example, a thin plate or the like. Here, it is preferable that the rectifying plate is not disposed at the thickest portion of the flow path so as not to interfere with the swirling flow.

[0054] 3. Third Embodiment (Fine Particle Separation Device 100)

[0055] FIGS. 12 and 14 to 16 are diagrams showing a form example of the connection portion C22 in the fine particle separation device 100 according to the third embodiment. In FIGS. 14 to 16, the vibration element C3 is omitted. In the present embodiment, the sheath liquid introduction connection portion C2 has a sheath liquid convergence portion C21 whose width and height gradually or partially decrease from the side where the vibration element C3 is attached toward the sheath liquid introduction portion M4 side, and between the sheath liquid convergence portion C21 and the sheath liquid introduction portion M4, a connection portion C22 having a tubular portion C221 communicating with the tip C211 of the sheath liquid convergence portion C21 is provided. Note that the present embodiment is the same as the above-described first embodiment except that the connection portion C22 is provided.

[0056] In the present embodiment, by providing the connection portion C22, it is possible to more efficiently connect the scale of the size of the vibration element C3 and the scale of the size of the flow path.

[0057] Examples of the material for forming the connection part C22 include an elastomer, a resin, a metal, or a combination of two or more of these.

[0058] Examples of the elastomer include a thermosetting elastomer, a thermoplastic elastomer, etc. Examples of the thermosetting elastomer include vulcanized rubbers such as natural rubber and synthetic rubber; resin-based elastomers such as silicone rubber and fluororubber; etc. Examples of the thermoplastic elastomer include thermoplastic elastomers such as polystyrene-based, olefin / alkene-based, polyvinyl chloride-based, polyurethane-based, polyester-based, and polyamide-based.

[0059] Examples of the resin include a thermoplastic resin, a thermosetting resin, etc. Examples of the thermoplastic resin include polyolefins such as polystyrene, polyethylene, and polypropylene; polyvinyl chloride; acrylic resin; ABS resin; AS resin; engineering plastics such as polyamide, polycarbonate, polyacetal, and polyethylene terephthalate; super engineering plastics such as polyethersulfone, polyetheretherketone (PEEK), and thermoplastic polyimide; etc. Examples of the thermosetting resin include phenol resin, melamine resin, polyurethane, unsaturated polyester resin, epoxy resin, etc.

[0060] Examples of the metal include aluminum alloy, titanium alloy, stainless steel, etc.

[0061] Hereinafter, specific form examples of the connection part C22 will be described in detail.

[0062] [Form Example 1 of Connection Part C22]

[0063] FIG. 12 is a diagram schematically showing a first exemplary form of the connection portion C22. In the first exemplary form, a tubular member C222 is inserted inside a tubular portion C221 made of a thermosetting elastomer. Thereby, when contacting the sheath liquid, it can deviate from the acoustic impedance of the sheath liquid (if the acoustic impedances are about the same, vibration diffuses and vibration energy dissipates). As a result, the vibration generated from the vibration element C3 is prevented from diffusing to the connection portion C22, and the vibration energy can be efficiently transmitted to the flow path in the microchip M.

[0064] Also, when the driving voltage applied to the vibration element C3 is the same as compared with the case where the tubular member C222 is not inserted, as shown in FIG. 13, a shorter BOP length can be obtained, or the same BOP length can be obtained at a lower driving voltage.

[0065] Examples of the material forming the tubular member C222 include the elastomer, resin, metal, or a combination of two or more of them described above. In the first exemplary form, among these, polystyrene, acrylic resin, aluminum alloy, titanium alloy, or stainless steel is particularly preferable, aluminum alloy, titanium alloy, or stainless steel is more preferable, and stainless steel is even more preferable.

[0066] Also, in the first exemplary form, it is preferable that at least a part of the tubular member C222 is made of the material described above, and it is more preferable that the entire tubular member C222 is made of the material described above. Further, in the first exemplary form, the tubular portion C221 may be formed of a thermosetting elastomer so as to function as a seal.

[0067] In the present embodiment, the longitudinal length of the tubular member C222 may be the same as the longitudinal length of the tubular portion C221, but may be slightly shorter in consideration of crushing for sealing. In addition, the size such as the inner and outer diameters of the tubular member C222 is not particularly limited, but the outer diameter of the tubular member C222 can be, for example, the same as the flow path diameter of the tubular portion C221 or slightly larger than the flow path diameter.

[0068] [Second exemplary form of connection part C22]

[0069] FIG. 14 is a diagram schematically showing a second exemplary form of connection part C22. In the second exemplary form, connection part C22 including tubular part 221 is formed by two-color molding with resin C22a and thermosetting elastomer C22b. Specifically, thermosetting elastomer C22b for sealing is fused to resin C22a by two-color molding. In this way, by forming at least a part of tubular part C221 of connection part C22 with resin C22a, when contacting the sheath liquid, it can deviate from the acoustic impedance of the sheath liquid and suppress the diffusion of vibration outside the flow path of tubular part C221 as compared with the case where the entire tubular part 221 is formed of a thermosetting elastomer. Here, examples of resin C22a include those described above, but in the second exemplary form, polystyrene or an acrylic resin is particularly preferable.

[0070] [Third exemplary form of connection part C22]

[0071] FIG. 15 is a diagram schematically showing a third exemplary form of connection part C22. In the third exemplary form, the entire connection part C22 including tubular part 221 is formed of metal, and O-rings C22c for sealing are arranged at both ends of connection part C22. In this way, by forming the entire connection part 22 of metal, the vibration generated from vibration element C3 can be prevented from diffusing to connection part C22, and the vibration energy can be efficiently transmitted to the flow path in microchip M. Here, examples of the metal include those described above, but in the third exemplary form, an aluminum alloy, a titanium alloy, or stainless steel is particularly preferable, and stainless steel is more preferable. Also, as the material for forming O-ring C22c, known materials in the art can be used. For example, the above-mentioned elastomers, resins, or combinations of two or more of them can be mentioned.

[0072] [Fourth exemplary form of connection part C22]

[0073] FIG. 16 is a diagram schematically showing a fourth exemplary form of the connection part C22. In the fourth exemplary form, a part of the connection part C22 including the sheath liquid converging part C21 and the tubular part 221 is formed of metal, and an O-ring C22c and a thermosetting elastomer C22b are arranged at both ends thereof for sealing. In this way, by forming up to the sheath liquid converging part C21 of metal, the diffusion of vibration generated from the vibration element C3 can be further suppressed. Here, examples of the metal forming the sheath liquid converging part C21 and a part of the connection part C22 include those described above. In the fourth exemplary form, among them, in particular, an aluminum alloy, a titanium alloy, or stainless steel is preferable, and stainless steel is more preferable. Also, examples of the material forming the O-ring C22c include those described above.

[0074] 4. Fourth Embodiment (Fine Particle Separation Device 100)

[0075] FIG. 10 is a diagram showing a configuration example of a fine particle separation device according to the fourth embodiment. The fine particle separation device 100 according to the present embodiment includes a main flow path M2 through which a liquid containing fine particles flows, a sheath liquid flow path M41 communicating with the main flow path M2 and supplying a sheath liquid, and a sheath liquid introduction part M4 for introducing the sheath liquid, a microchip, a light irradiation part 103 for irradiating the fine particles with light, a light detection part 104 for detecting light from the fine particles, and a processing part 105 for processing a signal obtained from the light detection part 104, and vibrates the sheath liquid flowing through the sheath liquid introduction part. Further, if necessary, a separation part 106, a storage part 107, a display part 108, an input part 109, a control part 110, etc. may be provided.

[0076] Since the microchip M is the same as that described above, the description thereof is omitted here. Also, since the method of vibrating the sheath liquid flowing through the sheath liquid introduction part M4 of the microchip M is the same as that described above, the description thereof is omitted here.

[0077] (1) Light Irradiation Part 103

[0078] The light irradiation unit 103 irradiates light (for example, excitation light, etc.) onto the microparticles to be sorted. The light irradiation unit 103 may include a light source that emits light and an objective lens that condenses the excitation light onto the microparticles flowing through the detection region. The light source may be appropriately selected by those skilled in the art according to the purpose of sorting. For example, it may be a laser diode, SHG laser, solid-state laser, gas laser, or high-brightness LED, or a combination of two or more of these. In addition to the light source and the objective lens, the light irradiation unit 103 may include other optical elements as necessary.

[0079] (2) Light detection unit 104

[0080] The light detection unit 104 detects the light (scattered light and / or fluorescence) generated from the microparticles by the irradiation of the light irradiation unit 103. The light detection unit 104 may include a condenser lens that condenses the fluorescence and / or scattered light generated from the microparticles and a photodetector. As the photodetector, a PMT, photodiode, CCD, CMOS, etc. may be used, but the present technology is not limited thereto. In addition to the condenser lens and the photodetector, the light detection unit 104 may include other optical elements as necessary. The light detection unit 104 may further include, for example, a spectroscopic unit. Examples of the optical components constituting the spectroscopic unit include a grating, a prism, an optical filter, etc. By the spectroscopic unit, for example, the light of the wavelength to be detected can be separated from the light of other wavelengths and detected.

[0081] The fluorescence detected by the light detection unit 104 may be fluorescence generated from the microparticles themselves and fluorescence generated from substances labeled on the microparticles, such as fluorescent substances, etc., but the present technology is not limited thereto. The scattered light detected by the light detection unit 104 may be forward scattered light, side scattered light, Rayleigh scattering, or Mie scattering, or a combination of these.

[0082] (3) Processing unit 105

[0083] The processing unit 105 is connected to the light detection unit 104 and processes the signal obtained from the light detection unit 104. For example, it can correct the detection value of the light received from the light detection unit 104 and calculate the characteristic amounts of each microparticle. More specifically, it can calculate the characteristic amounts indicating the size, shape, internal structure, etc. of the microparticles from the detection values of the received fluorescence, forward scattered light, and backward scattered light. Further, based on the calculated characteristic amounts and the fractionation conditions previously received from the input unit, etc., it can also make a fractionation determination and generate a fractionation control signal.

[0084] Based on the detection value of the light detected by the light detection unit 104, the processing unit 105 can also analyze the state of the microparticles, etc. using an external analysis device or the like. For example, the processing unit 105 may be implemented by a personal computer or a CPU. Furthermore, it can be stored as a program in hardware resources including a recording medium (non-volatile memory (such as a USB memory), HDD, CD, etc.) and be made to function by a personal computer or a CPU. Also, the processing unit 105 may be connected to each part of the microparticle fractionation device 100 via a network.

[0085] (4) Fractionation unit 106 (including the charging unit 106c)

[0086] The fractionation unit 106 has at least a deflection plate 106a for changing the charged droplets in a desired direction and a collection container 106b for collecting the droplets. The charging unit 106c, although defined separately in FIG. 4, is a part of the fractionation unit 106 and performs charging based on the fractionation control signal generated by the processing unit 105.

[0087] In the microparticle fractionation device 100 shown in FIG. 10, the vibration element C3 attached to the connection member C forms droplets by propagating vibration to the sheath liquid as described above. The charging unit 106c is connected to the electrode C4 inserted into the sheath liquid convergence unit C21 described above, and charges the droplets discharged from the orifice M1 of the microchip M positively or negatively based on the fractionation control signal generated by the processing unit 105. Then, the charged droplets have their paths changed in a desired direction by the deflection plate (opposing electrode) 106a to which a voltage is applied and are fractionated.

[0088] (5) Storage section 107

[0089] The memory unit 107 stores all items related to the measurement, such as the values ​​detected by the light detection unit 103, the feature amounts calculated by the processing unit 105, the fractionation control signal, and the fractionation conditions inputted by the input unit.

[0090] In the microparticle sorting device 100, the storage unit 107 is not essential, and an external storage device may be connected. For example, a hard disk or the like may be used as the storage unit 107. In addition, the recording unit 107 may be connected to each unit of the microparticle sorting device 100 via a network.

[0091] (6) Display section 108

[0092] The display unit 108 can display all items related to the measurement, such as the value detected by the light detection unit 103 and the characteristic amount calculated by the processing unit 105. The display unit 108 preferably displays the characteristic amount for each microparticle calculated by the processing unit 105 as a scattergram.

[0093] In the microparticle sorting device 100, the display unit 108 is not essential, and an external display device may be connected. For example, a display, a printer, or the like may be used as the display unit 110. In addition, the display unit 108 may be connected to each part of the microparticle sorting device 100 via a network.

[0094] (7) Input section 109

[0095] The input unit 109 is a portion for a user such as an operator to operate. The user can access a control unit 110, which will be described later, through the input unit 109 and control each part of the microparticle sorting device 100. The input unit 109 preferably sets a region of interest for the scattergram displayed on the display unit 108 and determines sorting conditions.

[0096] In the microparticle separation device 100, the input unit 109 is not essential, and an external operation device may be connected. As the input unit 109, for example, a mouse, a keyboard, or the like can be used. Further, the input unit 109 may be connected to each part of the microparticle separation device 100 via a network.

[0097] (8) Control unit 110

[0098] The control unit 110 is configured to be able to control each of the light irradiation unit 103, the light detection unit 104, the analysis unit 105, the separation unit 106, the charging unit 106c, the recording unit 107, the display unit 108, and the input unit 109. The control unit 110 may be separately arranged for each part of the microparticle separation device 100, or may be provided outside the microparticle separation device 100. For example, it may be implemented by a personal computer or a CPU, and further, it can be stored as a program in a hardware resource provided with a recording medium (non-volatile memory (such as a USB memory), HDD, CD, etc.), and can also be made to function by a personal computer or a CPU. Further, the control unit 110 may be connected to each part of the microparticle separation device 100 via a network.

[0099] 5. Fifth Embodiment (Microparticle Separation Method)

[0100] In the microchip including at least a main flow path through which a liquid containing microparticles flows, a sheath liquid flow path communicating with the main flow path and supplying a sheath liquid, and a sheath liquid introduction portion for introducing the sheath liquid, the sheath liquid flowing through the sheath liquid introduction portion is vibrated.

[0101] Since the microparticle separation method according to the present technology is the same as the method performed by the microparticle separation device according to the present technology described above, the description is omitted here.

Examples

[0102] Hereinafter, the present invention will be described in more detail based on examples. Note that the embodiments described below show an example of a representative embodiment of the present invention, and thus the scope of the present invention should not be construed narrowly.

[0103] In the prior art, a microchip was vibrated by a vibration element to form droplets. FIG. 11 is a graph showing the frequency characteristics of the break-off point when vibrating using this prior art and the break-off point when vibrating using the present technology.

[0104] As shown in FIG. 11, it can be seen that in the prior art, the BOP varies greatly depending on the frequency. This is considered to be because, as described above, the vibration frequency coincides with the eigenvalue of the microchip, and phenomena such as the microchip vibrating greatly (resonance, shorter BOP) or, conversely, canceling each other out and the vibration of the microchip becoming smaller (anti-resonance, longer BOP) occur. On the other hand, in the present technology, since the microchip is not involved during vibration and is not affected by its eigenvalue, it can be seen that the change in BOP due to frequency becomes gentle, and it is possible to stabilize the generation of droplets.

[0105] Note that in the present technology, the following configurations can also be adopted. [1] A microchip including a main flow path through which a liquid containing microparticles flows, a sheath liquid flow path communicating with the main flow path through which a sheath liquid flows, and a sheath liquid introduction portion for introducing the sheath liquid, A microparticle separation device that vibrates the sheath liquid flowing through the sheath liquid introduction portion. [2] The microparticle separation device according to [1], further comprising a connection member that can be attached to the microchip and has a sheath liquid introduction connection portion that connects to the sheath liquid introduction portion. [3] The microparticle separation device according to [2], wherein a vibration element is attached to the connection member. [4] The microparticle separation device according to [3], wherein the driving frequency of the vibration element is different from the resonance frequency of the flow path in the microchip. 〔5〕 The micro particle sorting device according to 〔4〕, wherein the driving frequency of the vibration element is within a range of ±10% from the resonance frequency of the flow path in the microchip. 〔6〕 The micro particle sorting device according to any one of 〔3〕 to 〔5〕, wherein the sheath liquid introduction connection part has a sheath liquid convergence part whose width gradually or partially narrows from the vibration element side toward the sheath liquid introduction part side. 〔7〕 The micro particle sorting device according to 〔6〕, wherein the sheath liquid convergence part gradually or partially decreases in height from the vibration element side toward the sheath liquid introduction part side. 〔8〕 The micro particle sorting device according to 〔7〕, further comprising a connection part having a tubular part communicating with the tip of the sheath liquid convergence part between the sheath liquid convergence part and the sheath liquid introduction part. 〔9〕 The micro particle sorting device according to 〔8〕, wherein a tubular member is inserted inside the tubular part. 〔10〕 The micro particle sorting device according to 〔9〕, wherein at least a part of the tubular part and / or the tubular member is made of any one or more selected from the group consisting of an elastomer, a resin, and a metal. 〔11〕 The micro particle sorting device according to any one of 〔6〕 to 〔10〕, wherein the sheath liquid convergence part is substantially conical, substantially polygonal pyramidal, or a solid of revolution of an exponential function or a parabola. 〔12〕 The micro particle sorting device according to any one of 〔6〕 to 〔11〕, wherein the sheath liquid convergence part is made of a transparent member or a metal. 〔13〕 The micro particle sorting device according to any one of 〔6〕 to 〔12〕, wherein an electrode is inserted into the sheath liquid convergence part. 〔14〕 The micro particle sorting device according to any one of 〔6〕 to 〔13〕, wherein a swirling flow for swirling the sheath liquid is generated in the sheath liquid convergence part. 〔15〕 The sheath liquid inlet for introducing the sheath liquid into the sheath liquid converging portion is located at a position away from the center of the sheath liquid converging portion, the microparticle separation device according to any one of [6] to

[14] . 〔16〕 A microchip comprising: a main flow path through which a liquid containing microparticles flows; a sheath liquid flow path communicating with the main flow path for supplying a sheath liquid; and a sheath liquid introduction portion for introducing the sheath liquid. A light irradiation portion for irradiating the microparticles with light. A light detection portion for detecting light from the microparticles. A processing portion for processing a signal obtained from the light detection portion. And having A microparticle separation device that vibrates the sheath liquid flowing through the sheath liquid introduction portion. 〔17〕 In a microchip comprising at least a main flow path through which a liquid containing microparticles flows, a sheath liquid flow path communicating with the main flow path for supplying a sheath liquid, and a sheath liquid introduction portion for introducing the sheath liquid. A microparticle separation method for vibrating the sheath liquid flowing through the sheath liquid introduction portion.

Explanation of Signs

[0106] 100: Microparticle separation device 101: Sheath liquid feeding portion 102 Sample liquid feeding portion 103: Light irradiation portion 104: Light detection portion 105: Processing portion 106: Separation portion 107: Storage portion 108: Display portion 109: Input portion 110: Control portion M: Microchip Ma, Mb: Substrate layer M1: Orifice M11: Notch M2: Main flow path M3: Sample liquid introduction portion M31: Sample liquid flow path M4: Sheath liquid introduction portion M41: Sheath liquid flow path M5: Suction opening M51: Suction flow path M52: Communication port M61, 62: Constriction part M7: Straight part L1: Diameter of notch M11 L2: Opening diameter of orifice M1 C: Connection member C1: Sample liquid introduction connection part C2: Sheath liquid introduction connection part C20: Sheath liquid inlet C21: Sheath liquid convergence part C211: Tip of sheath liquid convergence part C22: Connection part C22a: Resin C22b: Thermoplastic elastomer C22c: O-ring C221: Tubular part C222: Tubular member C3: Vibration element C4: Electrode

Claims

1. A microchip comprising a main flow path through which a liquid containing microparticles flows, a sheath liquid flow path communicating with the main flow path through which a sheath liquid flows, and a sheath liquid introduction part for introducing the sheath liquid. Further comprising a connection member that can be attached to the microchip and has a sheath liquid introduction connection part that connects to the sheath liquid introduction part. A vibration element is attached to the connection member. The vibration element vibrates the sheath liquid flowing through the sheath liquid introduction part, and The sheath liquid introduction connection part has a sheath liquid convergence part whose width gradually or partially narrows from the vibration element side toward the sheath liquid introduction part side, a microparticle separation device.

2. The microparticle separation device according to claim 1, wherein the driving frequency of the vibration element is different from the resonance frequency of the flow path in the microchip.

3. The microparticle separation device according to claim 2, wherein the driving frequency of the vibration element is within a range of ±10% from the resonance frequency of the flow path in the microchip.

4. The microparticle separation device according to claim 1, wherein the sheath liquid convergence part gradually or partially decreases in height from the vibration element side toward the sheath liquid introduction part side.

5. The microparticle separation device according to claim 4, further comprising a connection part having a tubular part communicating with the tip of the sheath liquid convergence part between the sheath liquid convergence part and the sheath liquid introduction part.

6. The microparticle separation device according to claim 5, wherein a tubular member is inserted inside the tubular part.

7. At least a part of the tubular part and / or the tubular member is made of any one or more selected from the group consisting of elastomer, resin, and metal, the microparticle separation device according to claim 6.

8. The sheath liquid converging portion is a substantially conical shape, a substantially polygonal pyramid shape, or a solid of revolution of an exponential function or a parabola, the microparticle sorting device according to claim 1.

9. The sheath liquid converging portion is made of resin, metal, or a transparent member, the microparticle sorting device according to claim 1.

10. An electrode is inserted into the sheath liquid converging portion, the microparticle sorting device according to claim 1.

11. In the sheath liquid converging portion, a swirling flow that swirls the sheath liquid is generated, the microparticle sorting device according to claim 1.

12. The sheath liquid inlet for introducing the sheath liquid into the sheath liquid converging portion is located at a position away from the center of the sheath liquid converging portion, the microparticle sorting device according to claim 1.

13. A microchip including a main flow path through which a liquid containing microparticles flows, a sheath liquid flow path communicating with the main flow path and supplying a sheath liquid, and a sheath liquid introduction portion for introducing the sheath liquid, A light irradiation unit that irradiates light on the microparticles, A light detection unit that detects light from the microparticles, A processing unit that processes a signal obtained from the light detection unit, having, A connection member that is attachable to the microchip and has a sheath liquid introduction connection portion that connects to the sheath liquid introduction portion, A vibration element is attached to the connection member, The vibration element vibrates the sheath liquid flowing through the sheath liquid introduction portion, and The sheath liquid introduction connection portion has a sheath liquid converging portion whose width gradually or partially narrows from the vibration element side toward the sheath liquid introduction portion side, a microparticle sorting device.

14. In a microchip including at least a main flow path through which a liquid containing fine particles flows, a sheath liquid flow path communicating with the main flow path and supplying a sheath liquid, and a sheath liquid introduction portion for introducing the sheath liquid, The microchip further includes a connecting member that can be attached to the microchip and has a sheath liquid introduction connecting portion that connects to the sheath liquid introduction portion, A vibration element is attached to the connecting member, The vibration element vibrates the sheath liquid flowing through the sheath liquid introduction portion, and The sheath liquid introduction connecting portion has a sheath liquid converging portion whose width gradually or partially narrows from the vibration element side toward the sheath liquid introduction portion side. A method for separating fine particles.

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