WO2024048648A1 - Magnetic bead arrangement method and magnetic bead arrangement device - Google Patents

Magnetic bead arrangement method and magnetic bead arrangement device Download PDF

Info

Publication number
WO2024048648A1
WO2024048648A1 PCT/JP2023/031502 JP2023031502W WO2024048648A1 WO 2024048648 A1 WO2024048648 A1 WO 2024048648A1 JP 2023031502 W JP2023031502 W JP 2023031502W WO 2024048648 A1 WO2024048648 A1 WO 2024048648A1
Authority
WO
WIPO (PCT)
Prior art keywords
main surface
wipe member
liquid
magnetic beads
magnetic
Prior art date
Application number
PCT/JP2023/031502
Other languages
French (fr)
Japanese (ja)
Inventor
真吾 上野
章一 土屋
Original Assignee
公益財団法人川崎市産業振興財団
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 公益財団法人川崎市産業振興財団 filed Critical 公益財団法人川崎市産業振興財団
Publication of WO2024048648A1 publication Critical patent/WO2024048648A1/en

Links

Images

Classifications

    • 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
    • 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 dispensing method and a magnetic bead distributing device.
  • This application claims priority based on Japanese Patent Application No. 2022-138140 filed in Japan on August 31, 2022, the contents of which are incorporated herein.
  • Examples of methods for forming an oil-sealed bead-containing microwell array include the following methods (1) and (2).
  • An aqueous solution containing beads is poured into the space between the lower layer and the upper layer in which the storage section is formed. This introduces the bead-containing aqueous solution into the storage section.
  • the accommodating portion is sealed with oil by introducing oil into the space (see, for example, Patent Document 1).
  • a plate or sheet is placed over the microwell array to seal the microwells.
  • An object of one aspect of the present invention is to provide a magnetic bead distributing method and a magnetic bead distributing device that are excellent in ease of handling a sample in a storage section.
  • the present invention includes the following aspects.
  • a substrate having a main surface on which a plurality of accommodating parts are formed, a wipe member having a shape along the main surface, and a magnet disposed on the opposite side of the substrate from the wipe member.
  • a hydrophilic liquid containing a plurality of magnetic beads is spread on the main surface, and the magnet is applied to the wipe member while applying magnetic force to the magnetic beads.
  • a method for distributing magnetic beads comprising the step of covering the surface of the hydrophilic liquid in the storage part with the hydrophobic liquid by spreading it over the main surface.
  • the method for distributing magnetic beads according to any one of [1] to [4], further comprising the step of forming.
  • a magnetic bead dispensing device that distributes magnetic beads to the accommodating portion of a substrate having a main surface on which a plurality of accommodating portions are formed, the wipe member having a shape along the main surface; a magnet disposed on the opposite side of the wipe member, the wipe member being movable forward along the main surface on which a hydrophilic liquid containing a plurality of magnetic beads is placed, the magnet comprising: A magnetic bead dispensing device capable of moving forward along with the wipe member while applying a magnetic force to the magnetic beads.
  • a magnetic bead distributing method and a magnetic bead distributing device that are excellent in ease of handling a sample in a storage section.
  • FIG. 2 is a front cross-sectional view of a magnetic bead distribution device and an array substrate used in a magnetic bead distribution method according to an embodiment.
  • FIG. 2 is a plan view of a portion of an array substrate used in the magnetic bead distribution method according to the embodiment.
  • FIG. 2 is a side sectional view of a magnetic bead distribution device and an array substrate used in a magnetic bead distribution method according to an embodiment.
  • FIG. 2 is an explanatory diagram of a magnetic bead distribution method according to an embodiment.
  • FIG. 2 is an explanatory diagram of a magnetic bead distribution method according to an embodiment.
  • FIG. 3 is a cross-sectional view of a stacking frame and an array substrate used to form a layer of a hydrophilic liquid.
  • FIG. 3 is an explanatory diagram of a method for forming a liquid layer of a hydrophilic liquid. It is a sectional view of a frame for liquid lamination.
  • 1 shows a microscopic image of an array substrate in which DNA-immobilized magnetic beads and a fluorescent aqueous solution were distributed and sealed in microwells in Example 1.
  • A is a bright field image.
  • B is a fluorescence image.
  • FIG. 7 is a diagram showing a procedure for laminating water on an oil layer on the surface of an array substrate using a water lamination frame in Example 2.
  • A This is an image at the start of the test when the water layer was not laminated on the oil layer.
  • B This is an image taken 10 minutes after the start of the test.
  • A This is an image at the start of the test when a water layer is laminated on an oil layer.
  • B This is an image taken 3 days after the start of the test.
  • FIG. 1 is a front cross-sectional view of a magnetic bead distribution device 100 and an array substrate 10 used in the magnetic bead distribution method according to the embodiment.
  • FIG. 2 is a plan view of a portion of the array substrate 10.
  • FIG. 3 is a side sectional view of the magnetic bead dispensing device 100 and the array substrate 10.
  • the magnetic bead distribution device 100 includes a wipe member 20 and a magnet 30. The magnetic bead distribution device 100 distributes magnetic beads to the array substrate 10.
  • 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 (see FIG. 2).
  • 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 X direction is a direction along a pair of opposing sides of the array substrate 10.
  • One of the X directions (the right direction in FIGS. 1 and 2) is the +X direction.
  • the direction opposite to the +X direction (the left direction in FIGS. 1 and 2) is the ⁇ X direction.
  • the Y direction is within a plane along the main surface 10a and is orthogonal to the X direction.
  • the Z direction is orthogonal to the X direction and the Y direction. Planar view means viewing from the Z direction.
  • the vertical positional relationship will be tentatively determined based on FIG.
  • the array substrate 10 is oriented with the main surface 10a facing upward.
  • the main surface 10a is horizontal.
  • 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 accommodating portion 11 has, for example, a cylindrical internal space.
  • the shape of the accommodating portion 11 is not particularly limited.
  • 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 housing portion 11 may have a shape in which the inner diameter decreases in the depth direction.
  • the plurality of storage units 11 are arranged, for example, in a two-dimensional matrix with multiple rows and multiple columns.
  • the plurality of accommodating parts 11 may constitute a plurality of accommodating part groups 11A.
  • the accommodating section group 11A is composed of a plurality of accommodating sections 11 arranged at regular intervals along the X direction.
  • the plurality of housing section groups 11A are arranged at regular intervals in the Y direction.
  • the accommodating parts 11 of the accommodating part groups 11A that are adjacent to each other in the Y direction are arranged in the Y direction, but the accommodating parts 11 of the neighboring accommodating part groups 11A do not have to be arranged in the Y direction.
  • the accommodating portions 11 may be arranged in a staggered manner when viewed from above.
  • the number and arrangement of the plurality of accommodating parts are not particularly limited.
  • the number of storage section groups may be one. That is, in the substrate, all the housing parts may be arranged in a line. Two or more of the plurality of accommodating parts are formed at different positions in the X direction.
  • the array substrate 10 may be a laminated structure including a flat base and a well structure formed on the base. A well (accommodating portion) is formed in the well structure portion.
  • the base is made of glass, quartz, silicon wafer, or the like.
  • the well structure portion 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 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 wipe member 20 has a cylindrical shape with a central axis C along the Y direction. A side surface (outer peripheral surface) of the wipe member 20 is in contact with the main surface 10a of the array substrate 10. Since the wipe member 20 has a cylindrical shape, it contacts the main surface 10a with a large contact area. Therefore, the liquid stopping properties of the wipe member 20 can be improved.
  • the lowermost portion 20a of the wipe member 20 is linear along the Y direction when viewed from the X direction. Since the lowermost portion 20a is linear, the wipe member 20 can contact the flat main surface 10a without any gaps. Therefore, it can be said that the wipe member 20 has a shape along the main surface 10a of the array substrate 10.
  • the lowermost portion 20a of the wipe member 20 covers the opening of the accommodating portion 11 in a side sectional view (a sectional view along the Y direction and the Z direction) of the array substrate 10.
  • the wipe member 20 includes a main body portion 21 and a covering layer 22.
  • the main body portion 21 has a cylindrical shape with a central axis C.
  • the main body portion 21 is made of, for example, metal, resin, or the like.
  • the covering layer 22 is formed on the outer circumferential surface of the main body portion 21.
  • the coating layer 22 covers the entire outer peripheral surface of the main body portion 21 .
  • the coating layer 22 has a lower hardness than the outer circumferential surface of the main body portion 21 .
  • Examples of the hardness include durometer hardness (based on JIS K6253 or JIS K7215).
  • the covering layer 22 is made of, for example, rubber (such as silicone rubber), thermoplastic resin, thermoplastic elastomer, thermosetting elastomer, or the like.
  • the physical properties of the coating layer 22 are selected in consideration of adhesion to the main surface 10a, liquid stopping properties, and the like.
  • the covering layer 22 can be formed, for example, by covering the main body portion 21 with a silicone rubber tube (silicone tube).
  • the magnet 30 is 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 rare earth magnet material e.g., neodymium iron boron (NdFeB), samarium cobalt (SmCo), etc.
  • a ceramic magnet material e.g., strontium ferrite, etc.
  • other magnetic material e.g., iron, cobalt, nickel, etc.
  • a neodymium magnet (NdFeB) is preferable.
  • the shape of the magnet 30 is determined so that magnetic force can be applied to the magnetic beads 1 included in the bead suspension 4 on the main surface 10a.
  • the magnet 30 can have a prismatic shape extending in the Y direction, for example. Since the magnet 30 has a length that spans the entire length of the array substrate 10 in the Y direction, it is possible to apply magnetic force to the magnetic beads 1 over a wide range in the Y direction.
  • one of the upper end and the lower end of the magnet 30 may be an N pole, and the other of the upper end and the lower end may be an S pole.
  • the magnet 30 is not limited to a prismatic shape, and may be, for example, cylindrical. Magnet 30 may be an electromagnet.
  • the magnet 30 is arranged on the opposite side of the array substrate 10 from the wipe member 20. Specifically, the magnet 30 is arranged on the lower surface side of the array substrate 10. The magnet 30 is located at a height close to the bottom surface of the array substrate 10. The magnet 30 is movable in the +X direction (to the right in FIG. 1).
  • Magnetic bead distribution method (First embodiment) Next, a magnetic bead distribution method according to the first embodiment will be explained.
  • the magnetic bead distributing method of this embodiment performs the first step (accommodating magnetic beads in the storage section) and the second step (covering the hydrophilic liquid in the storage section with a hydrophobic liquid) in one operation.
  • a wipe member 20 is arranged on the main surface 10a of the array substrate 10.
  • the wipe member 20 contacts the main surface 10a without any gaps along the length direction (Y direction).
  • the advancing direction of the wipe member 20 is the +X direction (to the right in FIG. 1).
  • the +X direction is the "forward” (first direction).
  • -X direction is "backward”.
  • a hydrophilic liquid 2 containing a plurality of magnetic beads 1 is placed on the main surface 10a and on the front side of the wipe member 20.
  • the magnetic beads 1 have, for example, immobilized biomolecules such as nucleic acids, peptides, and proteins.
  • Magnetic beads 1 are dispersed in a hydrophilic liquid 2.
  • the hydrophilic liquid 2 is, for example, water or an aqueous solution.
  • the hydrophilic liquid 2 containing the magnetic beads 1 is referred to as a "bead suspension 4.”
  • the bead suspension 4 is in contact with the front surface of the wipe member 20.
  • 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.
  • Hydrophobic liquid 3 is placed on the main surface 10a and on the rear side of the wipe member 20.
  • the hydrophobic liquid 3 is in contact with the rear surface of the wipe member 20.
  • Examples of the hydrophobic liquid 3 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 3 may also be hydrofluoroether (HFE), mineral oil, saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, perfluorocarbon, or the like.
  • FIGS. 4 and 5 are explanatory diagrams of the magnetic bead distribution method according to the embodiment.
  • the wipe member 20 is moved forward (+X direction) along the main surface 10a while maintaining its posture.
  • the wipe member 20 moves forward while maintaining contact with the main surface 10a along its length.
  • the bead suspension 4 is pushed forward and spread over the front region of the main surface 10a.
  • the direction of movement of the wipe member 20 is forward (+X direction).
  • the magnet 30 When the wipe member 20 moves forward, the magnet 30 is moved forward (+X direction) along with the wipe member 20. The magnet 30 moves forward at the same speed as the wipe member 20. Therefore, the magnet 30 moves forward without changing its relative position with respect to the wipe member 20.
  • the magnet 30 be located in front of the wipe member 20 in plan view.
  • the magnet 30 applies a downward magnetic force (attractive force) to the magnetic beads 1 contained in the bead suspension 4 in front of the wipe member 20 .
  • the magnet 30 moves forward while applying magnetic force to the magnetic beads 1. Since the magnetic beads 1 are arranged slightly forward of the wipe member 20 by the magnet 30, the magnetic beads 1 are not interfered with by the wipe member 20 and are easily introduced into the storage section 11.
  • a part of the bead suspension 4 is introduced into the storage section 11. Since the main surface 10a is made hydrophobic and the inner surface of the accommodating part 11 is made hydrophilic, the bead suspension 4 easily enters the accommodating part 11. For example, the magnetic beads 1 are distributed one by one into the storage section 11 .
  • the magnetic beads 1 in the container 11 can be operated from above the container 11 .
  • the operation of taking out a sample from the storage section 11 can be performed using an experimental instrument such as a capillary that approaches the storage section 11 from above.
  • samples that can be taken out from the container 11 using a capillary or the like include magnetic beads 1, hydrophilic liquid 2, and bead suspension 4.
  • the magnetic bead distribution method involves moving the wipe member 20 forward to spread the bead suspension 4 on the main surface 10a, and moving the magnet 30 forward while applying magnetic force to the magnetic beads 1 to suspend the beads.
  • the suspension 4 is stored in the storage section 11 (first step).
  • the hydrophobic liquid 3 on the rear side of the wipe member 20 is spread over the main surface 10a by the above-described movement of the wipe member 20, and covers the surface of the hydrophilic liquid 2 in the storage part 11 (second step).
  • the hydrophobic liquid 3 isolates the plurality of storage parts 11 from each other.
  • the bead suspension 4 is sealed within the container 11 .
  • the magnetic beads 1 are distributed to the storage section 11 by only one operation of moving the wipe member 20 and the magnet 30 forward, and the storage section 11 is sealed with the hydrophobic liquid 3. can do. Therefore, the work of distributing the magnetic beads 1 becomes easy. Moreover, since the hydrophilic liquid 2 in the storage part 11 is immediately covered with the hydrophobic liquid 3, the time period during which the hydrophilic liquid 2 in the storage part 11 is exposed can be shortened. Therefore, evaporation of the hydrophilic liquid 2 can be suppressed.
  • the liquid containing the magnetic beads 1 is distributed to the storage section 11 with the upper side of the array substrate 10 open. Therefore, unlike the method using the upper layer provided on the array substrate 10 (see Japanese Patent No. 5337324), the operation of taking out the sample from the storage section 11 is performed by inserting an experimental instrument such as a capillary into the storage section 11 from above. It can be done up close. Therefore, it is possible to improve the ease of handling the sample in the storage section 11.
  • the bead suspension 4 easily enters the accommodating part 11.
  • the wipe member 20 Since the wipe member 20 has a cylindrical shape, it contacts the main surface 10a with a large contact area. Therefore, the liquid stopping properties of the wipe member 20 can be improved. Therefore, it is possible to suppress the bead suspension 4 from leaking to the rear side of the wipe member 20 and the hydrophobic liquid 3 from entering the front side of the wipe member 20.
  • the wipe member 20 has the low-hardness coating layer 22, it is possible to improve the adhesion to the main surface 10a of the wipe member 20, the liquid-stopping property of the wipe member 20, and the like.
  • the magnetic beads 1 are distributed to the storage section 11 by just one operation of moving the wipe member 20 and the magnet 30 forward, and the storage section 11 is sealed with the hydrophobic liquid 3. can be stopped. Therefore, the work of distributing the magnetic beads 1 becomes easy. Moreover, since the hydrophilic liquid 2 in the storage part 11 is immediately covered with the hydrophobic liquid 3, the time period during which the hydrophilic liquid 2 in the storage part 11 is exposed can be shortened. Therefore, evaporation of the hydrophilic liquid 2 can be suppressed.
  • the magnetic bead distribution method according to the embodiment can perform layer formation of a hydrophobic liquid and a hydrophilic liquid following the above-described magnetic bead distribution method.
  • the magnetic bead distribution method according to the present embodiment includes the above-mentioned first step (accommodating magnetic beads in the storage section) and second step (covering the hydrophilic liquid in the storage section with a hydrophobic liquid). Then, the following third step, fourth step, and fifth step are performed.
  • a liquid stacking frame surrounding the accommodating portions is installed on the main surface of the substrate having a main surface on which a plurality of accommodating portions are formed.
  • the liquid stacking frame has an annular groove surrounding the storage portion.
  • a hydrophobic liquid layer made of a hydrophobic liquid is formed within the liquid stacking frame and on the main surface.
  • a hydrophilic liquid is introduced into the annular groove, and the hydrophilic liquid overflows from the annular groove to form a hydrophilic liquid layer on the hydrophobic liquid layer.
  • FIG. 6 is a cross-sectional view of the liquid stacking frame 110 and the array substrate 10 used in the magnetic bead distribution method according to the present embodiment.
  • FIG. 7 is an explanatory diagram of a liquid layer forming method.
  • FIG. 8 is a cross-sectional view of the liquid stacking frame 110.
  • the area in which the plurality of accommodating parts 11 are formed on the main surface 10a of the array substrate 10 is referred to as an "array area.”
  • the array area is an area that collectively includes a plurality of accommodating parts 11. Components common to other embodiments will be given the same reference numerals and descriptions will be omitted.
  • a liquid stacking frame 110 is installed on the main surface 10a of the array substrate 10.
  • the magnetic beads 1 and the hydrophilic liquid 2 are accommodated in the accommodating part 11 of the array substrate 10 by the above-described magnetic bead distribution method, and the accommodating part 11 is sealed with the hydrophobic liquid 3.
  • the liquid stacking frame 110 includes a bottom wall 111, an inner peripheral wall 112, and an outer peripheral wall 113.
  • the bottom wall 111 is formed in an annular shape.
  • the bottom wall 111 may have, for example, an annular shape, a rectangular annular shape, or the like.
  • the internal dimensions of the bottom wall 111 are determined so that the bottom wall 111 can surround the array area in plan view.
  • the opening in the bottom wall 111 encompasses the array area in plan view.
  • the inner dimension of the bottom wall 111 is the inner diameter of the bottom wall 111 when the bottom wall 111 is annular.
  • the internal dimensions of the bottom wall 111 are the lengths of the sides of the rectangular opening.
  • the bottom wall 111 is installed on the main surface 10a of the array substrate 10.
  • the inner peripheral wall 112 is erected on the inner peripheral edge of the bottom wall 111.
  • the inner peripheral wall 112 has a cylindrical shape corresponding to the shape of the inner peripheral edge of the bottom wall 111.
  • the inner peripheral wall 112 has a cylindrical shape.
  • the height of the inner peripheral wall 112 from the top surface of the bottom wall 111 is determined according to the thickness of the hydrophobic liquid layer 121 formed on the main surface 10a.
  • the height of the inner circumferential wall 112 is the same over the entire circumference.
  • the outer peripheral wall 113 is erected on the outer peripheral edge of the bottom wall 111.
  • the outer peripheral wall 113 has a cylindrical shape corresponding to the shape of the outer peripheral edge of the bottom wall 111.
  • the outer peripheral wall 113 has a cylindrical shape.
  • the inner diameter of the outer peripheral wall 113 is larger than the outer diameter of the inner peripheral wall 112.
  • the outer peripheral wall 113 is located outwardly away from the inner peripheral wall 112. Therefore, an annular groove 114 is formed between the inner peripheral wall 112 and the outer peripheral wall 113.
  • the height of the outer peripheral wall 113 from the top surface of the bottom wall 111 (the bottom surface of the annular groove 114) is greater than the height of the inner peripheral wall 112.
  • the height of the outer peripheral wall 113 is determined according to the thickness of the hydrophilic liquid layer 122 formed on the hydrophobic liquid layer 121.
  • the height H1 of the inner peripheral wall 112 from the bottom surface of the bottom wall 111 is, for example, 2 mm.
  • the height H2 of the inner peripheral wall 112 from the bottom surface of the annular groove 114 is, for example, 1.5 mm.
  • the height H3 of the outer peripheral wall 113 from the bottom surface of the annular groove 114 is, for example, 9.5 mm.
  • the height H4 of the outer peripheral wall 113 from the bottom surface of the bottom wall 111 is, for example, 10 mm.
  • the bottom wall 111 surrounds the array area with an annular peripheral area having a width of 1.5 mm.
  • the width W1 which is the inner dimension of the bottom wall 111, is equal to the outer dimension of the array area plus 3 mm.
  • the width W3 of the annular groove 114 is the distance between the inner circumferential wall 112 and the outer circumferential wall 113. Width W3 can be determined according to widths W1 and W2. Note that although the liquid stacking frame 110 includes the bottom wall 111, the liquid stacking frame may have a configuration without a bottom wall. In this case, it is desirable that the inner circumferential wall and the outer circumferential wall are connected to each other by connecting portions formed at one or more locations.
  • a hydrophobic liquid layer 121 is formed by introducing the hydrophobic liquid 203 into the space defined by the main surface 10a and the inner circumferential wall 112.
  • the hydrophobic liquid layer 121 is formed on the main surface 10a in a region that includes all the housing parts 11.
  • the thickness of the hydrophobic liquid layer 121 is, for example, equal to or smaller than the height of the inner peripheral wall 112.
  • Examples of the hydrophobic liquid 203 include fluorine oil and silicone oil. Examples of fluorine oil include perfluoropolyethers such as PFPE, PFAE, and PFPAE.
  • the hydrophobic liquid 203 may also be HFE, mineral oil, saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, perfluorocarbon, or the like.
  • the hydrophobic liquid 203 may have a higher viscosity than the hydrophobic liquid 3.
  • a hydrophilic liquid 202 is introduced into the annular groove 114.
  • the hydrophilic liquid 202 include water or an aqueous solution.
  • the hydrophilic liquid 202 overflows from the annular groove 114 and flows inward over the inner circumferential wall 112.
  • the inflow of the hydrophilic liquid 202 occurs in a wide range in the circumferential direction of the inner peripheral wall 112. For example, the hydrophilic liquid 202 flows inward over the inner circumferential wall 112 over the entire circumference of the inner circumferential wall 112 at the same time.
  • a hydrophilic liquid layer 122 is formed on the hydrophobic liquid layer 121.
  • Hydrophilic liquid layer 122 is formed inside outer peripheral wall 113 .
  • the hydrophilic liquid layer 122 can prevent the hydrophilic liquid 2 in the storage section 11 from evaporating through the hydrophobic liquid layer 121 .
  • the hydrophilic liquid 202 flows into the inner peripheral wall 112 over a wide range in the circumferential direction of the inner peripheral wall 112. Therefore, the load that the hydrophilic liquid 202 applies to the hydrophobic liquid layer 121 at the time of inflow is dispersed. Therefore, it is possible to prevent the hydrophilic liquid 202 from locally entering the hydrophobic liquid layer 121 and reaching the inside of the accommodating portion 11 . Thereby, a hydrophilic liquid layer 122 covering the entire hydrophobic liquid layer 121 can be formed. Therefore, it is possible to suppress the hydrophilic liquid 2 in the storage part 11 from evaporating. Therefore, a lid member for preventing evaporation of the hydrophilic liquid 2 is not necessary.
  • Example 1 ⁇ 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.
  • QIAquick registered trademark
  • QIAGEN PCR purification column
  • 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 in which the pieces were 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 of the array substrate where no microwells are present is referred to as "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 inside the microwell was hydrophilic and the surface (principal 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 between the contact portion of the wipe member and the end of the array area and below the array substrate.
  • 60 ⁇ L of the DNA-immobilized magnetic bead suspension was dropped onto the main surface of the array substrate (the front position of the wipe member: the position between the contact part of the wipe member and the end of the array area).
  • 60 ⁇ L of fluorine oil (trade name: Krytox (registered trademark) GPL104 (manufactured by Chemours) was dropped onto the main surface of the array substrate (at the rear position of the wipe member).
  • the wipe member and neodymium magnet were moved forward at the same time 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.
  • Figures 9(A) and 9(B) show microscopic images of an array substrate in which DNA-immobilized magnetic beads and a fluorescent aqueous solution are sealed in microwells using fluorinated oil (Figure 9(A): bright field image, 9(B): Fluorescent image). As shown in FIGS. 9(A) and 9(B), the fluorescent aqueous solution was distributed to all microwells in the array area, and DNA-immobilized magnetic beads were distributed to 67% of the microwells.
  • Example 2 ⁇ Formation of a water layer 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 water layering frame 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 main surface of the array substrate.
  • FIG. 11(A) is an image at the start of the test when the water layer was not laminated on the oil layer.
  • FIG. 11(B) is an image taken 10 minutes after the start of the test.
  • FIG. 12(A) is an image at the start of the test when a water layer is laminated on an oil layer.
  • FIG. 12(B) is an image taken 3 days after the start of the test.
  • the number of magnetic beads 1 housed in the housing 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 (inner diameter) 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 shape of the wipe member is not particularly limited.
  • the wipe member may be, for example, blade-shaped.
  • the wipe member preferably contacts the main surface of the substrate, but may be separated from the main surface by a small distance.
  • 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.

Abstract

The present invention uses a substrate (10) having a main surface (10a) on which a plurality of accommodation parts (11) are formed, a wiping member (20) having a shape that follows that of the main surface (10a), and a magnet (30) disposed on the side of the substrate (10) that is opposite from the wiping member (20). In a first step, the wiping member (20) is moved forward along the main surface (10a), whereby a hydrophilic liquid (2) containing a plurality of magnetic beads (1) is spread onto the main surface (10a). The magnet (30) is moved forward together with the wiping member (20) while magnetic force is caused to act on the magnetic beads (1), whereby the magnetic beads (1) and the hydrophilic liquid (2) are accommodated in the accommodation parts (11). In a second step, a hydrophobic liquid (3) arranged on the movement-direction rear side of the wiping member (20) is spread onto the main surface (10a) due to the movement of the wiping member (20), whereby the surface of the hydrophilic liquid (2) within the accommodation parts (11) is covered by the hydrophobic liquid (3).

Description

磁気ビーズ分配方法および磁気ビーズ分配装置Magnetic bead distribution method and magnetic bead distribution device
　本発明は、磁気ビーズ分配方法および磁気ビーズ分配装置に関する。
　本願は、２０２２年８月３１日に日本に出願された特願２０２２－１３８１４０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 dispensing method and a magnetic bead distributing device.
This application claims priority based on Japanese Patent Application No. 2022-138140 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。ビーズに固定した生体分子の機能観察・検出のために、ビーズは、例えば、アレイ化したマイクロウェルに分配される。一般的に、上記用途でのビーズおよびマイクロウェルのサイズはマイクロメートルオーダーであることから、ビーズを１粒子ずつ操作してマイクロウェルに配置することは非効率的である。そのため、複数のビーズを一度に複数のマイクロウェルに分配する方法が採用される。生体分子の機能観察・検出を行う方法としては、ビーズを分配した複数のマイクロウェルをオイルで封止して閉鎖空間を形成し、これらの閉鎖空間内での反応を指標にビーズ上の生体分子の機能観察・検出を行う方法がある。 There is a method of immobilizing biomolecules on beads for the purpose of functional observation and detection of biomolecules. For functional observation and detection of biomolecules immobilized on beads, beads are distributed, for example, into arrayed microwells. Generally, the sizes of beads and microwells in the above-mentioned applications are on the order of micrometers, so it is inefficient to manipulate beads one by one and place them in microwells. Therefore, a method is adopted in which multiple beads are distributed into multiple microwells at once. To observe and detect the functions of biomolecules, multiple microwells containing beads are sealed with oil to form a closed space, and the biomolecules on the beads are detected based on the reactions within these closed spaces. There is a method to observe and detect the function of
　オイル封止されたビーズ含有マイクロウェルアレイを形成する手法としては、例えば、次の（１）、（２）に示す方法がある。
（１）収容部が形成された下層部と上層部との間の空間に、ビーズを含む水溶液を流す。これによって、収容部にビーズ含有水溶液を導入する。次いで、空間にオイルを導入することによって収容部をオイルで封止する（例えば、特許文献１を参照）。
（２）ビーズ含有水溶液をマイクロウェルアレイ上に展開し、その上にオイルを積層した後、マイクロウェルアレイ上に板もしくはシートを被せてマイクロウェルを密閉する。 Examples of methods for forming an oil-sealed bead-containing microwell array include the following methods (1) and (2).
(1) An aqueous solution containing beads is poured into the space between the lower layer and the upper layer in which the storage section is formed. This introduces the bead-containing aqueous solution into the storage section. Next, the accommodating portion is sealed with oil by introducing oil into the space (see, for example, Patent Document 1).
(2) After spreading the bead-containing aqueous solution on the microwell array and layering oil thereon, a plate or sheet is placed over the microwell array to seal the microwells.
特許第５３３７３２４号公報Patent No. 5337324
　複数のマイクロウェル内の生体分子の機能観察・検出を行った後、マイクロウェル内の水溶液、もしくは、ビーズ上の生体分子について、二次解析を行う必要が生じることがある。
　しかしながら、前記技術では、マイクロウェルアレイの上面側が覆われているため、マイクロウェル（収容部）内の試料（ビーズ、水溶液など）を、キャピラリー等を用いて取り出すことが難しくなる可能性がある。 After functional observation and detection of biomolecules in a plurality of microwells, it may be necessary to perform secondary analysis on the aqueous solution in the microwells or the biomolecules on beads.
However, in the above technique, since the upper surface side of the microwell array is covered, it may be difficult to take out the sample (beads, aqueous solution, etc.) inside the microwell (accommodating part) using a capillary or the like.
　本発明の一態様は、収容部内の試料の取り扱いの容易性の点で優れた磁気ビーズ分配方法および磁気ビーズ分配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 distributing method and a magnetic bead distributing device that are excellent in ease of handling a sample in a storage section.
　本発明は、以下の態様を含む。
［１］複数の収容部が形成された主面を有する基板と、前記主面に沿う形状を有するワイプ部材と、前記基板に対して前記ワイプ部材とは反対側に配置した磁石と、を用い、前記主面に沿って前記ワイプ部材を前方移動させることで、複数の磁気ビーズを含む親水性液体を前記主面に広げるとともに、磁力を前記磁気ビーズに作用させつつ前記磁石を前記ワイプ部材に同伴させて前方移動させることによって、前記磁気ビーズおよび前記親水性液体を前記収容部に収容する工程と、前記ワイプ部材の移動方向の後ろ側に配した疎水性液体を、前記ワイプ部材の移動によって前記主面に広げることによって、前記収容部内の前記親水性液体の表面を前記疎水性液体で覆う工程と、を有する、磁気ビーズ分配方法。
［２］前記主面は、疎水化されており、前記収容部の内面は、親水化されている、［１］記載の磁気ビーズ分配方法。
［３］ワイプ部材は、前記主面に沿う中心軸を有する円柱状とされている、［１］記載の磁気ビーズ分配方法。
［４］前記ワイプ部材は、円柱状の本体部と、前記本体部の外周面に形成され、前記外周面より硬度が低い被覆層と、を備える、［３］記載の磁気ビーズ分配方法。
［５］前記収容部を囲む環状溝が形成され、前記収容部を包囲する液体積層用枠を前記主面に設置する工程と、前記液体積層用枠内であって前記主面の上に、疎水性液体からなる疎水性液体層を形成する工程と、前記環状溝に親水性液体を導入し、前記親水性液体を前記環状溝から溢れさせて前記疎水性液体層の上に親水性液体層を形成する工程と、をさらに有する、［１］～［４］のうちいずれか１つに記載の磁気ビーズ分配方法。 The present invention includes the following aspects.
[1] Using a substrate having a main surface on which a plurality of accommodating parts are formed, a wipe member having a shape along the main surface, and a magnet disposed on the opposite side of the substrate from the wipe member. , by moving the wipe member forward along the main surface, a hydrophilic liquid containing a plurality of magnetic beads is spread on the main surface, and the magnet is applied to the wipe member while applying magnetic force to the magnetic beads. A step of accommodating the magnetic beads and the hydrophilic liquid in the accommodating portion by moving the magnetic beads and the hydrophilic liquid forward together; A method for distributing magnetic beads, comprising the step of covering the surface of the hydrophilic liquid in the storage part with the hydrophobic liquid by spreading it over the main surface.
[2] The magnetic bead distribution method according to [1], wherein the main surface is made hydrophobic, and the inner surface of the accommodating part is made hydrophilic.
[3] The magnetic bead distribution method according to [1], wherein the wipe member has a cylindrical shape with a central axis along the main surface.
[4] The magnetic bead distribution method according to [3], wherein the wipe member includes a cylindrical main body and a coating layer formed on the outer peripheral surface of the main body and having a lower hardness than the outer peripheral surface.
[5] A step of installing a liquid stacking frame on the main surface in which an annular groove surrounding the storage part is formed and surrounding the storage part, and a step of: a step of forming a hydrophobic liquid layer made of a hydrophobic liquid; introducing a hydrophilic liquid into the annular groove and causing the hydrophilic liquid to overflow from the annular groove to form a hydrophilic liquid layer on the hydrophobic liquid layer; The method for distributing magnetic beads according to any one of [1] to [4], further comprising the step of forming.
［６］複数の収容部が形成された主面を有する基板の前記収容部に磁気ビーズを分配する磁気ビーズ分配装置であって、前記主面に沿う形状を有するワイプ部材と、前記基板に対して前記ワイプ部材とは反対側に配置した磁石と、を備え、前記ワイプ部材は、複数の磁気ビーズを含む親水性液体を載せた前記主面に沿って前方移動可能であり、前記磁石は、前記磁気ビーズに磁力を作用させつつ前記ワイプ部材に同伴させて前方移動可能である、磁気ビーズ分配装置。 [6] A magnetic bead dispensing device that distributes magnetic beads to the accommodating portion of a substrate having a main surface on which a plurality of accommodating portions are formed, the wipe member having a shape along the main surface; a magnet disposed on the opposite side of the wipe member, the wipe member being movable forward along the main surface on which a hydrophilic liquid containing a plurality of magnetic beads is placed, the magnet comprising: A magnetic bead dispensing device capable of moving forward along with the wipe member while applying a magnetic force to the magnetic beads.
　本発明の一態様によれば、収容部内の試料の取り扱いの容易性の点で優れた磁気ビーズ分配方法および磁気ビーズ分配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 distributing method and a magnetic bead distributing device that are excellent in ease of handling a sample in a storage section.
実施形態に係る磁気ビーズ分配方法に用いられる磁気ビーズ分配装置およびアレイ基板の正断面図である。FIG. 2 is a front cross-sectional view of a magnetic bead distribution device and an array substrate used in a magnetic bead distribution method according to an embodiment. 実施形態に係る磁気ビーズ分配方法に用いられるアレイ基板の一部の平面図である。FIG. 2 is a plan view of a portion of an array substrate used in the magnetic bead distribution method according to the embodiment. 実施形態に係る磁気ビーズ分配方法に用いられる磁気ビーズ分配装置およびアレイ基板の側断面図である。FIG. 2 is a side sectional view of a magnetic bead distribution device and an array substrate used in a magnetic bead distribution method according to an embodiment. 実施形態に係る磁気ビーズ分配方法の説明図である。FIG. 2 is an explanatory diagram of a magnetic bead distribution method according to an embodiment. 実施形態に係る磁気ビーズ分配方法の説明図である。FIG. 2 is an explanatory diagram of a magnetic bead distribution method according to an embodiment. 親水性液体の層形成に用いられる積層用枠およびアレイ基板の断面図である。FIG. 3 is a cross-sectional view of a stacking frame and an array substrate used to form a layer of a hydrophilic liquid. 親水性液体の液体層形成方法の説明図である。FIG. 3 is an explanatory diagram of a method for forming a liquid layer of a hydrophilic liquid. 液体積層用枠の断面図である。It is a sectional view of a frame for liquid lamination. 実施例１において、ＤＮＡ固定化磁気ビーズおよび蛍光水溶液をマイクロウェルに分配および封入した、アレイ基板の顕微鏡画像を示す。（Ａ）は明視野画像である。（Ｂ）は蛍光画像である。1 shows a microscopic image of an array substrate in which DNA-immobilized magnetic beads and a fluorescent aqueous solution were distributed and sealed in microwells in Example 1. (A) is a bright field image. (B) is a fluorescence image. 実施例２において、水積層用枠を用いて、アレイ基板表面のオイル層に水を積層する手順を示す図である。FIG. 7 is a diagram showing a procedure for laminating water on an oil layer on the surface of an array substrate using a water lamination frame in Example 2. （Ａ）オイル層上に水層を積層しなかった場合の、試験開始時の画像である。（Ｂ）試験開始から１０分間経過した時点の画像である。(A) This is an image at the start of the test when the water layer was not laminated on the oil layer. (B) This is an image taken 10 minutes after the start of the test. （Ａ）オイル層上に水層を積層した場合の、試験開始時の画像である。（Ｂ）試験開始から３日間経過した時点の画像である。(A) This is an image at the start of the test when a water layer is laminated on an oil layer. (B) This is an image taken 3 days after the start of the test.
　図１は、実施形態に係る磁気ビーズ分配方法に用いられる磁気ビーズ分配装置１００およびアレイ基板１０の正断面図である。図２は、アレイ基板１０の一部の平面図である。図３は、磁気ビーズ分配装置１００およびアレイ基板１０の側断面図である。
　図１に示すように、磁気ビーズ分配装置１００は、ワイプ部材２０と、磁石３０と、を備える。磁気ビーズ分配装置１００は、アレイ基板１０に対して磁気ビーズの分配を行う。 FIG. 1 is a front cross-sectional view of a magnetic bead distribution device 100 and an array substrate 10 used in the magnetic bead distribution method according to the embodiment. FIG. 2 is a plan view of a portion of the array substrate 10. As shown in FIG. FIG. 3 is a side sectional view of the magnetic bead dispensing device 100 and the array substrate 10.
As shown in FIG. 1, the magnetic bead distribution device 100 includes a wipe member 20 and a magnet 30. The magnetic bead distribution device 100 distributes magnetic beads to the array substrate 10.
［基板］
　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（図２参照）。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]
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 (see FIG. 2). 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.
　図２に示すように、Ｘ方向は、アレイ基板１０の向かい合う一対の辺に沿う方向である。Ｘ方向のうち一方向（図１および図２における右方向）は＋Ｘ方向である。＋Ｘ方向と反対の方向（図１および図２における左方向）は－Ｘ方向である。Ｙ方向は、主面１０ａに沿う面内にあってＸ方向に直交する。Ｚ方向は、Ｘ方向およびＹ方向に直交する。平面視は、Ｚ方向から見ることである。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。主面１０ａは水平とされている。 As shown in FIG. 2, the X direction is a direction along a pair of opposing sides of the array substrate 10. One of the X directions (the right direction in FIGS. 1 and 2) is the +X direction. The direction opposite to the +X direction (the left direction in FIGS. 1 and 2) is the −X direction. The Y direction is within a plane along the main surface 10a and is orthogonal to the X direction. The Z direction is orthogonal to the X direction and the Y direction. Planar view means viewing from the Z direction. 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. The main surface 10a is horizontal.
　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。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。収容部１１は、例えば、深さ方向に内径が小さくなる形状であってもよい。 The shape of the accommodating portion 11 in plan view is, for example, circular. The accommodating portion 11 has, for example, a cylindrical internal space.
Note that the shape of the accommodating portion 11 is not particularly limited. The shape of the accommodating portion 11 in plan view may be a polygonal shape such as a rectangular shape or a hexagonal shape. For example, the housing portion 11 may have a shape in which the inner diameter decreases in the depth direction.
　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。複数の収容部１１は、複数の収容部群１１Ａを構成してもよい。収容部群１１Ａは、Ｘ方向に沿って一定の間隔をおいて並んだ複数の収容部１１によって構成される。複数の収容部群１１Ａは、Ｙ方向に一定の間隔をおいて並んでいる。図２では、Ｙ方向に隣り合う収容部群１１Ａの収容部１１はＹ方向に並ぶ位置にあるが、隣り合う収容部群１１Ａの収容部１１は、Ｙ方向に並ぶ位置になくてもよい。例えば、収容部１１は、平面視において千鳥状に配置されていてもよい。 The plurality of storage units 11 are arranged, for example, in a two-dimensional matrix with multiple rows and multiple columns. The plurality of accommodating parts 11 may constitute a plurality of accommodating part groups 11A. The accommodating section group 11A is composed of a plurality of accommodating sections 11 arranged at regular intervals along the X direction. The plurality of housing section groups 11A are arranged at regular intervals in the Y direction. In FIG. 2, the accommodating parts 11 of the accommodating part groups 11A that are adjacent to each other in the Y direction are arranged in the Y direction, but the accommodating parts 11 of the neighboring accommodating part groups 11A do not have to be arranged in the Y direction. For example, the accommodating portions 11 may be arranged in a staggered manner when viewed from above.
　なお、複数の収容部の数および配列は特に限定されない。例えば、収容部群の数は１でもよい。すなわち、基板は、すべての収容部が一列に並んでいてもよい。複数の収容部のうち２以上は、Ｘ方向に位置を違えて形成されている。 Note that the number and arrangement of the plurality of accommodating parts are not particularly limited. For example, the number of storage section groups may be one. That is, in the substrate, all the housing parts may be arranged in a line. Two or more of the plurality of accommodating parts are formed at different positions in the X direction.
　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 portion) is formed in the well structure portion. The base is made of glass, quartz, silicon wafer, or the like. The well structure portion 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、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 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.
［磁気ビーズ分配装置］
　図１に示すように、ワイプ部材２０は、Ｙ方向に沿う中心軸Ｃを有する円柱状とされている。ワイプ部材２０の側面（外周面）はアレイ基板１０の主面１０ａに接する。ワイプ部材２０は、円柱状であるため、大きな接触面積で主面１０ａに接する。そのため、ワイプ部材２０の止液性を高めることができる。 [Magnetic bead distribution device]
As shown in FIG. 1, the wipe member 20 has a cylindrical shape with a central axis C along the Y direction. A side surface (outer peripheral surface) of the wipe member 20 is in contact with the main surface 10a of the array substrate 10. Since the wipe member 20 has a cylindrical shape, it contacts the main surface 10a with a large contact area. Therefore, the liquid stopping properties of the wipe member 20 can be improved.
　図３に示すように、ワイプ部材２０の最下部２０ａは、Ｘ方向から見て、Ｙ方向に沿う直線状である。最下部２０ａが直線状であるため、ワイプ部材２０は、平坦な主面１０ａに隙間なく接することができる。そのため、ワイプ部材２０は、アレイ基板１０の主面１０ａに沿う形状を有するといえる。ワイプ部材２０の最下部２０ａは、アレイ基板１０の側断面図（Ｙ方向とＺ方向に沿う断面図）において、収容部１１の開口を覆う。 As shown in FIG. 3, the lowermost portion 20a of the wipe member 20 is linear along the Y direction when viewed from the X direction. Since the lowermost portion 20a is linear, the wipe member 20 can contact the flat main surface 10a without any gaps. Therefore, it can be said that the wipe member 20 has a shape along the main surface 10a of the array substrate 10. The lowermost portion 20a of the wipe member 20 covers the opening of the accommodating portion 11 in a side sectional view (a sectional view along the Y direction and the Z direction) of the array substrate 10.
　図１に示すように、ワイプ部材２０は、本体部２１と、被覆層２２とを備える。本体部２１は、中心軸Ｃを有する円柱状とされている。本体部２１は、例えば、金属、樹脂などで構成されている。 As shown in FIG. 1, the wipe member 20 includes a main body portion 21 and a covering layer 22. The main body portion 21 has a cylindrical shape with a central axis C. The main body portion 21 is made of, for example, metal, resin, or the like.
　被覆層２２は、本体部２１の外周面に形成されている。被覆層２２は、本体部２１の外周面を全領域にわたって覆う。被覆層２２は、本体部２１の外周面より硬度が低い。硬度としては、デュロメータ硬さ（ＪＩＳ　Ｋ６２５３またはＪＩＳ　Ｋ７２１５に準拠）等が挙げられる。被覆層２２は、弾性体であることが好ましい。被覆層２２は、例えば、ゴム（シリコーンゴムなど）、熱可塑性樹脂、熱可塑性エラストマ、熱硬化性エラストマなどで形成されている。被覆層２２の物性は、主面１０ａに対する密着性、および止液性などを考慮して選択される。被覆層２２は、例えば、本体部２１にシリコーンゴム製のチューブ（シリコーンチューブ）を被せることによって形成することができる。 The covering layer 22 is formed on the outer circumferential surface of the main body portion 21. The coating layer 22 covers the entire outer peripheral surface of the main body portion 21 . The coating layer 22 has a lower hardness than the outer circumferential surface of the main body portion 21 . Examples of the hardness include durometer hardness (based on JIS K6253 or JIS K7215). It is preferable that the covering layer 22 is an elastic body. The covering layer 22 is made of, for example, rubber (such as silicone rubber), thermoplastic resin, thermoplastic elastomer, thermosetting elastomer, or the like. The physical properties of the coating layer 22 are selected in consideration of adhesion to the main surface 10a, liquid stopping properties, and the like. The covering layer 22 can be formed, for example, by covering the main body portion 21 with a silicone rubber tube (silicone tube).
　磁石３０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３０としては、ネオジウム磁石（ＮｄＦｅＢ）が好ましい。 The magnet 30 is 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 30, a neodymium magnet (NdFeB) is preferable.
　磁石３０の形状は、主面１０ａ上のビーズ懸濁液４に含まれる磁気ビーズ１に磁力を作用させることができるように定められる。図３に示すように、磁石３０は、例えば、Ｙ方向に延びる角柱状とすることができる。磁石３０は、アレイ基板１０のＹ方向の全長に及ぶ長さを有するため、Ｙ方向の広い範囲にわたって磁気ビーズ１に磁力を作用させることができる。 The shape of the magnet 30 is determined so that magnetic force can be applied to the magnetic beads 1 included in the bead suspension 4 on the main surface 10a. As shown in FIG. 3, the magnet 30 can have a prismatic shape extending in the Y direction, for example. Since the magnet 30 has a length that spans the entire length of the array substrate 10 in the Y direction, it is possible to apply magnetic force to the magnetic beads 1 over a wide range in the Y direction.
　磁石３０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３０は角柱状に限らず、例えば、円柱状であってもよい。磁石３０は電磁石であってもよい。 For example, one of the upper end and the lower end of the magnet 30 may be an N pole, and the other of the upper end and the lower end may be an S pole. The magnet 30 is not limited to a prismatic shape, and may be, for example, cylindrical. Magnet 30 may be an electromagnet.
　磁石３０は、アレイ基板１０に対してワイプ部材２０とは反対側に配置されている。詳しくは、磁石３０は、アレイ基板１０の下面側に配置されている。磁石３０は、アレイ基板１０の下面に近い高さ位置にある。磁石３０は、＋Ｘ方向（図１における右方）に移動可能である。 The magnet 30 is arranged on the opposite side of the array substrate 10 from the wipe member 20. Specifically, the magnet 30 is arranged on the lower surface side of the array substrate 10. The magnet 30 is located at a height close to the bottom surface of the array substrate 10. The magnet 30 is movable in the +X direction (to the right in FIG. 1).
［磁気ビーズ分配方法］（第１実施形態）
　次に、第１実施形態に係る磁気ビーズ分配方法について説明する。
　本実施形態の磁気ビーズ分配方法は、第１工程（収容部への磁気ビーズ収容）と、第２工程（収容部内の親水性液体を疎水性液体で覆う）と、を一度の操作で行う。 [Magnetic bead distribution method] (First embodiment)
Next, a magnetic bead distribution method according to the first embodiment will be explained.
The magnetic bead distributing method of this embodiment performs the first step (accommodating magnetic beads in the storage section) and the second step (covering the hydrophilic liquid in the storage section with a hydrophobic liquid) in one operation.
（第１工程：収容部への磁気ビーズ収容）
　図１に示すように、アレイ基板１０の主面１０ａに、ワイプ部材２０を配置する。ワイプ部材２０は、長さ方向（Ｙ方向）にわたって主面１０ａに隙間なく接触する。ワイプ部材２０の進行方向は＋Ｘ方向（図１における右方）である。＋Ｘ方向は「前方」（第１方向）である。－Ｘ方向は「後方」である。 (First step: Magnetic beads accommodation in the accommodation part)
As shown in FIG. 1, a wipe member 20 is arranged on the main surface 10a of the array substrate 10. The wipe member 20 contacts the main surface 10a without any gaps along the length direction (Y direction). The advancing direction of the wipe member 20 is the +X direction (to the right in FIG. 1). The +X direction is the "forward" (first direction). -X direction is "backward".
　主面１０ａ上であってワイプ部材２０の前側に、複数の磁気ビーズ１を含む親水性液体２を載せる。
　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固定化している。磁気ビーズ１は、親水性液体２に分散されている。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を含む親水性液体２を「ビーズ懸濁液４」という。ビーズ懸濁液４は、ワイプ部材２０の前面に接している。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。 A hydrophilic liquid 2 containing a plurality of magnetic beads 1 is placed on the main surface 10a and on the front side of the wipe member 20.
The magnetic beads 1 have, for example, immobilized biomolecules such as nucleic acids, peptides, and proteins. Magnetic beads 1 are dispersed in a hydrophilic liquid 2. The hydrophilic liquid 2 is, for example, water or an aqueous solution. The hydrophilic liquid 2 containing the magnetic beads 1 is referred to as a "bead suspension 4." The bead suspension 4 is in contact with the front surface of the wipe member 20. 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.
　主面１０ａ上であってワイプ部材２０の後側に、疎水性液体３を載せる。疎水性液体３は、ワイプ部材２０の後面に接している。
　疎水性液体３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３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。 Hydrophobic liquid 3 is placed on the main surface 10a and on the rear side of the wipe member 20. The hydrophobic liquid 3 is in contact with the rear surface of the wipe member 20.
Examples of the hydrophobic liquid 3 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 3 may also be hydrofluoroether (HFE), mineral oil, saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, perfluorocarbon, or the like.
　図４および図５は、実施形態に係る磁気ビーズ分配方法の説明図である。
　図４および図５に示すように、ワイプ部材２０を、その姿勢を維持したまま、主面１０ａに沿って前方（＋Ｘ方向）に移動させる。ワイプ部材２０は、長さ方向にわたって主面１０ａに接触した状態を維持しつつ前方移動する。ワイプ部材２０の前方移動によって、ビーズ懸濁液４は前方に押され、主面１０ａの前方領域に広げられる。ワイプ部材２０の移動方向は前方（＋Ｘ方向）である。 4 and 5 are explanatory diagrams of the magnetic bead distribution method according to the embodiment.
As shown in FIGS. 4 and 5, the wipe member 20 is moved forward (+X direction) along the main surface 10a while maintaining its posture. The wipe member 20 moves forward while maintaining contact with the main surface 10a along its length. By moving the wipe member 20 forward, the bead suspension 4 is pushed forward and spread over the front region of the main surface 10a. The direction of movement of the wipe member 20 is forward (+X direction).
　ワイプ部材２０が前方移動する際には、ワイプ部材２０に同伴させて磁石３０を前方（＋Ｘ方向）に移動させる。磁石３０は、ワイプ部材２０と同じ速度で前方移動する。そのため、磁石３０は、ワイプ部材２０に対する相対位置を変えずに前方移動する。 When the wipe member 20 moves forward, the magnet 30 is moved forward (+X direction) along with the wipe member 20. The magnet 30 moves forward at the same speed as the wipe member 20. Therefore, the magnet 30 moves forward without changing its relative position with respect to the wipe member 20.
　磁石３０は、平面視において、ワイプ部材２０に比べて前方に位置することが望ましい。磁石３０は、ワイプ部材２０の前方にあるビーズ懸濁液４に含まれる磁気ビーズ１に下向きの磁力（吸引力）を作用させる。磁石３０は、磁気ビーズ１に磁力を作用させつつ前方移動する。磁気ビーズ１は、磁石３０によって、ワイプ部材２０よりやや前方に配置されるため、ワイプ部材２０に干渉されず、収容部１１に導入されやすくなる。 It is desirable that the magnet 30 be located in front of the wipe member 20 in plan view. The magnet 30 applies a downward magnetic force (attractive force) to the magnetic beads 1 contained in the bead suspension 4 in front of the wipe member 20 . The magnet 30 moves forward while applying magnetic force to the magnetic beads 1. Since the magnetic beads 1 are arranged slightly forward of the wipe member 20 by the magnet 30, the magnetic beads 1 are not interfered with by the wipe member 20 and are easily introduced into the storage section 11.
　ビーズ懸濁液４の一部は、収容部１１に導入される。主面１０ａは疎水化され、収容部１１の内面は親水化されているため、ビーズ懸濁液４は収容部１１に入りやすくなる。磁気ビーズ１は、例えば、１個ずつ収容部１１に分配される。 A part of the bead suspension 4 is introduced into the storage section 11. Since the main surface 10a is made hydrophobic and the inner surface of the accommodating part 11 is made hydrophilic, the bead suspension 4 easily enters the accommodating part 11. For example, the magnetic beads 1 are distributed one by one into the storage section 11 .
（第２工程：収容部内の親水性液体を疎水性液体で覆う）
　図１および図４に示すように、ワイプ部材２０の後ろにある疎水性液体３は、ワイプ部材２０の前方移動の際、ワイプ部材２０に追従して主面１０ａに広がる。そのため、疎水性液体３は、収容部１１内の親水性液体２の表面を覆う層を形成する。これにより、疎水性液体３は複数の収容部１１を相互に隔離する。ビーズ懸濁液４は収容部１１内に封入される。 (Second step: Covering the hydrophilic liquid in the container with a hydrophobic liquid)
As shown in FIGS. 1 and 4, the hydrophobic liquid 3 behind the wipe member 20 follows the wipe member 20 and spreads over the main surface 10a when the wipe member 20 moves forward. Therefore, the hydrophobic liquid 3 forms a layer covering the surface of the hydrophilic liquid 2 in the storage section 11 . Thereby, the hydrophobic liquid 3 isolates the plurality of storage parts 11 from each other. The bead suspension 4 is sealed within the container 11 .
（試料の取り扱い）
　収容部１１内の磁気ビーズ１に対する操作は、収容部１１に対して上方から行うことができる。例えば、収容部１１から試料を取り出す操作は、キャピラリーなどの実験器具を上から収容部１１に近づけて用いて行うことができる。キャピラリーなどによって収容部１１から取り出すことができる試料は、例えば、磁気ビーズ１、親水性液体２、ビーズ懸濁液４などである。 (Handling of samples)
The magnetic beads 1 in the container 11 can be operated from above the container 11 . For example, the operation of taking out a sample from the storage section 11 can be performed using an experimental instrument such as a capillary that approaches the storage section 11 from above. Examples of samples that can be taken out from the container 11 using a capillary or the like include magnetic beads 1, hydrophilic liquid 2, and bead suspension 4.
［第１実施形態の磁気ビーズ分配方法および磁気ビーズ分配装置が奏する効果］
　本実施形態に係る磁気ビーズ分配方法は、ワイプ部材２０を前方移動させてビーズ懸濁液４を主面１０ａに広げるとともに、磁力を磁気ビーズ１に作用させつつ磁石３０を前方移動させてビーズ懸濁液４を収容部１１に収容する（第１工程）。ワイプ部材２０の後ろ側の疎水性液体３は、前述のワイプ部材２０の移動によって主面１０ａに広げられ、収容部１１内の親水性液体２の表面を覆う（第２工程）。疎水性液体３は複数の収容部１１を相互に隔離する。ビーズ懸濁液４は収容部１１内に封入される。 [Effects produced by the magnetic bead distribution method and magnetic bead distribution device of the first embodiment]
The magnetic bead distribution method according to the present embodiment involves moving the wipe member 20 forward to spread the bead suspension 4 on the main surface 10a, and moving the magnet 30 forward while applying magnetic force to the magnetic beads 1 to suspend the beads. The suspension 4 is stored in the storage section 11 (first step). The hydrophobic liquid 3 on the rear side of the wipe member 20 is spread over the main surface 10a by the above-described movement of the wipe member 20, and covers the surface of the hydrophilic liquid 2 in the storage part 11 (second step). The hydrophobic liquid 3 isolates the plurality of storage parts 11 from each other. The bead suspension 4 is sealed within the container 11 .
　本実施形態に係る磁気ビーズ分配方法では、ワイプ部材２０および磁石３０を前進移動させるという１つの操作だけで、磁気ビーズ１を収容部１１に分配し、収容部１１を疎水性液体３で封止することができる。よって、磁気ビーズ１を分配する作業は容易となる。また、収容部１１内の親水性液体２は直ちに疎水性液体３に覆われるため、収容部１１内の親水性液体２が露出する時間を短くできる。よって、親水性液体２の蒸散を抑制できる。 In the magnetic bead distribution method according to the present embodiment, the magnetic beads 1 are distributed to the storage section 11 by only one operation of moving the wipe member 20 and the magnet 30 forward, and the storage section 11 is sealed with the hydrophobic liquid 3. can do. Therefore, the work of distributing the magnetic beads 1 becomes easy. Moreover, since the hydrophilic liquid 2 in the storage part 11 is immediately covered with the hydrophobic liquid 3, the time period during which the hydrophilic liquid 2 in the storage part 11 is exposed can be shortened. Therefore, evaporation of the hydrophilic liquid 2 can be suppressed.
　本実施形態に係る磁気ビーズ分配方法では、アレイ基板１０の上側を開放した状態で、磁気ビーズ１を含む液を収容部１１に分配する。そのため、アレイ基板１０の上に設けられた上層部を用いる手法（特許第５３３７３２４号公報を参照）と異なり、収容部１１から試料を取り出す操作を、キャピラリーなどの実験器具を上から収容部１１に近づけて行うことができる。よって、収容部１１内の試料の取り扱いの容易性を高めることができる。 In the magnetic bead distribution method according to this embodiment, the liquid containing the magnetic beads 1 is distributed to the storage section 11 with the upper side of the array substrate 10 open. Therefore, unlike the method using the upper layer provided on the array substrate 10 (see Japanese Patent No. 5337324), the operation of taking out the sample from the storage section 11 is performed by inserting an experimental instrument such as a capillary into the storage section 11 from above. It can be done up close. Therefore, it is possible to improve the ease of handling the sample in the storage section 11.
　アレイ基板１０は、主面１０ａが疎水化され、収容部１１の内面は親水化されているため、ビーズ懸濁液４は収容部１１に入りやすくなる。 Since the main surface 10a of the array substrate 10 is made hydrophobic and the inner surface of the accommodating part 11 is made hydrophilic, the bead suspension 4 easily enters the accommodating part 11.
　ワイプ部材２０は、円柱状とされているため、大きな接触面積で主面１０ａに接する。そのため、ワイプ部材２０の止液性を高めることができる。よって、ビーズ懸濁液４がワイプ部材２０の後側に漏れたり、疎水性液体３がワイプ部材２０の前側に浸入するのを抑制できる。 Since the wipe member 20 has a cylindrical shape, it contacts the main surface 10a with a large contact area. Therefore, the liquid stopping properties of the wipe member 20 can be improved. Therefore, it is possible to suppress the bead suspension 4 from leaking to the rear side of the wipe member 20 and the hydrophobic liquid 3 from entering the front side of the wipe member 20.
　ワイプ部材２０は、低硬度の被覆層２２を有するため、ワイプ部材２０の主面１０ａに対する密着性、ワイプ部材２０の止液性などを高めることができる。 Since the wipe member 20 has the low-hardness coating layer 22, it is possible to improve the adhesion to the main surface 10a of the wipe member 20, the liquid-stopping property of the wipe member 20, and the like.
　本実施形態に係る磁気ビーズ分配装置１００では、ワイプ部材２０および磁石３０を前進移動させるという１つの操作だけで、磁気ビーズ１を収容部１１に分配し、収容部１１を疎水性液体３で封止することができる。よって、磁気ビーズ１を分配する作業は容易となる。また、収容部１１内の親水性液体２は直ちに疎水性液体３に覆われるため、収容部１１内の親水性液体２が露出する時間を短くできる。よって、親水性液体２の蒸散を抑制できる。 In the magnetic bead dispensing device 100 according to the present embodiment, the magnetic beads 1 are distributed to the storage section 11 by just one operation of moving the wipe member 20 and the magnet 30 forward, and the storage section 11 is sealed with the hydrophobic liquid 3. can be stopped. Therefore, the work of distributing the magnetic beads 1 becomes easy. Moreover, since the hydrophilic liquid 2 in the storage part 11 is immediately covered with the hydrophobic liquid 3, the time period during which the hydrophilic liquid 2 in the storage part 11 is exposed can be shortened. Therefore, evaporation of the hydrophilic liquid 2 can be suppressed.
［磁気ビーズ分配方法］（第２実施形態）
　実施形態に係る磁気ビーズ分配方法は、前述の磁気ビーズ分配方法に続いて、疎水性液体および親水性液体の層形成を行うことができる。詳しくは、本実施形態に係る磁気ビーズ分配方法は、前述の第１工程（収容部への磁気ビーズ収容）と、第２工程（収容部内の親水性液体を疎水性液体で覆う）とに続いて、次に示す第３工程と、第４工程と、第５工程とを行う。 [Magnetic bead distribution method] (Second embodiment)
The magnetic bead distribution method according to the embodiment can perform layer formation of a hydrophobic liquid and a hydrophilic liquid following the above-described magnetic bead distribution method. Specifically, the magnetic bead distribution method according to the present embodiment includes the above-mentioned first step (accommodating magnetic beads in the storage section) and second step (covering the hydrophilic liquid in the storage section with a hydrophobic liquid). Then, the following third step, fourth step, and fifth step are performed.
　第３工程では、複数の収容部が形成された主面を有する基板の前記主面に、前記収容部を包囲する液体積層用枠を設置する。前記液体積層用枠は、前記収容部を囲む環状溝が形成されている。
　第４工程では、前記液体積層用枠内であって前記主面の上に、疎水性液体からなる疎水性液体層を形成する。
　第５工程では、前記環状溝に親水性液体を導入し、前記親水性液体を前記環状溝から溢れさせて前記疎水性液体層の上に親水性液体層を形成する。
　以下、各工程を具体的に説明する。 In the third step, a liquid stacking frame surrounding the accommodating portions is installed on the main surface of the substrate having a main surface on which a plurality of accommodating portions are formed. The liquid stacking frame has an annular groove surrounding the storage portion.
In the fourth step, a hydrophobic liquid layer made of a hydrophobic liquid is formed within the liquid stacking frame and on the main surface.
In the fifth step, a hydrophilic liquid is introduced into the annular groove, and the hydrophilic liquid overflows from the annular groove to form a hydrophilic liquid layer on the hydrophobic liquid layer.
Each step will be specifically explained below.
　図６は、本実施形態に係る磁気ビーズ分配方法に用いられる液体積層用枠１１０およびアレイ基板１０の断面図である。図７は、液体層形成方法の説明図である。図８は、液体積層用枠１１０の断面図である。アレイ基板１０の主面１０ａにおいて複数の収容部１１が形成された領域を「アレイ領域」という。アレイ領域は、複数の収容部１１を一括して包含する領域である。他の実施形態との共通構成については、同じ符号を付して説明を省略する。 FIG. 6 is a cross-sectional view of the liquid stacking frame 110 and the array substrate 10 used in the magnetic bead distribution method according to the present embodiment. FIG. 7 is an explanatory diagram of a liquid layer forming method. FIG. 8 is a cross-sectional view of the liquid stacking frame 110. The area in which the plurality of accommodating parts 11 are formed on the main surface 10a of the array substrate 10 is referred to as an "array area." The array area is an area that collectively includes a plurality of accommodating parts 11. Components common to other embodiments will be given the same reference numerals and descriptions will be omitted.
（第３工程：液体積層用枠の設置）
　図６に示すように、アレイ基板１０の主面１０ａに、液体積層用枠１１０を設置する。アレイ基板１０の収容部１１には、前述の磁気ビーズ分配方法によって、磁気ビーズ１および親水性液体２が収容され、収容部１１は疎水性液体３で封止されている。 (Third step: Installation of liquid stacking frame)
As shown in FIG. 6, a liquid stacking frame 110 is installed on the main surface 10a of the array substrate 10. The magnetic beads 1 and the hydrophilic liquid 2 are accommodated in the accommodating part 11 of the array substrate 10 by the above-described magnetic bead distribution method, and the accommodating part 11 is sealed with the hydrophobic liquid 3.
　液体積層用枠１１０は、底壁１１１と、内周壁１１２と、外周壁１１３とを備える。　底壁１１１は、環状に形成されている。底壁１１１は、例えば、円環状、矩形環状などであってよい。底壁１１１の内形寸法は、底壁１１１が平面視においてアレイ領域を包囲できるように定められる。底壁１１１の開口は、平面視においてアレイ領域を包含する。底壁１１１の内形寸法は、底壁１１１が円環状の場合、底壁１１１の内径である。底壁１１１の内形寸法は、底壁１１１が矩形環状の場合、矩形状の開口の辺の長さである。底壁１１１は、アレイ基板１０の主面１０ａ上に設置される。 The liquid stacking frame 110 includes a bottom wall 111, an inner peripheral wall 112, and an outer peripheral wall 113. The bottom wall 111 is formed in an annular shape. The bottom wall 111 may have, for example, an annular shape, a rectangular annular shape, or the like. The internal dimensions of the bottom wall 111 are determined so that the bottom wall 111 can surround the array area in plan view. The opening in the bottom wall 111 encompasses the array area in plan view. The inner dimension of the bottom wall 111 is the inner diameter of the bottom wall 111 when the bottom wall 111 is annular. When the bottom wall 111 has a rectangular annular shape, the internal dimensions of the bottom wall 111 are the lengths of the sides of the rectangular opening. The bottom wall 111 is installed on the main surface 10a of the array substrate 10.
　内周壁１１２は、底壁１１１の内周縁に立設される。内周壁１１２は、底壁１１１の内周縁の形状に応じた筒状とされている。例えば、底壁１１１が円環状である場合、内周壁１１２は、円筒形状となる。底壁１１１の上面からの内周壁１１２の高さは、主面１０ａの上に形成される疎水性液体層１２１の厚さに合わせて定められる。内周壁１１２の高さは、全周にわたって同じである。 The inner peripheral wall 112 is erected on the inner peripheral edge of the bottom wall 111. The inner peripheral wall 112 has a cylindrical shape corresponding to the shape of the inner peripheral edge of the bottom wall 111. For example, when the bottom wall 111 has an annular shape, the inner peripheral wall 112 has a cylindrical shape. The height of the inner peripheral wall 112 from the top surface of the bottom wall 111 is determined according to the thickness of the hydrophobic liquid layer 121 formed on the main surface 10a. The height of the inner circumferential wall 112 is the same over the entire circumference.
　外周壁１１３は、底壁１１１の外周縁に立設される。外周壁１１３は、底壁１１１の外周縁の形状に応じた筒状とされている。例えば、底壁１１１が円環状である場合、外周壁１１３は、円筒形状となる。外周壁１１３の内径は、内周壁１１２の外径より大きい。外周壁１１３は、内周壁１１２に対して外方に離れた位置にある。そのため、内周壁１１２と外周壁１１３との間には、環状溝１１４が形成される。 The outer peripheral wall 113 is erected on the outer peripheral edge of the bottom wall 111. The outer peripheral wall 113 has a cylindrical shape corresponding to the shape of the outer peripheral edge of the bottom wall 111. For example, when the bottom wall 111 has an annular shape, the outer peripheral wall 113 has a cylindrical shape. The inner diameter of the outer peripheral wall 113 is larger than the outer diameter of the inner peripheral wall 112. The outer peripheral wall 113 is located outwardly away from the inner peripheral wall 112. Therefore, an annular groove 114 is formed between the inner peripheral wall 112 and the outer peripheral wall 113.
　底壁１１１の上面（環状溝１１４の底面）からの外周壁１１３の高さは、内周壁１１２の高さより大である。外周壁１１３の高さは、疎水性液体層１２１の上に形成される親水性液体層１２２の厚さに合わせて定められる。 The height of the outer peripheral wall 113 from the top surface of the bottom wall 111 (the bottom surface of the annular groove 114) is greater than the height of the inner peripheral wall 112. The height of the outer peripheral wall 113 is determined according to the thickness of the hydrophilic liquid layer 122 formed on the hydrophobic liquid layer 121.
　液体積層用枠１１０の構成の具体例を示す。図８に示すように、底壁１１１の底面からの内周壁１１２の高さＨ１は、例えば２ｍｍである。環状溝１１４の底面からの内周壁１１２の高さＨ２は、例えば１．５ｍｍである。環状溝１１４の底面からの外周壁１１３の高さＨ３は、例えば９．５ｍｍである。底壁１１１の底面からの外周壁１１３の高さＨ４は、例えば１０ｍｍである。底壁１１１は、幅１．５ｍｍの環状の周辺領域をおいてアレイ領域を囲む。そのため、底壁１１１の内形寸法である幅Ｗ１は、アレイ領域の外形寸法に３ｍｍを加えた寸法となる。底壁１１１の外形寸法である幅Ｗ２は、アレイ基板１０の外形寸法と同等とされる。環状溝１１４の幅Ｗ３は、内周壁１１２と外周壁１１３との間隔である。幅Ｗ３は、幅Ｗ１，Ｗ２に応じて定めることができる。
　なお、液体積層用枠１１０は底壁１１１を備えるが、液体積層用枠は、底壁がない構成であってもよい。この場合、内周壁と外周壁とは、１または複数箇所に形成された連結部によって互いに連結されることが望ましい。 A specific example of the structure of the liquid stacking frame 110 will be shown. As shown in FIG. 8, the height H1 of the inner peripheral wall 112 from the bottom surface of the bottom wall 111 is, for example, 2 mm. The height H2 of the inner peripheral wall 112 from the bottom surface of the annular groove 114 is, for example, 1.5 mm. The height H3 of the outer peripheral wall 113 from the bottom surface of the annular groove 114 is, for example, 9.5 mm. The height H4 of the outer peripheral wall 113 from the bottom surface of the bottom wall 111 is, for example, 10 mm. The bottom wall 111 surrounds the array area with an annular peripheral area having a width of 1.5 mm. Therefore, the width W1, which is the inner dimension of the bottom wall 111, is equal to the outer dimension of the array area plus 3 mm. The width W2, which is the outer dimension of the bottom wall 111, is equal to the outer dimension of the array substrate 10. The width W3 of the annular groove 114 is the distance between the inner circumferential wall 112 and the outer circumferential wall 113. Width W3 can be determined according to widths W1 and W2.
Note that although the liquid stacking frame 110 includes the bottom wall 111, the liquid stacking frame may have a configuration without a bottom wall. In this case, it is desirable that the inner circumferential wall and the outer circumferential wall are connected to each other by connecting portions formed at one or more locations.
（第４工程：疎水性液体層の形成）
　主面１０ａおよび内周壁１１２によって区画された空間に、疎水性液体２０３を導入することによって、疎水性液体層１２１を形成する。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の厚さは、例えば、内周壁１１２の高さと同等、または内周壁１１２の高さより小さい。疎水性液体２０３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、ＰＦＰＥ、ＰＦＡＥ、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２０３は、このほか、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。疎水性液体２０３は、疎水性液体３に比べて高粘度であってよい。 (4th step: Formation of hydrophobic liquid layer)
A hydrophobic liquid layer 121 is formed by introducing the hydrophobic liquid 203 into the space defined by the main surface 10a and the inner circumferential wall 112. The hydrophobic liquid layer 121 is formed on the main surface 10a in a region that includes all the housing parts 11. The thickness of the hydrophobic liquid layer 121 is, for example, equal to or smaller than the height of the inner peripheral wall 112. Examples of the hydrophobic liquid 203 include fluorine oil and silicone oil. Examples of fluorine oil include perfluoropolyethers such as PFPE, PFAE, and PFPAE. The hydrophobic liquid 203 may also be HFE, mineral oil, saturated hydrocarbon, unsaturated hydrocarbon, aromatic hydrocarbon, perfluorocarbon, or the like. The hydrophobic liquid 203 may have a higher viscosity than the hydrophobic liquid 3.
（第５工程：親水性液体層の形成）
　図６および図７に示すように、環状溝１１４に、親水性液体２０２を導入する。親水性液体２０２としては、例えば、水、または水溶液が挙げられる。親水性液体２０２が環状溝１１４を満たした後、さらに親水性液体２０２を導入すると、親水性液体２０２は環状溝１１４から溢れ、内周壁１１２を越えて内側に流入する。親水性液体２０２の流入は、内周壁１１２の周方向の広い範囲で起きる。例えば、親水性液体２０２は、内周壁１１２の全周にわたって同時に内周壁１１２を越えて内側に流入する。 (Fifth step: Formation of hydrophilic liquid layer)
As shown in FIGS. 6 and 7, a hydrophilic liquid 202 is introduced into the annular groove 114. Examples of the hydrophilic liquid 202 include water or an aqueous solution. When the hydrophilic liquid 202 is further introduced after the hydrophilic liquid 202 fills the annular groove 114, the hydrophilic liquid 202 overflows from the annular groove 114 and flows inward over the inner circumferential wall 112. The inflow of the hydrophilic liquid 202 occurs in a wide range in the circumferential direction of the inner peripheral wall 112. For example, the hydrophilic liquid 202 flows inward over the inner circumferential wall 112 over the entire circumference of the inner circumferential wall 112 at the same time.
　親水性液体２０２をさらに導入すると、疎水性液体層１２１の上に、親水性液体層１２２が形成される。親水性液体層１２２は、外周壁１１３の内側に形成される。親水性液体層１２２によって、収容部１１内の親水性液体２が疎水性液体層１２１を通して蒸散するのを抑制することができる。 When the hydrophilic liquid 202 is further introduced, a hydrophilic liquid layer 122 is formed on the hydrophobic liquid layer 121. Hydrophilic liquid layer 122 is formed inside outer peripheral wall 113 . The hydrophilic liquid layer 122 can prevent the hydrophilic liquid 2 in the storage section 11 from evaporating through the hydrophobic liquid layer 121 .
［第２実施形態の磁気ビーズ分配方法が奏する効果］
　本実施形態に係る磁気ビーズ分配方法によれば、内周壁１１２の内側への親水性液体２０２の流入は、内周壁１１２の周方向の広い範囲で起きる。そのため、流入時に親水性液体２０２が疎水性液体層１２１に加える荷重は分散される。したがって、親水性液体２０２が局所的に疎水性液体層１２１中に進入して収容部１１内に達するのを抑制できる。これにより、疎水性液体層１２１の全体を覆う親水性液体層１２２を形成することができる。そのため、収容部１１内の親水性液体２が蒸散するのを抑制できる。よって、親水性液体２の蒸散を防ぐための蓋部材は不要となる。 [Effects achieved by the magnetic bead distribution method of the second embodiment]
According to the magnetic bead distribution method according to the present embodiment, the hydrophilic liquid 202 flows into the inner peripheral wall 112 over a wide range in the circumferential direction of the inner peripheral wall 112. Therefore, the load that the hydrophilic liquid 202 applies to the hydrophobic liquid layer 121 at the time of inflow is dispersed. Therefore, it is possible to prevent the hydrophilic liquid 202 from locally entering the hydrophobic liquid layer 121 and reaching the inside of the accommodating portion 11 . Thereby, a hydrophilic liquid layer 122 covering the entire hydrophobic liquid layer 121 can be formed. Therefore, it is possible to suppress the hydrophilic liquid 2 in the storage part 11 from evaporating. Therefore, a lid member for preventing evaporation of the hydrophilic liquid 2 is not necessary.
　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.
［実施例１］
＜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。 [Example 1]
<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、後述の＜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> described 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」という。アレイ基板の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 in which the pieces were 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." The area of the array substrate where no microwells are present is referred to as "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が親水性で、マイクロウェル外部の表面（主面）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 inside the microwell was hydrophilic and the surface (principal 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であって、アレイ基板の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 between the contact portion of the wipe member and the end of the array area and below the array substrate.
　アレイ基板の主面（ワイプ部材の前方位置：ワイプ部材の接触部からアレイ領域端までの間の位置）に、６０μＬのＤＮＡ固定化磁気ビーズ懸濁液を滴下した。アレイ基板の主面（ワイプ部材の後方位置）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 (the front position of the wipe member: the position between the contact part of the wipe member and the end of the array area). 60 μL of fluorine oil (trade name: Krytox (registered trademark) GPL104 (manufactured by Chemours) was dropped onto the main surface of the array substrate (at the rear position of the wipe member).
　ワイプ部材およびネオジウム磁石を、０．１ｍｍ／ｓで前方に同時に動かし、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 moved forward at the same time 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.
　図９（Ａ）および図９（Ｂ）に、マイクロウェルにＤＮＡ固定化磁気ビーズと蛍光水溶液とがフッ素化オイルにより封入されたアレイ基板の顕微鏡画像（図９（Ａ）：明視野像、図９（Ｂ）：蛍光像）を示す。図９（Ａ）および図９（Ｂ）に示すように、アレイ領域内の全てのマイクロウェルに蛍光水溶液が分配され、６７％のマイクロウェルにＤＮＡ固定化磁気ビーズが分配されていた。 Figures 9(A) and 9(B) show microscopic images of an array substrate in which DNA-immobilized magnetic beads and a fluorescent aqueous solution are sealed in microwells using fluorinated oil (Figure 9(A): bright field image, 9(B): Fluorescent image). As shown in FIGS. 9(A) and 9(B), the fluorescent aqueous solution was distributed to all microwells in the array area, and DNA-immobilized magnetic beads were distributed to 67% of the microwells.
［実施例２］
＜アレイ基板表面のオイル層への水の層形成＞
　図１０（Ａ）に示すように、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、アレイ基板の主面に、フッ素オイルからなる疎水性液体層を形成した。 [Example 2]
<Formation of a water layer on the oil layer on the surface of the array substrate>
As shown in FIG. 10(A), a frame for water lamination (frame for liquid lamination) was produced using a 3D printer (trade name: From2, manufactured by Formlabs). A water layering frame 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 main surface of the array substrate.
　図１０（Ｂ）および図１０（Ｃ）に示すように、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の水をさらに環状溝に添加した。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。 As shown in FIG. 10(B) and FIG. 10(C), 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, 3 mL of water was further added to the annular groove. . 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.
　蛍光顕微鏡により、マイクロウェル内の蛍光水溶液の保持時間を確認した。
　図１１（Ａ）は、オイル層上に水層を積層しなかった場合の、試験開始時の画像である。図１１（Ｂ）は、試験開始から１０分間経過した時点の画像である。図１２（Ａ）は、オイル層上に水層を積層した場合の、試験開始時の画像である。図１２（Ｂ）は、試験開始から３日間経過した時点の画像である。 The retention time of the fluorescent aqueous solution in the microwell was confirmed using a fluorescence microscope.
FIG. 11(A) is an image at the start of the test when the water layer was not laminated on the oil layer. FIG. 11(B) is an image taken 10 minutes after the start of the test. FIG. 12(A) is an image at the start of the test when a water layer is laminated on an oil layer. FIG. 12(B) is an image taken 3 days after the start of the test.
　図１１（Ａ）および図１１（Ｂ）に示すように、オイル層上に水層を積層しなかった場合、マイクロウェル内の蛍光水溶液は試験開始から１０分間で消失した。図１２（Ａ）および図１２（Ｂ）に示すように、オイル層上に水の層を形成した場合、試験開始から３日間経過後も、蛍光水溶液はマイクロウェル内に保持されていた。 As shown in FIGS. 11(A) and 11(B), when the water layer was not stacked on the oil layer, the fluorescent aqueous solution in the microwell disappeared within 10 minutes from the start of the test. As shown in FIGS. 12(A) and 12(B), when a water layer was formed on the oil layer, the fluorescent aqueous solution was retained in the microwells even after 3 days had passed from the start of the test.
　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。
　例えば、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（内径）と深さのうち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。 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.
For example, the number of magnetic beads 1 housed in the housing 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 (inner diameter) 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.
　図１に示すワイプ部材は円柱状であるが、ワイプ部材の形状は特に限定されない。ワイプ部材は、例えば、ブレード状であってもよい。ワイプ部材は、基板の主面に対して接触することが好ましいが、主面からわずかな距離だけ離れていてもよい。
　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。 Although the wipe member shown in FIG. 1 has a cylindrical shape, the shape of the wipe member is not particularly limited. The wipe member may be, for example, blade-shaped. The wipe member preferably contacts the main surface of the substrate, but may be separated from the main surface by a small distance.
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.
　１　磁気ビーズ
　２，２０２　親水性液体
　３，２０３　疎水性液体
　１０　アレイ基板（基板）
　１０ａ　主面
　１１　収容部
　２０　ワイプ部材
　２１　本体部
　２２　被覆層
　３０　磁石
　１００　磁気ビーズ分配装置
　１１０　液体積層用枠
　１１４　環状溝
　１２１　疎水性液体層
　１２２　親水性液体層
　Ｃ…中心軸 1 Magnetic beads 2,202 Hydrophilic liquid 3,203 Hydrophobic liquid 10 Array substrate (substrate)
10a Main surface 11 Storage part 20 Wipe member 21 Main body part 22 Covering layer 30 Magnet 100 Magnetic bead distribution device 110 Liquid stacking frame 114 Annular groove 121 Hydrophobic liquid layer 122 Hydrophilic liquid layer C... Central axis

Claims (6)

  1. 　複数の収容部が形成された主面を有する基板と、前記主面に沿う形状を有するワイプ部材と、前記基板に対して前記ワイプ部材とは反対側に配置した磁石と、を用い、
    　前記主面に沿って前記ワイプ部材を前方移動させることで、複数の磁気ビーズを含む親水性液体を前記主面に広げるとともに、磁力を前記磁気ビーズに作用させつつ前記磁石を前記ワイプ部材に同伴させて前方移動させることによって、前記磁気ビーズおよび前記親水性液体を前記収容部に収容する工程と、
    　前記ワイプ部材の移動方向の後ろ側に配した疎水性液体を、前記ワイプ部材の移動によって前記主面に広げることによって、前記収容部内の前記親水性液体の表面を前記疎水性液体で覆う工程と、を有する、
    　磁気ビーズ分配方法。     A substrate having a main surface on which a plurality of accommodating portions are formed, a wipe member having a shape along the main surface, and a magnet disposed on the opposite side of the substrate from the wipe member,
    By moving the wipe member forward along the main surface, a hydrophilic liquid containing a plurality of magnetic beads is spread over the main surface, and the magnet is brought along with the wipe member while applying magnetic force to the magnetic beads. accommodating the magnetic beads and the hydrophilic liquid in the accommodating section by moving the magnetic beads and the hydrophilic liquid forward;
    a step of covering the surface of the hydrophilic liquid in the storage portion with the hydrophobic liquid by spreading the hydrophobic liquid arranged on the rear side in the moving direction of the wipe member over the main surface by the movement of the wipe member; , has
    Magnetic bead distribution method.
  2. 　前記主面は、疎水化されており、
    　前記収容部の内面は、親水化されている、
    　請求項１記載の磁気ビーズ分配方法。     The main surface is made hydrophobic,
    The inner surface of the accommodating portion is made hydrophilic.
    The method for distributing magnetic beads according to claim 1.
  3. 　前記ワイプ部材は、前記主面に沿う中心軸を有する円柱状とされている、
    　請求項１記載の磁気ビーズ分配方法。     The wipe member has a cylindrical shape having a central axis along the main surface.
    The method for distributing magnetic beads according to claim 1.
  4. 　前記ワイプ部材は、円柱状の本体部と、前記本体部の外周面に形成され、前記外周面より硬度が低い被覆層と、を備える、
    　請求項３記載の磁気ビーズ分配方法。     The wipe member includes a cylindrical main body and a coating layer formed on the outer circumferential surface of the main body and having a lower hardness than the outer circumferential surface.
    The magnetic bead distribution method according to claim 3.
  5. 　前記収容部を囲む環状溝が形成され、前記収容部を包囲する液体積層用枠を前記主面に設置する工程と、
    　前記液体積層用枠内であって前記主面の上に、疎水性液体からなる疎水性液体層を形成する工程と、
    　前記環状溝に親水性液体を導入し、前記親水性液体を前記環状溝から溢れさせて前記疎水性液体層の上に親水性液体層を形成する工程と、をさらに有する、
    　請求項１～４のうちいずれか１項に記載の磁気ビーズ分配方法。     an annular groove surrounding the accommodating part is formed, and a step of installing a liquid stacking frame surrounding the accommodating part on the main surface;
    forming a hydrophobic liquid layer made of a hydrophobic liquid on the main surface within the liquid stacking frame;
    Further comprising the step of introducing a hydrophilic liquid into the annular groove and causing the hydrophilic liquid to overflow from the annular groove to form a hydrophilic liquid layer on the hydrophobic liquid layer.
    The method for distributing magnetic beads according to any one of claims 1 to 4.
  6. 　複数の収容部が形成された主面を有する基板の前記収容部に磁気ビーズを分配する磁気ビーズ分配装置であって、
    　前記主面に沿う形状を有するワイプ部材と、
    　前記基板に対して前記ワイプ部材とは反対側に配置した磁石と、を備え、
    　前記ワイプ部材は、複数の磁気ビーズを含む親水性液体を載せた前記主面に沿って前方移動可能であり、
    　前記磁石は、前記磁気ビーズに磁力を作用させつつ前記ワイプ部材に同伴させて前方移動可能である、
    　磁気ビーズ分配装置。     A magnetic bead dispensing device for distributing magnetic beads to the accommodating parts of a substrate having a main surface on which a plurality of accommodating parts are formed,
    a wipe member having a shape along the main surface;
    a magnet disposed on the opposite side of the wipe member with respect to the substrate,
    The wipe member is movable forward along the main surface carrying a hydrophilic liquid containing a plurality of magnetic beads,
    The magnet is capable of moving forward along with the wipe member while applying a magnetic force to the magnetic beads.
    Magnetic bead dispensing device.
PCT/JP2023/031502 2022-08-31 2023-08-30 Magnetic bead arrangement method and magnetic bead arrangement device WO2024048648A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2022138140 2022-08-31
JP2022-138140 2022-08-31

Publications (1)

Publication Number Publication Date
WO2024048648A1 true WO2024048648A1 (en) 2024-03-07

Family

ID=90099664

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2023/031502 WO2024048648A1 (en) 2022-08-31 2023-08-30 Magnetic bead arrangement method and magnetic bead arrangement device

Country Status (1)

Country Link
WO (1) WO2024048648A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018508187A (en) * 2015-01-13 2018-03-29 ギルソン インコーポレイテッド Adapter for sliding magnetic particle separation
JP2019505761A (en) * 2015-12-01 2019-02-28 イラミーナ インコーポレーテッド Digital microfluidic system for single cell isolation and analyte characterization
JP2019536981A (en) * 2016-10-05 2019-12-19 アボット・ラボラトリーズＡｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ Device and method for sample analysis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018508187A (en) * 2015-01-13 2018-03-29 ギルソン インコーポレイテッド Adapter for sliding magnetic particle separation
JP2019505761A (en) * 2015-12-01 2019-02-28 イラミーナ インコーポレーテッド Digital microfluidic system for single cell isolation and analyte characterization
JP2019536981A (en) * 2016-10-05 2019-12-19 アボット・ラボラトリーズＡｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ Device and method for sample analysis

Similar Documents

Publication Publication Date Title
US10190082B2 (en) Multiwell plate
Kim et al. Soft lithography for microfluidics: a review
Ueda et al. Emerging applications of superhydrophilic‐superhydrophobic micropatterns
US6037168A (en) Microbiological assembly comprising resealable closure means
EP2911781B1 (en) Formation of array of membranes and apparatus therefor
MichelleáGrandin Micro-well arrays for 3D shape control and high resolution analysis of single cells
US9095852B2 (en) Multilayer high density microwells
US7790109B2 (en) Micro-fluid reaction vessel, method for manufacturing the same, and micro-fluid reaction method using the vessel
US11061043B2 (en) Loading station
WO2016149661A1 (en) Massively parallel on-chip coalescence of microemulsions
US20130071914A1 (en) Method for studying floating, living cells
KR20110137319A (en) Microarray with microchannels
Wu et al. High-throughput generation of durable droplet arrays for single-cell encapsulation, culture, and monitoring
US8470110B2 (en) Sample chamber and method for the production thereof
WO2005010147A2 (en) Device and method for contacting pocoliter volumes of fluids
KR20120071216A (en) Cell chip
WO2024048648A1 (en) Magnetic bead arrangement method and magnetic bead arrangement device
JP7464038B2 (en) Microfluidic device and sample analysis method
US9689878B2 (en) Assay method using encoded particle-based platform
JP2022547239A (en) Effect of magnetic beads on microfluidics
WO2024048660A1 (en) Magnetic bead recovery method and magnetic bead recovery device
Wang et al. A novel dual-well array chip for efficiently trapping single-cell in large isolated micro-well without complicated accessory equipment
JP6521432B2 (en) Cell incubator and cell culture method
JP2022531307A (en) Microfabrication equipment with hydrophilic microwells and hydrophobic interstitial spaces
JP6821947B2 (en) Particle storage method and particle encapsulation method

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23860410

Country of ref document: EP

Kind code of ref document: A1