WO2024048660A1 - Magnetic bead recovery method and magnetic bead recovery device - Google Patents

Magnetic bead recovery method and magnetic bead recovery device Download PDF

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Publication number
WO2024048660A1
WO2024048660A1 PCT/JP2023/031569 JP2023031569W WO2024048660A1 WO 2024048660 A1 WO2024048660 A1 WO 2024048660A1 JP 2023031569 W JP2023031569 W JP 2023031569W WO 2024048660 A1 WO2024048660 A1 WO 2024048660A1
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Prior art keywords
capillary
magnetic beads
hydrophilic liquid
magnetic
magnetic bead
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PCT/JP2023/031569
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French (fr)
Japanese (ja)
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真吾 上野
章一 土屋
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公益財団法人川崎市産業振興財団
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Publication of WO2024048660A1 publication Critical patent/WO2024048660A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices

Definitions

  • the present invention relates to a magnetic bead collection method and a magnetic bead collection device.
  • This application claims priority based on Japanese Patent Application No. 2022-138151 filed in Japan on August 31, 2022, the contents of which are incorporated herein.
  • the following method can be used to analyze the function of a functional protein. Beads with immobilized proteins and DNA are distributed into microwells (containing areas). Seal the microwells with oil. Analyze the function of proteins within microwells (see, for example, Patent Document 1).
  • An object of one aspect of the present invention is to provide a magnetic bead collection method and a magnetic bead collection device that can easily collect beads in a storage section.
  • the present invention includes the following aspects.
  • [1] When recovering the magnetic beads from the storage part that accommodates the magnetic beads and the first hydrophilic liquid and is covered with a hydrophobic liquid layer, a capillary whose tip is filled with the second hydrophilic liquid is used. , bringing the magnetic beads close to the storage part through the hydrophobic liquid layer and fusing the second hydrophilic liquid with the first hydrophilic liquid in the storage part; and a step of introducing the second hydrophilic liquid into the capillary.
  • [2] The method for collecting magnetic beads according to [1], wherein the second hydrophilic liquid is made to protrude from the tip when the capillary is brought close to the storage section.
  • a moving mechanism for moving the first hydrophilic liquid wherein a hydrophobic liquid layer covering the first hydrophilic liquid is formed in the accommodating part, and the moving mechanism is configured to move the second hydrophilic liquid in a range including the tip thereof.
  • a magnetic bead collection device in which a capillary is brought close to the storage part through the hydrophobic liquid layer.
  • FIG. 1 is a configuration diagram of a magnetic bead collection device according to an embodiment.
  • FIG. 3 is a process diagram of a magnetic bead collection method according to an embodiment.
  • FIG. 3 is a process diagram of a magnetic bead collection method according to an embodiment.
  • FIG. 3 is a process diagram of a magnetic bead collection method according to an embodiment.
  • FIG. 3 is a process diagram of a magnetic bead collection method according to an embodiment.
  • FIG. 3 is an explanatory diagram of a magnetic bead collection method according to a first comparative embodiment.
  • FIG. 7 is an explanatory diagram of a magnetic bead collection method according to a second comparative embodiment.
  • A A microscopic image of a microwell before magnetic bead collection.
  • B A microscopic image of the microwell after magnetic bead collection.
  • A Microscopic image of magnetic beads and a glass capillary before ejection.
  • B Microscopic image of the magnetic beads and the glass capillary
  • FIG. 1 is a configuration diagram of a magnetic bead collection device 200 according to an embodiment.
  • the array substrate 10 is oriented with the main surface 10a facing upward.
  • the array substrate 10 has a plurality of accommodating portions (microwells) 11 formed on one main surface 10a.
  • the array substrate 10 is, for example, rectangular in plan view.
  • the array substrate 10 has a flat plate shape.
  • the main surface 10a is flat.
  • the array substrate 10 is an example of a "substrate.” It is preferable that the array substrate 10 has transparency. When the array substrate 10 is transparent, it is easy to observe the sample inside the storage section 11. Note that the light transmittance of the array substrate 10 is not particularly limited.
  • the accommodating portion 11 is a recess formed in the main surface 10a.
  • the housing section 11 is capable of housing a sample. Since the array substrate 10 has a plurality of accommodating parts 11, it has the advantage that a plurality of samples can be processed at once.
  • the internal dimensions (for example, inner diameter) and depth of the storage section 11 are determined according to the size of the magnetic beads 1 to be stored. It is desirable that the internal dimensions and depth of the housing section 11 be slightly larger than the average particle diameter of the magnetic beads 1.
  • the internal dimensions and depth of the housing portion 11 may be, for example, more than one time and less than twice the average particle diameter of the magnetic beads 1. This makes it easier for the accommodating section 11 to accommodate only one magnetic bead 1.
  • the shape of the accommodating portion 11 in plan view is, for example, circular.
  • the housing portion 11 has, for example, a cylindrical internal space.
  • the shape of the accommodating portion 11 is not particularly limited.
  • the housing portion 11 may have a shape in which the inner diameter decreases in the depth direction.
  • the shape of the accommodating portion 11 in plan view may be a polygonal shape such as a rectangular shape or a hexagonal shape.
  • the plurality of storage units 11 are arranged, for example, in a two-dimensional matrix with a plurality of rows and a plurality of columns.
  • the array substrate 10 may be a laminated structure including a flat base and a well structure formed on the base. A well (accommodating part) is formed in the well structure part.
  • the base is made of glass, quartz, silicon wafer, or the like.
  • the well structure is formed of photoresist or the like.
  • the inner surface of the accommodating portion 11 be made hydrophilic.
  • hydrophilic treatment methods include oxygen plasma irradiation and ultraviolet-ozone treatment. If the inner surface of the accommodating part 11 is made hydrophilic, the first hydrophilic liquid 2 can be easily introduced into the accommodating part 11.
  • the main surface 10a be made hydrophobic.
  • the treatment agent for making the main surface 10a of the array substrate 10 hydrophobic include hexamethyldisilazane, organochlorosilane, polyorganosiloxane, and the like.
  • HMDS hexamethyldisilazane
  • the main surface 10a has a central region 10b and a peripheral region 10c.
  • the housing portion 11 is formed in the central region 10b.
  • a peripheral region 10c surrounds the central region 10b.
  • the magnetic beads 1 and the first hydrophilic liquid 2 are housed in the housing section 11 .
  • the magnetic beads 1 immobilize biomolecules such as nucleic acids, peptides, and proteins as targets.
  • the first hydrophilic liquid 2 is, for example, water or an aqueous solution.
  • the magnetic beads 1 are particles containing a magnetic material. Magnetic materials include iron, nickel, cobalt, or oxides thereof.
  • the average particle size of the magnetic beads 1 may be, for example, 1 ⁇ m to 10 ⁇ m.
  • the average particle diameter of the magnetic beads 1 is preferably 1 ⁇ m to 5 ⁇ m, more preferably 2 ⁇ m to 4 ⁇ m.
  • the magnetic bead collection device 200 includes an optical system 201, a photodetector 202, a stage 203, a water stacking frame (liquid stacking frame) 204, a capillary 5, a height adjustment mechanism (moving mechanism) 206, It includes a liquid feeding system 207 and a magnet 208.
  • the optical system 201 includes a light source 104, a half mirror 105, and an objective lens 106.
  • a light source 104 a mercury lamp, a halogen lamp, a laser light source, a xenon lamp, an LED, etc. can be used.
  • the wavelength of the emitted light L1 from the light source 104 is not particularly limited, but may include, for example, the visible light region (wavelengths from 360 nm to 780 nm).
  • the half mirror 105 reflects the emitted light L1 and directs it toward the array substrate 10.
  • the half mirror 105 can irradiate the array substrate 10 with the emitted light L1 from below (the side opposite to the capillary 5).
  • the objective lens 106 is installed below the array substrate 10 (on the opposite side from the capillary 5).
  • the optical system 201 irradiates the array substrate 10 on the stage 203 with the emitted light L1 from the light source 104 through the half mirror 105 and the objective lens 106.
  • the reflected light L2 which is the emitted light L1 reflected within the housing section 11, passes through the half mirror 105 and reaches the light detection section 202.
  • the light detection unit 202 includes, for example, an image sensor such as a CCD (Charge-Coupled Device) or a CMOS (Complementary MOS).
  • the array substrate 10 is placed on the stage 203.
  • Stage 203 is movable in any direction within a horizontal plane.
  • the water stacking frame 204 includes a bottom wall 111, an inner circumferential wall 112, and an outer circumferential wall 113.
  • the bottom wall 111 is annular.
  • the inner peripheral wall 112 is erected on the inner peripheral edge of the bottom wall 111.
  • the outer peripheral wall 113 is erected on the outer peripheral edge of the bottom wall 111.
  • An annular groove 114 is formed between the inner peripheral wall 112 and the outer peripheral wall 113.
  • the water stacking frame 204 is provided on the main surface 10a of the array substrate 10.
  • the water stacking frame 204 surrounds all the housing sections 11 in plan view.
  • the capillary 5 is a tubular body made of glass, resin, or the like.
  • the capillary 5 is, for example, shaped like a circular tube.
  • the capillary 5 is in a posture with its tip 5a facing downward.
  • the tip 5a faces the main surface 10a of the array substrate 10.
  • the capillary 5 is filled with a hydrophilic liquid (for example, water or an aqueous solution).
  • the capillary 5 is provided above the array substrate 10.
  • the inner diameter of the tip 5a of the capillary 5 can be determined, for example, according to the size of the particles to be collected (magnetic beads 1 in this embodiment).
  • the inner diameter of the tip 5a of the capillary 5 may be, for example, not less than one time and not more than twice the average particle diameter of the magnetic beads 1.
  • the height adjustment mechanism 206 is, for example, a manipulator that includes a holding arm 211 and a lifting mechanism 212.
  • the holding arm 211 holds the capillary 5.
  • the lifting mechanism 212 raises and lowers the capillary 5 via the holding arm 211.
  • the elevating mechanism 212 moves the capillary 5 toward and away from the array substrate 10 .
  • the height adjustment mechanism 206 can hold the capillary 5 at an arbitrary height position.
  • the height adjustment mechanism 206 may have the function of not only adjusting the height of the capillary 5 but also allowing the capillary 5 to be placed at an arbitrary position within a horizontal plane.
  • the liquid feeding system 207 includes a liquid feeding path 213, a pump 214, and a pressure gauge 215.
  • the liquid sending path 213 connects the capillary 5 and the pump 214.
  • the liquid feeding path 213 is, for example, a tube made of resin.
  • the liquid sending path 213 is filled with, for example, oil (hydrophobic liquid).
  • the pump 214 can adjust the pressure of the hydrophilic liquid in the capillary 5 by applying pressure to the oil in the liquid sending path 213.
  • the pressure gauge 215 measures the pressure within the liquid feeding path 213.
  • the magnet 208 may be made of, for example, a rare earth magnet material (e.g., neodymium iron boron (NdFeB), samarium cobalt (SmCo), etc.), a ceramic magnet material (e.g., strontium ferrite, etc.), or other magnetic material (e.g., iron, cobalt, nickel, etc.). , their alloys and oxides), etc.
  • a neodymium magnet (NdFeB) is preferable.
  • Magnet 208 is installed above array substrate 10 .
  • the magnet 208 is located above the housing portion 11 .
  • the magnet 208 can apply magnetic force (magnetic force in the attraction direction) to the magnetic beads 1 in the housing section 11 .
  • one of the upper and lower ends of the magnet 208 may be a north pole, and the other of the upper and lower ends may be a south pole.
  • Magnet 208 may be an electromagnet.
  • a hydrophobic liquid layer 121 is formed inside the inner peripheral wall 112 of the water lamination frame 204.
  • the hydrophobic liquid layer 121 is formed on the main surface 10a in a region that includes all the housing parts 11.
  • the hydrophobic liquid layer 121 covers the surface of the first hydrophilic liquid 2 within the storage section 11 .
  • Examples of the hydrophobic liquid constituting the hydrophobic liquid layer 121 include fluorine oil and silicone oil.
  • the fluorine oil include perfluoropolyethers such as perfluoropolyether (PFPE), perfluoroalkyl ether (PFAE), and perfluoropolyalkyl ether (PFPAE).
  • the hydrophobic liquid may also be hydrofluoroether (HFE), mineral oil, saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, perfluorocarbon, or the like.
  • a hydrophilic liquid layer 122 is formed on the hydrophobic liquid layer 121.
  • Examples of the hydrophilic liquid constituting the hydrophilic liquid layer 122 include water and an aqueous solution.
  • the hydrophilic liquid layer 122 is formed inside the outer peripheral wall 113 of the water lamination frame 204.
  • Magnetic bead collection method A bead collection method according to an embodiment will be described.
  • 2 to 5 are process diagrams of the magnetic bead collection method according to the embodiment.
  • the magnetic bead collection method of the present embodiment includes a first step (fusing the second hydrophilic liquid with the first hydrophilic liquid in the container), a second step (guiding the magnetic beads into the capillary), and a third step. (discharge of magnetic beads).
  • a second hydrophilic liquid 302 is introduced into the capillary 5.
  • the second hydrophilic liquid 302 inside the capillary 5 reaches the tip 5a.
  • the second hydrophilic liquid 302 is in a state of protruding downward from the tip 5a.
  • the second hydrophilic liquid 302 is, for example, water or an aqueous solution.
  • the portion of the second hydrophilic liquid 302 that protrudes from the tip 5a is referred to as "second hydrophilic liquid 302A.”
  • the second hydrophilic liquid 302A has, for example, a curved convex shape such as a partially spherical shape or a partially ellipsoidal spherical shape.
  • the magnet 208 applies a magnetic force (attractive force) upward to the magnetic beads 1 in the housing section 11. Therefore, the magnetic beads 1 in the container 11 rise within the first hydrophilic liquid 2.
  • the capillary 5 is arranged to face the accommodating part 11.
  • the capillary 5 is lowered using the height adjustment mechanism 206 (see FIG. 1).
  • a portion of the capillary 5 including the tip 5a is brought close to the accommodating portion 11 through the hydrophobic liquid layer 121.
  • a portion of the capillary 5 including the tip 5a is immersed in the hydrophobic liquid layer 121. It is desirable that the pressure within the capillary 5 is adjusted so that the second hydrophilic liquid 302A remains protruding from the tip 5a.
  • the pressure inside the capillary 5 can be adjusted by a pump 214 (see FIG. 1).
  • the magnetic beads 1 rise through the first hydrophilic liquid 2 and the second hydrophilic liquid 302 due to the magnetic force (attractive force) of the magnet 208 and are guided into the capillary 5.
  • the capillary 5 is raised using the height adjustment mechanism 206 (see FIG. 1).
  • the second hydrophilic liquid 302 in the capillary 5 is separated from the first hydrophilic liquid 2 in the storage section 11 . Thereby, the sample can be collected from the storage section 11 of the array substrate 10.
  • the sample to be collected is magnetic beads 1 or bead suspension (hydrophilic liquid 2 containing magnetic beads 1).
  • the second hydrophilic liquid 302 in the capillary 5 is fused with the first hydrophilic liquid 2 in the storage part 11, and the magnetic beads 1 in the storage part 11 are moved into the capillary 5 by magnetic force. lead to. Therefore, the magnetic beads 1 in the storage section 11 can be easily collected.
  • the second hydrophilic liquid 302A is made to protrude from the tip 5a. Therefore, the second hydrophilic liquid 302 easily fuses with the first hydrophilic liquid 2 in the storage section 11 .
  • FIG. 6 is an explanatory diagram of the magnetic bead collection method according to the first comparative embodiment.
  • the magnetic bead collection method of the first comparative embodiment there is no hydrophilic liquid in the capillary 5. Therefore, even if the pressure inside the capillary 5 is reduced by bringing the tip 5a of the capillary 5 closer to the accommodating part 11, the hydrophobic liquid around the capillary 5 will be introduced into the capillary 5, but the magnetic beads 1 in the accommodating part 11 will be introduced into the capillary 5. It is difficult to lead to capillary 5.
  • FIG. 6 is an explanatory diagram of the magnetic bead collection method according to the first comparative embodiment.
  • FIG. 7 is an explanatory diagram of a magnetic bead collection method according to a second comparative embodiment. As shown in FIG. 7, the magnetic bead collection method of the second comparative embodiment does not use a magnet. Therefore, even if the second hydrophilic liquid 302 fuses with the first hydrophilic liquid 2, the magnetic beads 1 are not guided into the capillary 5.
  • the magnetic bead collection device 200 of this embodiment includes a height adjustment mechanism 206 that brings the capillary 5 closer to the storage section 11 and a magnet 208 that applies magnetic force in the attraction direction to the magnetic beads 1. Therefore, the second hydrophilic liquid 302 in the capillary 5 can be fused with the first hydrophilic liquid 2 in the container 11, and the magnetic beads 1 in the container 11 can be guided into the capillary 5 by magnetic force. Therefore, the magnetic beads 1 in the storage section 11 can be easily collected.
  • PCR reaction solution 20pg/ ⁇ L template DNA 0.3 ⁇ M biotin-modified DNA primer 0.3 ⁇ M DNA primer 0.2 ⁇ M each dNTP Mix 0.025U/ ⁇ L PrimeSTAR (registered trademark) HS polymerase 1xPrimeSTAR (registered trademark) buffer (Takara Bio)
  • Magnetic beads streptavidin-modified magnetic beads (MS300/streptavidin, JSR; hereinafter referred to as “magnetic beads”) supernatant was removed, and 100 ⁇ L of binding buffer (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, 0.05% (w) /v) Tween 20, pH 7.4).
  • the magnetic beads were suspended in 170 ⁇ L of binding buffer in which 7 pmol of biotin-modified DNA had been dissolved, and stirred at room temperature for 30 minutes. This allowed the biotin-modified DNA to bind to the magnetic beads.
  • the magnetic beads were then washed 5 times with 100 ⁇ L of binding buffer.
  • the magnetic beads were then suspended in 30 ⁇ L of binding buffer.
  • the concentration of DNA-immobilized magnetic beads in the above suspension was measured using an automatic cell counter (trade name: Countess II, manufactured by ThermoFisher).
  • the supernatant of the DNA-immobilized magnetic bead suspension (5 x 10 7 DNA-immobilized magnetic beads) was removed, and the DNA-immobilized magnetic beads were mixed with 60 ⁇ L of a fluorescent aqueous solution (10 ⁇ M sodium fluorescein, 50 mM Tris-HCl, pH 7. 5).
  • a fluorescent aqueous solution (10 ⁇ M sodium fluorescein, 50 mM Tris-HCl, pH 7. 5
  • the STAVAX® substrate surface (30 mm x 30 mm x 5 mm) was plated with nickel phosphorus to a thickness of approximately 100 ⁇ m.
  • a cubic structure (4 ⁇ m x 4 ⁇ m x 4 ⁇ m) was formed on a plane (1.2 mm x 1.2 mm) by 1 x 10 6
  • a mold with individual pieces lined up was fabricated. The mold was attached to an injection molding machine, and a cycloolefin polymer (trade name: ZEONEX (registered trademark) 480, manufactured by Nippon Zeon) was injected.
  • an array substrate was produced in which 1 x 10 6 microwells (4 ⁇ m x 4 ⁇ m x 4 ⁇ m) were lined up on a region (1.2 mm x 1.2 mm) of the substrate surface (30 mm x 30 mm x 1 mm).
  • the area of the array substrate where the microwells are lined up will be referred to as the "array area”
  • the area where no microwells will be present will be referred to as the "outside the array area”.
  • ⁇ Surface treatment of array substrate> By irradiating the main surface of the array substrate with oxygen plasma, the main surface of the array substrate and the surface inside the microwell were made hydrophilic. Subsequently, a polypropylene film on which hexamethyldisilazane (HMDS) was vapor-deposited was pressed onto the surface of the array substrate, HMDS was transferred to the surface of the array substrate, and the substrate surface was made hydrophobic by treatment at 110° C. for 10 minutes. Through the above operations, an array substrate was produced in which the surface (principal surface) inside the microwell was hydrophilic and the surface outside the microwell was hydrophobic.
  • HMDS hexamethyldisilazane
  • a wipe member was prepared by inserting a stainless steel rod with a diameter of 3 mm into a silicone tube (outer diameter 4 mm, inner diameter 2 mm, length 50 mm). The wipe member was placed in contact with the outside of the array area of the array substrate. A neodymium magnet (20 x 5 x 10 mm) was placed under the array substrate between the contact portion of the wipe member and the edge of the array area.
  • 60 ⁇ L of the DNA-immobilized magnetic bead suspension was dropped onto the main surface of the array substrate at the edge of the array area from the contact portion of the wipe member.
  • 60 ⁇ L of fluorine oil (trade name: Krytox (registered trademark) GPL104 (manufactured by Chemours) was dropped onto the main surface of the array substrate on the opposite side from the array area from the contact surface of the wipe member.
  • the wipe member and neodymium magnet were simultaneously moved toward the array area at 0.1 mm/s, and the wipe member and neodymium magnet were stopped after passing through the array area. Through this operation, the DNA-immobilized magnetic beads and the fluorescent aqueous solution were distributed to the microwells and sealed with fluorine oil.
  • a frame for water lamination (frame for liquid lamination) was produced using a 3D printer (trade name: From2, manufactured by Formlabs).
  • a frame for water lamination was pasted onto an array substrate in which DNA-immobilized magnetic beads and a fluorescent aqueous solution were sealed in microwells.
  • 800 ⁇ L of fluorine oil (trade name: Krytox (registered trademark) GPL107 (manufactured by Chemours) was added to the inner peripheral wall of the water lamination frame.
  • a hydrophobic liquid layer made of fluorine oil was formed on the surface of the array substrate.
  • the liquid passage was filled with mineral oil.
  • a glass capillary (tip inner diameter 5.5 ⁇ m) was entirely filled with an aqueous solution (50 mM Tris-HCl, pH 7.5) and attached to a capillary holder. The tip of the glass capillary was oriented downward, and the tip of the glass capillary was observed using coaxial epi-illumination through a half mirror, and adjusted so that the tip of the glass capillary was located at the center of the field of view of the microscope.
  • a neodymium magnet (10 x 10 x 35 mm) was installed near the glass capillary.
  • the gauge pressure of the pressure gauge was set to -3 kPa by operating the syringe pump.
  • a manipulator was operated to lower the tip of the glass capillary toward the surface of the array substrate.
  • FIG. 8(A) is a microscopic image of the microwell before magnetic bead collection.
  • FIG. 8(B) is a microscopic image of the microwell after magnetic bead collection. It can be seen from FIGS. 8(A) and 8(B) that magnetic beads were collected from the microwells.
  • the DNA-immobilized magnetic beads were moved to the tip of the glass capillary by bringing it close to a neodymium magnet.
  • the glass capillary tip was lowered onto a plastic substrate of the same material and thickness as the array substrate and observed under coaxial epi-illumination, the presence of DNA-immobilized magnetic beads at the glass capillary tip was confirmed.
  • FIG. 9(A) is a microscopic image of the magnetic beads and the glass capillary before ejection.
  • FIG. 9(B) is a microscopic image of the magnetic beads and the glass capillary after ejection. It can be seen from FIGS. 9(A) and 9(B) that the magnetic beads were ejected from the glass capillary.
  • the embodiments of the present invention have been described above, but each configuration and combination thereof in the embodiments are merely examples, and additions, omissions, substitutions, and other changes to the configurations may be made without departing from the spirit of the present invention. is possible.
  • the number of magnetic beads 1 accommodated in the accommodation section 11 is not limited to one.
  • the number of magnetic beads 1 accommodated in the accommodation section 11 may be plural (any number greater than or equal to 2).
  • the number of magnetic beads 1 accommodated can be adjusted by at least one of the internal dimensions and depth of the accommodating portion 11. If a plurality of magnetic beads 1 are accommodated in the accommodating section 11, the number of beads to be analyzed per area of the array substrate 10 can be increased. Therefore, screening can be performed efficiently.
  • the contact angle of water on the hydrophobized surface is, for example, 60 degrees or more.
  • the contact angle of water on the hydrophilized surface is, for example, 40 degrees or less.
  • Nucleic acids and proteins may be immobilized on the magnetic beads. After evaluating the function of the protein within the container, the magnetic beads can be collected by the magnetic bead collection method of the embodiment. By analyzing the nucleic acids immobilized on the recovered magnetic beads, the amino acid sequence of the protein can be identified (for example, see International Publication No. 2020/095985).
  • Nucleic acids may be immobilized on the magnetic beads. After evaluating the function of the nucleic acid within the container, the magnetic beads can be collected by the magnetic bead collection method of the embodiment. By analyzing the nucleic acids immobilized on the recovered magnetic beads, the nucleic acid sequence can be identified (for example, see Aniela Wochner et al., Ribozyme-Catalyzed Transcription of an Active Ribozyme. Science 332, 209 (2011)) ).
  • Nucleic acids may be immobilized on the magnetic beads. After cells are lysed in the container and intracellular RNA is hybridized to the nucleic acid of the magnetic beads, the magnetic beads can be collected by the magnetic bead collection method of the embodiment. RNA hybridized to the nucleic acids immobilized on the collected magnetic beads can be analyzed (for example, Jinzhou Yuan et al., An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq; Sci. Rep. 6:33883 (2016)).
  • the magnetic beads may have antibodies immobilized thereon. After the cells are lysed in the container and the cell-derived protein is bound to the antibody of the magnetic beads, the magnetic beads can be collected by the magnetic bead collection method of the embodiment. Proteins bound to antibodies immobilized on recovered magnetic beads can be analyzed (e.g., Lucas Armbrecht et al., Single-cell protein profiling in microchambers with barcoded beads. Microsystems & Nanoengineering 5:55 (2019) ).
  • Magnetic beads 2 First hydrophilic liquid 5 Capillary 5a Tip 10 Array substrate (substrate) 10a Main surface (one side) 11 Storage part 121 Hydrophobic liquid layer 206 Height adjustment mechanism (moving mechanism) 208 Magnet 302 Second hydrophilic liquid

Abstract

In a magnetic bead recovery method, a magnetic bead (1) is recovered from a storage section (11) containing the magnetic bead (1) and a first hydrophilic liquid (2). The magnetic bead recovery method comprises: a step for bringing a capillary (5) filled with a second hydrophilic liquid (302) in a range including a tip (5a) close to the storage section (11) through a hydrophilic liquid layer (121) and coalescing the second hydrophilic liquid (302) with the first hydrophilic liquid (2) in the storage section (11); and a step for introducing the magnetic bead (1) by a magnetic force into the capillary (5) through the first hydrophilic liquid (2) and the second hydrophilic liquid (302).

Description

磁気ビーズ回収方法および磁気ビーズ回収装置Magnetic bead collection method and magnetic bead collection device
　本発明は、磁気ビーズ回収方法および磁気ビーズ回収装置に関する。
　本願は、２０２２年８月３１日に日本に出願された特願２０２２－１３８１５１号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to a magnetic bead collection method and a magnetic bead collection device.
This application claims priority based on Japanese Patent Application No. 2022-138151 filed in Japan on August 31, 2022, the contents of which are incorporated herein.
　生体分子の機能観察・検出を目的として、ビーズに生体分子を固定する方法がある。生体分子を解析するには、次の手法が有用である。エマルションのような閉鎖空間に、生体分子を固定したビーズを入れる。閉鎖空間内でビーズ上の生体分子の反応を行う。閉鎖空間からビーズを回収し、ビーズ上の生体分子を解析する（例えば、非特許文献１を参照）。
　一方、ビーズを含有する閉鎖反応空間として、オイルで封止されたマイクロウェルアレイを用いる技術がある。この技術によれば、並列測定が可能になり、解析効率を向上することができる（例えば、非特許文献２を参照）。 There is a method of immobilizing biomolecules on beads for the purpose of functional observation and detection of biomolecules. The following techniques are useful for analyzing biomolecules. Beads with immobilized biomolecules are placed in a closed space like an emulsion. Reactions of biomolecules on beads are carried out in a closed space. Beads are collected from the closed space and biomolecules on the beads are analyzed (see, for example, Non-Patent Document 1).
On the other hand, there is a technique that uses a microwell array sealed with oil as a closed reaction space containing beads. According to this technique, parallel measurement becomes possible and analysis efficiency can be improved (see, for example, Non-Patent Document 2).
　機能性タンパク質の機能を解析するには、次の手法を用いることができる。タンパク質とＤＮＡを固定化したビーズをマイクロウェル（収容部）に分配する。マイクロウェルをオイルで封止する。マイクロウェル内のタンパク質の機能を解析する（例えば、特許文献１を参照）。 The following method can be used to analyze the function of a functional protein. Beads with immobilized proteins and DNA are distributed into microwells (containing areas). Seal the microwells with oil. Analyze the function of proteins within microwells (see, for example, Patent Document 1).
国際公開第２０２０／０９５９８５号International Publication No. 2020/095985
　上記技術において、高機能性タンパク質の遺伝子を特定するためにビーズ上のＤＮＡを解析するには、オイルで封止されたマイクロウェル（収容部）からビーズを回収する必要がある。しかし、オイル封止されたマイクロウェル内のビーズを回収する操作は容易ではなかった。そのため、ビーズを容易に回収する方法が求められていた。 In the above technology, in order to analyze the DNA on the beads in order to identify the genes of highly functional proteins, it is necessary to collect the beads from the microwell (container) sealed with oil. However, it was not easy to collect the beads in the oil-sealed microwells. Therefore, there has been a need for a method for easily collecting beads.
　本発明の一態様は、収容部内のビーズを容易に回収できる磁気ビーズ回収方法および磁気ビーズ回収装置を提供することを課題とする。 An object of one aspect of the present invention is to provide a magnetic bead collection method and a magnetic bead collection device that can easily collect beads in a storage section.
　本発明は、以下の態様を含む。
［１］磁気ビーズおよび第１親水性液体が収容され、疎水性液体層で覆われた収容部から前記磁気ビーズを回収するにあたって、第２親水性液体が先端を含む範囲に充てんされたキャピラリーを、前記疎水性液体層を通して前記収容部に近づけ、前記第２親水性液体を前記収容部内の前記第１親水性液体に融合させる工程と、前記磁気ビーズを、磁力によって、前記第１親水性液体および前記第２親水性液体を通して前記キャピラリー内に導く工程と、を有する、磁気ビーズ回収方法。
［２］前記キャピラリーを前記収容部に近づけるにあたって、前記第２親水性液体を前記先端から突出させる、［１］記載の磁気ビーズ回収方法。
［３］前記収容部は、基板の一方の面に形成された凹部である、［１］記載の磁気ビーズ回収方法。
［４］前記キャピラリー内の前記磁気ビーズを前記キャピラリーから吐出する工程をさらに有する、［１］～［３］のうちいずれか１つに記載の磁気ビーズ回収方法。
［５］磁気ビーズおよび第１親水性液体が収容された収容部に対向するキャピラリーと、前記磁気ビーズに吸引方向の磁力を作用させる磁石と、前記キャピラリーを前記収容部に接近および離間する方向に移動させる移動機構と、を備え、前記収容部に、前記第１親水性液体を覆う疎水性液体層が形成され、前記移動機構は、第２親水性液体が先端を含む範囲に充てんされた前記キャピラリーを、前記疎水性液体層を通して前記収容部に近づける、磁気ビーズ回収装置。 The present invention includes the following aspects.
[1] When recovering the magnetic beads from the storage part that accommodates the magnetic beads and the first hydrophilic liquid and is covered with a hydrophobic liquid layer, a capillary whose tip is filled with the second hydrophilic liquid is used. , bringing the magnetic beads close to the storage part through the hydrophobic liquid layer and fusing the second hydrophilic liquid with the first hydrophilic liquid in the storage part; and a step of introducing the second hydrophilic liquid into the capillary.
[2] The method for collecting magnetic beads according to [1], wherein the second hydrophilic liquid is made to protrude from the tip when the capillary is brought close to the storage section.
[3] The magnetic bead collection method according to [1], wherein the storage section is a recess formed on one surface of the substrate.
[4] The method for collecting magnetic beads according to any one of [1] to [3], further comprising the step of discharging the magnetic beads in the capillary from the capillary.
[5] A capillary facing the storage section in which the magnetic beads and the first hydrophilic liquid are stored, a magnet that applies a magnetic force in the attracting direction to the magnetic beads, and a capillary that moves the capillary toward and away from the storage section. a moving mechanism for moving the first hydrophilic liquid, wherein a hydrophobic liquid layer covering the first hydrophilic liquid is formed in the accommodating part, and the moving mechanism is configured to move the second hydrophilic liquid in a range including the tip thereof. A magnetic bead collection device in which a capillary is brought close to the storage part through the hydrophobic liquid layer.
　本発明の一態様によれば、収容部内のビーズを容易に回収できる磁気ビーズ回収方法および磁気ビーズ回収装置を提供することができる。 According to one aspect of the present invention, it is possible to provide a magnetic bead collection method and a magnetic bead collection device that can easily collect beads in a storage section.
実施形態に係る磁気ビーズ回収装置の構成図である。FIG. 1 is a configuration diagram of a magnetic bead collection device according to an embodiment. 実施形態に係る磁気ビーズ回収方法の工程図である。FIG. 3 is a process diagram of a magnetic bead collection method according to an embodiment. 実施形態に係る磁気ビーズ回収方法の工程図である。FIG. 3 is a process diagram of a magnetic bead collection method according to an embodiment. 実施形態に係る磁気ビーズ回収方法の工程図である。FIG. 3 is a process diagram of a magnetic bead collection method according to an embodiment. 実施形態に係る磁気ビーズ回収方法の工程図である。FIG. 3 is a process diagram of a magnetic bead collection method according to an embodiment. 第１比較形態に係る磁気ビーズ回収方法の説明図である。FIG. 3 is an explanatory diagram of a magnetic bead collection method according to a first comparative embodiment. 第２比較形態に係る磁気ビーズ回収方法の説明図である。FIG. 7 is an explanatory diagram of a magnetic bead collection method according to a second comparative embodiment. （Ａ）磁気ビーズ回収前のマイクロウェルの顕微鏡画像である。（Ｂ）磁気ビーズ回収後のマイクロウェルの顕微鏡画像である。(A) A microscopic image of a microwell before magnetic bead collection. (B) A microscopic image of the microwell after magnetic bead collection. （Ａ）磁気ビーズと、吐出前のガラスキャピラリーの顕微鏡画像である。（Ｂ）磁気ビーズと吐出後のガラスキャピラリーの顕微鏡画像である。(A) Microscopic image of magnetic beads and a glass capillary before ejection. (B) Microscopic image of the magnetic beads and the glass capillary after ejection.
　図１は、実施形態に係る磁気ビーズ回収装置２００の構成図である。以下、図１に即して上下の位置関係を仮に定める。アレイ基板１０は、主面１０ａを上に向けた姿勢とされている。 FIG. 1 is a configuration diagram of a magnetic bead collection device 200 according to an embodiment. Hereinafter, the vertical positional relationship will be tentatively determined based on FIG. The array substrate 10 is oriented with the main surface 10a facing upward.
［基板］
　図１に示すように、アレイ基板１０は、一方の面である主面１０ａに、複数の収容部（マイクロウェル）１１が形成されている。アレイ基板１０は、例えば、平面視において矩形状とされている。アレイ基板１０は平板状とされている。主面１０ａは平坦である。アレイ基板１０は、「基板」の一例である。
　アレイ基板１０は、透明性を有することが好ましい。アレイ基板１０が透明性を有すると、収容部１１内の試料を観察しやすい。なお、アレイ基板１０の光透過性は特に限定されない。 [substrate]
As shown in FIG. 1, the array substrate 10 has a plurality of accommodating portions (microwells) 11 formed on one main surface 10a. The array substrate 10 is, for example, rectangular in plan view. The array substrate 10 has a flat plate shape. The main surface 10a is flat. The array substrate 10 is an example of a "substrate."
It is preferable that the array substrate 10 has transparency. When the array substrate 10 is transparent, it is easy to observe the sample inside the storage section 11. Note that the light transmittance of the array substrate 10 is not particularly limited.
　収容部１１は、主面１０ａに形成された凹部である。収容部１１は、試料を収容可能である。アレイ基板１０は、複数の収容部１１を有するため、複数の試料を一度に処理できるという利点がある。 The accommodating portion 11 is a recess formed in the main surface 10a. The housing section 11 is capable of housing a sample. Since the array substrate 10 has a plurality of accommodating parts 11, it has the advantage that a plurality of samples can be processed at once.
　収容部１１の内形寸法（例えば、内径）および深さは、収容する磁気ビーズ１の大きさに合わせて定められる。収容部１１の内形寸法および深さは、磁気ビーズ１の平均粒径よりやや大きい程度であることが望ましい。収容部１１の内形寸法および深さは、例えば、磁気ビーズ１の平均粒径の１倍を越え、かつ２倍未満であってよい。これにより、収容部１１は、磁気ビーズ１を１個のみ収容しやすくなる。 The internal dimensions (for example, inner diameter) and depth of the storage section 11 are determined according to the size of the magnetic beads 1 to be stored. It is desirable that the internal dimensions and depth of the housing section 11 be slightly larger than the average particle diameter of the magnetic beads 1. The internal dimensions and depth of the housing portion 11 may be, for example, more than one time and less than twice the average particle diameter of the magnetic beads 1. This makes it easier for the accommodating section 11 to accommodate only one magnetic bead 1.
　平面視における収容部１１の形状は、例えば、円形状である。収容部１１は、例えば、円柱状の内部空間を有する。
　なお、収容部１１の形状は特に限定されない。例えば、収容部１１は、深さ方向に内径が小さくなる形状であってもよい。収容部１１の平面視形状は、矩形状、六角形状などの多角形状でもよい。
　複数の収容部１１は、例えば、複数行および複数列の２次元マトリクス状に配列されている。 The shape of the accommodating portion 11 in plan view is, for example, circular. The housing portion 11 has, for example, a cylindrical internal space.
Note that the shape of the accommodating portion 11 is not particularly limited. For example, the housing portion 11 may have a shape in which the inner diameter decreases in the depth direction. The shape of the accommodating portion 11 in plan view may be a polygonal shape such as a rectangular shape or a hexagonal shape.
The plurality of storage units 11 are arranged, for example, in a two-dimensional matrix with a plurality of rows and a plurality of columns.
　アレイ基板１０の材料としては、樹脂、ガラス、石英などがある。樹脂としては、シクロオレフィンポリマー（ＣＯＰ）、ポリスチレン（ＰＳ）、ポリカーボネート（ＰＣ）、環状オレフィン・コポリマー（ＣＯＣ）、ポリジメチルシロキサン（ＰＤＭＳ）、ポリエチレンテレフタレート（ＰＥＴ）、アクリル樹脂（ＰＭＭＡなど）等が挙げられる。アレイ基板１０は、平板状の基体と、基体の上に形成されたウェル構造部とを備えた積層構造体であってもよい。ウェル構造部にはウェル（収容部）が形成されている。基体はガラス、石英、シリコンウエハなどで形成される。ウェル構造部はフォトレジストなどで形成される。 Materials for the array substrate 10 include resin, glass, quartz, etc. Examples of resins include cycloolefin polymer (COP), polystyrene (PS), polycarbonate (PC), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), and acrylic resin (PMMA, etc.). Can be mentioned. The array substrate 10 may be a laminated structure including a flat base and a well structure formed on the base. A well (accommodating part) is formed in the well structure part. The base is made of glass, quartz, silicon wafer, or the like. The well structure is formed of photoresist or the like.
　収容部１１の内面は、親水化されていることが望ましい。親水化処理方法としては、酸素プラズマ照射、紫外線－オゾン処理などが挙げられる。収容部１１の内面が親水化されていると、第１親水性液体２を収容部１１に容易に導入することができる。 It is desirable that the inner surface of the accommodating portion 11 be made hydrophilic. Examples of hydrophilic treatment methods include oxygen plasma irradiation and ultraviolet-ozone treatment. If the inner surface of the accommodating part 11 is made hydrophilic, the first hydrophilic liquid 2 can be easily introduced into the accommodating part 11.
　主面１０ａは、疎水化されていることが望ましい。アレイ基板１０の主面１０ａを疎水化する処理剤としては、ヘキサメチルジシラザン、オルガノクロロシラン、ポリオルガノシロキサン等が挙げられる。例えば、ヘキサメチルジシラザン（ＨＭＤＳ）を蒸着したポリプロピレンフィルムを主面１０ａに圧着させてＨＭＤＳを主面１０ａに転写することによって、疎水化された主面１０ａを有するアレイ基板１０が得られる。 It is desirable that the main surface 10a be made hydrophobic. Examples of the treatment agent for making the main surface 10a of the array substrate 10 hydrophobic include hexamethyldisilazane, organochlorosilane, polyorganosiloxane, and the like. For example, by pressing a polypropylene film on which hexamethyldisilazane (HMDS) has been vapor-deposited onto the main surface 10a and transferring HMDS onto the main surface 10a, the array substrate 10 having the hydrophobic main surface 10a can be obtained.
　主面１０ａは、中央領域１０ｂと、周辺領域１０ｃとを有する。収容部１１は、中央領域１０ｂに形成されている。周辺領域１０ｃは、中央領域１０ｂを囲む。
　収容部１１には、磁気ビーズ１および第１親水性液体２が収容されている。磁気ビーズ１は、例えば、核酸、ペプチド、タンパク質などの生体分子をターゲットとして固定化している。第１親水性液体２は、例えば、水、または水溶液である。磁気ビーズ１は磁性材料を含む粒子である。磁性材料としては、鉄、ニッケル、コバルト、またはそれらの酸化物が挙げられる。磁気ビーズ１の平均粒径は、例えば、１μｍ～１０μｍであってよい。磁気ビーズ１の平均粒径は、１μｍ～５μｍが好ましく、２μｍ～４μｍがさらに好ましい。 The main surface 10a has a central region 10b and a peripheral region 10c. The housing portion 11 is formed in the central region 10b. A peripheral region 10c surrounds the central region 10b.
The magnetic beads 1 and the first hydrophilic liquid 2 are housed in the housing section 11 . The magnetic beads 1 immobilize biomolecules such as nucleic acids, peptides, and proteins as targets. The first hydrophilic liquid 2 is, for example, water or an aqueous solution. The magnetic beads 1 are particles containing a magnetic material. Magnetic materials include iron, nickel, cobalt, or oxides thereof. The average particle size of the magnetic beads 1 may be, for example, 1 μm to 10 μm. The average particle diameter of the magnetic beads 1 is preferably 1 μm to 5 μm, more preferably 2 μm to 4 μm.
［磁気ビーズ回収装置］
　実施形態に係る磁気ビーズ回収装置について説明する。
　磁気ビーズ回収装置２００は、光学系２０１と、光検出部２０２と、ステージ２０３と、水積層用枠（液体積層用枠）２０４と、キャピラリー５と、高さ調整機構（移動機構）２０６と、送液系２０７と、磁石２０８と、を備える。 [Magnetic bead collection device]
A magnetic bead collection device according to an embodiment will be described.
The magnetic bead collection device 200 includes an optical system 201, a photodetector 202, a stage 203, a water stacking frame (liquid stacking frame) 204, a capillary 5, a height adjustment mechanism (moving mechanism) 206, It includes a liquid feeding system 207 and a magnet 208.
　光学系２０１は、光源１０４と、ハーフミラー１０５と、対物レンズ１０６と、を備える。
　光源１０４としては、水銀ランプ、ハロゲンランプ、レーザ光源、キセノンランプ、ＬＥＤなどを使用できる。光源１０４からの出射光Ｌ１の波長は特に限定されないが、例えば、可視光領域（波長３６０ｎｍ～７８０ｎｍ）を含んでいてよい。 The optical system 201 includes a light source 104, a half mirror 105, and an objective lens 106.
As the light source 104, a mercury lamp, a halogen lamp, a laser light source, a xenon lamp, an LED, etc. can be used. The wavelength of the emitted light L1 from the light source 104 is not particularly limited, but may include, for example, the visible light region (wavelengths from 360 nm to 780 nm).
　ハーフミラー１０５は、出射光Ｌ１を反射させ、アレイ基板１０に向ける。ハーフミラー１０５は、アレイ基板１０に対して下側（キャピラリー５とは反対側）から、出射光Ｌ１をアレイ基板１０に照射できる。
　対物レンズ１０６は、アレイ基板１０に対して下側（キャピラリー５とは反対側）に設置されている。 The half mirror 105 reflects the emitted light L1 and directs it toward the array substrate 10. The half mirror 105 can irradiate the array substrate 10 with the emitted light L1 from below (the side opposite to the capillary 5).
The objective lens 106 is installed below the array substrate 10 (on the opposite side from the capillary 5).
　光学系２０１は、光源１０４からの出射光Ｌ１を、ハーフミラー１０５および対物レンズ１０６を経て、ステージ２０３上のアレイ基板１０に照射する。出射光Ｌ１が収容部１１内で反射した反射光Ｌ２は、ハーフミラー１０５を透過して光検出部２０２に達する。 The optical system 201 irradiates the array substrate 10 on the stage 203 with the emitted light L1 from the light source 104 through the half mirror 105 and the objective lens 106. The reflected light L2, which is the emitted light L1 reflected within the housing section 11, passes through the half mirror 105 and reaches the light detection section 202.
　光検出部２０２は、例えば、ＣＣＤ（Ｃｈａｒｇｅ－Ｃｏｕｐｌｅｄ  Ｄｅｖｉｃｅ）、ＣＭＯＳ（Ｃｏｍｐｌｅｍｅｎｔａｒｙ  ＭＯＳ）等の撮像素子を含む。
　ステージ２０３は、アレイ基板１０が載置される。ステージ２０３は、水平面内で任意の方向に移動可能である。 The light detection unit 202 includes, for example, an image sensor such as a CCD (Charge-Coupled Device) or a CMOS (Complementary MOS).
The array substrate 10 is placed on the stage 203. Stage 203 is movable in any direction within a horizontal plane.
　水積層用枠２０４は、底壁１１１と、内周壁１１２と、外周壁１１３とを備える。底壁１１１は環状とされる。内周壁１１２は、底壁１１１の内周縁に立設される。外周壁１１３は、底壁１１１の外周縁に立設される。内周壁１１２と外周壁１１３との間には、環状溝１１４が形成される。水積層用枠２０４は、アレイ基板１０の主面１０ａ上に設けられる。水積層用枠２０４は、平面視においてすべての収容部１１を包囲する。 The water stacking frame 204 includes a bottom wall 111, an inner circumferential wall 112, and an outer circumferential wall 113. The bottom wall 111 is annular. The inner peripheral wall 112 is erected on the inner peripheral edge of the bottom wall 111. The outer peripheral wall 113 is erected on the outer peripheral edge of the bottom wall 111. An annular groove 114 is formed between the inner peripheral wall 112 and the outer peripheral wall 113. The water stacking frame 204 is provided on the main surface 10a of the array substrate 10. The water stacking frame 204 surrounds all the housing sections 11 in plan view.
　キャピラリー５は、ガラス、樹脂などで形成された管状体である。キャピラリー５は、例えば、円管状とされている。キャピラリー５は、先端５ａを下に向けた姿勢とされている。先端５ａはアレイ基板１０の主面１０ａに対向する。キャピラリー５には、親水性液体（例えば、水、水溶液）が充てんされている。キャピラリー５は、アレイ基板１０の上側に設けられている。 The capillary 5 is a tubular body made of glass, resin, or the like. The capillary 5 is, for example, shaped like a circular tube. The capillary 5 is in a posture with its tip 5a facing downward. The tip 5a faces the main surface 10a of the array substrate 10. The capillary 5 is filled with a hydrophilic liquid (for example, water or an aqueous solution). The capillary 5 is provided above the array substrate 10.
　キャピラリー５の先端５ａの内径は、例えば、回収対象となる粒子（本実施形態では磁気ビーズ１）の大きさに合わせて定めることができる。キャピラリー５の先端５ａの内径は、例えば、磁気ビーズ１の平均粒径の１倍以上、かつ２倍以下であってよい。 The inner diameter of the tip 5a of the capillary 5 can be determined, for example, according to the size of the particles to be collected (magnetic beads 1 in this embodiment). The inner diameter of the tip 5a of the capillary 5 may be, for example, not less than one time and not more than twice the average particle diameter of the magnetic beads 1.
　高さ調整機構２０６は、例えば、保持アーム２１１と、昇降機構２１２と、を備えるマニピュレーターである。保持アーム２１１は、キャピラリー５を保持する。昇降機構２１２は、保持アーム２１１を介してキャピラリー５を上昇および下降させる。昇降機構２１２は、キャピラリー５を、アレイ基板１０に接近および離間する方向に移動させる。高さ調整機構２０６は、キャピラリー５を任意の高さ位置に保持することができる。
　高さ調整機構２０６は、キャピラリー５の高さ調整だけでなく、キャピラリー５を水平面内で任意の位置に配置することができる機能を有していてもよい。 The height adjustment mechanism 206 is, for example, a manipulator that includes a holding arm 211 and a lifting mechanism 212. The holding arm 211 holds the capillary 5. The lifting mechanism 212 raises and lowers the capillary 5 via the holding arm 211. The elevating mechanism 212 moves the capillary 5 toward and away from the array substrate 10 . The height adjustment mechanism 206 can hold the capillary 5 at an arbitrary height position.
The height adjustment mechanism 206 may have the function of not only adjusting the height of the capillary 5 but also allowing the capillary 5 to be placed at an arbitrary position within a horizontal plane.
　送液系２０７は、送液路２１３と、ポンプ２１４と、圧力計２１５と、を備える。送液路２１３は、キャピラリー５とポンプ２１４とを接続する。送液路２１３は、例えば、樹脂製のチューブである。送液路２１３には、例えば、オイル（疎水性液体）が充てんされている。ポンプ２１４は、送液路２１３内のオイルに圧力を加えることによって、キャピラリー５内の親水性液体の圧力を調整することができる。圧力計２１５は、送液路２１３内の圧力を測定する。 The liquid feeding system 207 includes a liquid feeding path 213, a pump 214, and a pressure gauge 215. The liquid sending path 213 connects the capillary 5 and the pump 214. The liquid feeding path 213 is, for example, a tube made of resin. The liquid sending path 213 is filled with, for example, oil (hydrophobic liquid). The pump 214 can adjust the pressure of the hydrophilic liquid in the capillary 5 by applying pressure to the oil in the liquid sending path 213. The pressure gauge 215 measures the pressure within the liquid feeding path 213.
　磁石２０８は、例えば、希土類磁石材料（例えば、ネオジウム鉄ボロン（ＮｄＦｅＢ）、サマリウムコバルト（ＳｍＣｏ）など）、セラミック磁石材料（例えば、ストロンチウムフェライトなど）、その他の磁性材料（例えば、鉄、コバルト、ニッケル、それらの合金および酸化物など）等で構成される。磁石２０８としては、ネオジウム磁石（ＮｄＦｅＢ）が好ましい。
　磁石２０８は、アレイ基板１０の上方に設置される。磁石２０８は、収容部１１に対して上側にある。磁石２０８は、収容部１１内の磁気ビーズ１に磁力（吸引方向の磁力）を作用させることができる。 The magnet 208 may be made of, for example, a rare earth magnet material (e.g., neodymium iron boron (NdFeB), samarium cobalt (SmCo), etc.), a ceramic magnet material (e.g., strontium ferrite, etc.), or other magnetic material (e.g., iron, cobalt, nickel, etc.). , their alloys and oxides), etc. As the magnet 208, a neodymium magnet (NdFeB) is preferable.
Magnet 208 is installed above array substrate 10 . The magnet 208 is located above the housing portion 11 . The magnet 208 can apply magnetic force (magnetic force in the attraction direction) to the magnetic beads 1 in the housing section 11 .
　磁石２０８は、例えば、上端と下端のうち一方がＮ極であり、上端と下端のうち他方がＳ極であってよい。磁石２０８は、電磁石であってもよい。 For example, one of the upper and lower ends of the magnet 208 may be a north pole, and the other of the upper and lower ends may be a south pole. Magnet 208 may be an electromagnet.
　水積層用枠２０４の内周壁１１２の内側には、疎水性液体層１２１が形成されている。疎水性液体層１２１は、主面１０ａ上であって、すべての収容部１１を包含する領域に形成される。疎水性液体層１２１は収容部１１内の第１親水性液体２の表面を覆う。
　疎水性液体層１２１を構成する疎水性液体としては、例えば、フッ素オイル、シリコーンオイルなどが挙げられる。フッ素オイルとしては、パーフルオロポリエーテル（ＰＦＰＥ）、パーフルオロアルキルエーテル（ＰＦＡＥ）、パーフルオロポリアルキルエーテル（ＰＦＰＡＥ）などのパーフルオロポリエーテル類がある。疎水性液体は、このほか、ハイドロフルオロエーテル（ＨＦＥ）、ミネラルオイル、飽和炭化水素、不飽和炭化水素、芳香族炭化水素、パーフルオロカーボンなどでもよい。 A hydrophobic liquid layer 121 is formed inside the inner peripheral wall 112 of the water lamination frame 204. The hydrophobic liquid layer 121 is formed on the main surface 10a in a region that includes all the housing parts 11. The hydrophobic liquid layer 121 covers the surface of the first hydrophilic liquid 2 within the storage section 11 .
Examples of the hydrophobic liquid constituting the hydrophobic liquid layer 121 include fluorine oil and silicone oil. Examples of the fluorine oil include perfluoropolyethers such as perfluoropolyether (PFPE), perfluoroalkyl ether (PFAE), and perfluoropolyalkyl ether (PFPAE). The hydrophobic liquid may also be hydrofluoroether (HFE), mineral oil, saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, perfluorocarbon, or the like.
　疎水性液体層１２１の上には、親水性液体層１２２が形成される。親水性液体層１２２を構成する親水性液体としては、例えば、水、水溶液が挙げられる。親水性液体層１２２は、水積層用枠２０４の外周壁１１３の内側に形成される。 A hydrophilic liquid layer 122 is formed on the hydrophobic liquid layer 121. Examples of the hydrophilic liquid constituting the hydrophilic liquid layer 122 include water and an aqueous solution. The hydrophilic liquid layer 122 is formed inside the outer peripheral wall 113 of the water lamination frame 204.
［磁気ビーズ回収方法］
　実施形態に係るビーズ回収方法について説明する。
　図２～図５は、実施形態に係る磁気ビーズ回収方法の工程図である。
　本実施形態の磁気ビーズ回収方法は、第１工程（第２親水性液体を収容部内の第１親水性液体と融合）と、第２工程（磁気ビーズをキャピラリー内に導く）と、第３工程（磁気ビーズの吐出）と、を有する。 [Magnetic bead collection method]
A bead collection method according to an embodiment will be described.
2 to 5 are process diagrams of the magnetic bead collection method according to the embodiment.
The magnetic bead collection method of the present embodiment includes a first step (fusing the second hydrophilic liquid with the first hydrophilic liquid in the container), a second step (guiding the magnetic beads into the capillary), and a third step. (discharge of magnetic beads).
（第１工程：第２親水性液体を収容部内の第１親水性液体と融合）
　図２に示すように、第２親水性液体３０２をキャピラリー５内に導入する。キャピラリー５内の第２親水性液体３０２は先端５ａに達する。第２親水性液体３０２は先端５ａから下方に突出した状態とする。第２親水性液体３０２は、例えば、水、または水溶液である。先端５ａから突出した部分の第２親水性液体３０２を「第２親水性液体３０２Ａ」という。第２親水性液体３０２Ａは、例えば、部分球状、部分楕円球状などの湾曲凸形状を有する。 (First step: Fusing the second hydrophilic liquid with the first hydrophilic liquid in the storage part)
As shown in FIG. 2, a second hydrophilic liquid 302 is introduced into the capillary 5. The second hydrophilic liquid 302 inside the capillary 5 reaches the tip 5a. The second hydrophilic liquid 302 is in a state of protruding downward from the tip 5a. The second hydrophilic liquid 302 is, for example, water or an aqueous solution. The portion of the second hydrophilic liquid 302 that protrudes from the tip 5a is referred to as "second hydrophilic liquid 302A." The second hydrophilic liquid 302A has, for example, a curved convex shape such as a partially spherical shape or a partially ellipsoidal spherical shape.
　磁石２０８は、収容部１１内の磁気ビーズ１に上方に磁力（吸引力）を作用させる。そのため、収容部１１内の磁気ビーズ１は第１親水性液体２内で上昇する。 The magnet 208 applies a magnetic force (attractive force) upward to the magnetic beads 1 in the housing section 11. Therefore, the magnetic beads 1 in the container 11 rise within the first hydrophilic liquid 2.
　キャピラリー５を収容部１１に対向させて配置する。高さ調整機構２０６（図１参照）を用いてキャピラリー５を下降させる。キャピラリー５の先端５ａを含む部分を、疎水性液体層１２１を通して収容部１１に近づける。キャピラリー５の先端５ａを含む部分は、疎水性液体層１２１に浸漬される。
　キャピラリー５内の圧力は、第２親水性液体３０２Ａが先端５ａから突出した状態が維持されるように調整されることが望ましい。キャピラリー５内の圧力は、ポンプ２１４（図１参照）によって調整することができる。 The capillary 5 is arranged to face the accommodating part 11. The capillary 5 is lowered using the height adjustment mechanism 206 (see FIG. 1). A portion of the capillary 5 including the tip 5a is brought close to the accommodating portion 11 through the hydrophobic liquid layer 121. A portion of the capillary 5 including the tip 5a is immersed in the hydrophobic liquid layer 121.
It is desirable that the pressure within the capillary 5 is adjusted so that the second hydrophilic liquid 302A remains protruding from the tip 5a. The pressure inside the capillary 5 can be adjusted by a pump 214 (see FIG. 1).
　図３に示すように、第２親水性液体３０２Ａ（図２参照）が収容部１１内の第１親水性液体２に達すると、第２親水性液体３０２は第１親水性液体２と融合する。 As shown in FIG. 3, when the second hydrophilic liquid 302A (see FIG. 2) reaches the first hydrophilic liquid 2 in the storage section 11, the second hydrophilic liquid 302 fuses with the first hydrophilic liquid 2. .
（第２工程：磁気ビーズをキャピラリー内に導く）
　図４に示すように、磁石２０８による磁力（吸引力）によって、磁気ビーズ１は第１親水性液体２および第２親水性液体３０２を通して上昇し、キャピラリー５内に導かれる。　図５に示すように、高さ調整機構２０６（図１参照）を用いてキャピラリー５を上昇させる。キャピラリー５内の第２親水性液体３０２は、収容部１１内の第１親水性液体２から分離する。これによって、アレイ基板１０の収容部１１から試料を回収することができる。回収される試料は、磁気ビーズ１、またはビーズ懸濁液（磁気ビーズ１を含む親水性液体２）である。 (Second step: Guide the magnetic beads into the capillary)
As shown in FIG. 4, the magnetic beads 1 rise through the first hydrophilic liquid 2 and the second hydrophilic liquid 302 due to the magnetic force (attractive force) of the magnet 208 and are guided into the capillary 5. As shown in FIG. 5, the capillary 5 is raised using the height adjustment mechanism 206 (see FIG. 1). The second hydrophilic liquid 302 in the capillary 5 is separated from the first hydrophilic liquid 2 in the storage section 11 . Thereby, the sample can be collected from the storage section 11 of the array substrate 10. The sample to be collected is magnetic beads 1 or bead suspension (hydrophilic liquid 2 containing magnetic beads 1).
（第３工程：磁気ビーズの吐出）
　キャピラリー５を疎水性液体層１２１から引き上げた後、磁石の磁力（吸引力）を磁気ビーズ１に作用させることによって、磁気ビーズ１をキャピラリー５の先端５ａに移動させる。ポンプ２１４（図１参照）によってキャピラリー５内の圧力を高めることによって、磁気ビーズ１を、第２親水性液体３０２の一部とともに先端５ａから吐出することができる。 (Third step: Discharge of magnetic beads)
After the capillary 5 is pulled up from the hydrophobic liquid layer 121, the magnetic force (attractive force) of the magnet is applied to the magnetic beads 1, thereby moving the magnetic beads 1 to the tip 5a of the capillary 5. By increasing the pressure inside the capillary 5 using the pump 214 (see FIG. 1), the magnetic beads 1 can be discharged from the tip 5a along with a portion of the second hydrophilic liquid 302.
［実施形態の磁気ビーズ回収方法が奏する効果］
　本実施形態の磁気ビーズ回収方法では、キャピラリー５内の第２親水性液体３０２を収容部１１内の第１親水性液体２に融合させ、収容部１１内の磁気ビーズ１を磁力によってキャピラリー５内に導く。そのため、収容部１１内の磁気ビーズ１を容易に回収することができる。 [Effects achieved by the magnetic bead collection method of the embodiment]
In the magnetic bead collection method of this embodiment, the second hydrophilic liquid 302 in the capillary 5 is fused with the first hydrophilic liquid 2 in the storage part 11, and the magnetic beads 1 in the storage part 11 are moved into the capillary 5 by magnetic force. lead to. Therefore, the magnetic beads 1 in the storage section 11 can be easily collected.
　本実施形態の磁気ビーズ回収方法では、キャピラリー５を収容部１１に近づけるにあたって、第２親水性液体３０２Ａを先端５ａから突出させる。そのため、第２親水性液体３０２は、収容部１１内の第１親水性液体２と容易に融合する。 In the magnetic bead collection method of this embodiment, when the capillary 5 is brought close to the storage section 11, the second hydrophilic liquid 302A is made to protrude from the tip 5a. Therefore, the second hydrophilic liquid 302 easily fuses with the first hydrophilic liquid 2 in the storage section 11 .
　実施形態の磁気ビーズ回収方法の効果を明確にするため、比較形態の磁気ビーズ回収方法を以下に示す。
　図６は、第１比較形態に係る磁気ビーズ回収方法の説明図である。図６に示すように、第１比較形態の磁気ビーズ回収方法では、キャピラリー５内に親水性液体はない。そのため、キャピラリー５の先端５ａを収容部１１に近づけてキャピラリー５内を減圧したとしても、キャピラリー５の周囲の疎水性液体はキャピラリー５内に導入されるが、収容部１１内の磁気ビーズ１をキャピラリー５に導くのは難しい。
　図７は、第２比較形態に係る磁気ビーズ回収方法の説明図である。図７に示すように、第２比較形態の磁気ビーズ回収方法では、磁石を使用しない。そのため、第２親水性液体３０２が第１親水性液体２と融合しても、磁気ビーズ１はキャピラリー５内に導かれない。 In order to clarify the effects of the magnetic bead collection method of the embodiment, a comparative magnetic bead collection method is shown below.
FIG. 6 is an explanatory diagram of the magnetic bead collection method according to the first comparative embodiment. As shown in FIG. 6, in the magnetic bead collection method of the first comparative embodiment, there is no hydrophilic liquid in the capillary 5. Therefore, even if the pressure inside the capillary 5 is reduced by bringing the tip 5a of the capillary 5 closer to the accommodating part 11, the hydrophobic liquid around the capillary 5 will be introduced into the capillary 5, but the magnetic beads 1 in the accommodating part 11 will be introduced into the capillary 5. It is difficult to lead to capillary 5.
FIG. 7 is an explanatory diagram of a magnetic bead collection method according to a second comparative embodiment. As shown in FIG. 7, the magnetic bead collection method of the second comparative embodiment does not use a magnet. Therefore, even if the second hydrophilic liquid 302 fuses with the first hydrophilic liquid 2, the magnetic beads 1 are not guided into the capillary 5.
［実施形態の磁気ビーズ回収装置が奏する効果］
　本実施形態の磁気ビーズ回収装置２００は、キャピラリー５を収容部１１に近づける高さ調整機構２０６と、磁気ビーズ１に吸引方向の磁力を作用させる磁石２０８とを備える。そのため、キャピラリー５内の第２親水性液体３０２を収容部１１内の第１親水性液体２に融合させ、収容部１１内の磁気ビーズ１を磁力によってキャピラリー５内に導くことができる。よって、収容部１１内の磁気ビーズ１を容易に回収することができる。 [Effects produced by the magnetic bead collection device of the embodiment]
The magnetic bead collection device 200 of this embodiment includes a height adjustment mechanism 206 that brings the capillary 5 closer to the storage section 11 and a magnet 208 that applies magnetic force in the attraction direction to the magnetic beads 1. Therefore, the second hydrophilic liquid 302 in the capillary 5 can be fused with the first hydrophilic liquid 2 in the container 11, and the magnetic beads 1 in the container 11 can be guided into the capillary 5 by magnetic force. Therefore, the magnetic beads 1 in the storage section 11 can be easily collected.
　本発明を実施例に基づいて説明する。ただし、本発明の実施態様は、これら実施例の記載に限定されない。 The present invention will be explained based on examples. However, the embodiments of the present invention are not limited to the description of these Examples.
［ビーズを封入するオイル層上に水が積層されたアレイ基板の準備］
＜ビオチン修飾ＤＮＡ合成＞
　下記組成のＰＣＲ反応液を調製し、３０サイクル（９８℃，１０秒；５５℃，５秒；７２℃，２分）でＰＣＲを行った。ＰＣＲ産物をＱＩＡｑｕｉｃｋ（登録商標）ＰＣＲ　ｐｕｒｉｆｉｃａｔｉｏｎ　ｃｏｌｕｍｎ（ＱＩＡＧＥＮ）を用いて精製し、ビオチン修飾ＤＮＡを得た。 [Preparation of an array substrate in which water is layered on an oil layer that encapsulates beads]
<Biotin-modified DNA synthesis>
A PCR reaction solution having the following composition was prepared, and PCR was performed in 30 cycles (98°C, 10 seconds; 55°C, 5 seconds; 72°C, 2 minutes). The PCR product was purified using QIAquick (registered trademark) PCR purification column (QIAGEN) to obtain biotin-modified DNA.
（ＰＣＲ反応液）
　２０ｐｇ／μＬ　鋳型ＤＮＡ
　０．３μＭ　ビオチン修飾ＤＮＡプライマー
　０．３μＭ　ＤＮＡプライマー
　０．２μＭ　ｅａｃｈ　ｄＮＴＰ　Ｍｉｘ
　０．０２５Ｕ／μＬ　ＰｒｉｍｅＳＴＡＲ（登録商標）　ＨＳ　ｐｏｌｙｍｅｒａｓｅ　１ｘＰｒｉｍｅＳＴＡＲ（登録商標）　ｂｕｆｆｅｒ（タカラバイオ） (PCR reaction solution)
20pg/μL template DNA
0.3μM biotin-modified DNA primer 0.3μM DNA primer 0.2μM each dNTP Mix
0.025U/μL PrimeSTAR (registered trademark) HS polymerase 1xPrimeSTAR (registered trademark) buffer (Takara Bio)
＜ＤＮＡ固定化磁気ビーズの調製＞
　ストレプトアビジン修飾磁気ビーズ（ＭＳ３００／ｓｔｒｅｐｔａｖｉｄｉｎ，ＪＳＲ；以下、「磁気ビーズ」という）１２０μＬの上清を除去し、１００μＬの結合バッファー（１０ｍＭ　Ｔｒｉｓ－ＨＣｌ，１ｍＭ　ＥＤＴＡ，１Ｍ　ＮａＣｌ，０．０５％（ｗ／ｖ）　Ｔｗｅｅｎ（登録商標）２０、ｐＨ７．４）で磁気ビーズを洗浄した。７ｐｍｏｌのビオチン修飾ＤＮＡを溶解した１７０μＬの結合バッファーに、前記磁気ビーズを懸濁し、室温で３０分間攪拌した。これにより、ビオチン修飾ＤＮＡを磁気ビーズに結合させた。次いで、磁気ビーズを１００μＬの結合バッファーで５回洗浄した。その後、磁気ビーズを３０μＬの結合バッファーに懸濁した。 <Preparation of DNA immobilized magnetic beads>
120 μL of streptavidin-modified magnetic beads (MS300/streptavidin, JSR; hereinafter referred to as “magnetic beads”) supernatant was removed, and 100 μL of binding buffer (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, 0.05% (w) /v) Tween 20, pH 7.4). The magnetic beads were suspended in 170 μL of binding buffer in which 7 pmol of biotin-modified DNA had been dissolved, and stirred at room temperature for 30 minutes. This allowed the biotin-modified DNA to bind to the magnetic beads. The magnetic beads were then washed 5 times with 100 μL of binding buffer. The magnetic beads were then suspended in 30 μL of binding buffer.
　上記懸濁液におけるＤＮＡ固定化磁気ビーズの濃度を、自動セルカウンター（商品名：Ｃｏｕｎｔｅｓｓ　ＩＩ，ＴｈｅｒｍｏＦｉｓｈｅｒ製）を用いて測定した。ＤＮＡ固定化磁気ビーズ懸濁液（ＤＮＡ固定化磁気ビーズ　５　ｘ　１０７個分）の上清を除去し、ＤＮＡ固定化磁気ビーズを６０μＬの蛍光水溶液（１０μＭ　フルオレセインナトリウム，５０ｍＭ　Ｔｒｉｓ－ＨＣｌ，ｐＨ７．５）に懸濁した。このように調製したＤＮＡ固定化磁気ビーズ懸濁液を、下記＜アレイ基板へのビーズと水溶液の分配＞に用いた。 The concentration of DNA-immobilized magnetic beads in the above suspension was measured using an automatic cell counter (trade name: Countess II, manufactured by ThermoFisher). The supernatant of the DNA-immobilized magnetic bead suspension (5 x 10 7 DNA-immobilized magnetic beads) was removed, and the DNA-immobilized magnetic beads were mixed with 60 μL of a fluorescent aqueous solution (10 μM sodium fluorescein, 50 mM Tris-HCl, pH 7. 5). The thus prepared DNA-immobilized magnetic bead suspension was used in <Distribution of beads and aqueous solution to array substrate> below.
＜アレイ基板の作製＞
　ＳＴＡＶＡＸ（登録商標）基材表面（３０ｍｍ　ｘ　３０ｍｍ　ｘ　５ｍｍ）に、ニッケルリンを約１００μｍの厚さまでメッキした。そのニッケルリン層に刃幅６μｍの切削工具を用いて格子状に溝加工を行うことで、立方体構造（４μｍ　ｘ　４μｍ　ｘ　４μｍ）が平面（１．２ｍｍ　ｘ　１．２ｍｍ）上に１　ｘ　１０６個並んだ金型を作製した。その金型を射出成型機に装着し、シクロオレフィンポリマー（商品名：ＺＥＯＮＥＸ（登録商標）４８０、日本ゼオン製）を射出した。これにより、基板表面（３０ｍｍ　ｘ　３０ｍｍ　ｘ　１ｍｍ）の領域（１．２ｍｍ　ｘ　１．２ｍｍ）上にマイクロウェル（４μｍ　ｘ　４μｍ　ｘ　４μｍ）が１ｘ１０６個並んだアレイ基板を作製した。以下、アレイ基板のマイクロウェルが並んだ領域を「アレイ領域」、マイクロウェルが存在しない領域を「アレイ領域外」という。 <Preparation of array substrate>
The STAVAX® substrate surface (30 mm x 30 mm x 5 mm) was plated with nickel phosphorus to a thickness of approximately 100 μm. By cutting grooves in the nickel-phosphorus layer in a grid pattern using a cutting tool with a blade width of 6 μm, a cubic structure (4 μm x 4 μm x 4 μm) was formed on a plane (1.2 mm x 1.2 mm) by 1 x 10 6 A mold with individual pieces lined up was fabricated. The mold was attached to an injection molding machine, and a cycloolefin polymer (trade name: ZEONEX (registered trademark) 480, manufactured by Nippon Zeon) was injected. As a result, an array substrate was produced in which 1 x 10 6 microwells (4 μm x 4 μm x 4 μm) were lined up on a region (1.2 mm x 1.2 mm) of the substrate surface (30 mm x 30 mm x 1 mm). Hereinafter, the area of the array substrate where the microwells are lined up will be referred to as the "array area", and the area where no microwells will be present will be referred to as the "outside the array area".
＜アレイ基板の表面処理＞
　アレイ基板の主面に酸素プラズマ照射を行うことにより、アレイ基板の主面およびマイクロウェル内部の表面を親水化した。続いて、ヘキサメチルジシラザン（ＨＭＤＳ）を蒸着したポリプロピレンフィルムをアレイ基板表面に圧着させて、ＨＭＤＳをアレイ基板表面に転写し、１１０℃で１０分間処理することによって基板表面を疎水化した。以上の操作により、マイクロウェル内部の表面（主面）が親水性で、マイクロウェル外部の表面が疎水性であるアレイ基板を作製した。 <Surface treatment of array substrate>
By irradiating the main surface of the array substrate with oxygen plasma, the main surface of the array substrate and the surface inside the microwell were made hydrophilic. Subsequently, a polypropylene film on which hexamethyldisilazane (HMDS) was vapor-deposited was pressed onto the surface of the array substrate, HMDS was transferred to the surface of the array substrate, and the substrate surface was made hydrophobic by treatment at 110° C. for 10 minutes. Through the above operations, an array substrate was produced in which the surface (principal surface) inside the microwell was hydrophilic and the surface outside the microwell was hydrophobic.
＜アレイ基板へのビーズと水溶液の分配＞
　直径３ｍｍのステンレス製の棒をシリコーンチューブ（外径４ｍｍ，内径２ｍｍ，長さ５０ｍｍ）に挿入してワイプ部材を作製した。ワイプ部材をアレイ基板のアレイ領域外に接触させて配置した。ワイプ部材の接触部とアレイ領域端との間のアレイ基板下にネオジウム磁石（２０　ｘ　５　ｘ　１０ｍｍ）を配置した。 <Distribution of beads and aqueous solution to array substrate>
A wipe member was prepared by inserting a stainless steel rod with a diameter of 3 mm into a silicone tube (outer diameter 4 mm, inner diameter 2 mm, length 50 mm). The wipe member was placed in contact with the outside of the array area of the array substrate. A neodymium magnet (20 x 5 x 10 mm) was placed under the array substrate between the contact portion of the wipe member and the edge of the array area.
　ワイプ部材の接触部からアレイ領域端のアレイ基板の主面に、６０μＬのＤＮＡ固定化磁気ビーズ懸濁液を滴下した。ワイプ部材の接触面からアレイ領域とは逆側のアレイ基板の主面に６０μＬのフッ素オイル（商品名：Ｋｒｙｔｏｘ（登録商標）ＧＰＬ１０４（Ｃｈｅｍｏｕｒｓ社製）を滴下した。 60 μL of the DNA-immobilized magnetic bead suspension was dropped onto the main surface of the array substrate at the edge of the array area from the contact portion of the wipe member. 60 μL of fluorine oil (trade name: Krytox (registered trademark) GPL104 (manufactured by Chemours) was dropped onto the main surface of the array substrate on the opposite side from the array area from the contact surface of the wipe member.
　ワイプ部材およびネオジウム磁石を、アレイ領域側に、０．１ｍｍ／ｓで同時に動かし、アレイ領域を通過したところでワイプ部材およびネオジウム磁石を停止させた。この操作により、ＤＮＡ固定化磁気ビーズおよび蛍光水溶液のマイクロウェルへの分配と、フッ素オイルによる封入とを行った。 The wipe member and neodymium magnet were simultaneously moved toward the array area at 0.1 mm/s, and the wipe member and neodymium magnet were stopped after passing through the array area. Through this operation, the DNA-immobilized magnetic beads and the fluorescent aqueous solution were distributed to the microwells and sealed with fluorine oil.
＜アレイ基板表面のオイル層への水の積層＞
　水積層用枠（液体積層用枠）を３Ｄプリンター（商品名：Ｆｒｏｍ２，Ｆｏｒｍｌａｂｓ社製）で作製した。マイクロウェルにＤＮＡ固定化磁気ビーズおよび蛍光水溶液を封入したアレイ基板上に水積層用枠を貼付した。水積層用枠の内周壁内に、８００μＬのフッ素オイル（商品名：Ｋｒｙｔｏｘ（登録商標）ＧＰＬ１０７（Ｃｈｅｍｏｕｒｓ社製））を添加した。これにより、アレイ基板表面に、フッ素オイルからなる疎水性液体層を形成した。
　水積層用枠の内周壁と外周壁の間の環状溝を１ｍＬの水で満たした後、３ｍＬの水をさらに追加で添加した。これにより、環状溝内の水を溢れさせ、内周壁内のフッ素オイル（疎水性液体層）の上に水の層（親水性液体層）を形成した。 <Lamination of water on the oil layer on the surface of the array substrate>
A frame for water lamination (frame for liquid lamination) was produced using a 3D printer (trade name: From2, manufactured by Formlabs). A frame for water lamination was pasted onto an array substrate in which DNA-immobilized magnetic beads and a fluorescent aqueous solution were sealed in microwells. 800 μL of fluorine oil (trade name: Krytox (registered trademark) GPL107 (manufactured by Chemours)) was added to the inner peripheral wall of the water lamination frame. As a result, a hydrophobic liquid layer made of fluorine oil was formed on the surface of the array substrate.
After filling the annular groove between the inner peripheral wall and the outer peripheral wall of the water lamination frame with 1 mL of water, an additional 3 mL of water was added. This caused the water in the annular groove to overflow, forming a water layer (hydrophilic liquid layer) on the fluorine oil (hydrophobic liquid layer) in the inner peripheral wall.
［マイクロウェルからのＤＮＡ固定化磁気ビーズの回収］
　上記のように準備したアレイ基板を、倒立落射顕微鏡（Ｔｉ－Ｅ，ニコン社製）のステージに設置した。シリンジポンプ（ＹＭＣ社製）にガスタイトシリンジ（ハミルトン社製）および圧力計（商品名：ＫＤＭ３０，クローネ社製）を装着し、ポリエチレンチューブ（送液路）を介してキャピラリーホルダー（エッペンドルフ社製）を連結した。これを、倒立落射顕微鏡（Ｔｉ－Ｅ，ニコン社製）に設置したマニピュレーター（商品名：ＴｒａｎｓｆｅｒＭａｎ　ＮＫ２，エッペンドルフ社製）に装着した（図１参照）。 [Recovery of DNA-immobilized magnetic beads from microwells]
The array substrate prepared as described above was placed on the stage of an inverted epi-illumination microscope (Ti-E, manufactured by Nikon Corporation). A gastight syringe (manufactured by Hamilton) and a pressure gauge (product name: KDM30, manufactured by Krone) are attached to a syringe pump (manufactured by YMC), and a capillary holder (manufactured by Eppendorf) is attached via a polyethylene tube (liquid feeding path). were connected. This was attached to a manipulator (trade name: TransferMan NK2, manufactured by Eppendorf) installed on an inverted epi-illumination microscope (Ti-E, manufactured by Nikon Corporation) (see FIG. 1).
　送液路をミネラルオイルで充てんした。ガラスキャピラリー（先端内径５．５μｍ）の全体を、水溶液（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ，ｐＨ７．５）で満たし、キャピラリーホルダーに装着した。ガラスキャピラリーの先端を下向きにし、ハーフミラーを介した同軸落射照明でガラスキャピラリーの先端を観察し、顕微鏡の視野中心にガラスキャピラリー先端が位置するように調整した。 The liquid passage was filled with mineral oil. A glass capillary (tip inner diameter 5.5 μm) was entirely filled with an aqueous solution (50 mM Tris-HCl, pH 7.5) and attached to a capillary holder. The tip of the glass capillary was oriented downward, and the tip of the glass capillary was observed using coaxial epi-illumination through a half mirror, and adjusted so that the tip of the glass capillary was located at the center of the field of view of the microscope.
　ガラスキャピラリー近傍にネオジウム磁石（１０　ｘ　１０　ｘ　３５ｍｍ）を設置した。シリンジポンプを操作することで、圧力計のゲージ圧を－３ｋＰａに設定した。マニピュレーターを操作してガラスキャピラリー先端をアレイ基板表面に向けて降下させた。 A neodymium magnet (10 x 10 x 35 mm) was installed near the glass capillary. The gauge pressure of the pressure gauge was set to -3 kPa by operating the syringe pump. A manipulator was operated to lower the tip of the glass capillary toward the surface of the array substrate.
　同軸落射照明でマイクロウェルとキャピラリー先端を同時観察しながら、ＤＮＡ固定化磁気ビーズが封入されているマイクロウェルの直上にガラスキャピラリーを降下させた。ガラスキャピラリー先端の水溶液とマイクロウェル内の水溶液とが接触すると同時に、マイクロウェル内のＤＮＡ固定化磁気ビーズは上昇し、ガラスキャピラリー内に移動した。　図８（Ａ）は、磁気ビーズ回収前のマイクロウェルの顕微鏡画像である。図８（Ｂ）は、磁気ビーズ回収後のマイクロウェルの顕微鏡画像である。図８（Ａ）および図８（Ｂ）より、マイクロウェルから磁気ビーズが回収されたことがわかる。 While observing the microwell and capillary tip simultaneously using coaxial epi-illumination, the glass capillary was lowered directly above the microwell containing the DNA-immobilized magnetic beads. At the same time that the aqueous solution at the tip of the glass capillary and the aqueous solution in the microwell came into contact, the DNA-immobilized magnetic beads in the microwell rose and moved into the glass capillary. FIG. 8(A) is a microscopic image of the microwell before magnetic bead collection. FIG. 8(B) is a microscopic image of the microwell after magnetic bead collection. It can be seen from FIGS. 8(A) and 8(B) that magnetic beads were collected from the microwells.
　ガラスキャピラリーを上昇させ、ガラスキャピラリーをフッ素オイルの層から引き上げた後、ネオジウム磁石に近接させることでガラスキャピラリー先端にＤＮＡ固定化磁気ビーズを移動させた。ガラスキャピラリー先端をアレイ基板と同材質および同厚のプラスチック基板上に降下させ、同軸落射照明で観察したところ、ガラスキャピラリー先端にＤＮＡ固定化磁気ビーズの存在が確認された。 After raising the glass capillary and lifting it from the layer of fluorine oil, the DNA-immobilized magnetic beads were moved to the tip of the glass capillary by bringing it close to a neodymium magnet. When the glass capillary tip was lowered onto a plastic substrate of the same material and thickness as the array substrate and observed under coaxial epi-illumination, the presence of DNA-immobilized magnetic beads at the glass capillary tip was confirmed.
　シリンジポンプを操作することで、圧力計のゲージ圧を＋３ｋＰａに設定し、プラスチック基板上に滴下した水滴にガラスキャピラリー先端を挿入することで、ガラスキャピラリー先端からＤＮＡ固定化磁気ビーズを水滴中に吐出した。
　図９（Ａ）は、磁気ビーズと、吐出前のガラスキャピラリーの顕微鏡画像である。図９（Ｂ）は、磁気ビーズと吐出後のガラスキャピラリーの顕微鏡画像である。図９（Ａ）および図９（Ｂ）より、磁気ビーズがガラスキャピラリーから吐出されたことがわかる。 By operating the syringe pump, the gauge pressure of the pressure gauge is set to +3 kPa, and the tip of the glass capillary is inserted into the water droplet dropped on the plastic substrate, and the DNA-immobilized magnetic beads are discharged from the tip of the glass capillary into the water droplet. did.
FIG. 9(A) is a microscopic image of the magnetic beads and the glass capillary before ejection. FIG. 9(B) is a microscopic image of the magnetic beads and the glass capillary after ejection. It can be seen from FIGS. 9(A) and 9(B) that the magnetic beads were ejected from the glass capillary.
　以上、本発明の実施形態を説明したが、実施形態における各構成及びそれらの組み合わせ等は一例であり、本発明の趣旨から逸脱しない範囲内で、構成の付加、省略、置換、及びその他の変更が可能である。
　収容部１１に収容される磁気ビーズ１の数は１つに限らない。収容部１１に収容される磁気ビーズ１の数は、複数（２以上の任意の数）であってもよい。磁気ビーズ１の収容数は、収容部１１の内形寸法と深さの少なくとも一方によって調整できる。収容部１１に収容される磁気ビーズ１の数が複数であると、アレイ基板１０の面積あたりの解析対象ビーズを増やすことができる。よって、効率的にスクリーニングを行うことができる。
　疎水化された表面の水の接触角は、例えば６０度以上である。親水化された表面の水の接触角は、例えば４０度以下である。 The embodiments of the present invention have been described above, but each configuration and combination thereof in the embodiments are merely examples, and additions, omissions, substitutions, and other changes to the configurations may be made without departing from the spirit of the present invention. is possible.
The number of magnetic beads 1 accommodated in the accommodation section 11 is not limited to one. The number of magnetic beads 1 accommodated in the accommodation section 11 may be plural (any number greater than or equal to 2). The number of magnetic beads 1 accommodated can be adjusted by at least one of the internal dimensions and depth of the accommodating portion 11. If a plurality of magnetic beads 1 are accommodated in the accommodating section 11, the number of beads to be analyzed per area of the array substrate 10 can be increased. Therefore, screening can be performed efficiently.
The contact angle of water on the hydrophobized surface is, for example, 60 degrees or more. The contact angle of water on the hydrophilized surface is, for example, 40 degrees or less.
　磁気ビーズは、核酸およびタンパク質が固定化されていてもよい。収容部内でタンパク質の機能を評価した後、実施形態の磁気ビーズ回収方法によって磁気ビーズを回収することができる。回収された磁気ビーズに固定化された核酸を解析することによって、タンパク質のアミノ酸配列を特定できる（例えば、国際公開第２０２０／０９５９８５号を参照）。 Nucleic acids and proteins may be immobilized on the magnetic beads. After evaluating the function of the protein within the container, the magnetic beads can be collected by the magnetic bead collection method of the embodiment. By analyzing the nucleic acids immobilized on the recovered magnetic beads, the amino acid sequence of the protein can be identified (for example, see International Publication No. 2020/095985).
　磁気ビーズは、核酸が固定化されていてもよい。収容部内で核酸の機能を評価した後、実施形態の磁気ビーズ回収方法によって磁気ビーズを回収することができる。回収された磁気ビーズに固定化された核酸を解析することによって、核酸の配列を特定できる（例えば、Aniela Wochner et al., Ribozyme-Catalyzed Transcription of an Active Ribozyme. Science 332, 209 (2011)を参照）。 Nucleic acids may be immobilized on the magnetic beads. After evaluating the function of the nucleic acid within the container, the magnetic beads can be collected by the magnetic bead collection method of the embodiment. By analyzing the nucleic acids immobilized on the recovered magnetic beads, the nucleic acid sequence can be identified (for example, see Aniela Wochner et al., Ribozyme-Catalyzed Transcription of an Active Ribozyme. Science 332, 209 (2011)) ).
　磁気ビーズは、核酸が固定化されていてもよい。収容部内で細胞を溶解して細胞内ＲＮＡを磁気ビーズの核酸にハイブリダイズさせた後、実施形態の磁気ビーズ回収方法によって磁気ビーズを回収することができる。回収された磁気ビーズに固定化された核酸にハイブリダイズしたＲＮＡを解析することができる（例えば、Jinzhou Yuan et al., An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq;. Sci. Rep. 6:33883 (2016)を参照）。 Nucleic acids may be immobilized on the magnetic beads. After cells are lysed in the container and intracellular RNA is hybridized to the nucleic acid of the magnetic beads, the magnetic beads can be collected by the magnetic bead collection method of the embodiment. RNA hybridized to the nucleic acids immobilized on the collected magnetic beads can be analyzed (for example, Jinzhou Yuan et al., An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq; Sci. Rep. 6:33883 (2016)).
　磁気ビーズは、抗体が固定化されていてもよい。収容部内で細胞を溶解して細胞由来タンパク質を磁気ビーズの抗体に結合させた後、実施形態の磁気ビーズ回収方法によって磁気ビーズを回収することができる。回収された磁気ビーズに固定化された抗体に結合したタンパク質を解析することができる（例えば、Lucas Armbrecht et al., Single-cell protein profiling in microchambers with barcoded beads. Microsystems & Nanoengineering 5:55 (2019)を参照）。 The magnetic beads may have antibodies immobilized thereon. After the cells are lysed in the container and the cell-derived protein is bound to the antibody of the magnetic beads, the magnetic beads can be collected by the magnetic bead collection method of the embodiment. Proteins bound to antibodies immobilized on recovered magnetic beads can be analyzed (e.g., Lucas Armbrecht et al., Single-cell protein profiling in microchambers with barcoded beads. Microsystems & Nanoengineering 5:55 (2019) ).
　１　磁気ビーズ
　２　第１親水性液体
　５　キャピラリー
　５ａ　先端
　１０　アレイ基板（基板）
　１０ａ　主面（一方の面）
　１１　収容部
　１２１　疎水性液体層
　２０６　高さ調整機構（移動機構）
　２０８　磁石
　３０２　第２親水性液体 1 Magnetic beads 2 First hydrophilic liquid 5 Capillary 5a Tip 10 Array substrate (substrate)
10a Main surface (one side)
11 Storage part 121 Hydrophobic liquid layer 206 Height adjustment mechanism (moving mechanism)
208 Magnet 302 Second hydrophilic liquid

Claims (5)

  1. 　磁気ビーズおよび第１親水性液体が収容され、疎水性液体層で覆われた収容部からから前記磁気ビーズを回収するにあたって、
    　第２親水性液体が先端を含む範囲に充てんされたキャピラリーを、前記疎水性液体層を通して前記収容部に近づけ、前記第２親水性液体を前記収容部内の前記第１親水性液体に融合させる工程と、
    　前記磁気ビーズを、磁力によって、前記第１親水性液体および前記第２親水性液体を通して前記キャピラリー内に導く工程と、を有する、
    　磁気ビーズ回収方法。     In recovering the magnetic beads from the storage part containing the magnetic beads and the first hydrophilic liquid and covered with the hydrophobic liquid layer,
    A step of bringing a capillary whose tip is filled with a second hydrophilic liquid close to the housing part through the hydrophobic liquid layer, and fusing the second hydrophilic liquid with the first hydrophilic liquid in the housing part. and,
    guiding the magnetic beads through the first hydrophilic liquid and the second hydrophilic liquid into the capillary by magnetic force;
    Magnetic bead collection method.
  2. 　前記キャピラリーを前記収容部に近づけるにあたって、前記第２親水性液体を前記先端から突出させる、
    　請求項１記載の磁気ビーズ回収方法。     When the capillary is brought close to the housing part, the second hydrophilic liquid is made to protrude from the tip;
    The method for collecting magnetic beads according to claim 1.
  3. 　前記収容部は、基板の一方の面に形成された凹部である、
    　請求項１記載の磁気ビーズ回収方法。     The accommodating portion is a recess formed on one surface of the substrate,
    The method for collecting magnetic beads according to claim 1.
  4. 　前記キャピラリー内の前記磁気ビーズを前記キャピラリーから吐出する工程をさらに有する、
    　請求項１～３のうちいずれか１項に記載の磁気ビーズ回収方法。     further comprising the step of discharging the magnetic beads in the capillary from the capillary,
    The method for collecting magnetic beads according to any one of claims 1 to 3.
  5. 　磁気ビーズおよび第１親水性液体が収容された収容部に対向するキャピラリーと、
    　前記磁気ビーズに吸引方向の磁力を作用させる磁石と、
    　前記キャピラリーを前記収容部に接近および離間する方向に移動させる移動機構と、を備え、
    　前記収容部に、前記第１親水性液体を覆う疎水性液体層が形成され、
    　前記移動機構は、第２親水性液体が先端を含む範囲に充てんされた前記キャピラリーを、前記疎水性液体層を通して前記収容部に近づける、　
    　磁気ビーズ回収装置。     a capillary facing a housing portion containing magnetic beads and a first hydrophilic liquid;
    a magnet that applies magnetic force in the attraction direction to the magnetic beads;
    a moving mechanism that moves the capillary in a direction toward and away from the housing part,
    A hydrophobic liquid layer covering the first hydrophilic liquid is formed in the housing part,
    The moving mechanism moves the capillary, which is filled with the second hydrophilic liquid in a range including the tip thereof, toward the accommodation part through the hydrophobic liquid layer.
    Magnetic bead collection device.
PCT/JP2023/031569 2022-08-31 2023-08-30 Magnetic bead recovery method and magnetic bead recovery device WO2024048660A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004011146A1 (en) * 2002-07-29 2004-02-05 Campbell Andrew T A device and method for manipulating magnetic particles
US20100170852A1 (en) * 2004-12-03 2010-07-08 Chris Suh Method and Device for Gravity Flow Chromatography
JP2013015532A (en) * 2006-11-24 2013-01-24 Agency For Science Technology & Research Device for processing sample in liquid droplet, apparatus, and method of using the same
US20160186166A1 (en) * 2014-12-09 2016-06-30 Life Technologies Corporation High efficiency, small volume nucleic acid synthesis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004011146A1 (en) * 2002-07-29 2004-02-05 Campbell Andrew T A device and method for manipulating magnetic particles
US20100170852A1 (en) * 2004-12-03 2010-07-08 Chris Suh Method and Device for Gravity Flow Chromatography
JP2013015532A (en) * 2006-11-24 2013-01-24 Agency For Science Technology & Research Device for processing sample in liquid droplet, apparatus, and method of using the same
US20160186166A1 (en) * 2014-12-09 2016-06-30 Life Technologies Corporation High efficiency, small volume nucleic acid synthesis

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