[go: up one dir, main page]

WO2017131216A1 - Biopolymer fractionation chip, and biopolymer fractionation method and biopolymer analysis method using same - Google Patents

Biopolymer fractionation chip, and biopolymer fractionation method and biopolymer analysis method using same Download PDF

Info

Publication number
WO2017131216A1
WO2017131216A1 PCT/JP2017/003069 JP2017003069W WO2017131216A1 WO 2017131216 A1 WO2017131216 A1 WO 2017131216A1 JP 2017003069 W JP2017003069 W JP 2017003069W WO 2017131216 A1 WO2017131216 A1 WO 2017131216A1
Authority
WO
WIPO (PCT)
Prior art keywords
biopolymer
opening
chip
fractionation
wall
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2017/003069
Other languages
French (fr)
Japanese (ja)
Inventor
博文 新宅
卓哉 藁谷
想太郎 上村
祐伴 小口
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Tokyo NUC
Kyoto University NUC
Original Assignee
University of Tokyo NUC
Kyoto University NUC
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 University of Tokyo NUC, Kyoto University NUC filed Critical University of Tokyo NUC
Priority to JP2017557217A priority Critical patent/JP6338262B2/en
Publication of WO2017131216A1 publication Critical patent/WO2017131216A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a biopolymer fractionation chip, a biopolymer fractionation method using the chip, and a biopolymer analysis method.
  • Patent Document 1 a method for fractionating nucleic acid from a sample containing target cells, etc., a cell membrane of the target cells is disrupted by applying a voltage to the sample and a liquid containing molecules having a molecular sieve function, and electrophoresis is performed.
  • Patent Document 1 a method for fractionating nuclear nucleic acid and cytoplasmic nucleic acid is known.
  • the fractionated biopolymers contain molecules having the molecular sieve function. Further, the molecule having the molecular sieve function interferes with the analysis of the biopolymer such as the nucleic acid. For this reason, when analyzing the fractionated biopolymer, there is a problem that the analysis accuracy is lowered.
  • the present invention provides, for example, a new biopolymer fractionation chip that can fractionate biopolymers such as nucleic acids even in liquids that do not contain molecules having the molecular sieve function, and separation of biopolymers using the same.
  • An object of the present invention is to provide a drawing method and a biopolymer analysis method.
  • the biopolymer fractionation chip of the present invention (hereinafter also referred to as “chip”) is: Having a substrate, The substrate has a separation channel for separating cytoplasmic biopolymers of target cells, and one or more openings.
  • the one or more openings have a first opening into which a sample containing the target cells can be introduced into the separation channel, The first opening communicates with the separation channel;
  • the separation channel is in the channel and has a wall in the cross-sectional direction, The wall has an opening.
  • the biopolymer fractionation method of the present invention includes a trap step of trapping target cells in an opening of a wall having an opening, The method includes a release step of releasing a cytoplasmic biopolymer from the target cell by destroying a cell membrane of the target cell, and a separation step of separating the released cytoplasmic biopolymer.
  • the biopolymer analysis method of the present invention includes a fractionation step of fractionating a cytoplasmic biopolymer from a target cell, and the cytoplasmic biopolymer and the cytoplasmic biomolecule height.
  • a biopolymer such as a nucleic acid can be fractionated even in a liquid system that does not contain a molecule having the molecular sieve function.
  • FIG. 1A is a top view showing the configuration of the device of Embodiment 1
  • FIG. 1B is a cross-sectional view taken along the II direction of FIG. 1A
  • FIG. It is sectional drawing seen from the II-II direction.
  • FIG. 2A is a top view showing the configuration of the device of Embodiment 2
  • FIG. 2B is an enlarged view (top view) of a region surrounded by a two-dot chain line shown in FIG.
  • C) is a cross-sectional view as viewed from the II direction of (B)
  • (D) is an enlarged view of a region surrounded by a two-dot chain line (D) of (A).
  • FIG. 3 is a top view illustrating the configuration of the device according to the third embodiment.
  • FIG. 4 is a top view showing the configuration of the device of the first modification.
  • FIG. 5 is a top view illustrating the configuration of the device according to the fourth embodiment.
  • FIG. 6 is a top view illustrating the configuration of the device according to the fifth embodiment.
  • FIG. 7 is a top view illustrating the configuration of the device according to the sixth embodiment.
  • FIG. 8 is a photograph showing separation of nucleic acids during voltage application in Example 2.
  • FIG. 9 is a photograph showing the recovery of other nucleic acids in Example 3.
  • FIG. 10 is a photograph showing separation of nucleic acids during voltage application in Example 4.
  • FIG. 11 is a graph showing the fluorescence intensity after correction in Example 5.
  • FIG. 12 is a graph showing the fluorescence intensity in Example 5.
  • FIG. 13 is a photograph showing separation of nucleic acids during voltage application in Example 7.
  • FIG. 14 is a graph showing the types of sequences included in each library in Example 8.
  • FIG. 15 is a graph showing the origin of each library in Example 8.
  • FIG. 16 is a graph showing the yield in Example 9.
  • FIG. 17 is a graph showing simulation results and approximate expressions in Example 10.
  • the wall has two or more openings.
  • the diameter (w 1 ) of the opening of the wall is smaller than the diameter (w t ) of the target cell.
  • the ratio (w 1 : w t ) of the diameter (w 1 ) of the opening of the wall and the diameter (w t ) of the target cell is 1: 2 or more.
  • the diameter (w 1 ) of the opening of the wall is smaller than the diameter (w n ) of the remaining target cell after separation of the cytoplasmic biopolymer.
  • the ratio (w t : w 2 ) of the target cell diameter (w t ) to the separation channel diameter (w 2 ) is in the range of 1: 1 to 1: 100. It is.
  • the ratio (S 1 : S 2 ) of the sectional area (S 1 ) of the opening of the wall and the sectional area (S 2 ) of the separation channel is 1: 2 or more. .
  • the first opening portion is tapered from the outer surface to the inner surface of the substrate.
  • the biopolymer fractionation chip further includes a suction / discharge unit capable of sucking the liquid in the separation channel and / or discharging the liquid into the separation channel.
  • a suction / discharge unit capable of sucking the liquid in the separation channel and / or discharging the liquid into the separation channel.
  • the one or more suction / discharge portions are arranged to connect to the separation channel between the wall and an end portion in a direction opposite to the first opening with respect to the wall.
  • the biopolymer fractionation chip further has a capture section for the biopolymer,
  • the capture part is disposed between the wall and the connection part of the suction / discharge part in the separation channel.
  • the biopolymer fractionation chip has two or more suction / discharge sections,
  • the capture part is disposed between the connection parts of the suction and discharge parts in the separation channel.
  • the chip of the present invention for example, the biopolymer fractionation chip further has an electrode, The electrode is disposed in the separation channel between the wall and an end portion in a direction opposite to the first opening with respect to the wall.
  • the one or more openings further include a second opening capable of collecting the cytoplasmic biopolymer,
  • the second opening communicates with the separation channel;
  • the wall is disposed between the first opening and the second opening in the separation channel.
  • the biopolymer fractionation chip further includes a liquid movement control unit that controls movement of the liquid between the wall and the second opening,
  • the liquid movement control unit is disposed between the wall and the second opening in the separation channel.
  • the chip of the present invention further includes, for example, an adjustment channel that adjusts the movement of the target cell, and a third opening.
  • the third opening and the separation channel are communicated with the adjustment channel,
  • the separation channel communicates with the adjustment channel on the second opening side from the wall of the separation channel.
  • the chip of the present invention further includes, for example, a connection channel that communicates the separation channel and the adjustment channel,
  • the connection channel communicates with the separation channel on the first opening side from the wall of the separation channel.
  • the cross-sectional area and the length of the connection channel are such that the flow rate of the sample (F 1 ) flowing through the wall opening in a state where the wall opening does not trap the target cell.
  • the ratio (F 1 : F c ) of the flow rate (F c ) of the sample flowing through the connection flow path satisfies the cross-sectional area and length in the range of 1: 1 to 20: 1.
  • the chip of the present invention further has, for example, an electrode system,
  • the electrode system comprises one or more electrodes;
  • the one or more electrodes are in the first opening, between the wall and the first opening in the separation channel, in the second opening, and the wall in the separation channel. It arrange
  • the chip of the present invention is, for example, a biopolymer fractionation chip in which the biopolymer fractionation chip has the third opening,
  • the one or more electrodes are further disposed so as to be located in at least one of the third opening and the adjustment channel.
  • the biopolymer is at least one selected from the group consisting of nucleic acids, sugars, proteins, and lipids.
  • the cytoplasmic biopolymer is at least one of a biopolymer of a cytoplasmic matrix and a biopolymer of an organelle.
  • the biopolymer of the organelle is at least one selected from the group consisting of chloroplast nucleic acid, mitochondrial nucleic acid, and liposome nucleic acid.
  • the cell membrane of the target cell is electrically or chemically destroyed.
  • a cytoplasmic biopolymer is released from the target cell by electrically destroying a cell membrane of the target cell
  • the separation step the released cytoplasmic biopolymer is separated by an electrical separation method.
  • the fractionation method of the present invention further includes, for example, at least one of the separated cytoplasmic biopolymer and the biopolymer contained in the remainder of the target cell after fractionation of the cytoplasmic biopolymer in the separation step.
  • a recovery step for recovery is included.
  • the wall has two or more openings.
  • the diameter (w 1 ) of the opening of the wall is smaller than the diameter (w t ) of the target cell.
  • the ratio (w 1 : w t ) between the diameter (w 1 ) of the wall opening and the diameter (w t ) of the target cell is 1: 2 or more.
  • the diameter (w 1 ) of the opening of the wall is smaller than the diameter (w n ) of the remaining portion of the target cell after separation of the cytoplasmic biopolymer.
  • the fractionation method of the present invention uses, for example, the biopolymer fractionation chip of the present invention, An introducing step of introducing a sample containing the target cells from the first opening; A trapping step for trapping the target cell in the opening of the wall; A release step of releasing a biopolymer from the target cell by applying a voltage to the separation channel; and the wall; and an end opposite to the first opening with respect to the wall; A separation step of separating a cytoplasmic biopolymer of the target cell in the separation channel between the two.
  • the biopolymer fractionation chip is a biopolymer fractionation chip having the second opening
  • the method further includes a step of recovering the cytoplasmic biopolymer from the second opening.
  • the fractionation method of the present invention further includes, for example, a step of recovering the biopolymer contained in the remainder of the target cell after separation of the cytoplasmic biopolymer from the first opening.
  • the fractionation method of the present invention further includes, for example, a step of preparing a sample containing the target cells.
  • the cytoplasmic biopolymer is at least one of a cytosolic biopolymer and a cell organelle biopolymer.
  • the biopolymer of the organelle is at least one selected from the group consisting of chloroplast nucleic acid, mitochondrial nucleic acid, and liposome nucleic acid.
  • the analysis method of the present invention includes, for example, a step of recovering at least one of the analyzed cytoplasmic biopolymer and the remainder of the target cell after fractionation (separation) of the cytoplasmic biopolymer. .
  • the analysis method of the present invention maintains the fractionated state of the biopolymer contained in the fractionated cytoplasmic biopolymer and the remainder of the target cell after fractionation (separation) of the cytoplasmic biopolymer. Process.
  • the biopolymer is a nucleic acid
  • the biopolymer fractionation chip of the present invention has a substrate as described above, and the substrate has a separation channel for separating cytoplasmic biopolymers of target cells, and one or more openings.
  • the one or more openings have a first opening through which the sample containing the target cells can be introduced into the separation channel, and the first opening is the separation channel.
  • the separation channel is in the channel and has a wall in a cross-sectional direction, and the wall has an opening.
  • the chip of the present invention is characterized in that the separation channel is in the channel and has a wall in a cross-sectional direction, and the wall has an opening, and other configurations and conditions are particularly limited. Not.
  • the chip of the present invention has a wall in the cross-sectional direction in the separation channel, and the wall has an opening. For this reason, the chip of the present invention, for example, destroys the cell membrane of the target cell in a state where the target cell introduced from the first opening is trapped in the opening of the wall, or the cell membrane of the target cell After the destruction of the cytoplasmic biopolymer, by trapping the remainder of the target cell after separation of the cytoplasmic biopolymer in the opening of the wall, the cytoplasmic biopolymer such as the cytoplasmic nucleic acid is passed through the opening of the wall,
  • the separation channel can be separated into the separation channel between the wall and the end portion in the direction opposite to the first opening direction with respect to the wall, and the target cell after separation of the cytoplasmic biopolymer can be separated.
  • the remaining biopolymer (hereinafter also referred to as “residual biopolymer”) can be trapped in the opening of the wall.
  • a biopolymer such as a nucleic acid can be separated even in a liquid system that does not contain a molecule having the molecular sieve function.
  • the cytoplasmic biopolymer can be separated in a state where the remaining biopolymer is trapped in the opening of the wall.
  • the quality that can be used for next-generation sequencing Samples with (eg high purity) can be prepared.
  • the chip of the present invention can trap the target cells with high accuracy at the opening of the wall when, for example, one target cell is introduced into the separation channel. Therefore, according to the chip of the present invention, for example, a cytoplasmic biopolymer can be separated from one target cell. Therefore, the chip of the present invention can also be referred to as a chip that separates a cytoplasmic biopolymer from one target cell, for example.
  • the separation channel has a wall having the opening in the channel. For this reason, the chip of the present invention has, for example, a separation channel that does not have a wall having the opening when a voltage is applied to the separation channel (for example, the separation channel in Patent Document 1).
  • the current density around the wall opening where the target cells are trapped can be increased. Therefore, for example, when electrically destroying the cell membrane of the target cell, according to the chip of the present invention, in the separation channel of Patent Document 1, compared with the voltage when crushing the cell membrane of the target cell Then, the cell membrane of the target cell can be crushed with a lower voltage (for example, a voltage of 1/10 or less).
  • a lower voltage for example, a voltage of 1/10 or less.
  • the chip of the present invention can be used at a lower voltage, for example, the generation of Joule heat during voltage application can be reduced, and the influence of denaturation of biopolymers such as nucleic acids due to the Joule heat can be reduced.
  • the chip of the present invention can be used at a lower voltage, for example, a highly conductive solution containing an electrolyte can be used as a separation liquid used for biopolymer separation.
  • the cell membrane of the target cell may be electrically broken as described above, or other methods as described later. Thus, the cell membrane of the target cell may be destroyed.
  • the chip of the present invention is a chip that can be used for the fractionation method of the present invention, and the description of the fractionation method of the present invention described later can be used.
  • the “chip” includes, for example, a capillary tube.
  • the substrate means, for example, the outer wall of the capillary tube.
  • tip is demonstrated, the following description can be used for description of the form of a capillary tube.
  • the size of the chip is not particularly limited, and can be appropriately determined according to, for example, the number of the separation channels arranged on the substrate.
  • the maximum length of the chip is, for example, 50 to 100 mm
  • the maximum width of the chip is, for example, 10 to 50 mm
  • the maximum thickness of the chip is 3 to 30 mm.
  • “length” is the distance in the longitudinal direction of the chip
  • the maximum length of the chip is the distance of the longest portion in the longitudinal direction of the chip
  • “width” is the longitudinal direction of the chip.
  • the maximum width of the chip is the distance of the longest part in the width direction of the chip, and "thickness (depth, height)" It is the distance in the direction perpendicular to the longitudinal direction and the width direction of the chip (thickness direction, height direction), and the maximum thickness of the chip is the distance of the longest part in the thickness direction of the chip.
  • the target cell is not particularly limited and can be any cell.
  • the target cell may be, for example, one cell or two or more cells.
  • examples of the target cell include a cell mass.
  • examples of the cells include living body-derived cells and cultured cells.
  • Examples of the cell mass include a fertilized egg.
  • the origin of the cell is not particularly limited, and examples thereof include humans, non-human animals other than humans, plants, prokaryotes, eukaryotes, and the like.
  • Examples of the non-human animal include non-human animals excluding humans. Examples of the non-human animal include monkeys, mice, rats, dogs, rabbits, sheep, horses, guinea pigs and the like.
  • Examples of the eukaryote include Euglena.
  • the separation channel is a channel communicating with the first opening, and the inside is a void (hollow).
  • a direction perpendicular to the axial direction of the separation channel is referred to as a “cross-sectional direction”, and the first opening with the first opening side as the center and the first opening with the wall as a center.
  • the direction opposite to the part direction is referred to as the downstream side
  • the separation channel between the first opening and the wall is referred to as the upstream channel
  • the first reference is made on the basis of the wall and the wall.
  • the separation channel between the end opposite to the opening direction and the separation channel between the wall and the second opening described later are referred to as downstream channels.
  • upstream and downstream are expressions for indicating the positional relationship in the separation channel, and the moving direction of the liquid (for example, the sample) introduced into the separation channel.
  • the “cross-sectional area of the flow path” means the cross-sectional area of the void inside the flow path in the cross-sectional direction
  • the “length of the flow path” means the flow path This means the length in the axial direction.
  • the shape of the separation channel is not particularly limited, and the shape of the cross section may be a circle such as a circle, a perfect circle, or an ellipse; a semicircle; a polygon such as a triangle, a quadrangle, a square, or a rectangle.
  • the upstream channel and the downstream channel may have the same or different cross-sectional shapes, for example.
  • the size of the separation channel (eg, width, depth, diameter, cross-sectional area, etc.) is not particularly limited, and may be any size as long as the target cell can move to the opening of the wall. It can be determined appropriately according to the size of the target cell.
  • the size of the upstream channel is preferably such a size that the target cell can move to the opening of the wall.
  • the upstream channel and the downstream channel may be the same size or different sizes, for example.
  • the size of the target cell (w t) and the ratio of the diameter of the separation channel (w 2) (w t: w 2) are, for example, 1: 1 or greater, preferably, 1 1 to 1: 100, 1: 2 to 1: 100.
  • the diameter of the target cell is, for example, the short diameter of the target cell.
  • the diameter of the separation channel is, for example, the short diameter of the separation channel, and when the cross-sectional shape of the separation channel is other than a circle, for example, in the cross section of the separation channel , The shortest distance. In the separation channel, it is preferable that the diameter of the upstream channel satisfies the ratio.
  • the separation channel preferably contains, for example, a separation liquid for separating the biopolymer.
  • a separation liquid for separating the biopolymer examples include Tris buffer solution, Bis-Tris buffer solution, Tris-HEPES buffer solution, imidazole buffer solution, phosphate buffer solution buffer medium, and the like.
  • the concentration of the buffer is not particularly limited and is, for example, 1 to 500 mmol / L.
  • the separation liquid examples include saccharides such as sucrose and mannitol, surfactants such as Triton (registered trademark) X100 and Tween (registered trademark) 20, proteins such as bovine serum albumin (BSA) and acetylated BSA, carrier RNA, Includes nucleic acid adsorbents such as carrier DNA, solvents such as DMSO and pluronic (registered trademark) F-127 (Sigma Aldrich), inhibitors such as RNase inhibitor, proteases such as protease K, nucleases such as DNase and RNase, etc. But you can.
  • the pH of the separation liquid is, for example, pH 6-9.
  • the separation channel is in the channel and has a wall in the cross-sectional direction.
  • the position of the wall is not particularly limited, and examples thereof include the first opening end, the second opening end, and other positions.
  • the wall is provided at the end of the first opening, for example, it is not necessary to introduce the target cell into the separation channel, so that the size of the separation channel can be freely set.
  • the wall is of the other positions, the position of the wall, the ratio of the length of said upstream channel and (l u), the length of the downstream flow path (l d) (l u: l d) Is a position in the range of 1:10 to 1: 500, for example.
  • the ratio (l u: l d) are, for example, because it can separate the nucleic acid of higher purity the cytoplasm of, preferably, 1: 100-200 The position is in the range of 1: 500.
  • the length (thickness, l w ) of the wall in the axial direction of the separation channel is not particularly limited.
  • the wall thickness is, for example, 1 to 20 ⁇ m. By shortening the thickness of the wall, for example, the cell membrane of the target cell can be crushed at a lower voltage when the cell membrane of the target cell is electrically destroyed.
  • the wall has an opening (orifice).
  • the upstream flow channel and the downstream flow channel are communicated with each other through an opening of the wall.
  • the number of openings in the wall is 1 or more, and is preferably 2 or more, more preferably 2 to 3 because the remaining biopolymer can be collected more easily.
  • the shape of the opening of the wall is not particularly limited, and the shape of the cross section may be a circle such as a circle, a perfect circle or an ellipse; a semicircle; a polygon such as a triangle, a rectangle, a square or a rectangle.
  • each opening may have, for example, the same cross-sectional shape or different cross-sectional shapes.
  • the size of the opening in the wall is not particularly limited as long as the target cell can be trapped.
  • the diameter (w 1 ) of the opening of the wall is preferably smaller than the diameter (w t ) of the target cell because, for example, the target cell can be trapped more accurately.
  • the ratio (w 1 : w t ) of the diameter (w 1 ) of the wall opening to the diameter (w t ) of the target cell is 1: 2 or more, The range is 1:50, 1:10 to 1:50.
  • Diameter of the target cells (w t) is, for example, a minor axis of said target cells.
  • the diameter (w 1 ) of the opening of the wall is, for example, the short diameter of the opening of the wall, and when the sectional shape of the opening of the wall is other than a circle, for example, in the section of the opening of the wall , The shortest distance.
  • the diameter (w 1 ) of the wall opening is preferably a target cell after separation of the cytoplasmic biopolymer. Smaller than the diameter (w n ) of the remaining portion (target cell from which the cytoplasmic biopolymer has been separated, hereinafter also referred to as “remaining portion of the target cell”).
  • the diameter (w n ) of the remaining portion of the target cell is, for example, the short diameter of the remaining portion of the target cell.
  • the diameter (w 1 ) of the opening of the wall is, for example, the short diameter of the opening of the wall, and when the sectional shape of the opening of the wall is other than a circle, for example, in the section of the opening of the wall , The shortest distance.
  • the diameter (w 1 ) of the opening of the wall preferably satisfies the following formula (2). If the cross-sectional shape of the opening of the wall is circular, the diameter of the opening of the wall (w 1) preferably satisfies the following formula (3).
  • the diameter (w 1 ) of the opening of the wall satisfies the following formula (2) or (3), for example, the cell membrane of the target cell is destroyed in a state where the target cell is trapped in the opening of the wall And the destruction of the nuclear membrane of the target cell can be suppressed, so that the cytoplasmic biopolymer with higher purity can be separated.
  • Sectional area of the opening of the wall (S 1) for example, electrically by lower voltage when destroying the cell membrane of target cells, wherein since the cell membrane of the target cell can be disrupted, cross-sectional area of the opening of the wall (S 1 ) and the sectional area (S 2 ) of the separation channel (S 1 : S 2 ) are preferably 1: 2 or more, in the range of 1: 2 to 1: 100, 1: 3 to 1 : 100 range, 1: 10-1: 100 range, more preferably, 1: 3-1: 100 range, 1: 10-1: 100 range.
  • the total cross-sectional area of the two or more openings preferably satisfies the above ratio.
  • the minimum cross-sectional area in the opening of the wall satisfies the above ratio.
  • the cross-sectional area of the cross-sectional area of the separation channel (S 2) is changed, the in separation channel, the maximum cross-sectional area, it is preferable to satisfy the above ratio.
  • the size of each opening may be the same or different.
  • the target cells can be trapped with higher accuracy and the remaining biopolymer can be more easily collected. Therefore, all the openings have the size of the opening of the wall. It is preferable to satisfy the conditions.
  • the opening having the largest cross-sectional area traps the target cell, for example, and the other openings allow the sample to pass through. For this reason, the opening having the maximum cross-sectional area can also be called a trap port, and the other openings can also be called bypass ports.
  • the wall comprises said trap inlet and said bypass port
  • the ratio of the diameter of the trap opening diameter (w c) and the bypass port (w b) (w c: w b) for example, 0
  • the range is from 1: 1 to 3: 1.
  • the ratio (S t : S b ) of the cross-sectional area (S t ) of the trap port and the cross-sectional area (S b ) of the bypass port is, for example, in the range of 0.1: 1 to 3: 1. .
  • the size of the opening of the wall may be the same or different from the upstream channel side to the downstream channel side of the wall opening, for example.
  • the opening of the wall is, for example, an opening that is tapered from the upstream flow path side to the downstream flow path side of the opening, from the upstream flow path side of the opening and the downstream flow path side of the opening.
  • the opening etc. which are taper-shaped are mentioned to the center of this.
  • the size of the trap port is different from the upstream channel side to the downstream channel side of the trap port, and the size of the bypass port is the same. Preferably there is.
  • the first opening is an opening into which a sample containing the target cell can be introduced.
  • the first opening portion traps the target cell on the upstream flow path side of the opening of the wall, and separates the cytoplasmic biopolymer into the downstream flow path.
  • the first opening can also be referred to as an opening capable of recovering the remaining biopolymer.
  • the first opening traps the target cell on the downstream channel side of the opening of the wall and separates the cytoplasmic biopolymer into the upstream channel, After separation, the cytoplasmic biopolymer can be recovered by aspiration from the first opening.
  • the first opening can also be referred to as an opening capable of recovering the cytoplasmic biopolymer, for example.
  • the first opening may be used to introduce or lead the separation liquid into the separation channel. It is preferable that the first opening communicates with an end of the separation channel, for example.
  • the chip of the present invention may further have a second opening.
  • the second opening is an opening capable of recovering the cytoplasmic biopolymer.
  • the first opening is an opening capable of recovering the cytoplasmic biopolymer
  • the second opening may be replaced with the remaining biopolymer instead of the cytoplasmic biopolymer, for example.
  • recovered may be sufficient. With such an opening, when the target cell is trapped on the downstream flow path side of the opening of the wall and the cytoplasmic biopolymer is separated into the upstream flow path, after the separation, The remaining biopolymer can be recovered by aspiration from the opening.
  • the second opening is in communication with the separation channel, for example.
  • the wall is disposed between the first opening and the second opening in the separation channel.
  • the second opening may be used, for example, to introduce or lead the separation liquid or the like into the separation channel. It is preferable that the second opening is communicated with, for example, an end of the separation channel.
  • the shape of the first opening is not particularly limited as long as the sample containing the target cell can be introduced.
  • the shape of the second opening is not particularly limited as long as the cytoplasmic biopolymer can be recovered.
  • Examples of the shapes of the first opening and the second opening include a polygonal column shape such as a triangular column shape and a quadrangular column shape, a cylindrical shape such as a true column shape and an elliptic column shape, and a cone shape.
  • the first opening and the second opening are preferably tapered from the outer surface to the inner surface of the substrate, and more preferably in an inverted conical shape.
  • the remaining biopolymer is removed from the first opening by using a suction means such as a micromanipulator or a micropipette. Can be easily recovered.
  • a suction means such as a micromanipulator or a micropipette.
  • the cytoplasmic biopolymer can be easily recovered using the suction means after separating the cytoplasmic biopolymer.
  • the first opening and the second opening may have the same shape or different shapes, for example.
  • the opening can store, for example, a separation liquid.
  • the said opening part can also be called a reservoir, for example.
  • the size of the first opening is not particularly limited as long as the sample can be introduced.
  • the size of the second opening is not particularly limited.
  • each of the first opening and the second opening has an outer surface diameter of 3 to 10 mm, for example, and the inner surface diameter of the substrate is 0, for example. 0.01-0.2 mm, the height is, for example, 5-25 mm, and the volume is, for example, 5-500 ⁇ L, 5-60 ⁇ L.
  • the chip of the present invention may further include a suction / discharge section (means) capable of sucking the liquid in the separation channel and / or discharging the liquid into the separation channel.
  • the chip of the present invention can control the movement of the liquid in the separation channel, for example, by having the suction / discharge section, for example, from the upstream channel to the downstream channel direction or from the downstream channel The liquid in the separation channel can be moved in the upstream channel direction.
  • the suction / discharge section is not particularly limited, and for example, known suction means, discharge means, and the like can be used. Specific examples include a micropump, a pump, a means using volume change of a separation channel, and a liquid by surface tension. Means for drawing in, means for using a pressure difference, and the like.
  • the chip of the present invention may have, for example, one suction discharge unit or two or more.
  • the means using the change in volume of the separation channel include a flexible substrate that forms the outer wall of the separation channel. In this case, the suction / discharge section is pressed from the outside of the substrate toward the inside of the substrate, thereby discharging the liquid in the separation channel and being released from the press, thereby separating the separation Liquid is sucked into the flow path.
  • Examples of the means using the pressure difference include a negative pressure chamber or a positive pressure chamber arranged in the separation channel. In the negative pressure chamber, for example, the pressure in the chamber is lower (for example, vacuum) than the other part of the separation channel.
  • the positive pressure chamber has a higher pressure in the chamber than, for example, other portions of the separation channel. For this reason, by destroying the wall of the positive pressure chamber, the liquid moves from the negative pressure chamber toward the other part of the separation channel, for example, the first opening. Can be discharged.
  • the suction / discharge section is arranged so as to suck the liquid in the separation channel and / or discharge the liquid into the separation channel, and specifically, the separation channel. It arrange
  • the suction / discharge section may be disposed in the upstream flow path, may be disposed in the downstream flow path, or may be disposed in both.
  • the chip of the present invention includes two or more suction / discharge sections
  • the two or more suction / discharge sections may be disposed in one of the upstream flow path and the downstream flow path, or may be disposed in both. Good.
  • the chip of the present invention may further have a capture section for the biopolymer.
  • the chip of the present invention can capture the remaining biopolymer in the upstream channel and the cytoplasmic polymer in the downstream channel, for example, by disposing the capture unit in the separation channel. Therefore, the separated biopolymer can be easily recovered.
  • the capture unit is not particularly limited, and for example, a known biopolymer adsorption means can be used, for example, a filter that specifically and / or non-specifically adsorbs the biopolymer, and a specific biopolymer.
  • the chip of the present invention may have, for example, one capture unit or two or more.
  • the capture unit is disposed in the separation channel so as to capture the biopolymer, and specifically, introduced into the separation channel in the separation channel.
  • the liquid is arranged so as to be in contact with the liquid.
  • the capture unit may be disposed in the upstream flow channel, may be disposed in the downstream flow channel, or may be disposed in both.
  • tip of this invention contains two or more capture parts, two or more capture parts may be arrange
  • the capture unit is preferably arranged in combination with the suction / discharge unit.
  • the chip of the present invention can capture the biopolymer more efficiently and more easily collect the separated biopolymer by combining the capture unit and the suction / discharge unit. it can.
  • the capturing unit may be combined with one suction / discharge unit, or may be combined with two or more suction / discharge units.
  • the capture unit may be configured such that the first opening or the wall is used as a reference between the wall and the connection portion of the suction / discharge unit. It arrange
  • the capture section may be disposed, for example, between the connection sections of the respective suction / discharge sections.
  • the capture unit and the suction / discharge unit are preferably disposed in the downstream flow path.
  • the chip having the second opening may have at least one of the suction / discharge section and the capture section.
  • the suction discharge section and the capture section for example, “end portion in the direction opposite to the first opening portion with respect to the wall” is read as “second opening portion”, and Explanation can be used.
  • the chip of the present invention When the chip of the present invention has the second opening, the chip of the present invention preferably has a liquid movement control unit that controls the movement of the liquid in the separation channel.
  • the liquid movement control unit may control the movement of the liquid between the first opening and the wall, or the movement of the liquid between the wall and the second opening. You may control.
  • the liquid movement control unit may control the liquid so that the liquid can move in one direction.
  • the liquid movement control unit may be able to control the movement of the liquid from the wall toward the second opening.
  • the movement of the liquid from the wall toward the first opening may be controllable.
  • the movement of the liquid from the second opening toward the wall may be suppressed or stopped.
  • the movement of the liquid from the first opening toward the wall may be suppressed or stopped.
  • the liquid movement control unit may be capable of controlling the movement of the liquid in both directions. Specifically, the liquid movement from the second opening to the wall and the wall from the first opening to the wall. The movement of the liquid in the direction may be suppressed or stopped. Thereby, for example, when the liquid movement control unit is arranged in the downstream flow path, the cytoplasmic biopolymer is separated from the liquid movement control unit to the second opening side in the separation flow path.
  • the liquid movement control unit is not particularly limited, and examples thereof include known valves such as valves and microvalves, and walls of a separation channel formed by a flexible substrate.
  • the liquid movement control unit is a wall of a separation channel formed by the flexible substrate, when the liquid movement control unit is pressed from the outside of the substrate toward the inside of the substrate, the separation channel is narrowed, The movement of the liquid in the separation channel can be suppressed or stopped.
  • the wall of the separation channel is formed by the flexible substrate, the whole or part of the wall of the separation channel is formed by the flexible substrate.
  • the chip of the present invention may have, for example, one liquid movement control unit or two or more.
  • the liquid movement control unit may be disposed in the upstream channel, may be disposed in the downstream channel, or may be disposed in both. It is preferable to arrange
  • the chip of the present invention may have, for example, a bypass channel that communicates the upstream channel and the downstream channel.
  • the target cell can be introduced into the downstream channel via the bypass channel, and the cytoplasmic biopolymer can be separated to the upstream channel side. Since the cytoplasmic biopolymer is present in the upstream flow path and the remaining biopolymer is trapped in the opening of the wall, the cytoplasmic biopolymer is recovered from the first opening. In this case, the remaining biopolymer can be collected before the remaining biopolymer, and contamination of the remaining biopolymer can be reduced. Therefore, the cytoplasmic biopolymer with higher purity can be prepared.
  • the bypass channel only needs to communicate with the upstream channel and the downstream channel, and the position thereof is not particularly limited.
  • the size of the bypass channel is not particularly limited, and for example, description of the size of the separation channel can be cited.
  • the chip of the present invention When the chip of the present invention has the bypass flow path, the chip preferably further includes a second liquid movement control unit that controls the movement of the liquid in the bypass flow path.
  • the second liquid movement control unit controls, for example, ON / OFF of liquid movement via the bypass flow path.
  • Examples of the second liquid movement control unit include known valves such as the microvalve.
  • the number of the second liquid movement control units is not particularly limited, and may be one, for example, or two or more.
  • the second liquid movement control unit is disposed in the bypass channel, for example.
  • the location of the second liquid movement control unit in the bypass channel is not particularly limited and can be any location.
  • the second liquid movement control unit includes an adjacent part of a connection part between the bypass channel and the upstream channel, and the bypass channel. It is preferable to arrange in the adjacent part of the connection part with the downstream flow path. By arranging in this way, ON / OFF of the movement of the liquid in the bypass channel can be controlled with higher accuracy.
  • the chip of the present invention may further include an adjustment channel for adjusting the movement of the target cell.
  • the chip of the present invention has a third opening, the third opening and the separation channel are communicated with the adjustment channel, and the separation channel is It is preferable that the downstream flow path (for example, the second opening side from the wall of the separation flow path) communicates with the adjustment flow path.
  • the chip can adjust the movement of the target cells in the chip, for example, and can prevent the target cells from detaching from the opening of the wall, for example.
  • the chip replaces the solution in the chip by, for example, introducing the separation liquid into the adjustment channel after trapping the target cells. Can do. For this reason, according to the chip having the adjustment channel, for example, the target cells can be washed, labeled, and biopolymers derived from cells other than the target cells can be removed.
  • the shape of the adjustment channel is not particularly limited, and for example, description of the cross-sectional shape of the separation channel can be cited.
  • the shape of the adjustment channel and the separation channel may be the same or different.
  • the size (for example, width, depth, diameter, cross-sectional area, etc.) of the adjustment channel is not particularly limited, and may be any size as long as the target cell is movable, for example, the target cell. It can be determined as appropriate according to the size.
  • the adjustment channel communicates with the second opening, that is, the downstream channel from the wall of the separation channel, for example.
  • the adjustment channel may be in communication with the downstream channel so that the movement of the target cell can be adjusted, for example.
  • the adjustment channel communicates in the vicinity of the wall of the downstream channel, for example.
  • the third opening is, for example, an opening used to introduce or lead the separation liquid into the adjustment channel.
  • the shape of the third opening is not particularly limited, and for example, description of the shapes of the first opening and the second opening can be cited.
  • the chip of the present invention further has a connection channel that communicates the separation channel and the adjustment channel.
  • the connection channel communicates with the separation channel on the first opening side from the wall of the separation channel.
  • the chip having the connection flow path can separate one target cell from a plurality of target cells.
  • the chip having the connection channel can also be referred to as a chip for separating one target cell, for example.
  • the chip can replace the solution in the chip by, for example, introducing the separation liquid or the like into the separation channel after trapping the target cell. it can.
  • the target cell can be washed, labeled, and the biopolymer derived from cells other than the target cell can be removed.
  • connection channel is not particularly limited, and for example, description of the cross-sectional shape of the separation channel can be cited.
  • the shapes of the connection channel and the separation channel may be the same or different.
  • the size (for example, width, depth, diameter, cross-sectional area, etc.) of the connection channel is not particularly limited, and may be any size as long as the target cell can move, for example, the size of the target cell. It can be determined appropriately according to Since the cross-sectional area and the length of the connection channel can trap, for example, one target cell in the opening of the wall with higher accuracy, the wall opening is not trapped in the target cell.
  • the ratio (F 1 : F c ) of the flow rate (F 1 ) of the sample flowing through the opening of the sample and the flow rate (F c ) of the sample flowing through the connection channel is preferably 1: 1 to 20: 1.
  • the range is 2: 1 to 20: 1 because one target cell can be trapped more accurately in the opening of the wall and the remaining biopolymer can be more easily recovered.
  • the ratio of the flow rate of the sample flowing through the opening of the wall and the flow rate of the sample flowing through the connection channel is, for example, from the cross-sectional area and length of the opening and the cross-sectional area and length of the connection channel. For example, it can be approximated by the following equation (1).
  • F 1 / F c (S 1 / l 1 ) / (S c / l c ) (1)
  • F 1 Flow rate of the sample flowing through the wall opening (m 3 / sec)
  • F c flow rate of the sample flowing through the connection channel (m 3 / sec)
  • S 1 sectional area of the opening (m 2 ) l 1 : length of opening (length of wall) (m)
  • S c cross-sectional area of connection channel (m 2 ) l c : length of connection channel (m)
  • connection flow path communicates with the first opening side, that is, the upstream flow path from the wall of the separation flow path, for example.
  • the connection channel may be in communication with the upstream channel so that, for example, cells that are not trapped in the opening of the wall can be moved to the adjustment channel via the connection channel.
  • the connection channel communicates, for example, in the vicinity of the wall of the upstream channel, and more specifically, from the wall of the upstream channel, approximately the same as the diameter of the target cell. Communication is performed at a distant position (for example, 10 to 30 ⁇ m).
  • connection flow path communicates with the adjustment flow path at an arbitrary position of the adjustment flow path, for example.
  • connection flow path is communicated at a position that is approximately the same as the diameter of the adjustment flow path from a communication portion between the downstream flow path and the adjustment flow path.
  • the chip of the present invention includes the adjustment flow path and the connection flow path
  • the chip includes, for example, a plurality of flow path groups of the separation flow path, the adjustment flow path, and the connection flow path.
  • the channel group may be the continuously connected chips.
  • the chip is, for example, a chip in which the third opening of the adjustment channel of one channel group also serves as the first opening of the separation channel of another channel group. In this case, for example, the chip omits the opening serving as the first opening and the third opening, and directly communicates the adjustment channel and the separation channel. Also good.
  • the chip for example, when a plurality of target cells are introduced, each has one target at the opening of the separation flow channel wall in each flow channel group. Can trap cells. For this reason, according to the chip including the plurality of flow channel groups, even if a plurality of target cells are introduced, the number of target cells that are not subjected to biopolymer separation can be reduced.
  • the chip itself may include an electrode, or a device for setting the chip may include an electrode.
  • the analysis chip of the present invention may further include an electrode system, for example.
  • the electrode system has one or more electrodes.
  • the electrode system may include, for example, one electrode or two or more electrodes.
  • the arrangement position of the electrode is not particularly limited, and examples thereof include the first opening, the upstream flow path, and the downstream flow path.
  • the electrodes may be arranged at one place or at two or more places.
  • the electrode may be disposed in the second opening.
  • the plurality of electrodes are arranged so as to be located in the first opening and the second opening, respectively.
  • a part of the electrode of the first opening may be disposed in the upstream flow path.
  • a part of the electrode of the second opening may be disposed in the downstream flow path, for example.
  • the electrode is preferably, for example, a solid electrode that can be inserted into the chip, and specific examples include a line electrode and a rod electrode.
  • the electrode is, for example, one of the first opening, the upstream flow path, and the downstream flow path that is provided in the chip itself, and the other electrode sets the chip. It is good also as an electrode with which the apparatus to perform.
  • the electrode includes, for example, one of the electrode in the first opening and the electrode of the second opening included in the chip itself.
  • the other electrode may be an electrode provided in a device for setting the chip.
  • the electrode is fixed to an inner wall of the first opening, for example.
  • the electrode is fixed to an inner wall of the upstream flow path or the downstream flow path, for example.
  • the said electrode is being fixed to the inner wall of the edge part in the reverse direction to the said wall in the said downstream flow path.
  • the electrode is fixed to an inner wall of the second opening, for example.
  • the material of the electrode is not particularly limited as long as it is a solid conductive material, such as platinum, gold, carbon, zinc, brass, copper, stainless steel, iron, silver / silver chloride, palladium, platinum black, and the like. It is done.
  • the plurality of electrodes may be further disposed in at least one of the third opening and the adjustment channel.
  • the plurality of electrodes may be further disposed in at least one of the third opening and the adjustment channel.
  • the electrode of the third opening may be controlled, and the amount of cytoplasmic biopolymer recovered can be improved.
  • a part of the electrode of the third opening may be disposed in the adjustment channel.
  • the third electrode may be included in the chip itself, or a device for setting the chip may include an electrode. In the former case, it is preferable that the electrode is fixed to the inner wall of the third opening.
  • the number of the separation channels disposed on the chip substrate is not particularly limited, and may be, for example, one or two or more. In the latter case, the number of separation channels is, for example, 4 to 400, more specifically 4, 6, 8, 16, 48, 96, 384, and the like.
  • the sample may include the target cell.
  • the sample include a cell fluid containing the target cells, a cell fluid containing isolated target cells, and the like.
  • the sample may include, for example, one target cell or two or more target cells.
  • the sample preferably includes one target cell.
  • the volume of the sample is not particularly limited and is, for example, 0.1 to 5 ⁇ L.
  • the cytoplasmic biopolymer is not particularly limited as long as it is a biopolymer present in the cytoplasm.
  • Specific examples of the cytoplasmic biopolymer include a cytosolic biopolymer and a cell organelle biopolymer.
  • Examples of the biopolymer of the organelle include a chloroplast biopolymer, a mitochondrial biopolymer, a liposome biopolymer, a vesicle biopolymer, an endosome biopolymer, and a Golgi apparatus.
  • the cytoplasmic biopolymer fractionated in the chip of the present invention may be, for example, one type or two or more types.
  • the cytoplasmic biopolymer fractionated in the chip of the present invention may be a biopolymer composed of the cytoplasmic biopolymer or a biopolymer containing the cytoplasmic biopolymer. In the latter case, the cytoplasmic biopolymer may include, for example, nuclear RNA.
  • a desired cytoplasmic biopolymer By adjusting the voltage applied to the chip, for example, a desired cytoplasmic biopolymer can be fractionated.
  • the biopolymer means, for example, a macromolecular organic compound present in the body, and specific examples thereof include nucleic acids, sugars (polysaccharides), proteins, lipids and the like.
  • the type of the nucleic acid is not particularly limited, and may be, for example, DNA or RNA.
  • the lipid include the cell membrane.
  • the material of the chip of the present invention is not particularly limited.
  • an inner wall of the chip is formed of an insulating material except for an electrode, and more preferably, the entire chip is insulating except for an electrode. It is preferably formed from a material.
  • the chip manufacturing method of the present invention is not particularly limited, and for example, a molded body having the flow path may be manufactured by injection molding or the like, or the flow path or the like is formed on a substrate such as a plate. Also good.
  • a method for forming the flow path and the like is not particularly limited, and examples thereof include lithography and cutting.
  • the insulating material is not particularly limited, and examples thereof include resin, silicone, glass, ceramics, and rubber.
  • the resin include polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyethylene terephthalate, polymethacrylate, polyamide, saturated polyester resin, thermoplastic resin such as acrylic resin, urea resin, melamine resin, phenol resin, fluororesin glass epoxy, etc. And thermosetting resins such as epoxy resins and unsaturated polyester resins.
  • the silicone include polydimethylsiloxane.
  • the biopolymer fractionation device of the present invention includes the biopolymer fractionation chip of the present invention.
  • the fractionation device of the present invention is characterized by including the chip of the present invention, and other configurations and conditions are not particularly limited.
  • a biopolymer can be fractionated even in a liquid system that does not contain a molecule having the molecular sieve function.
  • the description of the chip of the present invention can be cited for the fractionation apparatus of the present invention.
  • the fractionation device of the present invention preferably includes voltage application means.
  • the voltage application means is not particularly limited, and may be any voltage as long as a voltage can be applied to the electrode system of the chip, and a voltage device or the like can be used as a known means.
  • the fractionation device of the present invention preferably further includes an electrode system.
  • an electrode system For example, the above description can be used for the arrangement and materials of the electrode system.
  • the fractionation method of the present invention is characterized in that the cytoplasmic biopolymer is fractionated using the wall having the opening, and other configurations and conditions are not particularly limited.
  • a cytoplasmic biopolymer can be fractionated from one target cell with high accuracy.
  • the description of the chip and fractionation apparatus of the present invention can be cited.
  • the number of the opening of the wall, the diameter of the opening of the wall, the diameter of the target cell and the diameter of the remainder of the target cell after separation of the cytoplasmic biopolymer are as follows: The description of the chip of the present invention can be cited.
  • the trapping step is a step of trapping the target cell in the opening of the wall having the opening.
  • the method for trapping the target cell in the opening of the wall is not particularly limited, and the flow is directed from one of the openings in the wall to the other (for example, the downstream end of the downstream flow path or the second opening). And a method for trapping the target cells by the flow.
  • the flow can be generated using, for example, osmotic pressure difference, electroosmotic flow, dielectrophoresis, capillary action and the like.
  • the releasing step is a step of releasing a cytoplasmic biopolymer from the target cell by destroying a cell membrane of the target cell.
  • the method for destroying the cell membrane of the target cell is not particularly limited, and can be performed by a known cell membrane destruction method. Specific examples include an electrical destruction method, a chemical destruction method, a heat destruction method, and cooling.
  • the destruction method using a sound wave, the destruction method using a sound wave or an ultrasonic wave, the destruction method using a laser, the mechanical destruction method by a flow or pressing, etc. are mention
  • Examples of the electrical destruction method include a method in which a pair of electrodes are arranged so as to sandwich the opening of the wall and a voltage is applied to the pair of electrodes for destruction.
  • the description of the voltage in the description of the discharge step and the separation step in the fractionation method using the chip of the present invention described later can be used.
  • the chemical destruction method include a method of destroying by bringing a surfactant and the target cell into contact with each other, a method using osmotic pressure, and the like.
  • the separation step is a step of separating the released cytoplasmic biopolymer.
  • the released cytoplasmic biopolymer is separated from the remainder of the target cell after separation of the cytoplasmic biopolymer, that is, the remaining biopolymer.
  • the method for separating the cytoplasmic biopolymer is not particularly limited. For example, in the state where the remaining biopolymer is trapped in the opening of the wall, the cytoplasmic biopolymer is removed from the remaining biopolymer.
  • the method can be separated, and specific examples include a known electrical separation method such as electrophoresis. Examples of the electrical separation method include a method of arranging a pair of electrodes so as to sandwich the opening of the wall and applying a voltage to the pair of electrodes.
  • the description of the voltage in the description of the discharge step and the separation step in the fractionation method using the chip of the present invention described later can be used.
  • the method for separating the cytoplasmic biopolymer for example, a flow around the wall, specifically, from one side of the wall to the other is generated, and the cytoplasmic biopolymer is separated by this flow. How to do.
  • the order of the trapping step and the discharging step is not particularly limited.
  • the discharging step may be performed after the trapping step, or the trapping step may be performed after the discharging step.
  • Good In the discharging step, when the cell membrane of the target cell is destroyed by the electrical destruction method, the current density around the opening of the wall where the target cell is trapped can be increased, and the wall having the opening Since the cell membrane of the target cell can be destroyed at a lower voltage compared to a separation channel (for example, the separation channel disclosed in Patent Document 1) that does not have a cell, the release step is performed after the trap step. It is preferable to do.
  • the fractionation method of the present invention can be carried out, for example, using the chip of the present invention.
  • the fractionation method of the present invention includes, for example, an introducing step of introducing a sample containing the target cell from the first opening, a trapping step of trapping the target cell in the opening of the wall, the target cell And a separation step of separating the cytoplasmic biopolymer of the target cell into the separation channel in a direction opposite to the side on which the target cell is trapped with respect to the wall. including.
  • the separation step for example, the cytoplasmic biopolymer is separated into the downstream channel.
  • the separation step for example, the cytoplasmic biopolymer is separated into the upstream channel.
  • the discharge step and the separation step are performed by trapping on the upstream channel side of the opening of the wall and applying a voltage to the separation channel will be described as an example. This is not a limitation.
  • the fractionation method of the present invention may further include a step of supplying the separation liquid to the separation channel.
  • the separation liquid for example, the above description can be used for the separation liquid.
  • the introduction step is a step of introducing a sample containing the target cells from the first opening.
  • the method for introducing the sample is not particularly limited, and for example, a known dispensing means can be used.
  • the above description can be used for the volume of the sample to be introduced, for example.
  • the trapping step is a step of trapping the target cell at the opening of the wall.
  • the target cell is, for example, in the separation channel by the flow of the sample or the separation liquid. Is moved in the direction of the wall and trapped in the opening of the wall.
  • the target cell is, for example, from the first opening to the downstream channel direction ( For example, by generating an electroosmotic flow in the direction of the second opening), the separation channel is moved in the direction of the wall and trapped in the opening of the wall.
  • the electroosmotic flow applies a voltage to, for example, the electrode of the first opening and the electrode on the downstream channel side (for example, the electrode of the downstream channel, the electrode of the second opening, etc.). Caused by.
  • the chip has the third opening, for example, by the flow of the sample or the separation liquid from the first opening to the third opening, the electroosmotic flow, or the like,
  • the target cell may move in the direction of the wall in the separation channel and be trapped in the opening of the wall.
  • the releasing step is a step of releasing the biopolymer from the target cell, for example, by applying a voltage to the separation channel.
  • the separation step is a step of separating the cytoplasmic biopolymer of the target cell, for example, on the downstream flow channel side (for example, the second opening side).
  • a voltage is applied to the electrode of the first opening and the electrode on the downstream channel side (for example, the electrode of the downstream channel, the electrode of the second opening, etc.) Can be implemented.
  • the separation step can be performed by applying a voltage to the electrode of the first opening and the electrode on the downstream flow path side.
  • the voltage application to the separation channel can be performed by, for example, a voltage application unit.
  • the above-mentioned explanation can be used for the voltage application means, for example.
  • the voltage applied to the electrode of the first opening and the electrode on the downstream flow path side is not particularly limited as long as it is a voltage that can disrupt the cell membrane of the target cell.
  • ) of the difference between the voltage (V 1 ) of the electrode of the first opening and the voltage (V 2 ) of the electrode on the downstream channel side is, for example, , 50 to 1000V.
  • the combination of V 1 and V 2 is not particularly limited.
  • V 1 is, for example, ⁇ 50 to ⁇ 1000 V
  • V 2 is For example, it is 50 to 1000V.
  • a voltage may be applied to the electrode of the third opening.
  • the voltage of the electrode of the third opening is, for example, that the anion in the liquid in the adjustment channel flows from the separation channel into the adjustment channel by flowing into the separation channel. As long as the outflow of anions to the can be suppressed, it can be set as appropriate.
  • the voltage of the electrode of the first opening and the voltage of the electrode on the downstream flow path side are in the range of the specific example, the voltage of the electrode of the third opening can be set to ⁇ 50 to ⁇ 1000 V, for example.
  • the voltage application time to the electrode is, for example, a time for the current value after the voltage application to reach an equilibrium state, and can be set to 10 to 500 seconds as a specific example.
  • the fractionation method of the present invention preferably further includes a step of purifying the cytoplasmic biopolymer.
  • the purification step for example, any one or two or more of the biopolymers contained in the cytoplasmic biopolymer are purified.
  • the fractionation method of the present invention can separate the released protein and the cytoplasmic nucleic acid, for example, by disrupting the cell membrane of the target cell. For example, the cytoplasm having higher purity can be separated. Can be fractionated.
  • the method for purifying the cytoplasmic nucleic acid is not particularly limited, and a known fractionation method of protein and nucleic acid can be used. For example, it can be performed by isotachophoresis or the like.
  • the fractionation method of the present invention may further transport the separated cytoplasmic biopolymer to a predetermined position in the separation step.
  • the predetermined position include a first opening, an upstream flow channel, a downstream flow channel, and a second opening in the chip of the present invention.
  • the transport can be performed, for example, in the same manner as the separation method.
  • the fractionation method of the present invention may further include, for example, a recovery step of recovering at least one of the cytoplasmic biopolymer and the remaining biopolymer.
  • the cytoplasmic biopolymer and the remaining biopolymer can be collected using, for example, a suction means such as a micromanipulator or a micropipette.
  • the fractionation method of the present invention uses the chip of the present invention to separate the cytoplasmic biopolymer to the downstream flow channel side, the cytoplasmic biopolymer and the remaining biopolymer are, for example, the first It can collect
  • the cytoplasmic biopolymer and the remaining biopolymer are, for example, the second opening and the first opening, respectively. Can be recovered from. In the fractionation method of the present invention, for example, only the remaining biopolymer may be recovered. The remaining biopolymer can be recovered from the first opening using, for example, the suction means.
  • the chip of the present invention includes the capture unit, for example, by collecting the capture unit, at least one of the cytoplasmic biopolymer captured by the capture unit and the remaining biopolymer Biopolymers can be recovered.
  • the fractionation method of the present invention may include, for example, an amplification step of amplifying at least one of the separated cytoplasmic nucleic acid and the remaining nucleic acid.
  • the fractionation method of the present invention may include a reverse transcription step of synthesizing cDNA from the RNA prior to the amplification step.
  • the fractionation method of the present invention can prepare a nucleic acid sample for use in the nucleic acid analysis method from the separated cytoplasmic nucleic acid and other nucleic acids.
  • the nucleic acid sample can also be referred to as, for example, a cDNA library.
  • the amplification and reverse transcription of the nucleic acid can be performed, for example, by a known nucleic acid amplification method and nucleic acid reverse transcription method.
  • the fractionation method of the present invention further includes a holding step of retaining the fractionated state of the biopolymer contained in the fractionated cytoplasmic biopolymer and the remainder of the target cell after separation of the cytoplasmic biopolymer. But you can. Thereby, it is possible to prevent the fractionated cytoplasmic biopolymer and the remaining biopolymer from being mixed again.
  • the maintenance of the fractionated state is, for example, at least of the cytoplasmic biopolymer and the remaining biopolymer to such an extent that re-mixing of the fractionated cytoplasmic biopolymer and the remaining biopolymer can be prevented. It means to suppress the movement of one biopolymer.
  • the fractionation state can be maintained, for example, by gelling or solidifying the liquid between the fractionated cytoplasmic biopolymer and the remaining biopolymer.
  • the fractionated responsive gelling agent such as PEG-DA (Poly (ethylene glycol) diacrylate), Pluronic (registered trademark) F127, gelatin methacrylate is separated.
  • the fractional state can be maintained by mixing or substituting the liquid between the cytoplasmic biopolymer and the remaining biopolymer and applying a stimulus.
  • the chip of the present invention having the liquid movement control unit is used and the fractionation method of the present invention is performed, the fractionation state can be maintained, for example, by the liquid movement control unit.
  • the liquid movement control unit is used as the liquid movement control unit.
  • the cytoplasmic biopolymer is transported downstream from the liquid movement control unit in the separation step. Thereby, the fractionated state of the biopolymer contained in the fractionated cytoplasmic biopolymer and the remainder of the target cell after separation of the cytoplasmic biopolymer can be maintained.
  • the method for analyzing a biopolymer of the present invention includes a fractionation step of fractionating a cytoplasmic biopolymer from a target cell, and after fractionation of the cytoplasmic biopolymer and the cytoplasmic biopolymer.
  • the analysis method of the present invention is characterized in that the fractionation step is performed by the fractionation method of the present invention, and other steps and conditions are not particularly limited.
  • a cytoplasmic biopolymer can be accurately fractionated from one target cell, and the cytoplasmic biopolymer and the remaining biopolymer can be analyzed.
  • the description of the fractionation method of the present invention can be used for the analysis method of the present invention.
  • the analysis includes, for example, any meaning of qualitative analysis and quantitative analysis.
  • the analysis method of the present invention may further include a step of preparing a sample containing the target cells.
  • the method for preparing the sample is not particularly limited, and can be appropriately determined according to the type of the target cell.
  • the sample can be prepared, for example, by separating a desired single target cell using a flow cytometer.
  • the said sample can be prepared by isolate
  • fractionation step for example, the description of the biopolymer fractionation method can be cited.
  • the analysis step at least one of the cytoplasmic biopolymer and the biopolymer contained in the remainder of the target cell after fractionation (separation) of the cytoplasmic biopolymer is analyzed.
  • the analysis step for example, either the cytoplasmic biopolymer or the remaining biopolymer may be analyzed, or both may be analyzed.
  • the remaining biopolymer is, for example, a biopolymer other than the biopolymer fractionated in the fractionation step, and specifically includes a nuclear biopolymer.
  • the fractionation step when a part of the nucleic acid of the cytoplasmic biopolymer is fractionated, the remaining biopolymer may contain, for example, another cytoplasmic biopolymer that has not been fractionated.
  • the method of analyzing the cytoplasmic biopolymer and the remaining biopolymer is not particularly limited, and can be appropriately determined according to the analysis target and purpose.
  • the biopolymer is a nucleic acid and the presence / absence of the nucleic acid to be analyzed is analyzed
  • a probe that hybridizes to the nucleic acid to be analyzed can be used and analyzed by a melting curve method or the like.
  • analyzing the expression level of the nucleic acid to be analyzed for example, it can be analyzed by PCR, qRT-PCR or the like.
  • RNA-Seq Ribonucleic acid
  • DNA microarray analysis DNA microarray analysis or the like.
  • the biopolymer when analyzing the expression patterns of a plurality of nucleic acids to be analyzed, it can be analyzed by, for example, transcriptome analysis such as RNA-Seq, DNA microarray analysis or the like.
  • the biopolymer when the biopolymer is a sugar and the molecular weight, structure, etc. of the sugar are analyzed, it can be analyzed by a liquid chromatograph-time-of-flight mass spectrometer (LC-TOF-MS).
  • LC-TOF-MS liquid chromatograph-time-of-flight mass spectrometer
  • the biopolymer is a protein and the presence / absence, amount, etc. of the protein to be analyzed are analyzed, it can be analyzed by Western plotting, extended ligation assay, proximity ligation assay, etc.
  • the analysis method of the present invention may further include a step of collecting at least one of the analyzed cytoplasmic biopolymer and the remaining biopolymer.
  • the method for recovering the analyzed cytoplasmic biopolymer and the remaining biopolymer is not particularly limited, and for example, the description of the recovery step in the fractionation method of the present invention can be cited.
  • the analysis method of the present invention may further include, for example, an amplification step, a reverse transcription step, and / or a holding step in the fractionation method of the present invention.
  • the description of the fractionation method of the present invention can be used for the amplification step, the reverse transcription step, and the holding step.
  • FIG. 1 is a schematic view showing an example of the chip of the present invention, (A) is a top view, (B) is a cross-sectional view taken along the II direction of (A), and (C) is the above-mentioned FIG. It is sectional drawing seen from the II-II direction of (A).
  • the chip 10 has a substrate 1 composed of an upper substrate 1a and a lower substrate 1b.
  • the upper substrate 1a has two through holes 12 and 13 and concave portions 14, 15a, 15b, 15c, 16 on the lower surface, which are respectively formed by stacking the upper substrate 1a and the lower substrate 1b.
  • the upper substrate 1a has two convex portions 17a, 17b between the concave portions 15a, 15b, 15c on the lower surface, and these are formed by laminating the upper substrate 1a and the lower substrate 1b to form the walls 17a, 17b. It is composed.
  • the first opening 12 and the second opening 13 communicate with each other in the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16.
  • the size of the chip 10 is not particularly limited, and the following conditions can be exemplified.
  • First opening 12 Diameter 3-10mm (eg 5mm) Volume 5 to 500 ⁇ L, 5 to 60 ⁇ L (for example, 50, 10 ⁇ L)
  • Second opening 13 Diameter 3-10mm (eg 5mm) Volume 5 to 500 ⁇ L, 5 to 60 ⁇ L (for example, 50, 10 ⁇ L)
  • Upstream flow path 14 Length 10-5000 ⁇ m (for example, 200 ⁇ m) Width 20-500 ⁇ m (for example, 50 ⁇ m) Depth 15-40 ⁇ m (for example, 25 ⁇ m) Trap mouth 15a Length 1-20 ⁇ m (for example, 10 ⁇ m) Width 1 ⁇ 10 ⁇ m (eg 3 ⁇ m) Depth 0.1-40 ⁇ m, 15-40 ⁇ m (for example, 25 ⁇ m)
  • the separation liquid 11 is filled into the separation channel 11 by introducing the separation liquid into the first opening 12 of the chip 10. This also causes the separation liquid to flow from the first opening 12 to the second opening 13. Then, the sample is introduced into the first opening 12. The sample moves in the direction of the second opening 13 by the flow of the separation liquid, and target cells in the sample are trapped in the trap port 15a. After the trap, for example, the solution in the first opening 12 may be replaced with the separation liquid.
  • the chip 10 can separate the biopolymer by using, for example, a fractionation device including a voltage application unit.
  • a fractionation device including a voltage application unit.
  • an electrode system for applying a voltage may be provided in the fractionation device or the chip 10.
  • the electrode system of the fractionation device may be inserted into the first opening 12 and the second opening 13 of the chip 10.
  • the cell membrane of the target cell is disrupted by applying the voltage by the voltage applying means of the fractionation device.
  • the biopolymer is released from the target, and the cytoplasmic biopolymer of the target cell is separated on the second opening 13 side.
  • the remaining biopolymer that has not been separated such as nuclear biopolymer, is trapped in the trap port 15a.
  • the cytoplasmic biopolymer is recovered from the second opening 13, and the remaining biopolymer is recovered from the first opening 12.
  • FIG. 2A and 2B are schematic views showing an example of the chip of the present invention, where FIG. 2A is a top view, and FIG. 2B is an enlarged view of a region surrounded by a two-dot chain line shown in FIG. (Top view), (C) is a cross-sectional view as viewed from the II direction of (B), and (D) is an enlarged view of a region surrounded by a two-dot chain line (D) of (A).
  • FIG. the chip 20 of this embodiment further includes an adjustment channel 21, a third opening 22, and a connection channel 23, except that the wall 17 has one opening 15. 1 has the same configuration as the chip shown in FIG.
  • the adjustment channel 21 communicates with the downstream channel 16 in the vicinity of the wall 17 of the downstream channel 16. Further, one end of the connection channel 23 communicates with the upstream channel 14 near the wall 17 of the upstream channel 14, and the other end communicates with the adjustment channel 21.
  • the size of the chip 20 is not particularly limited, and the following conditions can be exemplified.
  • First opening 12 Diameter 3-10mm (eg 5mm) Volume 5 to 500 ⁇ L, 5 to 60 ⁇ L (for example, 50, 10 ⁇ L)
  • Second opening 13 Diameter 3-10mm (eg 5mm) Volume 5 to 500 ⁇ L, 5 to 60 ⁇ L (for example, 50, 10 ⁇ L)
  • Upstream flow path 14 Length 10-5000 ⁇ m (for example, 200 ⁇ m) Width 20-500 ⁇ m (for example, 50 ⁇ m) Depth 15-40 ⁇ m (for example, 25 ⁇ m) Opening 15 Length 1-20 ⁇ m (for example, 10 ⁇ m) Width 1 ⁇ 10 ⁇ m (eg 3 ⁇ m) Depth 0.1-40 ⁇ m, 15-40 ⁇ m (for example, 25 ⁇ m)
  • Downstream channel 16 Length 5000-20000 ⁇ m (eg 20000 ⁇ m) Width 10 to 300 ⁇ m (for example, 50 ⁇ m) Depth 5
  • the separation liquid 11 is introduced into the first opening 12 and the second opening 13 of the chip 20 and sucked from the third opening 22, whereby the separation channel 11, the adjustment channel 21,
  • the connection channel 23 is filled with the separation liquid. This also causes the separation liquid to flow from the first opening 12 to the third opening 22.
  • the sample is introduced into the first opening 12.
  • the sample moves toward the third opening 22 by the flow of the separation liquid, and target cells in the sample are trapped in the opening 15.
  • the separation liquid is introduced into the third opening 22 to relieve the flow from the first opening 12 to the third opening 22.
  • the chip 20 can separate the biopolymer by using, for example, a fractionation device including a voltage applying unit.
  • a fractionation device including a voltage applying unit an electrode system for applying a voltage may be provided in the fractionation device or the chip 20.
  • the electrode system of the fractionation device may be inserted into the first opening 12 and the second opening 13 of the chip 20.
  • an electrode may be arrange
  • FIG. 3 is a top view showing an example of the chip of the present invention.
  • the chip 30 includes a separation channel 11, a first opening 12, a suction / discharge unit 31, and a capturing unit 32.
  • the separation channel 11 has an upstream channel 14, an opening 17, and a downstream channel 16, and the wall 17 has walls 17 a and 17 b and an opening 15.
  • a capture unit 32 is disposed in the downstream flow channel 16, and a suction / discharge unit 31 is connected to an end of the downstream flow channel 16 in the direction opposite to the first opening 12.
  • the first opening 12 communicates with the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16.
  • the size of the chip 30 is not particularly limited, and the following conditions can be exemplified. Further, in the chip 30, the capture part 32 is arranged at a position of 0 to 20000 ⁇ m from the opening 15 in the downstream flow path 16.
  • First opening 12 Diameter 3-10mm (eg 5mm) Volume 5 to 500 ⁇ L, 5 to 60 ⁇ L (for example, 50, 10 ⁇ L)
  • Upstream flow path 14 Length 10-5000 ⁇ m (for example, 200 ⁇ m) Width 20-500 ⁇ m (for example, 50 ⁇ m) Depth 15-40 ⁇ m (for example, 25 ⁇ m) Opening 15 Length 1-20 ⁇ m (for example, 10 ⁇ m) Width 1 ⁇ 10 ⁇ m (eg 3 ⁇ m) Depth 0.1-40 ⁇ m, 15-40 ⁇ m (for example, 25 ⁇ m)
  • Downstream channel 16 Length 5000-20000 ⁇ m (eg 20000 ⁇ m) Width 10 to 300 ⁇ m (for example, 50 ⁇ m) Depth 5-40 ⁇ m (for
  • the sample is introduced into the first opening 12 of the chip 30.
  • the introduced sample moves from the upstream flow path 14 to the downstream flow path 16 through the opening 15 in the separation flow path 11. At this time, target cells in the sample are trapped in the opening 15.
  • the chip 30 can destroy the cell membrane of the target cell trapped in the opening 15 by introducing, for example, a solution containing a surfactant from the first opening 12, and thereby the biopolymer from the target. To release. Then, by sucking with the suction / discharge unit 31, the cytoplasmic biopolymer of the target cell is separated on the downstream channel 16 side, so that the cytoplasmic living body height is transferred to the capturing unit 32 arranged in the downstream channel 16. Molecules are captured. On the other hand, the remaining biopolymers such as nuclear biopolymers are trapped in the openings 15. For example, the remaining biopolymer trapped in the opening 15 is first recovered by sucking from the first opening 12 using the suction means. Further, for example, the cytoplasmic biopolymer trapped in the capture unit 32 is first recovered by suction from the first opening 12 using the suction means.
  • FIG. 4 is a top view showing an example of the chip of the present invention.
  • the chip 40 of the first modification has suction / discharge portions 31 a and 31 b as the suction / discharge portion 31. Further, in the chip 40, the suction / discharge portions 31 a and 31 b are connected to the downstream flow path 16.
  • a suction / discharge part 31 a is connected to the end of the downstream flow path 16 in the direction opposite to the first opening 12.
  • the suction / discharge part 31b is connected to the opening 15 side of the connection part between the suction / discharge part 31a and the downstream flow path 16.
  • a capture unit 32 is disposed between a connection portion between the suction / discharge portion 31 a and the downstream flow channel 16 and a connection portion between the suction / discharge portion 31 b and the downstream flow channel 16. Except for this point, the chip 40 of the first modification has the same configuration as the chip 30 of the third embodiment, and the description thereof can be used.
  • the sample is introduced into the first opening 12 of the chip 40.
  • the introduced sample moves from the upstream flow path 14 to the downstream flow path 16 through the opening 15 in the separation flow path 11. At this time, target cells in the sample are trapped in the opening 15.
  • the chip 40 can destroy the cell membrane of the target cell trapped in the opening 15 by introducing, for example, a solution containing a surfactant from the first opening 12, and thereby the biopolymer from the target. To release. Then, the cytoplasmic living body high polymer is separated into the capturing part 32 disposed in the downstream flow path 16 by separating the cytoplasmic biopolymer of the target cell on the downstream flow path 16 side by being sucked by the suction / discharge section 31a. Molecules are captured. On the other hand, the remaining biopolymers such as nuclear biopolymers are trapped in the openings 15.
  • the remaining biopolymer trapped in the opening 15 is opened by generating a flow from the downstream flow path 16 to the first opening 12 by being discharged by the suction discharge section 31b.
  • the remaining biopolymer is recovered by sucking from the first opening 12 using the suction means.
  • the cytoplasmic biopolymer trapped in the capture unit 32 is discharged from the capture unit 32 by generating a flow from the downstream flow path 16 to the first opening 12 by being discharged by the suction / discharge unit 31a. Move to the first opening 12. Then, for example, the cytoplasmic biopolymer is recovered by sucking from the first opening 12 using the suction means.
  • FIG. 5 is a top view showing an example of the chip of the present invention.
  • the chip 50 includes a separation channel 11, a first opening 12, and an electrode 33.
  • the separation channel 11 has an upstream channel 14, a wall 17, and a downstream channel 16, and the wall 17 has walls 17 a and 17 b and an opening 15.
  • an electrode 33 is disposed at the end of the downstream flow path 16 in the direction opposite to the first opening 12.
  • the first opening 12 communicates with the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16.
  • the size of the chip 50 is not particularly limited, and for example, description of the size of the chip 30 can be used.
  • the sample is introduced into the first opening 12 of the chip 50.
  • the introduced sample moves from the upstream flow path 14 to the downstream flow path 16 through the opening 15 in the separation flow path 11. At this time, target cells in the sample are trapped in the opening 15.
  • the chip 50 can separate the biopolymer by using, for example, a fractionation device including a voltage application unit.
  • the electrode system for applying the voltage may be provided in the fractionation device or the chip 50.
  • the electrode system of the fractionation device may be inserted into the first opening 12 of the chip 50.
  • the cell membrane of the target cell is disrupted by applying the voltage by the voltage applying means of the fractionation device.
  • the biopolymer is released from the target, and the cytoplasmic biopolymer of the target cell is separated on the electrode 33 side.
  • the remaining biopolymer that has not been separated such as nuclear biopolymer, is trapped in the opening 15.
  • the remaining biopolymer trapped in the opening 15 is first recovered by sucking from the first opening 12 using the suction means.
  • the cytoplasmic biopolymer is recovered by sucking from the first opening 12 again using the suction means, for example.
  • FIG. 6 is a top view showing an example of the chip of the present invention.
  • the chip 60 includes a separation channel 11, a first opening 12, a suction / discharge unit 31, a liquid movement control unit 34, a bypass channel 35, and a second liquid movement. And controllers 36a and 36b.
  • the separation channel 11 has an upstream channel 14, a wall 17, and a downstream channel 16, and the wall 17 has walls 17 a and 17 b and an opening 15.
  • a suction / discharge unit 31 is connected to an end of the downstream flow path 16 in the direction opposite to the first opening 12.
  • the liquid movement control unit 34 is disposed between the opening 15 and the connection portion between the upstream flow path 14 and the bypass flow path 35.
  • the second liquid movement control unit 36a is disposed so as to be adjacent to a connection part (communication part) between the bypass flow path 35 and the upstream flow path 14.
  • the second liquid movement control unit 36 b is disposed so as to be adjacent to the connection portion between the bypass channel 35 and the downstream channel 16.
  • the upstream flow path 14 and the downstream flow path 16 are communicated with the bypass flow path 35.
  • the first opening 12 communicates with the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16.
  • the size of the chip 60 is not particularly limited, and for example, description of the size of the chip 30 can be used.
  • the liquid is prevented from passing through the liquid movement control unit 34. Further, the liquid is allowed to pass through the second liquid movement control units 36a and 36b. Next, the sample is introduced into the first opening 12. Then, the target cells in the sample are introduced into the downstream flow path 16 via the bypass flow path 35 by being sucked by the suction / discharge section 31.
  • the liquid is allowed to pass through the liquid movement control unit 34, and the liquid is prevented from passing through the second liquid movement control units 36a and 36b. And it discharges by the suction discharge part 31, and the flow from the downstream flow path 16 to the 1st opening part 12 is produced. At this time, the target cell is trapped in the opening 15.
  • the chip 60 can destroy the cell membrane of the target cell trapped in the opening 15 by, for example, introducing a solution containing a surfactant from the first opening 12, and thereby the biopolymer from the target. To release. Then, by discharging by the suction / discharge unit 31, the biopolymer in the cytoplasm of the target cell is separated to the upstream flow path 14 side, and further moved to the first opening 12. Then, for example, the cytoplasmic biopolymer is recovered by sucking from the first opening 12 using the suction means. Further, the remaining biopolymer trapped in the opening 15 is moved from the opening 15 to the downstream flow path 16 by being sucked by the suction and discharge section 31.
  • the liquid is prevented from passing through the liquid movement control unit 34, and the liquid is allowed to pass through the second liquid movement control units 36a and 36b.
  • the remaining biopolymer moves to the first opening 12 via the bypass channel 35 and the upstream channel 14 by being discharged by the suction / discharge unit 31.
  • the remaining biopolymer is recovered by sucking from the first opening 12 using the suction means.
  • FIG. 7 is a top view showing an example of the chip of the present invention.
  • the chip 70 includes a separation channel 11, a first opening 12, a second opening 13, and a liquid movement control unit 34.
  • the separation channel 11 has an upstream channel 14, a wall 17, and a downstream channel 16, and the wall 17 has walls 17 a and 17 b and an opening 15.
  • a liquid movement control unit 34 is disposed in the downstream flow path 16.
  • the first opening 12 and the second opening 13 communicate with the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16.
  • the liquid movement control unit 34 is, for example, the microvalve.
  • the size of the chip 70 is not particularly limited, and the following conditions can be exemplified. Further, in the chip 70, the liquid movement control unit 34 is disposed at a position of 0 to 20000 ⁇ m from the opening 15 in the downstream flow path 16.
  • First opening 12 Diameter 3-10mm (eg 5mm) Volume 5 to 500 ⁇ L, 5 to 60 ⁇ L (for example, 50, 10 ⁇ L)
  • Second opening 13 Diameter 3-10mm (eg 5mm) Volume 5-60 ⁇ L (for example, 10 ⁇ L)
  • Upstream flow path 14 Length 10-5000 ⁇ m (for example, 200 ⁇ m) Width 20-500 ⁇ m (for example, 50 ⁇ m) Depth 15-40 ⁇ m (for example, 25 ⁇ m) Opening 15 Length 1-20 ⁇ m (for example, 10 ⁇ m) Width 1 ⁇ 10 ⁇ m (eg 3 ⁇ m) Depth 0.1-40 ⁇ m, 15-40 ⁇ m (for example, 25 ⁇ m)
  • Downstream channel 16 Length 5000-20000 ⁇ m (e
  • the liquid movement control unit 34 is released to enable liquid passage.
  • the sample is introduced into the first opening 12 of the chip 70.
  • the introduced sample moves from the upstream flow path 14 to the downstream flow path 16 through the opening 15 in the separation flow path 11. At this time, target cells in the sample are trapped in the opening 15.
  • the chip 70 can separate the biopolymer by using, for example, a fractionation device including a voltage applying unit.
  • a fractionation device including a voltage applying unit.
  • an electrode system for applying a voltage may be provided in the fractionation device or the chip 70.
  • the electrode system of the fractionation device may be inserted into the first opening 12 and the second opening 13 of the chip 70.
  • the chip 70 may destroy the cell membrane of the target cell trapped in the opening 15 by introducing a solution containing a surfactant from the first opening 12.
  • the cytoplasmic biopolymer is separated between the liquid movement control unit 34 and the second opening 13 in the downstream channel 16. Further, after the separation, the liquid movement control unit 34 is closed so that liquid cannot pass therethrough. For example, the cytoplasmic biopolymer is recovered from the second opening 13 and the remaining biopolymer is recovered from the first opening 12 using the suction means.
  • Example 1 The chip of the present invention was produced, and it was confirmed that the nucleic acid could be fractionated.
  • Chip A chip 10 shown in FIG. 1 was produced.
  • the size of each part of the chip 10 was as follows.
  • First opening 12 Cylindrical shape with a diameter of 5 mm
  • volume 10 ⁇ L Second opening 13 Cylindrical shape with a diameter of 5 mm
  • volume 10 ⁇ L Upstream flow path 14
  • Length 4600 ⁇ m width 50 ⁇ m
  • depth 25 ⁇ m Trap mouth
  • 15a Length 5 ⁇ m width 3 ⁇ m
  • Bypass ports 15b
  • Downstream channel 16 Length 20000 ⁇ m, width 50 ⁇ m, depth 25 ⁇ m
  • Liquid PDMS (Sylgard 184, manufactured by Dow Corning) was poured into a microchannel mold, degassed, and heated in an oven at 150 ° C. for 30 minutes to solidify PDMS. After the solidified PDMS is cut out using a razor, the first opening 12 and the second opening 13 are formed at both ends of the separation channel 11 using a punch, and a channel structure is formed. Obtained. The obtained flow path structure was sealed to a glass substrate using plasma bonding.
  • K562 cells as a sample suspension cells (obtained from National Institute of Biomedical Innovation) to 37 ° C., and cultured under the conditions of 5% CO 2.
  • As the culture solution RPMI-1640 culture solution (Sigma Aldrich) containing 10% FBS and 1% penicillin / streptomycin was used. After the culture, the cells were collected and made into one cell by stirring with a pipette. Next, after centrifugation at 1000 rpm for 3 minutes, the supernatant was removed and dispersed in a cell dispersion.
  • the composition of the cell dispersion was 50 mmol / L Tris, 25 mmol / L HEPES, and 175 mmol / L sucrose, and the pH was 8.3.
  • the dispersed cells Prior to introduction into the first inlet 12 of the chip 10 to be described later, the dispersed cells are further diluted 20 times or more with the cell dispersion, and the diluted cell suspension is used as a sample. did.
  • the composition of the separation liquid 2 was 50 mmol / L Tris and 25 mmol / L HEPES, and the pH was 8.2.
  • a platinum wire electrode (diameter 0.8 mm) into the first opening 12 and the second opening 13
  • -150V is applied to the electrode of the first opening 12
  • 0V is applied to the electrode of the second opening 13.
  • a voltage was applied.
  • the current supplied from the first opening 12 and the second opening 13 was measured in accordance with the voltage application.
  • the cytoplasmic biopolymer was separated by applying a voltage until the measured current value became a constant value. After the separation, it was confirmed by the optical microscope that cells (the remainder of the target cells) whose cell membrane was crushed were trapped in the trap port 15a.
  • the entire amount of the separation liquid 1 in the chip 10 was recovered from the second opening 13.
  • reverse transcription kit TaqMan RNA-to-Ct 1-Step Kit, Thermo Fisher Fisher Scientific
  • RT-PCR kit gene expression assay, Thermo Fisher Scientific
  • thermal cycler LightCycler96
  • a GAPDH primer Hs02758991_g1, Applied Biosystems
  • a total of 20 ⁇ L of the reaction solution containing 9 ⁇ L of the recovered separation solution 1 was subjected to reverse transcription at 48 ° C. for 15 minutes, and further incubated at 95 ° C.
  • cytoplasmic nucleic acid can be fractionated by the chip of the present invention.
  • Example 2 The chip of the present invention was produced, and it was confirmed that the nucleic acid could be fractionated.
  • Chip A chip 20 shown in FIG. 2 was produced.
  • the size of each part of the chip 20 was as follows.
  • tip 20 was produced like Example 1 (1).
  • the adjustment channel 21 was communicated with the downstream channel 16 on the downstream side of the wall 17 by 30 ⁇ m.
  • First opening 12 5mm diameter inverted cone, volume 10 ⁇ L Second opening 13 5mm diameter inverted cone, volume 10 ⁇ L Upstream flow path 14 Length 4600 ⁇ m, width 50 ⁇ m, depth 25 ⁇ m Opening 15 Length 5 ⁇ m, width 3 ⁇ m, depth 25 ⁇ m Downstream channel 16 Length 20000 ⁇ m, width 50 ⁇ m, depth 25 ⁇ m Adjustment flow path 21 Length 1100 ⁇ m, width 50 ⁇ m, depth 25 ⁇ m
  • Third opening 22 5mm diameter cylindrical shape, volume 60 ⁇ L Connection channel 23 Length 7310 ⁇ m, width 25 ⁇ m, depth 25 ⁇ m
  • the cells move in the direction of the third opening 22 due to the flow of the separation liquid, one K562 cell is trapped in the opening 15, and the other cells flow through the connection flow path 23 to adjust flow. Moved to Road 21. It was confirmed with the optical microscope that the single K562 cell was trapped in the opening 15. Then, 69 ⁇ L of the separation liquid 2 was introduced into the third opening 22.
  • a platinum wire electrode (diameter 0.8 mm) is inserted into the first opening 12, the second opening 13, and the third opening 22, -150V is applied to the electrode of the first opening 12, and the second opening A voltage of 0 V was applied to the electrode of the portion 13, and a voltage of ⁇ 130 V was applied to the electrode of the third opening 22.
  • the current supplied from the first opening 12 and the third opening 22 was measured. And voltage was applied until the measured electric current value became a fixed value under observation of the fluorescence microscope.
  • FIG. 8 is a photograph showing separation of nucleic acids during voltage application.
  • 8A is a photograph of the opening 15 and the downstream flow path 16 at the start of voltage application
  • FIG. 8B is a photograph of the opening 15 and the downstream flow path 16 5 seconds after the voltage application
  • C) is a photograph of the downstream flow path 16 11.5 seconds after voltage application.
  • the K562 cells were trapped in the opening 15 at the start of voltage application.
  • the fluorescence intensity of the K562 cells trapped in the opening 15 was reduced, and cytoplasmic nucleic acids were separated.
  • FIG. 8C the cytoplasmic nucleic acid was separated into the downstream flow path 16.
  • a chip having a width of the opening 15 of 2 to 5 ⁇ m, a chip having a length of the opening 15 of 8 to 14 ⁇ m, or a length of the connection channel 23 is 4.9 to 10 mm.
  • a voltage was applied in the same manner except that the chip was used, and observation was performed with the fluorescence microscope. As a result, it was found that the cytoplasmic nucleic acid can be separated also in these chips. From the above, it was found that according to the chip of the present invention, nucleic acids can be separated.
  • Example 3 After producing the chip of the present invention and fractionating the nucleic acid, it was confirmed that the remaining nucleic acid (hereinafter also referred to as “other nucleic acid”) could be recovered.
  • the cytoplasmic nucleic acid was fractionated by applying a voltage in the same manner as in Example 2 except that the separation solution 1 containing Hoechst® 33258 (manufactured by Sigma-Aldrich) was used. After the fractionation, the cytoplasmic nucleic acid was recovered by recovering the solution in the chip 20 from the second opening 13. Next, under the observation of the fluorescence microscope, the other nucleic acid was recovered by sucking the solution in the chip 20 from the first opening 12 using a micropipette.
  • FIG. 9 is a photograph showing the recovery of the other nucleic acid.
  • (A) shows a photograph of the opening 15 before suction
  • (B) shows a photograph of the opening 15 after suction.
  • the other nucleic acid trapped in the opening 15 was collected by aspiration from the first opening 12. From these facts, it was found that the nucleic acid was fractionated and other nucleic acids could be recovered using the chip of the present invention.
  • Chip A chip was manufactured in the same manner as in the chip 20 of Example 2 (1) except that the side surface of the opening 15 was tapered from the downstream flow path 16 to the upstream flow path 14.
  • the size of the opening 15 was as follows. Opening 15 Length 42 ⁇ m, upstream flow path 14 side width 3 ⁇ m, downstream flow path 16 side width 50 ⁇ m, depth 25 ⁇ m
  • FIG. 10 is a photograph showing separation of nucleic acids during voltage application.
  • (A) is a photograph of the opening 15 at the start of voltage application
  • (B) is a photograph of the downstream flow path 16 after voltage application.
  • the K562 cells were trapped in the opening 15 at the start of voltage application.
  • 10B the cytoplasmic nucleic acid was separated into the downstream channel 16 after voltage application.
  • the cytoplasmic nucleic acid was recovered from the second opening 13 and the remaining nucleic acid was recovered from the first opening 12. From these facts, it was found that the nucleic acid can be fractionated and the nucleic acid can be recovered using a chip having different openings 15.
  • Example 5 Using the chip of the present invention, it was confirmed that cytoplasmic nucleic acids other than mitochondrial nucleic acids can be fractionated and that separated nucleic acids can be analyzed.
  • FIG. 11 is a graph showing the fluorescence intensity after correction. As shown in FIG. 11, the corrected fluorescence intensity was constant, and mitochondria were trapped in the opening 15. From these, it was found that cytoplasmic nucleic acids other than mitochondrial nucleic acids can be fractionated, that is, only specific nucleic acids can be fractionated among cytoplasmic nucleic acids.
  • cytoplasmic nucleic acids other than the mitochondrial nucleic acids and the other nucleic acids were recovered in the same manner as in Example 3.
  • the fluorescence intensity during the incubation was measured in the same manner as in Example 1 (3).
  • the other nucleic acids were carried out using a qPCR kit (TaqMan Copy Number Assay, manufactured by Thermo Fisher Scientific) and the thermal cycler according to the attached protocol.
  • the GAPDH primer was used as the primer.
  • FIG. 12 is a graph showing fluorescence intensity.
  • the horizontal axis indicates the number of cycles
  • the vertical axis indicates the fluorescence intensity.
  • the fluorescence intensity increases depending on the number of cycles
  • GAPDH mRNA is different from the other nucleic acid.
  • GAPDH genomic DNA was confirmed to contain GAPDH genomic DNA. From the above, it was found that cytoplasmic nucleic acids other than mitochondrial nucleic acids can be fractionated and fractionated nucleic acids can be analyzed using the chip of the present invention.
  • Example 1 (1) (obtained from ATCC, CRL-2522) sample BJ cells 37 ° C., and cultured under the conditions of 5% CO 2.
  • a DMEM culture solution (Sigma Aldrich) containing 10% FBS and 1% penicillin / streptomycin was used. After the culturing, the cells were dispersed in the cell dispersion in the same manner as in Example 1 (1) except that the cells were collected using TrypLE (manufactured by Thermo Fisher Scientific).
  • Example 2 The chip used in Example 2 was used. First, 20 ⁇ L of the separation liquid 3 was introduced into the first opening 12 and 20 ⁇ L of the separation liquid 2 was introduced into the second opening 13 into the chip 20. The composition of the separation liquid 3 was 300 mmol / L Tris and 150 mmol / L HCl, and the pH was 8.2. In addition, what added SYBR (trademark) Green (TM) II was used for the separation liquid 3 of a present Example. Next, a flow of the separation liquid from the first opening 12 to the third opening 22 was generated by suction from the third opening 22. Next, 20 ⁇ L of the separation liquid 2 was introduced into the first opening 12, and 20 ⁇ L of the separation liquid 3 was introduced into the second opening 13.
  • SYBR trademark
  • a platinum wire electrode (diameter 0.8 mm) is inserted into the first opening 12, the second opening 13, and the third opening 22, -300V is applied to the electrode of the first opening 12, and the second opening
  • the voltage of 0 V and the voltage of the electrode of the third opening 22 -260 V were applied to the electrode of the portion 13.
  • the current supplied from the first opening 12 and the third opening 22 was measured.
  • a voltage was applied until the measured current value became a constant value.
  • a voltage was applied in the same manner under the observation of the fluorescence microscope except that the Euglena was not introduced.
  • FIG. 13 is a photograph showing separation of nucleic acids during voltage application.
  • (A) is a photograph of the opening 15 at the start of voltage application
  • (B) is a photograph of the opening 15 during voltage application
  • (C) is the downstream flow path 16 during voltage application
  • (D) is a photograph of the opening 15 after voltage application
  • (E) is a photograph of the downstream flow path 16 during voltage application in the control.
  • the Euglena was trapped in the opening 15 at the start of voltage application.
  • chloroplast nucleic acids were separated during voltage application. Further, as shown by the arrow in FIG.
  • Example 8 After preparing a library from nucleic acids fractionated using the chip of the present invention, it was confirmed that the cytoplasmic nucleic acid and the non-cytoplasmic nucleic acid could be fractionated with high precision by analyzing with a next-generation sequencing technique.
  • the cytoplasmic nucleic acid and the other nucleic acid are recovered after fractionating the cytoplasmic nucleic acid and the other nucleic acid in the same manner as in Example 3 except that the K562 is used instead of the BJ cell. did.
  • a cDNA generation kit (SMART-Seq (registered trademark) v4 Ultra (registered trademark) Low Input RNA Kit for Sequencing (Clontech) and a thermal cycler (S1000, Bio-rad) based on the attached protocol
  • CDNA was prepared from the obtained solution containing each nucleic acid.
  • a total of 10.5 ⁇ L of a reaction solution containing 1 ⁇ L of the buffer for the cDNA preparation kit and 9.5 ⁇ L of the collected solution was prepared.
  • the reaction solution was reacted at room temperature (about 25 ° C.) for 5 minutes.
  • 2 ⁇ L of a primer solution (3 ′ SMART-Seq CDS Primer II A, manufactured by Clontech) was added to 10.5 ⁇ L of the reaction solution, followed by incubation at 72 ° C. for 3 minutes.
  • a total of 20 ⁇ L of the reverse transcription reaction solution containing the reaction solution after the incubation and 7.5 ⁇ L of Master mix of the cDNA preparation kit was prepared.
  • the reverse transcription reaction solution was incubated at 42 ° C. for 90 minutes, and then reverse transcription was performed at 70 ° C. for 10 minutes. Then, a total of 50 ⁇ L of the amplification reaction solution containing the reverse transcription reaction solution after reverse transcription and 30 ⁇ L of the PCR Master Mix of the cDNA preparation kit was prepared. Furthermore, after incubating the amplification reaction solution at 95 ° C. for 1 minute, the amplification reaction was carried out 18 times, further comprising 98 ° C. for 10 seconds, 65 ° C. for 30 seconds, and 68 ° C. for 3 minutes. Went. After the cycle, the amplification reaction was completed by further incubation at 72 ° C. for 10 minutes.
  • the amplified cDNA was purified from the 50 ⁇ L amplification reaction solution using AMPure® XP® Kit (manufactured by Beckman® Coulter) based on the attached protocol to obtain a 17 ⁇ L cDNA solution. Furthermore, a library was prepared using TruSeq ⁇ ⁇ ⁇ ⁇ ⁇ ChIP Sample Prep Kit (manufactured by Illumina) based on the attached protocol. The prepared library was subjected to sequence analysis using HiSeq (manufactured by Illumina) under the conditions of Paired End, 100 bases / 1 read, and 5 million read pairs.
  • the output data was sorted by base call, filtering, and index sequence to obtain a FASTQ format data file containing the read sequence and the quality data of each base.
  • the data file is mapped using STAR (A. Dobin et al., "STAR: ultrafast universal RNA-seq aligner", Bioinformatics, 2012, vol.29, No.1, pp.15-21), Cufflinks (C. Trapnell et al., “Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.”, Nat. Protoc., 2012, Vol.7, No.3, pp562-578) Went.
  • STAR A. Dobin et al., "STAR: ultrafast universal RNA-seq aligner", Bioinformatics, 2012, vol.29, No.1, pp.15-21
  • Cufflinks C. Trapnell et al., “Differential gene and transcript expression analysis of RNA-seq experiments with TopH
  • FIG. 14 shows the result of the types of sequences included in each library.
  • FIG. 14 is a graph showing the types of sequences included in each library.
  • 14A shows the results of the cytoplasmic library
  • FIG. 14B shows the results of the nuclear library.
  • FIG. 14 (A) in the cytoplasmic library, RNA containing introns hardly existed, and mature RNA occupied the majority.
  • FIG. 14 (B) in the nuclear library, RNA containing introns accounted for half, and many immature RNAs before splicing existed.
  • FIG. 15 is a graph showing the origin (chromosome name) of each library.
  • the horizontal axis indicates the origin (chromosome name) of the cDNA constituting each library.
  • each bar indicates the cytoplasmic library, the nuclear library, the cytoplasmic library, and the nuclear library from the left.
  • the cytoplasmic library and the nuclear library both contained autosomal cDNA.
  • the cytoplasmic library contained cDNA derived from mitochondrial DNA, whereas the nuclear library contained almost no cytoplasmic library.
  • cytochip nucleic acids and other nucleic acids can be obtained with extremely high precision (purity) by the chip and fractionation method of the present invention. I found that it can be fractionated. In addition, by using the chip and the fractionation method of the present invention, a sample that is fractionated with extremely high accuracy and rich in cytoplasmic nucleic acids can be obtained. It has been found that samples with quality available for singing can be prepared.
  • Example 9 After fractionating nucleic acids using the chip of the present invention, it was confirmed that nucleic acid amplification and library preparation were possible within the chip.
  • the cytoplasmic nucleic acid and the other nucleic acids were fractionated in the same manner as in Example 3 except that a chip having the following size was used as the chip 20 shown in FIG.
  • a PEG-DA solution (containing Poly (ethylene glycol) diacrylate (MW575), 1% 2,2-Dimethoxy-2-phenylacetophenone) is transferred from the third opening 22 to the separation channel 11 of the chip 20.
  • PEG-DA was gelled by irradiating the separation channel 11 with ultraviolet rays.
  • a cDNA library was prepared in the first opening 12 and the second opening 13 using a cDNA library preparation kit (REPLI-g WTA Single Cell Kit, manufactured by Qiagen).
  • First opening 12 5mm diameter inverted cone, 35 ⁇ L capacity Second opening 13 5mm diameter inverted cone, 35 ⁇ L capacity Upstream flow path 14 Length 4600 ⁇ m, width 50 ⁇ m, depth 25 ⁇ m Opening 15 Length 5 ⁇ m, width 3 ⁇ m, depth 25 ⁇ m Downstream channel 16 Length 20000 ⁇ m, width 50 ⁇ m, depth 25 ⁇ m Adjustment flow path 21 Length 1100 ⁇ m, width 50 ⁇ m, depth 25 ⁇ m Third opening 22 Cylindrical shape with a diameter of 5 mm, volume of 100 ⁇ L Connection channel 23 Length 7310 ⁇ m, width 25 ⁇ m, depth 25 ⁇ m
  • reaction was stopped on the conditions for 95 degreeC for 5 minutes . After the reaction was stopped, 30 ⁇ L of amplification reaction solution was added and incubated at 30 ° C. for 2 hours to obtain a cDNA library.
  • the yield was measured for a molecular spectrometer (Qubit (registered trademark) fluorometer, Thermo Fisher Fisher Scientific) and a nucleic acid quantification kit (Qubit (registered)). (Trademark) dsDNA-HS-Assay-kit, Thermo-Fisher-Scientific).
  • FIG. 16 is a graph showing the yield of cDNA library.
  • the horizontal axis represents the type of library, and the vertical axis represents the yield.
  • the cDNA library obtained from both the first opening 12 and the second opening 13 had a sufficient yield for further analysis using the cDNA library.
  • the total yield of the cDNA library obtained from both the first opening 12 and the second opening 13 was generally equal to or higher than the yield of the cDNA library obtained from one cell. From these facts, it is possible to amplify cytoplasmic nucleic acids and other nucleic acids in the chip after fractionating the nucleic acids using the chip of the present invention. It was found that a library derived from nucleic acid can be prepared.
  • Example 10 In the chip of the present invention, in the state where the target cell is trapped in the opening of the wall, the cell membrane of the target cell can be destroyed, and the destruction of the nuclear membrane of the target cell can be further suppressed.
  • the relational expression between w 1 ) and the shortest distance (d) between the opening of the wall and the nuclear membrane of the target cell was calculated.
  • Simulation software (COMSOL, COMSOL Inc. Co.) was used, set the following conditions, the electric field (current density, E nuc) of the nuclear membrane at the position of the opening of the wall distance d o of the walls of the opening of the field ( The ratio to the current density (E orifice ) was simulated.
  • E nuc E orifice ⁇ w 1 / ( ⁇ d + w 1) ⁇ (4) E nuc: field of nuclear envelope E Orifice: field wall of the opening w 1: diameter of the wall of the opening [pi: pi d: minimum distance between the opening of the wall, and the nuclear membrane of the target cell
  • FIG. 17 is a graph showing the simulation result and the approximate expression.
  • the horizontal axis represents the distance d o from the opening of the wall
  • the vertical axis represents the electric field of the wall of the opening of the electric field of the nuclear membrane in the opening of the wall at a distance d o (E nuc) (E orifice ) represents the ratio (E nuc / E orifice) for.
  • the symbol in the figure indicates the simulation result
  • the solid line indicates the approximate expression of the expression (4)
  • the number indicated by the arrow in the figure indicates the diameter (w 1 ) of the wall opening. Indicates.
  • the approximate expression of the expression (4) can approximate the simulation result with high correlation.
  • the present inventors have satisfied that the target cell is trapped in the opening of the wall by satisfying the following formulas (5) and (6), respectively, in the electric field of the wall opening and the nuclear membrane: It has been found that the cell membrane of the target cell can be destroyed, and the destruction of the nuclear membrane of the target cell can be further suppressed.
  • E nuc field of nuclear envelope
  • E Orifice field wall opening
  • the diameter (w 1 ) of the opening of the wall can satisfy the following formula (2), thereby destroying the cell membrane of the target cell, And it was found that the destruction of the nuclear membrane of the target cell can be suppressed.
  • the diameter (w 1 ) of the opening of the wall satisfies the following formula (3), so that the cell membrane of the target cell can be destroyed, and the target cell It was found that the destruction of the nuclear membrane can be further suppressed.
  • the relationship between the diameter (w 1 ) of the opening of the wall and the shortest distance (d) between the opening of the wall and the nuclear membrane of the target cell is expressed by the above formula (2) or ( 3), it was found that the cell membrane of the target cell can be destroyed while the target cell is trapped in the opening of the wall, and the destruction of the nuclear membrane of the target cell can be suppressed.
  • a biopolymer such as a nucleic acid can be fractionated even in a liquid system that does not contain a molecule having the molecular sieve function.
  • the cytoplasmic biopolymer can be separated in a state where the remaining biopolymer is trapped in the opening of the wall. For example, the quality that can be used for next-generation sequencing Can be prepared.
  • the chip of the present invention can trap the target cells with high accuracy at the opening of the wall when, for example, one target cell is introduced into the separation channel. Therefore, according to the chip of the present invention, for example, a cytoplasmic biopolymer can be separated from one target cell.
  • the separation channel has a wall having the opening in the channel.
  • the chip of the present invention has, for example, a separation channel that does not have a wall having the opening when a voltage is applied to the separation channel (for example, the separation channel in Patent Document 1).
  • a voltage for example, 1/10 or less
  • a lower voltage for example, 1/10 or less
  • the voltage at the time of crushing the cell membrane of the target cell. Voltage can disrupt the cell membrane of the target cell.
  • the chip of the present invention can be used at a lower voltage, for example, generation of Joule heat at the time of voltage application can be reduced, and the influence of denaturation of the biopolymer due to the Joule heat can be reduced. Furthermore, since the chip of the present invention can be used at a lower voltage, for example, a highly conductive solution containing an electrolyte can be used as a separation liquid used for biopolymer separation. For this reason, the present invention is extremely useful in the clinical field, medical field, life science field, and the like.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Sustainable Development (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Provided are a novel biopolymer fractionation chip, which can fractionate biopolymers such as nucleic acids, even in liquids that do not contain molecules having a molecular sieve function, a biopolymer fractionation method and a biopolymer analysis method. This biopolymer fractionation chip is characterized by having a substrate, in which the substrate has a separation flow path for separating biopolymers in the cytoplasm of target cells and one or more openings; the one or more openings have a first opening, on the separation flow path, into which a sample, which includes the target cells, can be introduced; the first opening communicates with the separation flow path; the separation flow path has a wall that is in the flow path and in the cross-sectional direction; and the wall has an opening.

Description

生体高分子分画用チップ、それを用いた生体高分子の分画方法、および生体高分子の分析方法Biopolymer fractionation chip, biopolymer fractionation method using the same, and biopolymer analysis method

　本発明は、生体高分子分画用チップ、それを用いた生体高分子の分画方法、および生体高分子の分析方法に関する。 The present invention relates to a biopolymer fractionation chip, a biopolymer fractionation method using the chip, and a biopolymer analysis method.

　ターゲット細胞等を含む試料から核酸を分画する方法として、前記試料および分子篩機能を有する分子を含む液体に対し電圧を印加することにより、前記ターゲット細胞の細胞膜を破砕し、さらに、電気泳動を実施することで、核の核酸と、細胞質の核酸とを分画する方法が知られている（特許文献１）。 As a method for fractionating nucleic acid from a sample containing target cells, etc., a cell membrane of the target cells is disrupted by applying a voltage to the sample and a liquid containing molecules having a molecular sieve function, and electrophoresis is performed. Thus, a method for fractionating nuclear nucleic acid and cytoplasmic nucleic acid is known (Patent Document 1).

国際公開第２０１５／１０６１４６号International Publication No. 2015/106146

　しかしながら、前記分子篩機能を有する分子を含む液体系（分画系）で核酸等の生体高分子を分画するため、分画された生体高分子は、前記分子篩機能を有する分子を含む。また、前記分子篩機能を有する分子は、前記核酸等の生体高分子の分析に干渉する。このため、分画された生体高分子を分析する場合、分析精度が低下するという問題があった。 However, in order to fractionate biopolymers such as nucleic acids in a liquid system (fractionation system) containing molecules having the molecular sieve function, the fractionated biopolymers contain molecules having the molecular sieve function. Further, the molecule having the molecular sieve function interferes with the analysis of the biopolymer such as the nucleic acid. For this reason, when analyzing the fractionated biopolymer, there is a problem that the analysis accuracy is lowered.

　そこで、本発明は、例えば、前記分子篩機能を有する分子を含まない液体においても、核酸等の生体高分子を分画できる新たな生体高分子分画用チップ、それを用いた生体高分子の分画方法、および生体高分子の分析方法の提供を目的とする。 Therefore, the present invention provides, for example, a new biopolymer fractionation chip that can fractionate biopolymers such as nucleic acids even in liquids that do not contain molecules having the molecular sieve function, and separation of biopolymers using the same. An object of the present invention is to provide a drawing method and a biopolymer analysis method.

　前記目的を達成するために、本発明の生体高分子分画用チップ（以下、「チップ」ともいう）は、
基板を有し、
前記基板は、ターゲット細胞が有する細胞質の生体高分子を分離する分離用流路と、１以上の開口部とを有し、
前記１以上の開口部は、前記分離用流路に前記ターゲット細胞を含む試料を導入可能な第１の開口部を有し、
前記第１の開口部は、前記分離用流路に連通され、
前記分離用流路は、その流路内であり、且つ断面方向に壁を有し、
前記壁は、開口を有することを特徴とする。 In order to achieve the above object, the biopolymer fractionation chip of the present invention (hereinafter also referred to as “chip”) is:
Having a substrate,
The substrate has a separation channel for separating cytoplasmic biopolymers of target cells, and one or more openings.
The one or more openings have a first opening into which a sample containing the target cells can be introduced into the separation channel,
The first opening communicates with the separation channel;
The separation channel is in the channel and has a wall in the cross-sectional direction,
The wall has an opening.

　本発明の生体高分子の分画方法（以下、「分画方法」ともいう）は、ターゲット細胞を、開口を有する壁の開口にトラップするトラップ工程、
前記ターゲット細胞の細胞膜を破壊することにより、前記ターゲット細胞から細胞質の生体高分子を放出させる放出工程、および
放出された細胞質の生体高分子を分離する分離工程を含むことを特徴とする。 The biopolymer fractionation method of the present invention (hereinafter, also referred to as “fractionation method”) includes a trap step of trapping target cells in an opening of a wall having an opening,
The method includes a release step of releasing a cytoplasmic biopolymer from the target cell by destroying a cell membrane of the target cell, and a separation step of separating the released cytoplasmic biopolymer.

　本発明の生体高分子の分析方法（以下、「分析方法」ともいう）は、ターゲット細胞から細胞質の生体高分子を分画する分画工程、および
前記細胞質の生体高分子および前記細胞質の生体高分子の分画後のターゲット細胞の残部が含む生体高分子の少なくとも一方を分析する分析工程を含み、
前記分画工程が、前記本発明の生体高分子の分画方法により実施されることを特徴とする。 The biopolymer analysis method of the present invention (hereinafter also referred to as “analysis method”) includes a fractionation step of fractionating a cytoplasmic biopolymer from a target cell, and the cytoplasmic biopolymer and the cytoplasmic biomolecule height. An analysis step of analyzing at least one of the biopolymers contained in the remainder of the target cell after molecular fractionation,
The fractionation step is performed by the biopolymer fractionation method of the present invention.

　本発明によれば、例えば、前記分子篩機能を有する分子を含まない液体系においても、核酸等の生体高分子を分画できる。 According to the present invention, for example, a biopolymer such as a nucleic acid can be fractionated even in a liquid system that does not contain a molecule having the molecular sieve function.

図１（Ａ）は、実施形態１のデバイスの構成を示す上面図であり、（Ｂ）は、（Ａ）のＩ－Ｉ方向からみた断面図であり、（Ｃ）は、（Ａ）のII－II方向からみた断面図である。FIG. 1A is a top view showing the configuration of the device of Embodiment 1, FIG. 1B is a cross-sectional view taken along the II direction of FIG. 1A, and FIG. It is sectional drawing seen from the II-II direction. 図２（Ａ）は、実施形態２のデバイスの構成を示す上面図であり、（Ｂ）は、前記（Ａ）の（Ｂ）で示す二点鎖線で囲った領域の拡大図（上面図）であり、（Ｃ）は、前記（Ｂ）のＩ－Ｉ方向からみた断面図であり、（Ｄ）は、前記（Ａ）の（Ｄ）で示す二点鎖線で囲った領域の拡大図（上面図）である。FIG. 2A is a top view showing the configuration of the device of Embodiment 2, and FIG. 2B is an enlarged view (top view) of a region surrounded by a two-dot chain line shown in FIG. (C) is a cross-sectional view as viewed from the II direction of (B), and (D) is an enlarged view of a region surrounded by a two-dot chain line (D) of (A). FIG. 図３は、実施形態３のデバイスの構成を示す上面図である。FIG. 3 is a top view illustrating the configuration of the device according to the third embodiment. 図４は、変形例１のデバイスの構成を示す上面図である。FIG. 4 is a top view showing the configuration of the device of the first modification. 図５は、実施形態４のデバイスの構成を示す上面図である。FIG. 5 is a top view illustrating the configuration of the device according to the fourth embodiment. 図６は、実施形態５のデバイスの構成を示す上面図である。FIG. 6 is a top view illustrating the configuration of the device according to the fifth embodiment. 図７は、実施形態６のデバイスの構成を示す上面図である。FIG. 7 is a top view illustrating the configuration of the device according to the sixth embodiment. 図８は、実施例２における電圧印加中の核酸の分離を示す写真である。FIG. 8 is a photograph showing separation of nucleic acids during voltage application in Example 2. 図９は、実施例３における他の核酸の回収を示す写真である。FIG. 9 is a photograph showing the recovery of other nucleic acids in Example 3. 図１０は、実施例４における電圧印加中の核酸の分離を示す写真である。FIG. 10 is a photograph showing separation of nucleic acids during voltage application in Example 4. 図１１は、実施例５における補正後の蛍光強度を示すグラフである。FIG. 11 is a graph showing the fluorescence intensity after correction in Example 5. 図１２は、実施例５における蛍光強度を示すグラフである。FIG. 12 is a graph showing the fluorescence intensity in Example 5. 図１３は、実施例７における電圧印加中の核酸の分離を示す写真である。FIG. 13 is a photograph showing separation of nucleic acids during voltage application in Example 7. 図１４は、実施例８における各ライブラリが含む配列の種類を示すグラフである。FIG. 14 is a graph showing the types of sequences included in each library in Example 8. 図１５は、実施例８における各ライブラリの由来を示すグラフである。FIG. 15 is a graph showing the origin of each library in Example 8. 図１６は、実施例９における収量を示すグラフである。FIG. 16 is a graph showing the yield in Example 9. 図１７は、実施例１０におけるシミュレーション結果および近似式を示すグラフである。FIG. 17 is a graph showing simulation results and approximate expressions in Example 10.

　本発明のチップは、例えば、前記壁が、２以上の開口を有する。 In the chip of the present invention, for example, the wall has two or more openings.

　本発明のチップは、例えば、前記壁の開口の径（ｗ１）が、前記ターゲット細胞の径（ｗｔ）より小さい。 In the chip of the present invention, for example, the diameter (w 1 ) of the opening of the wall is smaller than the diameter (w t ) of the target cell.

　本発明のチップは、例えば、前記壁の開口の径（ｗ１）と前記ターゲット細胞の径（ｗｔ）との比（ｗ１：ｗｔ）が、１：２以上である。 In the chip of the present invention, for example, the ratio (w 1 : w t ) of the diameter (w 1 ) of the opening of the wall and the diameter (w t ) of the target cell is 1: 2 or more.

　本発明のチップは、例えば、前記壁の開口の径（ｗ１）が、前記細胞質の生体高分子の分離後のターゲット細胞の残部の径（ｗｎ）より小さい。 In the chip of the present invention, for example, the diameter (w 1 ) of the opening of the wall is smaller than the diameter (w n ) of the remaining target cell after separation of the cytoplasmic biopolymer.

　本発明のチップは、例えば、前記ターゲット細胞の径（ｗｔ）と前記分離用流路の径（ｗ２）との比（ｗｔ：ｗ２）が、１：１～１：１００の範囲である。 In the chip of the present invention, for example, the ratio (w t : w 2 ) of the target cell diameter (w t ) to the separation channel diameter (w 2 ) is in the range of 1: 1 to 1: 100. It is.

　本発明のチップは、例えば、前記壁の開口の断面積（Ｓ１）と前記分離用流路の断面積（Ｓ２）との比（Ｓ１：Ｓ２）が、１：２以上である。 In the chip of the present invention, for example, the ratio (S 1 : S 2 ) of the sectional area (S 1 ) of the opening of the wall and the sectional area (S 2 ) of the separation channel is 1: 2 or more. .

　本発明のチップは、例えば、前記第１の開口部が、前記基板の外側面から内側面に向かってテーパー状である。 In the chip of the present invention, for example, the first opening portion is tapered from the outer surface to the inner surface of the substrate.

　本発明のチップは、例えば、前記生体高分子分画用チップが、さらに、前記分離用流路内の液体を吸引および／または前記分離用流路内へ液体を吐出可能な吸引吐出部を１以上有し、
前記１以上の吸引吐出部は、前記壁と、前記壁を基準として前記第１の開口部とは逆方向の端部との間で、前記分離用流路に接続するように配置される。 In the chip of the present invention, for example, the biopolymer fractionation chip further includes a suction / discharge unit capable of sucking the liquid in the separation channel and / or discharging the liquid into the separation channel. Have
The one or more suction / discharge portions are arranged to connect to the separation channel between the wall and an end portion in a direction opposite to the first opening with respect to the wall.

　本発明のチップは、例えば、さらに、前記生体高分子分画用チップが、前記生体高分子の捕獲部を有し、
前記捕獲部は、前記分離用流路内において、前記壁と、前記吸引吐出部の接続部との間に配置される。 In the chip of the present invention, for example, the biopolymer fractionation chip further has a capture section for the biopolymer,
The capture part is disposed between the wall and the connection part of the suction / discharge part in the separation channel.

　本発明のチップは、例えば、前記生体高分子分画用チップが、２以上の吸引吐出部を有し、
前記捕獲部が、前記分離用流路内において、前記各吸引吐出部の接続部間に配置される。 In the chip of the present invention, for example, the biopolymer fractionation chip has two or more suction / discharge sections,
The capture part is disposed between the connection parts of the suction and discharge parts in the separation channel.

　本発明のチップは、例えば、前記生体高分子分画用チップが、さらに、電極を有し、
前記電極は、前記壁と、前記壁を基準として前記第１の開口部とは逆方向の端部との間で、前記分離用流路内に配置される。 The chip of the present invention, for example, the biopolymer fractionation chip further has an electrode,
The electrode is disposed in the separation channel between the wall and an end portion in a direction opposite to the first opening with respect to the wall.

　本発明のチップは、例えば、さらに、前記１以上の開口部が、前記細胞質の生体高分子を回収可能な第２の開口部を有し、
前記第２の開口部が、前記分離用流路に連通され、
前記壁は、前記分離用流路において、前記第１の開口部と前記第２の開口部との間に配置される。 In the chip of the present invention, for example, the one or more openings further include a second opening capable of collecting the cytoplasmic biopolymer,
The second opening communicates with the separation channel;
The wall is disposed between the first opening and the second opening in the separation channel.

　本発明のチップは、例えば、さらに、前記生体高分子分画用チップが、前記壁と前記第２の開口部との間の液体の移動を制御する液体移動制御部を有し、
前記液体移動制御部が、前記分離用流路において、前記壁と前記第２の開口部との間に配置される。 In the chip of the present invention, for example, the biopolymer fractionation chip further includes a liquid movement control unit that controls movement of the liquid between the wall and the second opening,
The liquid movement control unit is disposed between the wall and the second opening in the separation channel.

　本発明のチップは、例えば、さらに、前記ターゲット細胞の移動を調整する調整用流路と、第３の開口部とを有し、
前記第３の開口部と前記分離用流路とが、前記調整用流路に連通され、
前記分離用流路は、前記分離用流路の前記壁から前記第２の開口部側において、前記調整用流路に連通される。 The chip of the present invention further includes, for example, an adjustment channel that adjusts the movement of the target cell, and a third opening.
The third opening and the separation channel are communicated with the adjustment channel,
The separation channel communicates with the adjustment channel on the second opening side from the wall of the separation channel.

　本発明のチップは、例えば、さらに、前記分離用流路および前記調整用流路を連通する接続流路を有し、
前記接続流路は、前記分離用流路の前記壁から前記第１の開口部側において、前記分離用流路に連通される。 The chip of the present invention further includes, for example, a connection channel that communicates the separation channel and the adjustment channel,
The connection channel communicates with the separation channel on the first opening side from the wall of the separation channel.

　本発明のチップは、例えば、前記接続流路の断面積および長さが、前記壁の開口が前記ターゲット細胞をトラップしていない状態において、前記壁の開口を流れる前記試料の流量（Ｆ１）と、前記接続流路を流れる前記試料の流量（Ｆｃ）との比（Ｆ１：Ｆｃ）が、１：１～２０：１の範囲となる断面積および長さを満たす。 In the chip of the present invention, for example, the cross-sectional area and the length of the connection channel are such that the flow rate of the sample (F 1 ) flowing through the wall opening in a state where the wall opening does not trap the target cell. And the ratio (F 1 : F c ) of the flow rate (F c ) of the sample flowing through the connection flow path satisfies the cross-sectional area and length in the range of 1: 1 to 20: 1.

　本発明のチップは、例えば、さらに、電極系を有し、
前記電極系が、１以上の電極を有し、
前記１以上の電極が、前記第１の開口部内、前記分離用流路における前記壁と前記第１の開口部との間、前記第２の開口部内、および前記分離用流路における前記壁と前記第２開口部との間からなる群から選択された少なくとも１つに位置するように配置されている。 The chip of the present invention further has, for example, an electrode system,
The electrode system comprises one or more electrodes;
The one or more electrodes are in the first opening, between the wall and the first opening in the separation channel, in the second opening, and the wall in the separation channel. It arrange | positions so that it may be located in at least 1 selected from the group which consists of between said 2nd opening parts.

　本発明のチップは、例えば、前記生体高分子分画用チップが前記第３の開口部を有する生体高分子分画用チップであり、
前記１以上の電極が、さらに、前記第３の開口部内および前記調整用流路内の少なくとも一方に位置するように配置されている。 The chip of the present invention is, for example, a biopolymer fractionation chip in which the biopolymer fractionation chip has the third opening,
The one or more electrodes are further disposed so as to be located in at least one of the third opening and the adjustment channel.

　本発明のチップは、例えば、前記生体高分子が、核酸、糖、タンパク質、および脂質からなる群から選択された少なくとも１つである。 In the chip of the present invention, for example, the biopolymer is at least one selected from the group consisting of nucleic acids, sugars, proteins, and lipids.

　本発明のチップは、例えば、前記細胞質の生体高分子が、細胞質基質の生体高分子および細胞小器官の生体高分子の少なくとも一方である。 In the chip of the present invention, for example, the cytoplasmic biopolymer is at least one of a biopolymer of a cytoplasmic matrix and a biopolymer of an organelle.

　本発明のチップは、例えば、前記細胞小器官の生体高分子が、葉緑体の核酸、ミトコンドリアの核酸、およびリポソームの核酸からなる群から選択された少なくとも１つである。 In the chip of the present invention, for example, the biopolymer of the organelle is at least one selected from the group consisting of chloroplast nucleic acid, mitochondrial nucleic acid, and liposome nucleic acid.

　本発明の分画方法は、例えば、前記放出工程において、前記ターゲット細胞の細胞膜を電気的または化学的に破壊する。 In the fractionation method of the present invention, for example, in the release step, the cell membrane of the target cell is electrically or chemically destroyed.

　本発明の分画方法は、例えば、前記放出工程において、前記ターゲット細胞の細胞膜を電気的に破壊することにより、前記ターゲット細胞から細胞質の生体高分子を放出させ、
前記分離工程において、電気的分離方法により、前記放出された細胞質の生体高分子を分離する。 In the fractionation method of the present invention, for example, in the release step, a cytoplasmic biopolymer is released from the target cell by electrically destroying a cell membrane of the target cell,
In the separation step, the released cytoplasmic biopolymer is separated by an electrical separation method.

　本発明の分画方法は、例えば、さらに、前記分離工程において、分離された細胞質の生体高分子および前記細胞質の生体高分子の分画後のターゲット細胞の残部が含む生体高分子の少なくとも一方を回収する回収工程を含む。 The fractionation method of the present invention further includes, for example, at least one of the separated cytoplasmic biopolymer and the biopolymer contained in the remainder of the target cell after fractionation of the cytoplasmic biopolymer in the separation step. A recovery step for recovery is included.

　本発明の分画方法は、例えば、前記壁が、２以上の開口を有する。 In the fractionation method of the present invention, for example, the wall has two or more openings.

　本発明の分画方法は、例えば、前記壁の開口の径（ｗ１）が、前記ターゲット細胞の径（ｗｔ）より小さい。 In the fractionation method of the present invention, for example, the diameter (w 1 ) of the opening of the wall is smaller than the diameter (w t ) of the target cell.

　本発明の分画方法は、例えば、前記壁の開口の径（ｗ１）と前記ターゲット細胞の径（ｗｔ）との比（ｗ１：ｗｔ）が、１：２以上である。 In the fractionation method of the present invention, for example, the ratio (w 1 : w t ) between the diameter (w 1 ) of the wall opening and the diameter (w t ) of the target cell is 1: 2 or more.

　本発明の分画方法は、例えば、前記壁の開口の径（ｗ１）が、前記細胞質の生体高分子の分離後のターゲット細胞の残部の径（ｗｎ）より小さい。 In the fractionation method of the present invention, for example, the diameter (w 1 ) of the opening of the wall is smaller than the diameter (w n ) of the remaining portion of the target cell after separation of the cytoplasmic biopolymer.

　本発明の分画方法は、例えば、前記本発明の生体高分子分画用チップを用い、
前記第１の開口部から前記ターゲット細胞を含む試料を導入する導入工程、
前記ターゲット細胞を、前記壁の開口にトラップするトラップ工程、
前記分離用流路に電圧を印加することにより、前記ターゲット細胞から生体高分子を放出させる放出工程、および
前記壁と、前記壁を基準として前記第１の開口部とは逆方向の端部との間の前記分離用流路に、前記ターゲット細胞の細胞質の生体高分子を分離する分離工程を含む。 The fractionation method of the present invention uses, for example, the biopolymer fractionation chip of the present invention,
An introducing step of introducing a sample containing the target cells from the first opening;
A trapping step for trapping the target cell in the opening of the wall;
A release step of releasing a biopolymer from the target cell by applying a voltage to the separation channel; and the wall; and an end opposite to the first opening with respect to the wall; A separation step of separating a cytoplasmic biopolymer of the target cell in the separation channel between the two.

　本発明の分画方法は、例えば、前記生体高分子分画用チップが、前記第２の開口部を有する生体高分子分画用チップであり、
さらに、前記第２の開口部から、前記細胞質の生体高分子を回収する工程を含む。 In the fractionation method of the present invention, for example, the biopolymer fractionation chip is a biopolymer fractionation chip having the second opening,
The method further includes a step of recovering the cytoplasmic biopolymer from the second opening.

　本発明の分画方法は、例えば、さらに、前記第１の開口部から、前記細胞質の生体高分子の分離後のターゲット細胞の残部が含む生体高分子を回収する工程を含む。 The fractionation method of the present invention further includes, for example, a step of recovering the biopolymer contained in the remainder of the target cell after separation of the cytoplasmic biopolymer from the first opening.

　本発明の分画方法は、例えば、さらに、前記ターゲット細胞を含む試料を調製する工程を含む。 The fractionation method of the present invention further includes, for example, a step of preparing a sample containing the target cells.

　本発明の分画方法は、例えば、前記細胞質の生体高分子が、細胞質基質の生体高分子および細胞小器官の生体高分子の少なくとも一方である。 In the fractionation method of the present invention, for example, the cytoplasmic biopolymer is at least one of a cytosolic biopolymer and a cell organelle biopolymer.

　本発明の分画方法は、例えば、前記細胞小器官の生体高分子が、葉緑体の核酸、ミトコンドリアの核酸、およびリポソームの核酸からなる群から選択された少なくとも１つである。 In the fractionation method of the present invention, for example, the biopolymer of the organelle is at least one selected from the group consisting of chloroplast nucleic acid, mitochondrial nucleic acid, and liposome nucleic acid.

　本発明の分析方法は、例えば、前記分析された細胞質の生体高分子および細胞質の生体高分子の分画（分離）後のターゲット細胞の残部が含む生体高分子の少なくとも一方を回収する工程を含む。 The analysis method of the present invention includes, for example, a step of recovering at least one of the analyzed cytoplasmic biopolymer and the remainder of the target cell after fractionation (separation) of the cytoplasmic biopolymer. .

　本発明の分析方法は、例えば、前記分画された細胞質の生体高分子および細胞質の生体高分子の分画（分離）後のターゲット細胞の残部が含む生体高分子の分画状態を保持する保持工程を含む。 The analysis method of the present invention, for example, maintains the fractionated state of the biopolymer contained in the fractionated cytoplasmic biopolymer and the remainder of the target cell after fractionation (separation) of the cytoplasmic biopolymer. Process.

　本発明の分析方法は、例えば、前記生体高分子が、核酸であり、
前記分画された細胞質の生体高分子および細胞質の生体高分子の分画（分離）後のターゲット細胞の残部が含む生体高分子の少なくとも一方を増幅する増幅工程を含む。 In the analysis method of the present invention, for example, the biopolymer is a nucleic acid,
An amplification step of amplifying at least one of the fractionated cytoplasmic biopolymer and the remainder of the target cell after fractionation (separation) of the cytoplasmic biopolymer.

＜生体高分子分画用チップ＞
　本発明の生体高分子分画用チップは、前述のように、基板を有し、前記基板は、ターゲット細胞が有する細胞質の生体高分子を分離する分離用流路と、１以上の開口部とを有し、前記１以上の開口部は、前記分離用流路に前記ターゲット細胞を含む試料を導入可能な第１の開口部を有し、前記第１の開口部は、前記分離用流路に連通され、前記分離用流路は、その流路内であり、且つ断面方向に壁を有し、前記壁は、開口を有することを特徴とする。本発明のチップは、前記分離用流路が、その流路内であり、且つ断面方向に壁を有し、前記壁が、開口を有することを特徴とし、その他の構成および条件は、特に制限されない。 <Chip for biopolymer fractionation>
The biopolymer fractionation chip of the present invention has a substrate as described above, and the substrate has a separation channel for separating cytoplasmic biopolymers of target cells, and one or more openings. The one or more openings have a first opening through which the sample containing the target cells can be introduced into the separation channel, and the first opening is the separation channel. The separation channel is in the channel and has a wall in a cross-sectional direction, and the wall has an opening. The chip of the present invention is characterized in that the separation channel is in the channel and has a wall in a cross-sectional direction, and the wall has an opening, and other configurations and conditions are particularly limited. Not.

　本発明のチップは、前記分離用流路において、その流路内であり、且つ断面方向に壁を有し、前記壁が、開口を有する。このため、本発明のチップは、例えば、前記第１の開口部から導入したターゲット細胞を前記壁の開口にトラップした状態で、前記ターゲット細胞の細胞膜を破壊することにより、または前記ターゲット細胞の細胞膜の破壊後、前記細胞質の生体高分子の分離後のターゲット細胞の残部を前記壁の開口にトラップすることにより、前記細胞質の核酸等の細胞質の生体高分子を、前記壁の開口を通過させ、前記壁と、前記壁を基準として前記第１の開口部方向とは逆方向の端部との間の前記分離用流路に分離でき、且つ前記細胞質の生体高分子の分離後のターゲット細胞の残部が含む生体高分子（以下、「残部の生体高分子」ともいう）を、前記壁の開口にトラップできる。このため、本発明のチップによれば、前記分子篩機能を有する分子を含まない液体系においても、核酸等の生体高分子を分離できる。また、本発明のチップによれば、前記残部の生体高分子を、前記壁の開口にトラップした状態で、前記細胞質の生体高分子を分離できるため、例えば、次世代シークエンシングに利用可能な品質（例えば、高い純度）を有するサンプルを調製できる。さらに、本発明のチップは、例えば、１個のターゲット細胞を前記分離用流路に導入した際に、前記壁の開口で、前記ターゲット細胞を精度よくトラップできる。このため、本発明のチップによれば、例えば、１個のターゲット細胞から細胞質の生体高分子を分離できる。したがって、本発明のチップは、例えば、１個のターゲット細胞から細胞質の生体高分子を分離するチップということもできる。また、本発明のチップは、前記分離用流路が、前記開口を有する壁をその流路内に有する。このため、本発明のチップは、例えば、前記分離用流路に電圧を印加した際に、前記開口を有する壁を有さない分離用流路（例えば、前記特許文献１の分離用流路）と比較して、前記ターゲット細胞がトラップされる前記壁の開口の周囲の電流密度を増加させることができる。このため、例えば、前記ターゲット細胞の細胞膜を電気的に破壊する場合、本発明のチップによれば、前記特許文献１の分離用流路において、前記ターゲット細胞の細胞膜を破砕する際の電圧と比較し、より低い電圧（例えば、１／１０以下の電圧）で、前記ターゲット細胞の細胞膜を破砕できる。また、本発明のチップは、例えば、より低い電圧で使用できることから、電圧印加時のジュール熱の発生を低減でき、前記ジュール熱による核酸等の生体高分子の変性等の影響を低減できる。さらに、本発明のチップは、例えば、より低い電圧で使用できることから、生体高分子の分離に使用する分離液として、電解質を含む高伝導性溶液が使用できる。なお、本発明のチップを使用して、前記細胞質の生体高分子を分離する場合、上述のように、電気的にターゲット細胞の細胞膜を破壊してもよいし、後述するように、その他の方法により、ターゲット細胞の細胞膜を破壊してもよい。 The chip of the present invention has a wall in the cross-sectional direction in the separation channel, and the wall has an opening. For this reason, the chip of the present invention, for example, destroys the cell membrane of the target cell in a state where the target cell introduced from the first opening is trapped in the opening of the wall, or the cell membrane of the target cell After the destruction of the cytoplasmic biopolymer, by trapping the remainder of the target cell after separation of the cytoplasmic biopolymer in the opening of the wall, the cytoplasmic biopolymer such as the cytoplasmic nucleic acid is passed through the opening of the wall, The separation channel can be separated into the separation channel between the wall and the end portion in the direction opposite to the first opening direction with respect to the wall, and the target cell after separation of the cytoplasmic biopolymer can be separated. The remaining biopolymer (hereinafter also referred to as “residual biopolymer”) can be trapped in the opening of the wall. For this reason, according to the chip of the present invention, a biopolymer such as a nucleic acid can be separated even in a liquid system that does not contain a molecule having the molecular sieve function. In addition, according to the chip of the present invention, the cytoplasmic biopolymer can be separated in a state where the remaining biopolymer is trapped in the opening of the wall. For example, the quality that can be used for next-generation sequencing Samples with (eg high purity) can be prepared. Furthermore, the chip of the present invention can trap the target cells with high accuracy at the opening of the wall when, for example, one target cell is introduced into the separation channel. Therefore, according to the chip of the present invention, for example, a cytoplasmic biopolymer can be separated from one target cell. Therefore, the chip of the present invention can also be referred to as a chip that separates a cytoplasmic biopolymer from one target cell, for example. In the chip of the present invention, the separation channel has a wall having the opening in the channel. For this reason, the chip of the present invention has, for example, a separation channel that does not have a wall having the opening when a voltage is applied to the separation channel (for example, the separation channel in Patent Document 1). Compared with, the current density around the wall opening where the target cells are trapped can be increased. Therefore, for example, when electrically destroying the cell membrane of the target cell, according to the chip of the present invention, in the separation channel of Patent Document 1, compared with the voltage when crushing the cell membrane of the target cell Then, the cell membrane of the target cell can be crushed with a lower voltage (for example, a voltage of 1/10 or less). In addition, since the chip of the present invention can be used at a lower voltage, for example, the generation of Joule heat during voltage application can be reduced, and the influence of denaturation of biopolymers such as nucleic acids due to the Joule heat can be reduced. Furthermore, since the chip of the present invention can be used at a lower voltage, for example, a highly conductive solution containing an electrolyte can be used as a separation liquid used for biopolymer separation. When the chip of the present invention is used to separate the cytoplasmic biopolymer, the cell membrane of the target cell may be electrically broken as described above, or other methods as described later. Thus, the cell membrane of the target cell may be destroyed.

　本発明のチップは、前記本発明の分画方法に使用可能なチップであり、後述する本発明の分画方法における説明を援用できる。 The chip of the present invention is a chip that can be used for the fractionation method of the present invention, and the description of the fractionation method of the present invention described later can be used.

　本発明において、「チップ」は、例えば、キャピラリー管を含む。この場合、前記基板は、例えば、前記キャピラリー管の外壁を意味する。以下、チップの形態を説明するが、以下の説明は、キャピラリー管の形態の説明に援用できる。 In the present invention, the “chip” includes, for example, a capillary tube. In this case, the substrate means, for example, the outer wall of the capillary tube. Hereinafter, although the form of a chip | tip is demonstrated, the following description can be used for description of the form of a capillary tube.

　本発明において、前記チップの大きさは、特に制限されず、例えば、前記基板上に配置する前記分離用流路の数に応じて、適宜決定できる。前記チップが１つの分離用流路を含む場合、前記チップの最大長は、例えば、５０～１００ｍｍであり、前記チップの最大幅は、例えば、１０～５０ｍｍであり、前記チップの最大の厚みは、３～３０ｍｍである。本発明において、「長さ」は、前記チップの長手方向の距離であり、前記チップの最大長は、前記チップの長手方向の最長部の距離であり、「幅」は、前記チップの長手方向に対する垂直方向であり、且つ平面方向（幅方向）の距離であり、前記チップの最大幅は、前記チップの幅方向の最長部の距離であり、「厚み（深さ、高さ）」は、前記チップの長手方向および幅方向に対する垂直方向（厚み方向、高さ方向）の距離であり、前記チップの最大の厚みは、前記チップの厚み方向の最長部の距離である。 In the present invention, the size of the chip is not particularly limited, and can be appropriately determined according to, for example, the number of the separation channels arranged on the substrate. When the chip includes one separation channel, the maximum length of the chip is, for example, 50 to 100 mm, the maximum width of the chip is, for example, 10 to 50 mm, and the maximum thickness of the chip is 3 to 30 mm. In the present invention, “length” is the distance in the longitudinal direction of the chip, the maximum length of the chip is the distance of the longest portion in the longitudinal direction of the chip, and “width” is the longitudinal direction of the chip. Is the distance in the plane direction (width direction), the maximum width of the chip is the distance of the longest part in the width direction of the chip, and "thickness (depth, height)" It is the distance in the direction perpendicular to the longitudinal direction and the width direction of the chip (thickness direction, height direction), and the maximum thickness of the chip is the distance of the longest part in the thickness direction of the chip.

　本発明のチップにおいて、前記ターゲット細胞は、特に制限されず、任意の細胞とできる。前記ターゲット細胞は、例えば、１個の細胞でもよいし、２個以上の細胞でもよい。後者の場合、前記ターゲット細胞は、例えば、細胞塊があげられる。前記細胞は、例えば、生体由来の細胞、培養細胞等があげられる。前記細胞塊は、例えば、受精卵等があげられる。前記細胞の由来は、特に制限されず、例えば、ヒト、ヒトを除く非ヒト動物、植物、原核生物、真核生物等があげられる。前記非ヒト動物は、例えば、ヒトを除く非ヒト動物があげられる。前記非ヒト動物は、例えば、サル、マウス、ラット、イヌ、ウサギ、ヒツジ、ウマ、モルモット等があげられる。前記真核生物は、例えば、ユーグレナ等があげられる。 In the chip of the present invention, the target cell is not particularly limited and can be any cell. The target cell may be, for example, one cell or two or more cells. In the latter case, examples of the target cell include a cell mass. Examples of the cells include living body-derived cells and cultured cells. Examples of the cell mass include a fertilized egg. The origin of the cell is not particularly limited, and examples thereof include humans, non-human animals other than humans, plants, prokaryotes, eukaryotes, and the like. Examples of the non-human animal include non-human animals excluding humans. Examples of the non-human animal include monkeys, mice, rats, dogs, rabbits, sheep, horses, guinea pigs and the like. Examples of the eukaryote include Euglena.

　本発明において、前記分離用流路は、前記第１の開口部に連通する流路であり、その内部が空隙（中空）である。本発明において、前記分離用流路の軸方向に対する垂直方向を、「断面方向」といい、前記壁を中心として、前記第１の開口部側を上流、前記壁を基準として前記第１の開口部方向とは逆方向側を下流といい、前記第１の開口部と前記壁との間の分離用流路を、上流流路といい、前記壁と、前記壁を基準として前記第１の開口部方向とは逆方向の端部との間の前記分離用流路および前記壁と後述する第２の開口部との間の分離用流路を、下流流路という。なお、本発明において、「上流」および「下流」は、前記分離用流路における位置関係を示すための表現であり、前記分離用流路に導入される液体（例えば、前記試料）の移動方向は、特に制限されない。また、本発明において、特に言及しない限り、「流路の断面積」は、断面方向における、前記流路の内部の空隙の断面積を意味し、「流路の長さ」は、前記流路の軸方向の長さを意味する。 In the present invention, the separation channel is a channel communicating with the first opening, and the inside is a void (hollow). In the present invention, a direction perpendicular to the axial direction of the separation channel is referred to as a “cross-sectional direction”, and the first opening with the first opening side as the center and the first opening with the wall as a center. The direction opposite to the part direction is referred to as the downstream side, the separation channel between the first opening and the wall is referred to as the upstream channel, and the first reference is made on the basis of the wall and the wall. The separation channel between the end opposite to the opening direction and the separation channel between the wall and the second opening described later are referred to as downstream channels. In the present invention, “upstream” and “downstream” are expressions for indicating the positional relationship in the separation channel, and the moving direction of the liquid (for example, the sample) introduced into the separation channel. Is not particularly limited. In the present invention, unless otherwise specified, the “cross-sectional area of the flow path” means the cross-sectional area of the void inside the flow path in the cross-sectional direction, and the “length of the flow path” means the flow path This means the length in the axial direction.

　前記分離用流路の形状は、特に制限されず、その断面の形状は、円、真円、楕円等の円形；半円形；三角形、四角形、正方形および長方形等の多角形等があげられる。前記分離用流路において、前記上流流路および前記下流流路の断面形状は、例えば、同じでもよいし、異なってもよい。 The shape of the separation channel is not particularly limited, and the shape of the cross section may be a circle such as a circle, a perfect circle, or an ellipse; a semicircle; a polygon such as a triangle, a quadrangle, a square, or a rectangle. In the separation channel, the upstream channel and the downstream channel may have the same or different cross-sectional shapes, for example.

　前記分離用流路の大きさ（例えば、幅、深さ、径、断面積等）は、特に制限されず、例えば、前記ターゲット細胞が前記壁の開口まで移動可能な大きさであればよく、前記ターゲット細胞の大きさに応じて適宜決定できる。前記分離用流路において、前記上流流路の大きさが、前記ターゲット細胞が前記壁の開口まで移動可能な大きさであることが好ましい。前記分離用流路において、前記上流流路および前記下流流路は、例えば、同じ大きさでもよいし、異なる大きさでもよい。 The size of the separation channel (eg, width, depth, diameter, cross-sectional area, etc.) is not particularly limited, and may be any size as long as the target cell can move to the opening of the wall. It can be determined appropriately according to the size of the target cell. In the separation channel, the size of the upstream channel is preferably such a size that the target cell can move to the opening of the wall. In the separation channel, the upstream channel and the downstream channel may be the same size or different sizes, for example.

　具体例として、前記ターゲット細胞の径（ｗｔ）と前記分離用流路の径（ｗ２）との比（ｗｔ：ｗ２）は、例えば、１：１以上であり、好ましくは、１：１～１：１００の範囲、１：２～１：１００の範囲である。前記ターゲット細胞の径は、例えば、前記ターゲット細胞の短径である。また、前記分離用流路の径は、例えば、前記分離用流路の短径であり、前記分離用流路の断面形状が円以外の場合には、例えば、前記分離用流路の断面において、最も短い距離である。前記分離用流路において、前記上流流路の径が、前記比を満たすことが好ましい。 As a specific example, the size of the target cell (w t) and the ratio of the diameter of the separation channel (w 2) (w t: w 2) are, for example, 1: 1 or greater, preferably, 1 1 to 1: 100, 1: 2 to 1: 100. The diameter of the target cell is, for example, the short diameter of the target cell. Further, the diameter of the separation channel is, for example, the short diameter of the separation channel, and when the cross-sectional shape of the separation channel is other than a circle, for example, in the cross section of the separation channel , The shortest distance. In the separation channel, it is preferable that the diameter of the upstream channel satisfies the ratio.

　前記分離用流路は、例えば、前記生体高分子を分離するための分離液を含むことが好ましい。前記分離液は、例えば、Ｔｒｉｓ緩衝液、Ｂｉｓ－Ｔｒｉｓ緩衝液、Ｔｒｉｓ－ＨＥＰＥＳ緩衝液、イミダゾール緩衝液、リン酸緩衝液等の緩衝液、培地等があげられる。前記緩衝液の濃度は、特に制限されず、例えば、１～５００ｍｍｏｌ／Ｌである。前記分離液は、例えば、スクロース、マンニトール等の糖類、Ｔｒｉｔｏｎ（登録商標）Ｘ１００、Ｔｗｅｅｎ（登録商標）２０等の界面活性剤、牛血清アルブミン（ＢＳＡ）、アセチル化ＢＳＡ等のタンパク質、carrier RNA、carrier DNA等の核酸吸着剤、ＤＭＳＯ、pluronic（登録商標）F-127（Sigma Aldrich社製）等の溶媒、RNase inhibitor等の阻害剤、protease K等のプロテアーゼ、DNase、RNase等のヌクレアーゼ等を含んでもよい。前記分離液のｐＨは、例えば、ｐＨ６～９である。 The separation channel preferably contains, for example, a separation liquid for separating the biopolymer. Examples of the separation solution include Tris buffer solution, Bis-Tris buffer solution, Tris-HEPES buffer solution, imidazole buffer solution, phosphate buffer solution buffer medium, and the like. The concentration of the buffer is not particularly limited and is, for example, 1 to 500 mmol / L. Examples of the separation liquid include saccharides such as sucrose and mannitol, surfactants such as Triton (registered trademark) X100 and Tween (registered trademark) 20, proteins such as bovine serum albumin (BSA) and acetylated BSA, carrier RNA, Includes nucleic acid adsorbents such as carrier DNA, solvents such as DMSO and pluronic (registered trademark) F-127 (Sigma Aldrich), inhibitors such as RNase inhibitor, proteases such as protease K, nucleases such as DNase and RNase, etc. But you can. The pH of the separation liquid is, for example, pH 6-9.

　前記分離用流路は、その流路内であり、且つ断面方向に壁を有する。前記分離用流路において、前記壁の位置は、特に制限されず、例えば、前記第１の開口部端、前記第２の開口部端、またはその他の位置があげられる。前記第１の開口部端に前記壁を有する場合、例えば、前記分離用流路に前記ターゲット細胞を導入する必要がないため、前記分離用流路の大きさを自由に設定できる。前記壁が前記その他の位置の場合、前記壁の位置は、前記上流流路の長さ（ｌｕ）と、前記下流流路の長さ（ｌｄ）との比（ｌｕ：ｌｄ）が、例えば、１：１０～１：５００の範囲となる位置である。前記細胞質の生体高分子として、前記細胞質の核酸を分離する場合、前記比（ｌｕ：ｌｄ）は、例えば、より高い純度の前記細胞質の核酸を分離できることから、好ましくは、１：１００～１：５００の範囲となる位置である。 The separation channel is in the channel and has a wall in the cross-sectional direction. In the separation channel, the position of the wall is not particularly limited, and examples thereof include the first opening end, the second opening end, and other positions. When the wall is provided at the end of the first opening, for example, it is not necessary to introduce the target cell into the separation channel, so that the size of the separation channel can be freely set. If the wall is of the other positions, the position of the wall, the ratio of the length of said upstream channel and (l u), the length of the downstream flow path (l d) (l u: l d) Is a position in the range of 1:10 to 1: 500, for example. As biopolymers of the cytoplasm, when separating the nucleic acids of the cytoplasmic, the ratio (l u: l d) are, for example, because it can separate the nucleic acid of higher purity the cytoplasm of, preferably, 1: 100-200 The position is in the range of 1: 500.

　前記分離用流路の軸方向における、前記壁の長さ（厚さ、ｌｗ）は、特に制限されない。前記壁の厚さは、例えば、１～２０μｍである。前記壁の厚さを短くすることで、例えば、電気的にターゲット細胞の細胞膜を破壊する際により低い電圧で、前記ターゲット細胞の細胞膜を破砕できる。 The length (thickness, l w ) of the wall in the axial direction of the separation channel is not particularly limited. The wall thickness is, for example, 1 to 20 μm. By shortening the thickness of the wall, for example, the cell membrane of the target cell can be crushed at a lower voltage when the cell membrane of the target cell is electrically destroyed.

　前記壁は、開口（オリフィス）を有する。前記チップにおいて、前記上流流路および前記下流流路は、前記壁の開口により連通されている。前記壁の開口の数は、１以上であり、前記残部の生体高分子をより簡便に回収できることから、好ましくは、２以上であり、より好ましくは、２～３個である。 The wall has an opening (orifice). In the chip, the upstream flow channel and the downstream flow channel are communicated with each other through an opening of the wall. The number of openings in the wall is 1 or more, and is preferably 2 or more, more preferably 2 to 3 because the remaining biopolymer can be collected more easily.

　前記壁の開口の形状は、特に制限されず、その断面の形状は、円、真円、楕円等の円形；半円形；三角形、四角形、正方形および長方形等の多角形等があげられる。前記壁が２以上の開口を有する場合、各開口は、例えば、同じ断面形状でもよいし、異なる断面形状でもよい。 The shape of the opening of the wall is not particularly limited, and the shape of the cross section may be a circle such as a circle, a perfect circle or an ellipse; a semicircle; a polygon such as a triangle, a rectangle, a square or a rectangle. When the wall has two or more openings, each opening may have, for example, the same cross-sectional shape or different cross-sectional shapes.

　前記壁の開口の大きさは、特に制限されず、前記ターゲット細胞をトラップできる大きさであればよい。具体例として、前記壁の開口の径（ｗ１）は、例えば、前記ターゲット細胞をより精度よくトラップできることから、好ましくは、前記ターゲット細胞の径（ｗｔ）より小さく、前記ターゲット細胞をさらに精度よくトラップできることから、より好ましくは、前記壁の開口の径（ｗ１）と前記ターゲット細胞の径（ｗｔ）との比（ｗ１：ｗｔ）が、１：２以上、１：２～１：５０の範囲、１：１０～１：５０の範囲である。前記ターゲット細胞の径（ｗｔ）は、例えば、前記ターゲット細胞の短径である。また、前記壁の開口の径（ｗ１）は、例えば、前記壁の開口の短径であり、前記壁の開口の断面形状が円以外の場合には、例えば、前記壁の開口の断面において、最も短い距離である。 The size of the opening in the wall is not particularly limited as long as the target cell can be trapped. As a specific example, the diameter (w 1 ) of the opening of the wall is preferably smaller than the diameter (w t ) of the target cell because, for example, the target cell can be trapped more accurately. More preferably, the ratio (w 1 : w t ) of the diameter (w 1 ) of the wall opening to the diameter (w t ) of the target cell is 1: 2 or more, The range is 1:50, 1:10 to 1:50. Diameter of the target cells (w t) is, for example, a minor axis of said target cells. Further, the diameter (w 1 ) of the opening of the wall is, for example, the short diameter of the opening of the wall, and when the sectional shape of the opening of the wall is other than a circle, for example, in the section of the opening of the wall , The shortest distance.

　前記壁の開口は、例えば、前記細胞質の生体高分子をより高い純度で分離できることから、前記壁の開口の径（ｗ１）が、好ましくは、前記細胞質の生体高分子の分離後のターゲット細胞の残部（細胞質の生体高分子が分離されたターゲット細胞、以下、「ターゲット細胞の残部」ともいう）の径（ｗｎ）より小さい。前記ターゲット細胞の残部の径（ｗｎ）は、例えば、前記ターゲット細胞の残部の短径である。また、前記壁の開口の径（ｗ１）は、例えば、前記壁の開口の短径であり、前記壁の開口の断面形状が円以外の場合には、例えば、前記壁の開口の断面において、最も短い距離である。 Since the opening of the wall can separate the cytoplasmic biopolymer with higher purity, for example, the diameter (w 1 ) of the wall opening is preferably a target cell after separation of the cytoplasmic biopolymer. Smaller than the diameter (w n ) of the remaining portion (target cell from which the cytoplasmic biopolymer has been separated, hereinafter also referred to as “remaining portion of the target cell”). The diameter (w n ) of the remaining portion of the target cell is, for example, the short diameter of the remaining portion of the target cell. Further, the diameter (w 1 ) of the opening of the wall is, for example, the short diameter of the opening of the wall, and when the sectional shape of the opening of the wall is other than a circle, for example, in the section of the opening of the wall , The shortest distance.

　前記壁の開口の断面形状が長方形等の矩形である場合、前記壁の開口の径（ｗ１）は、下記式（２）を満たすことが好ましい。前記壁の開口の断面形状が円形である場合、前記壁の開口の径（ｗ１）は、下記式（３）を満たすことが好ましい。前記壁の開口の径（ｗ１）が、下記式（２）または（３）を満たすことで、例えば、前記ターゲット細胞が前記壁の開口にトラップされた状態で、前記ターゲット細胞の細胞膜を破壊でき、かつ前記ターゲット細胞の核膜の破壊を抑制できるため、より高い純度の前記細胞質の生体高分子を分離できる。
　　　ｗ１≦πｄ　　　　・・・（２）
　　　ｗ１ ２≦８ｄ２　　　・・・（３）
　　　　π：円周率
　　　　ｄ：壁の開口と、ターゲット細胞の核膜との最短距離 When the cross-sectional shape of the opening of the wall is a rectangle such as a rectangle, the diameter (w 1 ) of the opening of the wall preferably satisfies the following formula (2). If the cross-sectional shape of the opening of the wall is circular, the diameter of the opening of the wall (w 1) preferably satisfies the following formula (3). When the diameter (w 1 ) of the opening of the wall satisfies the following formula (2) or (3), for example, the cell membrane of the target cell is destroyed in a state where the target cell is trapped in the opening of the wall And the destruction of the nuclear membrane of the target cell can be suppressed, so that the cytoplasmic biopolymer with higher purity can be separated.
w 1 ≦ πd (2)
w 1 2 ≦ 8d 2 (3)
π: Circumference d: Shortest distance between wall opening and target cell nuclear membrane

　前記壁の開口の断面積（Ｓ１）は、例えば、電気的にターゲット細胞の細胞膜を破壊する際により低い電圧で、前記ターゲット細胞の細胞膜を破砕できることから、前記壁の開口の断面積（Ｓ１）と前記分離用流路の断面積（Ｓ２）との比（Ｓ１：Ｓ２）が、好ましくは、１：２以上、１：２～１：１００の範囲、１：３～１：１００の範囲、１：１０～１：１００の範囲、より好ましくは、１：３～１：１００の範囲、１：１０～１：１００の範囲を満たすように設定する。前記壁が２以上の開口を含む場合、２以上の開口の合計の断面積が、上記比を満たすことが好ましい。また、前記壁の開口の断面積が変化する場合、前記壁の開口における、最小の断面積が、上記比を満たすことが好ましい。また、前記分離用流路の断面積（Ｓ２）の断面積が変化する場合、前記分離用流路における、最大の断面積が、上記比を満たすことが好ましい。 Sectional area of the opening of the wall (S 1), for example, electrically by lower voltage when destroying the cell membrane of target cells, wherein since the cell membrane of the target cell can be disrupted, cross-sectional area of the opening of the wall (S 1 ) and the sectional area (S 2 ) of the separation channel (S 1 : S 2 ) are preferably 1: 2 or more, in the range of 1: 2 to 1: 100, 1: 3 to 1 : 100 range, 1: 10-1: 100 range, more preferably, 1: 3-1: 100 range, 1: 10-1: 100 range. When the wall includes two or more openings, the total cross-sectional area of the two or more openings preferably satisfies the above ratio. Moreover, when the cross-sectional area of the opening of the wall changes, it is preferable that the minimum cross-sectional area in the opening of the wall satisfies the above ratio. Further, the case where the cross-sectional area of the cross-sectional area of the separation channel (S 2) is changed, the in separation channel, the maximum cross-sectional area, it is preferable to satisfy the above ratio.

　前記壁が２以上の開口を含む場合、各開口の大きさは、同じでもよいし、異なってもよい。前記壁が２以上の開口を含む場合、より精度よく前記ターゲット細胞をトラップし、且つ前記残部の生体高分子をより簡易に回収できることから、全ての開口が、前述の壁の開口の大きさの条件を満たすことが好ましい。また、前記壁が異なる大きさの２以上の開口を含む場合、最大の断面積を有する開口は、例えば、前記ターゲット細胞をトラップし、その他の開口は、前記試料を通過させる。このため、前記最大の断面積を有する開口を、トラップ口、その他の開口を、バイパス口ということもできる。 When the wall includes two or more openings, the size of each opening may be the same or different. When the wall includes two or more openings, the target cells can be trapped with higher accuracy and the remaining biopolymer can be more easily collected. Therefore, all the openings have the size of the opening of the wall. It is preferable to satisfy the conditions. When the wall includes two or more openings having different sizes, the opening having the largest cross-sectional area traps the target cell, for example, and the other openings allow the sample to pass through. For this reason, the opening having the maximum cross-sectional area can also be called a trap port, and the other openings can also be called bypass ports.

　前記壁が前記トラップ口および前記バイパス口を含む場合、また、前記トラップ口の径（ｗｃ）と前記バイパス口の径（ｗｂ）との比（ｗｃ：ｗｂ）は、例えば、０．１：１～３：１の範囲である。また、前記トラップ口の断面積（Ｓｔ）と前記バイパス口の断面積（Ｓｂ）との比（Ｓｔ：Ｓｂ）は、例えば、０．１：１～３：１の範囲である。 If the wall comprises said trap inlet and said bypass port, also, the ratio of the diameter of the trap opening diameter (w c) and the bypass port (w b) (w c: w b) , for example, 0 The range is from 1: 1 to 3: 1. The ratio (S t : S b ) of the cross-sectional area (S t ) of the trap port and the cross-sectional area (S b ) of the bypass port is, for example, in the range of 0.1: 1 to 3: 1. .

　前記壁の開口の大きさは、例えば、前記壁の開口の上流流路側から下流流路側にかけて、同じでもよいし、異なってもよい。後者の場合、具体例として、前記壁の開口は、例えば、前記開口の上流流路側から下流流路側にかけてテーパー状である開口、前記開口の上流流路側および前記開口の下流流路側から、前記開口の中央にかけてテーパー状である開口等があげられる。また、前記開口が前記トラップ口と前記バイパス口とを含む場合、前記トラップ口の大きさが、前記トラップ口の上流流路側から下流流路側にかけて、異なり、前記バイパス口の大きさが、同じであることが好ましい。 The size of the opening of the wall may be the same or different from the upstream channel side to the downstream channel side of the wall opening, for example. In the latter case, as a specific example, the opening of the wall is, for example, an opening that is tapered from the upstream flow path side to the downstream flow path side of the opening, from the upstream flow path side of the opening and the downstream flow path side of the opening. The opening etc. which are taper-shaped are mentioned to the center of this. Further, when the opening includes the trap port and the bypass port, the size of the trap port is different from the upstream channel side to the downstream channel side of the trap port, and the size of the bypass port is the same. Preferably there is.

　前記第１の開口部は、前記ターゲット細胞を含む試料を導入可能な開口部である。前記第１の開口部は、後述するように、例えば、前記壁の開口の上流流路側に前記ターゲット細胞をトラップし、前記細胞質の生体高分子を前記下流流路に分離した場合、前記分離後、前記第１の開口部から吸引することにより、前記壁の開口にトラップされた前記残部の生体高分子を回収することができる。このため、前記第１の開口部は、例えば、前記残部の生体高分子を回収可能な開口部ということもできる。また、前記第１の開口部は、後述するように、例えば、前記壁の開口の下流流路側に前記ターゲット細胞をトラップし、前記細胞質の生体高分子を前記上流流路に分離した場合、前記分離後、前記第１の開口部から吸引することにより、前記細胞質の生体高分子を回収することができる。このため、前記第１の開口部は、例えば、前記細胞質の生体高分子を回収可能な開口部ということもできる。さらに、前記第１の開口部は、前記分離用流路に前記分離液を導入または導出するのに用いてもよい。前記第１の開口部は、例えば、前記分離用流路の端部で連通されていることが好ましい。 The first opening is an opening into which a sample containing the target cell can be introduced. As described later, for example, the first opening portion traps the target cell on the upstream flow path side of the opening of the wall, and separates the cytoplasmic biopolymer into the downstream flow path. By sucking from the first opening, the remaining biopolymer trapped in the opening of the wall can be recovered. For this reason, the first opening can also be referred to as an opening capable of recovering the remaining biopolymer. Further, as described later, for example, when the first opening traps the target cell on the downstream channel side of the opening of the wall and separates the cytoplasmic biopolymer into the upstream channel, After separation, the cytoplasmic biopolymer can be recovered by aspiration from the first opening. For this reason, the first opening can also be referred to as an opening capable of recovering the cytoplasmic biopolymer, for example. Furthermore, the first opening may be used to introduce or lead the separation liquid into the separation channel. It is preferable that the first opening communicates with an end of the separation channel, for example.

　本発明のチップは、さらに、第２の開口部を有してもよい。前記第２の開口部は、前記細胞質の生体高分子を回収可能な開口部である。前記第１の開口部が前記細胞質の生体高分子を回収可能な開口部である場合、前記第２の開口部は、例えば、前記細胞質の生体高分子に代えて、前記残部の生体高分子を回収可能な開口部であってもよい。このような開口部とすることにより、前記壁の開口の下流流路側に前記ターゲット細胞をトラップし、前記細胞質の生体高分子を前記上流流路に分離した場合、前記分離後、前記第２の開口部から吸引することにより、前記残部の生体高分子を回収することができる。前記第２の開口部は、例えば、前記分離用流路に連通されている。また、この場合、前記壁は、前記分離用流路において、前記第１の開口部と前記第２の開口部との間に配置されている。前記第２の開口部は、例えば、前記分離用流路に前記分離液等を導入または導出するのに用いてもよい。前記第２の開口部は、例えば、前記分離用流路の端部で連通されていることが好ましい。 The chip of the present invention may further have a second opening. The second opening is an opening capable of recovering the cytoplasmic biopolymer. When the first opening is an opening capable of recovering the cytoplasmic biopolymer, the second opening may be replaced with the remaining biopolymer instead of the cytoplasmic biopolymer, for example. The opening which can be collect | recovered may be sufficient. With such an opening, when the target cell is trapped on the downstream flow path side of the opening of the wall and the cytoplasmic biopolymer is separated into the upstream flow path, after the separation, The remaining biopolymer can be recovered by aspiration from the opening. The second opening is in communication with the separation channel, for example. In this case, the wall is disposed between the first opening and the second opening in the separation channel. The second opening may be used, for example, to introduce or lead the separation liquid or the like into the separation channel. It is preferable that the second opening is communicated with, for example, an end of the separation channel.

　前記第１の開口部の形状は、特に制限されず、前記ターゲット細胞を含む試料を導入可能であればよい。また、前記第２の開口部の形状は、特に制限されず、前記細胞質の生体高分子を回収可能であればよい。前記第１の開口部および前記第２の開口部の形状は、例えば、三角柱状、四角柱状等の多角柱状、真円柱状、楕円柱状等の円柱状、錐体状等があげられる。前記第１の開口部および前記第２の開口部は、前記基板の外側面から内側面に向かってテーパー状であることが好ましく、逆円錐状であることがより好ましい。このような形状とすることで、例えば、前記第１の開口部に前記試料を導入した際に、前記試料に圧等を加えずに、前記分離用流路に前記試料を導入できる。また、このような形状とすることで、例えば、前記細胞質の生体高分子を分離後、マイクロマニピュレータ、マイクロピペット等の吸引手段を用いて、前記第１の開口部から、前記残部の生体高分子を簡易に回収することができる。また、前記第２の開口部を有する場合、前記細胞質の生体高分子を分離後、前記吸引手段を用いて、前記細胞質の生体高分子を簡易に回収することができる。前記第１の開口部および前記第２の開口部は、例えば、同じ形状でもよいし、異なる形状でもよい。本発明において、前記開口部は、例えば、分離液等を貯留することができる。このため、前記開口部は、例えば、リザーバーということもできる。 The shape of the first opening is not particularly limited as long as the sample containing the target cell can be introduced. The shape of the second opening is not particularly limited as long as the cytoplasmic biopolymer can be recovered. Examples of the shapes of the first opening and the second opening include a polygonal column shape such as a triangular column shape and a quadrangular column shape, a cylindrical shape such as a true column shape and an elliptic column shape, and a cone shape. The first opening and the second opening are preferably tapered from the outer surface to the inner surface of the substrate, and more preferably in an inverted conical shape. By adopting such a shape, for example, when the sample is introduced into the first opening, the sample can be introduced into the separation channel without applying pressure or the like to the sample. In addition, by adopting such a shape, for example, after separating the cytoplasmic biopolymer, the remaining biopolymer is removed from the first opening by using a suction means such as a micromanipulator or a micropipette. Can be easily recovered. When the second opening is provided, the cytoplasmic biopolymer can be easily recovered using the suction means after separating the cytoplasmic biopolymer. The first opening and the second opening may have the same shape or different shapes, for example. In the present invention, the opening can store, for example, a separation liquid. For this reason, the said opening part can also be called a reservoir, for example.

　前記第１の開口部の大きさは、特に制限されず、前記試料を導入できればよい。また、前記第２の開口部の大きさは、特に制限されない。具体例として、前記第１の開口部および前記第２の開口部は、それぞれ、前記基板の外側面の直径が、例えば、３～１０ｍｍであり、前記基板の内側面の直径が、例えば、０．０１～０．２ｍｍであり、高さが、例えば、５～２５ｍｍであり、容積が、例えば、５～５００μＬ、５～６０μＬである。 The size of the first opening is not particularly limited as long as the sample can be introduced. Further, the size of the second opening is not particularly limited. As a specific example, each of the first opening and the second opening has an outer surface diameter of 3 to 10 mm, for example, and the inner surface diameter of the substrate is 0, for example. 0.01-0.2 mm, the height is, for example, 5-25 mm, and the volume is, for example, 5-500 μL, 5-60 μL.

　本発明のチップは、さらに、前記分離用流路内の液体を吸引および／または前記分離用流路内へ液体を吐出可能な吸引吐出部（手段）を有してもよい。本発明のチップは、前記吸引吐出部を有することにより、例えば、前記分離用流路内での液体の動きを制御でき、例えば、前記上流流路から前記下流流路方向または前記下流流路から前記上流流路方向へ前記分離用流路内の液体を移動させることができる。前記吸引吐出部は、特に制限されず、例えば、公知の吸引手段、吐出手段等が使用でき、具体例として、マイクロポンプ、ポンプ、分離用流路の容積変化を用いる手段、表面張力により液体を引き込む手段、圧力差を用いる手段等があげられる。本発明のチップは、例えば、前記吸引吐出部を１つ有してもよいし、２つ以上有してもよい。前記分離用流路の容積変化を用いる手段としては、例えば、前記分離用流路の外壁を形成する可撓性の基板があげられる。この場合、前記吸引吐出部は、前記基板の外側から前記基板の内側方向に押圧されることで、前記分離用流路内の液体を吐出し、前記押圧から解放されることにより、前記分離用流路内に液体を吸引する。前記圧力差を用いる手段としては、例えば、前記分離用流路内に配置された陰圧室または陽圧室があげられる。前記陰圧室は、例えば、前記分離用流路の他の部分と比較して、その室内の圧力が低い（例えば、真空）。このため、前記陰圧室の壁を破壊することにより、前記分離用流路の他の部分から前記陰圧室に向かって液体が移動するため、前記液体を吸引できる。また、前記陽圧室は、例えば、前記分離用流路の他の部分と比較して、その室内の圧力が高い。このため、前記陽圧室の壁を破壊することにより、前記陰圧室から前記分離用流路の他の部分、例えば、第１の開口部等に向かって液体が移動するため、前記液体を吐出できる。 The chip of the present invention may further include a suction / discharge section (means) capable of sucking the liquid in the separation channel and / or discharging the liquid into the separation channel. The chip of the present invention can control the movement of the liquid in the separation channel, for example, by having the suction / discharge section, for example, from the upstream channel to the downstream channel direction or from the downstream channel The liquid in the separation channel can be moved in the upstream channel direction. The suction / discharge section is not particularly limited, and for example, known suction means, discharge means, and the like can be used. Specific examples include a micropump, a pump, a means using volume change of a separation channel, and a liquid by surface tension. Means for drawing in, means for using a pressure difference, and the like. The chip of the present invention may have, for example, one suction discharge unit or two or more. Examples of the means using the change in volume of the separation channel include a flexible substrate that forms the outer wall of the separation channel. In this case, the suction / discharge section is pressed from the outside of the substrate toward the inside of the substrate, thereby discharging the liquid in the separation channel and being released from the press, thereby separating the separation Liquid is sucked into the flow path. Examples of the means using the pressure difference include a negative pressure chamber or a positive pressure chamber arranged in the separation channel. In the negative pressure chamber, for example, the pressure in the chamber is lower (for example, vacuum) than the other part of the separation channel. For this reason, by destroying the wall of the negative pressure chamber, the liquid moves from the other part of the separation channel toward the negative pressure chamber, so that the liquid can be sucked. The positive pressure chamber has a higher pressure in the chamber than, for example, other portions of the separation channel. For this reason, by destroying the wall of the positive pressure chamber, the liquid moves from the negative pressure chamber toward the other part of the separation channel, for example, the first opening. Can be discharged.

　前記吸引吐出部は、例えば、前記分離用流路内の液体を吸引および／または前記分離用流路内へ液体を吐出可能なように配置されており、具体的には、前記分離用流路内または前記分離用流路に接続（例えば、隣接）するように配置されている。前記吸引吐出部は、例えば、前記上流流路に配置されてもよいし、前記下流流路に配置されてもよいし、両者に配置されてもよい。本発明のチップが２以上の吸引吐出部を含む場合、２以上の吸引吐出部は、例えば、前記上流流路および前記下流流路の一方に配置されてもよいし、両方に配置されてもよい。 For example, the suction / discharge section is arranged so as to suck the liquid in the separation channel and / or discharge the liquid into the separation channel, and specifically, the separation channel. It arrange | positions so that it may connect to the inside or the said flow path for separation (for example, adjacent). For example, the suction / discharge section may be disposed in the upstream flow path, may be disposed in the downstream flow path, or may be disposed in both. When the chip of the present invention includes two or more suction / discharge sections, the two or more suction / discharge sections may be disposed in one of the upstream flow path and the downstream flow path, or may be disposed in both. Good.

　本発明のチップは、さらに、前記生体高分子の捕獲部を有してもよい。本発明のチップは、例えば、前記分離用流路に前記捕獲部を配置することにより、前記上流流路において残部の生体高分子を、前記下流流路において前記細胞質の高分子を捕獲することができるため、分離した生体高分子を簡易に回収することができる。前記捕獲部は、特に制限されず、例えば、公知の生体高分子の吸着手段が利用でき、例えば、前記生体高分子を特異的および／または非特異的に吸着するフィルター、前記生体高分子を特異的および／または非特異的に吸着する表面修飾を施された前記分離用流路の流路表面、前記生体高分子を特異的および／または非特異的に吸着する表面修飾が施されたビーズ、ゲル、高分子ポリマー、界面活性剤、およびミセル等があげられる。本発明のチップは、例えば、前記捕獲部を１つ有してもよいし、２つ以上有してもよい。 The chip of the present invention may further have a capture section for the biopolymer. The chip of the present invention can capture the remaining biopolymer in the upstream channel and the cytoplasmic polymer in the downstream channel, for example, by disposing the capture unit in the separation channel. Therefore, the separated biopolymer can be easily recovered. The capture unit is not particularly limited, and for example, a known biopolymer adsorption means can be used, for example, a filter that specifically and / or non-specifically adsorbs the biopolymer, and a specific biopolymer. A surface of the separation channel subjected to surface modification that adsorbs specifically and / or non-specifically, a bead subjected to surface modification that adsorbs the biopolymer specifically and / or non-specifically, Examples thereof include gels, polymer polymers, surfactants, and micelles. The chip of the present invention may have, for example, one capture unit or two or more.

　前記捕獲部は、例えば、前記分離用流路内に、前記生体高分子を捕獲可能なように配置されており、具体的には、前記分離用流路内に、前記分離用流路に導入された液体と接液可能なように配置されている。前記捕獲部は、例えば、前記上流流路に配置されてもよいし、前記下流流路に配置されてもよいし、両者に配置されてもよい。本発明のチップが２以上の捕獲部を含む場合、２以上の捕獲部は、例えば、前記上流流路および前記下流流路の一方に配置されてもよいし、両方に配置されてもよい。 For example, the capture unit is disposed in the separation channel so as to capture the biopolymer, and specifically, introduced into the separation channel in the separation channel. The liquid is arranged so as to be in contact with the liquid. For example, the capture unit may be disposed in the upstream flow channel, may be disposed in the downstream flow channel, or may be disposed in both. When the chip | tip of this invention contains two or more capture parts, two or more capture parts may be arrange | positioned at one of the said upstream flow path and the said downstream flow path, for example, and may be arrange | positioned at both.

　前記捕獲部は、前記吸引吐出部と組合せて配置することが好ましい。本発明のチップは、前記捕獲部と前記吸引吐出部とを組合せることにより、より効率良く、前記捕獲部で前記生体高分子を捕獲することができ、分離した生体高分子をより簡易に回収できる。前記捕獲部は、例えば、１つの吸引吐出部と組合せてもよいし、２つ以上の吸引吐出部と組合せてもよい。前者の場合、前記捕獲部は、例えば、前記分離用流路において、前記壁と、前記吸引吐出部の接続部との間、または前記第１の開口部もしくは前記壁を基準として前記第１の開口部とは逆方向の端部と、前記吸引吐出部の接続部との間とに配置される。このように配置することにより、さらに効率良く、前記捕獲部で前記生体高分子を捕獲することができ、分離した生体高分子をさらに簡易に回収できる。また、後者の場合、前記捕獲部は、１つの吸引吐出部と組合せた場合の配置に加え、例えば、前記各吸引吐出部の接続部間に配置されてもよい。このように配置することにより、さらに効率良く、前記捕獲部で前記生体高分子を捕獲することができ、分離した生体高分子をさらに簡易に回収できる。また、本発明のチップにおいて、前記捕獲部と前記吸引吐出部とを組合せて配置する場合、前記捕獲部と前記吸引吐出部とは、前記下流流路に配置することが好ましい。このように配置することにより、前記下流流路に配置された前記捕獲部に前記細胞質の生体高分子を捕獲後、前記第１の開口から前記分離用流路内の液体を回収することで、前記細胞質の生体高分子を前記捕獲部に分離した状態で、前記残部の生体高分子をより簡易に回収することができるため、精度よく前記残部の生体高分子を回収できる。 The capture unit is preferably arranged in combination with the suction / discharge unit. The chip of the present invention can capture the biopolymer more efficiently and more easily collect the separated biopolymer by combining the capture unit and the suction / discharge unit. it can. For example, the capturing unit may be combined with one suction / discharge unit, or may be combined with two or more suction / discharge units. In the former case, for example, in the separation flow path, the capture unit may be configured such that the first opening or the wall is used as a reference between the wall and the connection portion of the suction / discharge unit. It arrange | positions between the edge part of a reverse direction to an opening part, and the connection part of the said suction discharge part. By arranging in this way, the biopolymer can be captured more efficiently by the capture unit, and the separated biopolymer can be recovered more easily. In the latter case, in addition to the arrangement when combined with one suction / discharge section, the capture section may be disposed, for example, between the connection sections of the respective suction / discharge sections. By arranging in this way, the biopolymer can be captured more efficiently by the capture unit, and the separated biopolymer can be recovered more easily. In the chip of the present invention, when the capture unit and the suction / discharge unit are disposed in combination, the capture unit and the suction / discharge unit are preferably disposed in the downstream flow path. By arranging in this way, after capturing the cytoplasmic biopolymer in the capture unit disposed in the downstream flow path, by collecting the liquid in the separation flow path from the first opening, In the state where the cytoplasmic biopolymer is separated into the capture part, the remaining biopolymer can be collected more easily, and thus the remaining biopolymer can be collected with high accuracy.

　本発明において、前記第２の開口部を有するチップが、前記吸引吐出部および前記捕獲部の少なくとも一方を有してもよい。この場合、前記吸引吐出部および前記捕獲部の説明において、例えば、「前記壁を基準として前記第１の開口部とは逆方向の端部」を「第２の開口部」に読み替えて、その説明を援用できる。 In the present invention, the chip having the second opening may have at least one of the suction / discharge section and the capture section. In this case, in the description of the suction discharge section and the capture section, for example, “end portion in the direction opposite to the first opening portion with respect to the wall” is read as “second opening portion”, and Explanation can be used.

　本発明のチップが前記第２の開口部を有する場合、本発明のチップは、前記分離用流路内の液体の移動を制御する液体移動制御部を有することが好ましい。前記液体移動制御部は、例えば、前記第１の開口部と前記壁との間の液体の移動を制御してもよいし、前記壁と前記第２の開口部との間の液体の移動を制御してもよい。前記液体移動制御部は、例えば、一方向に液体が移動できるように制御してもよく、具体的には、前記壁から前記第２の開口部方向への液体の移動を制御可能としてもよいし、前記壁から前記第１の開口部方向への液体の移動を制御可能としてもよく、具体例として、前記第２の開口部から前記壁方向への液体の移動を抑制もしくは停止可能でもよいし、または第１の開口部から前記壁方向への液体の移動を抑制もしくは停止可能でもよい。前記液体移動制御部は、例えば、両方向の液体の移動を制御可能としてもよく、具体的には、前記第２の開口部から前記壁方向への液体の移動および第１の開口部から前記壁方向への液体の移動を抑制もしくは停止可能としてもよい。これにより、例えば、前記液体移動制御部を下流流路に配置した場合、前記細胞質の生体高分子を、前記分離用流路において、前記液体移動制御部より前記第２の開口部側に分離すると、前記分離後に、前記壁の開口の残部の生体高分子と、前記細胞質の生体高分子とが、再度混合することを防止できるため、より純度の高い前記細胞質の生体高分子および前記残部の生体高分子を回収できるようになる。前記液体移動制御部は、特に制限されず、例えば、弁、マイクロバルブ等の公知のバルブ、可撓性の基板により形成された分離用流路の壁等があげられる。前記液体移動制御部が前記可撓性の基板により形成された分離用流路の壁の場合、前記基板の外側から前記基板の内側方向に押圧されると、前記分離用流路が狭窄し、前記分離用流路内の液体の移動を抑制または停止できる。前記可撓性の基板により前記分離用流路の壁を形成する場合、前記分離用流路の壁の全体または一部を前記可撓性の基板で形成する。本発明のチップは、例えば、前記液体移動制御部を１つ有してもよいし、２つ以上有してもよい。 When the chip of the present invention has the second opening, the chip of the present invention preferably has a liquid movement control unit that controls the movement of the liquid in the separation channel. For example, the liquid movement control unit may control the movement of the liquid between the first opening and the wall, or the movement of the liquid between the wall and the second opening. You may control. For example, the liquid movement control unit may control the liquid so that the liquid can move in one direction. Specifically, the liquid movement control unit may be able to control the movement of the liquid from the wall toward the second opening. The movement of the liquid from the wall toward the first opening may be controllable. As a specific example, the movement of the liquid from the second opening toward the wall may be suppressed or stopped. Alternatively, the movement of the liquid from the first opening toward the wall may be suppressed or stopped. For example, the liquid movement control unit may be capable of controlling the movement of the liquid in both directions. Specifically, the liquid movement from the second opening to the wall and the wall from the first opening to the wall. The movement of the liquid in the direction may be suppressed or stopped. Thereby, for example, when the liquid movement control unit is arranged in the downstream flow path, the cytoplasmic biopolymer is separated from the liquid movement control unit to the second opening side in the separation flow path. Since the biopolymer in the remaining part of the opening of the wall and the biopolymer in the cytoplasm can be prevented from being mixed again after the separation, the cytoplasmic biopolymer having a higher purity and the living body in the remaining part can be prevented. The polymer can be recovered. The liquid movement control unit is not particularly limited, and examples thereof include known valves such as valves and microvalves, and walls of a separation channel formed by a flexible substrate. In the case where the liquid movement control unit is a wall of a separation channel formed by the flexible substrate, when the liquid movement control unit is pressed from the outside of the substrate toward the inside of the substrate, the separation channel is narrowed, The movement of the liquid in the separation channel can be suppressed or stopped. When the wall of the separation channel is formed by the flexible substrate, the whole or part of the wall of the separation channel is formed by the flexible substrate. The chip of the present invention may have, for example, one liquid movement control unit or two or more.

　前記液体移動制御部は、前記分離用流路において、前記上流流路に配置されてもよいし、前記下流流路に配置されてもよし、両者に配置されてもよいが、前記下流流路に配置されることが好ましい。 In the separation channel, the liquid movement control unit may be disposed in the upstream channel, may be disposed in the downstream channel, or may be disposed in both. It is preferable to arrange | position.

　本発明のチップは、例えば、前記上流流路と前記下流流路とを連通するバイパス流路を有してもよい。これにより、例えば、前記バイパス流路を介して、前記下流流路に前記ターゲット細胞を導入することができ、前記上流流路側に前記細胞質の生体高分子を分離することができる。そして、前記細胞質の生体高分子が前記上流流路に存在し、前記残部の生体高分子が前記壁の開口にトラップされているため、前記第１の開口部から前記細胞質の生体高分子を回収する際に、前記残部の生体高分子より先に回収でき、且つ前記残部の生体高分子の混入を低減できる。このため、より純度の高い前記細胞質の生体高分子を調製できる。前記バイパス流路は、前記上流流路および前記下流流路と連通していればよく、その位置は、特に制限されない。前記バイパス流路の大きさは、特に制限されず、例えば、前記分離用流路の大きさの説明を援用できる。 The chip of the present invention may have, for example, a bypass channel that communicates the upstream channel and the downstream channel. Thereby, for example, the target cell can be introduced into the downstream channel via the bypass channel, and the cytoplasmic biopolymer can be separated to the upstream channel side. Since the cytoplasmic biopolymer is present in the upstream flow path and the remaining biopolymer is trapped in the opening of the wall, the cytoplasmic biopolymer is recovered from the first opening. In this case, the remaining biopolymer can be collected before the remaining biopolymer, and contamination of the remaining biopolymer can be reduced. Therefore, the cytoplasmic biopolymer with higher purity can be prepared. The bypass channel only needs to communicate with the upstream channel and the downstream channel, and the position thereof is not particularly limited. The size of the bypass channel is not particularly limited, and for example, description of the size of the separation channel can be cited.

　本発明のチップが前記パイパス流路を有する場合、前記チップは、さらに、前記バイパス流路における液体の移動を制御する第２の液体移動制御部を有することが好ましい。前記第２の液体移動制御部は、例えば、前記バイパス流路を介した液体の移動のＯＮ／ＯＦＦを制御する。前記第２の液体移動制御部は、例えば、前記マイクロバルブ等の公知のバルブがあげられる。 When the chip of the present invention has the bypass flow path, the chip preferably further includes a second liquid movement control unit that controls the movement of the liquid in the bypass flow path. The second liquid movement control unit controls, for example, ON / OFF of liquid movement via the bypass flow path. Examples of the second liquid movement control unit include known valves such as the microvalve.

　前記第２の液体移動制御部の数は、特に制限されず、例えば、１つでもよいし、２つ以上でもよい。前記第２の液体移動制御部は、例えば、前記バイパス流路内に配置される。前記バイパス流路における前記第２の液体移動制御部の配置箇所は、特に制限されず、任意の箇所とできる。前記チップが２つ以上の第２の液体移動制御部を有する場合、前記第２の液体移動制御部は、前記バイパス流路と前記上流流路との接続部の隣接部および前記バイパス流路と前記下流流路との接続部の隣接部に配置されることが好ましい。このように配置されることで、前記バイパス流路における液体の移動のＯＮ／ＯＦＦをより精度よく制御できる。 The number of the second liquid movement control units is not particularly limited, and may be one, for example, or two or more. The second liquid movement control unit is disposed in the bypass channel, for example. The location of the second liquid movement control unit in the bypass channel is not particularly limited and can be any location. When the chip has two or more second liquid movement control units, the second liquid movement control unit includes an adjacent part of a connection part between the bypass channel and the upstream channel, and the bypass channel. It is preferable to arrange in the adjacent part of the connection part with the downstream flow path. By arranging in this way, ON / OFF of the movement of the liquid in the bypass channel can be controlled with higher accuracy.

　本発明のチップは、さらに、前記ターゲット細胞の移動を調整する調整用流路を含んでもよい。この場合、本発明のチップは、第３の開口部を有し、前記第３の開口部と前記分離用流路とが、前記調整用流路に連通され、前記分離用流路は、前記下流流路（例えば、前記分離用流路の前記壁から前記第２の開口部側）において、前記調整用流路に連通されることが好ましい。前記調整用流路を有することで、前記チップは、例えば、前記チップ内のターゲット細胞の移動を調整でき、これにより、例えば、前記ターゲット細胞の前記壁の開口からの離脱を防止できる。また、前記調整用流路を有することで、前記チップは、例えば、前記ターゲット細胞をトラップ後、前記分離液等を前記調整用流路に導入することにより、前記チップ内の溶液を置換することができる。このため、前記調整用流路を有するチップによれば、例えば、前記ターゲット細胞の洗浄、標識、前記ターゲット細胞以外の細胞由来の生体高分子の除去等ができる。 The chip of the present invention may further include an adjustment channel for adjusting the movement of the target cell. In this case, the chip of the present invention has a third opening, the third opening and the separation channel are communicated with the adjustment channel, and the separation channel is It is preferable that the downstream flow path (for example, the second opening side from the wall of the separation flow path) communicates with the adjustment flow path. By having the adjustment flow path, the chip can adjust the movement of the target cells in the chip, for example, and can prevent the target cells from detaching from the opening of the wall, for example. In addition, by having the adjustment channel, the chip replaces the solution in the chip by, for example, introducing the separation liquid into the adjustment channel after trapping the target cells. Can do. For this reason, according to the chip having the adjustment channel, for example, the target cells can be washed, labeled, and biopolymers derived from cells other than the target cells can be removed.

　前記調整用流路の形状は、特に制限されず、例えば、前記分離用流路の断面形状の説明を援用できる。前記調整用流路および前記分離用流路の形状は、同じでもよいし、異なってもよい。また、前記調整用流路の大きさ（例えば、幅、深さ、径、断面積等）は、特に制限されず、例えば、前記ターゲット細胞が移動可能な大きさであればよく、前記ターゲット細胞の大きさに応じて適宜決定できる。 The shape of the adjustment channel is not particularly limited, and for example, description of the cross-sectional shape of the separation channel can be cited. The shape of the adjustment channel and the separation channel may be the same or different. Further, the size (for example, width, depth, diameter, cross-sectional area, etc.) of the adjustment channel is not particularly limited, and may be any size as long as the target cell is movable, for example, the target cell. It can be determined as appropriate according to the size.

　前記調整用流路は、例えば、前記分離用流路の前記壁から前記第２の開口部側、すなわち、前記下流流路と連通している。前記調整用流路は、例えば、前記ターゲット細胞の移動を調整できるように、前記下流流路と連通していればよい。具体例として、前記調整用流路は、例えば、前記下流流路の前記壁の近傍で連通している。 The adjustment channel communicates with the second opening, that is, the downstream channel from the wall of the separation channel, for example. The adjustment channel may be in communication with the downstream channel so that the movement of the target cell can be adjusted, for example. As a specific example, the adjustment channel communicates in the vicinity of the wall of the downstream channel, for example.

　前記第３の開口部は、例えば、前記調整用流路に前記分離液等を導入または導出するのに用いる開口部である。前記第３の開口部の形状は、特に制限されず、例えば、前記第１の開口部および第２の開口部の形状の説明を援用できる。 The third opening is, for example, an opening used to introduce or lead the separation liquid into the adjustment channel. The shape of the third opening is not particularly limited, and for example, description of the shapes of the first opening and the second opening can be cited.

　本発明のチップは、さらに、前記分離用流路および前記調整用流路を連通する接続流路を有することが好ましい。この場合、前記接続流路は、前記分離用流路の前記壁から前記第１の開口部側において、前記分離用流路に連通されることが好ましい。前記接続流路を有することにより、前記チップは、例えば、複数のターゲット細胞を含む試料を前記第１の開口部に導入した際に、１個のターゲット細胞を、前記壁の開口にトラップする。そして、前記チップは、例えば、前記接続流路を介して、トラップされなかった細胞を前記調整用流路に移動させることができ、前記分離用流路内を１個のターゲット細胞が存在する状態にすることができる。このため、前記接続流路を有するチップによれば、例えば、複数のターゲットを含む試料を用いても、１個のターゲット細胞から生体高分子を分離することができる。前記接続流路を有するチップは、例えば、複数のターゲット細胞から、１個のターゲット細胞を分離することができる。このため、前記接続流路を有するチップは、例えば、１個のターゲット細胞を分離するチップということもできる。また、前記接続流路を有することにより、前記チップは、例えば、前記ターゲット細胞をトラップ後、前記分離液等を前記分離用流路に導入することにより、前記チップ内の溶液を置換することができる。このため、前記接続流路を有するチップによれば、例えば、前記ターゲット細胞の洗浄、標識、前記ターゲット細胞以外の細胞由来の生体高分子の除去等ができる。 It is preferable that the chip of the present invention further has a connection channel that communicates the separation channel and the adjustment channel. In this case, it is preferable that the connection channel communicates with the separation channel on the first opening side from the wall of the separation channel. By having the connection channel, the chip traps one target cell in the opening of the wall when, for example, a sample containing a plurality of target cells is introduced into the first opening. The chip can move, for example, cells that have not been trapped to the adjustment flow path via the connection flow path, and one target cell exists in the separation flow path. Can be. For this reason, according to the chip having the connection channel, for example, even when a sample including a plurality of targets is used, the biopolymer can be separated from one target cell. For example, the chip having the connection flow path can separate one target cell from a plurality of target cells. For this reason, the chip having the connection channel can also be referred to as a chip for separating one target cell, for example. In addition, by having the connection channel, the chip can replace the solution in the chip by, for example, introducing the separation liquid or the like into the separation channel after trapping the target cell. it can. For this reason, according to the chip having the connection channel, for example, the target cell can be washed, labeled, and the biopolymer derived from cells other than the target cell can be removed.

　前記接続流路の形状は、特に制限されず、例えば、前記分離用流路の断面形状の説明を援用できる。前記接続流路および前記分離用流路の形状は、同じでもよいし、異なってもよい。 The shape of the connection channel is not particularly limited, and for example, description of the cross-sectional shape of the separation channel can be cited. The shapes of the connection channel and the separation channel may be the same or different.

　前記接続流路の大きさ（例えば、幅、深さ、径、断面積等）は、特に制限されず、例えば、前記ターゲット細胞が移動可能な大きさであればよく、前記ターゲット細胞の大きさに応じて適宜決定できる。前記接続流路の断面積および長さは、例えば、前記壁の開口に１個のターゲット細胞をより精度よくトラップできることから、前記壁の開口が前記ターゲット細胞をトラップしていない状態において、前記壁の開口を流れる前記試料の流量（Ｆ１）と、前記接続流路を流れる前記試料の流量（Ｆｃ）との比（Ｆ１：Ｆｃ）が、好ましくは、１：１～２０：１の範囲、前記壁の開口に１個のターゲット細胞をより精度よくトラップでき、且つ前記残部の生体高分子をより簡便に回収できることから、より好ましくは、２：１～２０：１の範囲となる断面積および長さを満たす。前記壁の開口を流れる前記試料の流量と、前記接続流路を流れる前記試料の流量との比は、例えば、前記開口の断面積および長さ、ならびに前記接続流路の断面積および長さから近似することができ、例えば、下記式（１）で近似できる。 The size (for example, width, depth, diameter, cross-sectional area, etc.) of the connection channel is not particularly limited, and may be any size as long as the target cell can move, for example, the size of the target cell. It can be determined appropriately according to Since the cross-sectional area and the length of the connection channel can trap, for example, one target cell in the opening of the wall with higher accuracy, the wall opening is not trapped in the target cell. The ratio (F 1 : F c ) of the flow rate (F 1 ) of the sample flowing through the opening of the sample and the flow rate (F c ) of the sample flowing through the connection channel is preferably 1: 1 to 20: 1. More preferably, the range is 2: 1 to 20: 1 because one target cell can be trapped more accurately in the opening of the wall and the remaining biopolymer can be more easily recovered. Fills the cross-sectional area and length. The ratio of the flow rate of the sample flowing through the opening of the wall and the flow rate of the sample flowing through the connection channel is, for example, from the cross-sectional area and length of the opening and the cross-sectional area and length of the connection channel. For example, it can be approximated by the following equation (1).

　Ｆ１／Ｆｃ＝（Ｓ１／ｌ１）／（Ｓｃ／ｌｃ）・・・（１）
　Ｆ１：壁の開口を流れる試料の流量（ｍ３／ｓｅｃ）
　Ｆｃ：接続流路を流れる試料の流量（ｍ３／ｓｅｃ）
　Ｓ１：開口の断面積（ｍ２）
　ｌ１：開口の長さ（壁の長さ）（ｍ）
　Ｓｃ：接続流路の断面積（ｍ２）
　ｌｃ：接続流路の長さ（ｍ） F 1 / F c = (S 1 / l 1 ) / (S c / l c ) (1)
F 1 : Flow rate of the sample flowing through the wall opening (m 3 / sec)
F c : flow rate of the sample flowing through the connection channel (m 3 / sec)
S 1 : sectional area of the opening (m 2 )
l 1 : length of opening (length of wall) (m)
S c : cross-sectional area of connection channel (m 2 )
l c : length of connection channel (m)

　前記接続流路は、例えば、前記分離用流路の前記壁から前記第１の開口部側、すなわち、前記上流流路と連通している。前記接続流路は、例えば、前記接続流路を介して、前記壁の開口にトラップされなかった細胞を前記調整用流路に移動させられるように、前記上流流路と連通していればよい。具体例として、前記接続流路は、例えば、前記上流流路の前記壁の近傍で連通しており、より具体的には、前記上流流路の前記壁から、前記ターゲット細胞の径と同程度離れた位置（例えば、１０～３０μｍ）で連通している。 The connection flow path communicates with the first opening side, that is, the upstream flow path from the wall of the separation flow path, for example. The connection channel may be in communication with the upstream channel so that, for example, cells that are not trapped in the opening of the wall can be moved to the adjustment channel via the connection channel. . As a specific example, the connection channel communicates, for example, in the vicinity of the wall of the upstream channel, and more specifically, from the wall of the upstream channel, approximately the same as the diameter of the target cell. Communication is performed at a distant position (for example, 10 to 30 μm).

　前記接続流路は、例えば、前記調整用流路の任意の位置で前記調整用流路と連通する。前記接続流路は、例えば、前記下流流路と前記調整用流路との連通部から、前記調整用流路の径と同程度離れた位置で連通している。 The connection flow path communicates with the adjustment flow path at an arbitrary position of the adjustment flow path, for example. For example, the connection flow path is communicated at a position that is approximately the same as the diameter of the adjustment flow path from a communication portion between the downstream flow path and the adjustment flow path.

　本発明のチップが前記調整用流路と前記接続流路とを含む場合、前記チップは、例えば、前記分離用流路と前記調整用流路と前記接続流路との流路群を複数含み、前記流路群が、前記連続的に接続されたチップでもよい。具体的には、前記チップは、例えば、ある流路群の調整用流路の第３の開口部が、別の流路群の分離用流路の第１の開口部を兼ねるチップである。この場合、前記チップは、例えば、前記第１の開口部と前記第３の開口部とを兼ねる開口部を省略し、前記調整用流路と前記分離用流路とを直接的に連通してもよい。このように複数の流路群を含むことにより、前記チップは、例えば、複数のターゲット細胞を導入した際に、各流路群における分離用流路の壁の開口に、それぞれ、１個のターゲット細胞をトラップできる。このため、前記複数の流路群を含むチップによれば、複数のターゲット細胞を導入しても、生体高分子の分離に供されないターゲット細胞の数を低減できる。 When the chip of the present invention includes the adjustment flow path and the connection flow path, the chip includes, for example, a plurality of flow path groups of the separation flow path, the adjustment flow path, and the connection flow path. The channel group may be the continuously connected chips. Specifically, the chip is, for example, a chip in which the third opening of the adjustment channel of one channel group also serves as the first opening of the separation channel of another channel group. In this case, for example, the chip omits the opening serving as the first opening and the third opening, and directly communicates the adjustment channel and the separation channel. Also good. By including a plurality of flow channel groups in this way, the chip, for example, when a plurality of target cells are introduced, each has one target at the opening of the separation flow channel wall in each flow channel group. Can trap cells. For this reason, according to the chip including the plurality of flow channel groups, even if a plurality of target cells are introduced, the number of target cells that are not subjected to biopolymer separation can be reduced.

　本発明のチップは、例えば、前記チップ自体が電極を備えてもよいし、前記チップをセットする装置が電極を備えてもよい。前記チップ自体が電極を備える場合、本発明の分析チップは、例えば、さらに、電極系を有してもよい。この場合、前記電極系が、１以上の電極を有する。前記電極系は、例えば、１つの電極を含んでもよいし、２つ以上の電極を含んでもよい。本発明のチップにおいて、前記電極の配置箇所は、特に制限されず、例えば、前記第１の開口部、上流流路、下流流路等があげられる。前記電極は、例えば、１箇所に配置してもよいし、２箇所以上に配置してもよい。また、本発明のチップが前記第２の開口部を有する場合、前記電極は、前記第２の開口部に配置してもよい。この場合、複数の電極が、それぞれ、前記第１の開口部内および前記第２の開口部内に位置するように配置されていることが好ましい。前記第１の開口部の電極は、例えば、前記上流流路内にその一部が配置されていてもよい。また、前記第２の開口部の電極は、例えば、前記下流流路内にその一部が配置されていてもよい。前記装置が電極を備える場合、前記電極は、例えば、前記チップへの挿入が可能な固体電極が好ましく、具体例として、線電極、棒電極等があげられる。本発明のチップにおいて、前記電極は、例えば、前記第１の開口部、前記上流流路および前記下流流路のいずれかが、前記チップ自体が備える電極とし、他方の電極が、前記チップをセットする装置が備える電極としてもよい。また、本発明のチップが前記第２の開口部を有する場合、前記電極は、例えば、前記第１の開口部内の電極および前記第２の開口部の電極の一方を、前記チップ自体が備える電極とし、他方の電極が、前記チップをセットする装置が備える電極としてもよい。 In the chip of the present invention, for example, the chip itself may include an electrode, or a device for setting the chip may include an electrode. When the chip itself includes an electrode, the analysis chip of the present invention may further include an electrode system, for example. In this case, the electrode system has one or more electrodes. The electrode system may include, for example, one electrode or two or more electrodes. In the chip of the present invention, the arrangement position of the electrode is not particularly limited, and examples thereof include the first opening, the upstream flow path, and the downstream flow path. For example, the electrodes may be arranged at one place or at two or more places. When the chip of the present invention has the second opening, the electrode may be disposed in the second opening. In this case, it is preferable that the plurality of electrodes are arranged so as to be located in the first opening and the second opening, respectively. For example, a part of the electrode of the first opening may be disposed in the upstream flow path. Further, a part of the electrode of the second opening may be disposed in the downstream flow path, for example. When the device includes an electrode, the electrode is preferably, for example, a solid electrode that can be inserted into the chip, and specific examples include a line electrode and a rod electrode. In the chip of the present invention, the electrode is, for example, one of the first opening, the upstream flow path, and the downstream flow path that is provided in the chip itself, and the other electrode sets the chip. It is good also as an electrode with which the apparatus to perform. In addition, when the chip of the present invention has the second opening, the electrode includes, for example, one of the electrode in the first opening and the electrode of the second opening included in the chip itself. The other electrode may be an electrode provided in a device for setting the chip.

　前記チップ自体が、前記第１の開口部内に電極を備える場合、前記電極は、例えば、前記第１の開口部の内壁に固定化されていることが好ましい。前記チップ自体が、前記上流流路または前記下流流路に電極を備える場合、前記電極は、例えば、前記上流流路または前記下流流路の内壁に固定化されていることが好ましい。また、前記電極が前記下流流路に配置される場合、前記電極は、前記下流流路における前記壁と逆方向の端部の内壁に固定化されていることが好ましい。また、前記チップ自体が、前記第２の開口部内に電極を備える場合、前記電極は、例えば、前記第２の開口部の内壁に固定化されていることが好ましい。 When the chip itself includes an electrode in the first opening, it is preferable that the electrode is fixed to an inner wall of the first opening, for example. In the case where the chip itself includes an electrode in the upstream flow path or the downstream flow path, it is preferable that the electrode is fixed to an inner wall of the upstream flow path or the downstream flow path, for example. Moreover, when the said electrode is arrange | positioned in the said downstream flow path, it is preferable that the said electrode is being fixed to the inner wall of the edge part in the reverse direction to the said wall in the said downstream flow path. In addition, when the chip itself includes an electrode in the second opening, it is preferable that the electrode is fixed to an inner wall of the second opening, for example.

　前記電極の材料は、特に制限されず、固形の導電材料であればよく、例えば、白金、金、炭素、亜鉛、真鍮、銅、ステンレス、鉄、銀／塩化銀、パラジウム、白金黒等があげられる。 The material of the electrode is not particularly limited as long as it is a solid conductive material, such as platinum, gold, carbon, zinc, brass, copper, stainless steel, iron, silver / silver chloride, palladium, platinum black, and the like. It is done.

　本発明のチップが前記第３の開口部を備える場合、前記複数の電極が、さらに、前記第３の開口部内および前記調整用流路内の少なくとも一方に位置するように配置されていることが好ましい。これにより、例えば、前記第３の開口部の電極に電圧を印加することで、前記細胞質の生体高分子の移動、前記分離用流路内のターゲット細胞の移動、および前記ターゲット細胞の前記壁の開口へのトラップを制御でき、前記細胞質の生体高分子の回収量を向上できる。前記第３の開口部の電極は、例えば、前記調整用流路内にその一部が配置されていてもよい。前記第３の電極は、例えば、前記チップ自体が備えてもよいし、前記チップをセットする装置が電極を備えてもよい。前者の場合、前記電極は、前記第３の開口部の内壁に固定化されていることが好ましい。 When the chip of the present invention includes the third opening, the plurality of electrodes may be further disposed in at least one of the third opening and the adjustment channel. preferable. Thereby, for example, by applying a voltage to the electrode of the third opening, the movement of the cytoplasmic biopolymer, the movement of the target cell in the separation channel, and the wall of the target cell The trap to the opening can be controlled, and the amount of cytoplasmic biopolymer recovered can be improved. For example, a part of the electrode of the third opening may be disposed in the adjustment channel. For example, the third electrode may be included in the chip itself, or a device for setting the chip may include an electrode. In the former case, it is preferable that the electrode is fixed to the inner wall of the third opening.

　本発明のチップにおいて、前記チップの基板上に配置される前記分離用流路の数は、特に制限されず、例えば、１個でもよいし、２個以上でもよい。後者の場合、前記分離用流路数は、例えば、４～４００個、より具体的には、４、６、８、１６、４８、９６、３８４個等があげられる。 In the chip of the present invention, the number of the separation channels disposed on the chip substrate is not particularly limited, and may be, for example, one or two or more. In the latter case, the number of separation channels is, for example, 4 to 400, more specifically 4, 6, 8, 16, 48, 96, 384, and the like.

　本発明のチップにおいて、前記試料は、前記ターゲット細胞を含めばよい。前記試料は、例えば、前記ターゲット細胞を含む細胞液、単離したターゲット細胞を含む細胞液等があげられる。前記試料は、例えば、１個のターゲット細胞を含んでもよいし、２個以上のターゲット細胞を含んでもよい。前記チップが接続流路を有さない場合、前記試料は、１個のターゲット細胞を含むことが好ましい。前記試料の体積は、特に制限されず、例えば、０．１～５μＬである。 In the chip of the present invention, the sample may include the target cell. Examples of the sample include a cell fluid containing the target cells, a cell fluid containing isolated target cells, and the like. The sample may include, for example, one target cell or two or more target cells. When the chip does not have a connection channel, the sample preferably includes one target cell. The volume of the sample is not particularly limited and is, for example, 0.1 to 5 μL.

　本発明のチップにおいて、前記細胞質の生体高分子は、特に制限されず、細胞質中に存在する生体高分子であればよい。具体例として、前記細胞質の生体高分子は、例えば、細胞質基質の生体高分子および細胞小器官の生体高分子があげられる。前記細胞小器官の生体高分子は、例えば、葉緑体の生体高分子、ミトコンドリアの生体高分子、リポソームの生体高分子、小胞（ベシクル）の生体高分子、エンドソームの生体高分子、ゴルジ体の生体高分子、ペロキシソームの生体高分子、リソソームの生体高分子、ファゴソームの生体高分子、オートファゴソームの生体高分子、エンラージオソームの生体高分子等があげられる。本発明のチップにおいて分画される前記細胞質の生体高分子は、例えば、１種類でもよいし、２種類以上でもよい。また、本発明のチップにおいて分画される前記細胞質の生体高分子は、前記細胞質の生体高分子からなる生体高分子でもよいし、前記細胞質の生体高分子を含む生体高分子でもよい。後者の場合、前記細胞質の生体高分子は、例えば、核内ＲＮＡを含んでもよい。前記チップに印加する電圧を調整することで、例えば、所望の細胞質の生体高分子を分画できる。前記生体高分子は、例えば、体内に存在する高分子の有機化合物を意味し、具体例として、核酸、糖（多糖）、タンパク質、脂質等があげられる。前記核酸の種類は、特に制限されず、例えば、ＤＮＡでもよいし、ＲＮＡでもよい。前記脂質は、例えば、前記細胞膜等があげられる。 In the chip of the present invention, the cytoplasmic biopolymer is not particularly limited as long as it is a biopolymer present in the cytoplasm. Specific examples of the cytoplasmic biopolymer include a cytosolic biopolymer and a cell organelle biopolymer. Examples of the biopolymer of the organelle include a chloroplast biopolymer, a mitochondrial biopolymer, a liposome biopolymer, a vesicle biopolymer, an endosome biopolymer, and a Golgi apparatus. Biopolymers of peroxisomes, biopolymers of lysosomes, biopolymers of phagosomes, biopolymers of autophagosomes, biopolymers of enlargiosomes, and the like. The cytoplasmic biopolymer fractionated in the chip of the present invention may be, for example, one type or two or more types. The cytoplasmic biopolymer fractionated in the chip of the present invention may be a biopolymer composed of the cytoplasmic biopolymer or a biopolymer containing the cytoplasmic biopolymer. In the latter case, the cytoplasmic biopolymer may include, for example, nuclear RNA. By adjusting the voltage applied to the chip, for example, a desired cytoplasmic biopolymer can be fractionated. The biopolymer means, for example, a macromolecular organic compound present in the body, and specific examples thereof include nucleic acids, sugars (polysaccharides), proteins, lipids and the like. The type of the nucleic acid is not particularly limited, and may be, for example, DNA or RNA. Examples of the lipid include the cell membrane.

　本発明のチップの材料は、特に制限されず、例えば、電極を除き、前記チップの内壁が絶縁性材料で形成されていることが好ましく、より好ましくは、電極を除き、前記チップ全体が絶縁性材料から形成されていることが好ましい。本発明のチップの製造方法は、特に制限されず、例えば、射出成型等により、前記流路等を有する成型体を製造してもよいし、プレート等の基材に流路等を形成してもよい。前記流路等の形成方法は、特に制限されず、例えば、リソグラフィ、切削加工等があげられる。 The material of the chip of the present invention is not particularly limited. For example, it is preferable that an inner wall of the chip is formed of an insulating material except for an electrode, and more preferably, the entire chip is insulating except for an electrode. It is preferably formed from a material. The chip manufacturing method of the present invention is not particularly limited, and for example, a molded body having the flow path may be manufactured by injection molding or the like, or the flow path or the like is formed on a substrate such as a plate. Also good. A method for forming the flow path and the like is not particularly limited, and examples thereof include lithography and cutting.

　前記絶縁性材料は、特に制限されず、例えば、樹脂、シリコーン、ガラス、セラミックス、ゴム等があげられる。前記樹脂は、例えば、ポリエチレン、ポリプロピレン、ポリスチレン、ポリ塩化ビニル、ポリエチレンテレフタレート、ポリメタクリレート、ポリアミド、飽和ポリエステル樹脂、アクリル樹脂等の熱可塑性樹脂、尿素樹脂、メラミン樹脂、フェノール樹脂、フッ素樹脂ガラスエポキシ等のエポキシ樹脂、不飽和ポリエステル樹脂等の熱硬化性樹脂等があげられる。前記シリコーンは、例えば、ポリジメチルシロキサン等があげられる。 The insulating material is not particularly limited, and examples thereof include resin, silicone, glass, ceramics, and rubber. Examples of the resin include polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyethylene terephthalate, polymethacrylate, polyamide, saturated polyester resin, thermoplastic resin such as acrylic resin, urea resin, melamine resin, phenol resin, fluororesin glass epoxy, etc. And thermosetting resins such as epoxy resins and unsaturated polyester resins. Examples of the silicone include polydimethylsiloxane.

＜生体高分子分画装置＞
　本発明の生体高分子分画装置（以下、「分画装置」ともいう）は、前記本発明の生体高分子分画用チップを備えることを特徴とする。本発明の分画装置は、前記本発明のチップを備えることが特徴であり、その他の構成および条件は、特に制限されない。本発明の分画装置によれば、例えば、前記分子篩機能を有する分子を含まない液体系においても、生体高分子を分画できる。本発明の分画装置は、例えば、前記本発明のチップの説明を援用できる。 <Biopolymer fractionator>
The biopolymer fractionation device of the present invention (hereinafter also referred to as “fractionation device”) includes the biopolymer fractionation chip of the present invention. The fractionation device of the present invention is characterized by including the chip of the present invention, and other configurations and conditions are not particularly limited. According to the fractionation apparatus of the present invention, for example, a biopolymer can be fractionated even in a liquid system that does not contain a molecule having the molecular sieve function. For example, the description of the chip of the present invention can be cited for the fractionation apparatus of the present invention.

　本発明の分画装置は、電圧印加手段を備えることが好ましい。前記電圧印加手段は、特に制限されず、例えば、前記チップの電極系に電圧を印加できればよく、公知の手段として電圧器等が使用できる。 The fractionation device of the present invention preferably includes voltage application means. The voltage application means is not particularly limited, and may be any voltage as long as a voltage can be applied to the electrode system of the chip, and a voltage device or the like can be used as a known means.

　前記チップが電極系を備えない場合、本発明の分画装置は、さらに、電極系を備えることが好ましい。前記電極系の配置、材料等は、例えば、前述の説明を援用できる。 When the chip does not include an electrode system, the fractionation device of the present invention preferably further includes an electrode system. For example, the above description can be used for the arrangement and materials of the electrode system.

＜生体高分子の分画方法＞
　本発明の生体高分子の分画方法は、前述のように、ターゲット細胞を、開口を有する壁の開口にトラップするトラップ工程、前記ターゲット細胞の細胞膜を破壊することにより、前記ターゲット細胞から細胞質の生体高分子を放出させる放出工程、および放出された細胞質の生体高分子を分離する分離工程を含むことを特徴とする。本発明の分画方法は、前記開口を有する壁を用いて、前記細胞質の生体高分子を分画することが特徴であり、その他の構成および条件は、特に制限されない。本発明の分画方法によれば、例えば、１個のターゲット細胞から、精度よく細胞質の生体高分子を分画できる。本発明の分画方法は、例えば、前記本発明のチップ、分画装置の説明を援用できる。 <Method of fractionating biopolymer>
In the biopolymer fractionation method of the present invention, as described above, the trapping step of trapping the target cell in the opening of the wall having the opening, the cell membrane of the target cell is destroyed, and thereby the cytoplasm of the target cell is separated from the target cell. It includes a release step of releasing a biopolymer and a separation step of separating the released cytoplasmic biopolymer. The fractionation method of the present invention is characterized in that the cytoplasmic biopolymer is fractionated using the wall having the opening, and other configurations and conditions are not particularly limited. According to the fractionation method of the present invention, for example, a cytoplasmic biopolymer can be fractionated from one target cell with high accuracy. For the fractionation method of the present invention, for example, the description of the chip and fractionation apparatus of the present invention can be cited.

　本発明の分画方法において、前記壁の開口の数、ならびに前記壁の開口の径と、前記ターゲット細胞の径および前記細胞質の生体高分子の分離後のターゲット細胞の残部の径との説明は、前記本発明のチップの説明を援用できる。 In the fractionation method of the present invention, the number of the opening of the wall, the diameter of the opening of the wall, the diameter of the target cell and the diameter of the remainder of the target cell after separation of the cytoplasmic biopolymer are as follows: The description of the chip of the present invention can be cited.

　前記トラップ工程は、前記ターゲット細胞を、前記開口を有する壁の開口にトラップする工程である。前記ターゲット細胞を前記壁の開口にトラップする方法は、特に制限されず、前記壁の開口の一方から他方（例えば、下流流路の下流側の端部または第２の開口部）にむけた流れを生じさせ、前記流れにより前記ターゲット細胞をトラップする方法があげられる。前記流れは、例えば、浸透圧差、電気浸透流、誘電泳動、毛管現象等を利用し、生じさせることができる。 The trapping step is a step of trapping the target cell in the opening of the wall having the opening. The method for trapping the target cell in the opening of the wall is not particularly limited, and the flow is directed from one of the openings in the wall to the other (for example, the downstream end of the downstream flow path or the second opening). And a method for trapping the target cells by the flow. The flow can be generated using, for example, osmotic pressure difference, electroosmotic flow, dielectrophoresis, capillary action and the like.

　前記放出工程は、前記ターゲット細胞の細胞膜を破壊することにより、前記ターゲット細胞から細胞質の生体高分子を放出させる工程である。前記ターゲット細胞の細胞膜を破壊する方法は、特に制限されず、公知の細胞膜の破壊方法により実施でき、具体例として、電気的な破壊方法、化学的な破壊方法、熱を用いた破壊方法、冷却を用いた破壊方法、音波あるいは超音波を用いた破壊方法、レーザーを用いた破壊方法、流れあるいは押し付けによる力学的な破壊方法等があげられる。前記電気的な破壊方法は、例えば、前記壁の開口を挟むように一対の電極を配置し、前記一対の電極に電圧を印加することにより、破壊する方法があげられる。前記一対の電極に印加する電圧は、例えば、後述する本発明のチップを用いた分画方法における放出工程および分離工程の説明における電圧の説明を援用できる。前記化学的な破壊方法は、例えば、界面活性剤と前記ターゲット細胞とを接触させることにより破壊する方法、浸透圧を利用する方法等があげられる。 The releasing step is a step of releasing a cytoplasmic biopolymer from the target cell by destroying a cell membrane of the target cell. The method for destroying the cell membrane of the target cell is not particularly limited, and can be performed by a known cell membrane destruction method. Specific examples include an electrical destruction method, a chemical destruction method, a heat destruction method, and cooling. The destruction method using a sound wave, the destruction method using a sound wave or an ultrasonic wave, the destruction method using a laser, the mechanical destruction method by a flow or pressing, etc. are mention | raise | lifted. Examples of the electrical destruction method include a method in which a pair of electrodes are arranged so as to sandwich the opening of the wall and a voltage is applied to the pair of electrodes for destruction. For the voltage applied to the pair of electrodes, for example, the description of the voltage in the description of the discharge step and the separation step in the fractionation method using the chip of the present invention described later can be used. Examples of the chemical destruction method include a method of destroying by bringing a surfactant and the target cell into contact with each other, a method using osmotic pressure, and the like.

　前記分離工程は、放出された細胞質の生体高分子を分離する工程である。前記分離工程では、放出された細胞質の生体高分子と、前記細胞質の生体高分子の分離後のターゲット細胞の残部、すなわち、残部の生体高分子とを分離する。前記細胞質の生体高分子の分離方法は、特に制限されず、例えば、前記残部の生体高分子が、前記壁の開口にトラップされた状態で、前記細胞質の生体高分子を前記残部の生体高分子から分離可能な方法があげられ、具体例として、電気泳動等の公知の電気的分離方法があげられる。前記電気的分離方法は、例えば、前記壁の開口を挟むように一対の電極を配置し、前記一対の電極に電圧を印加することにより、分離する方法があげられる。前記一対の電極に印加する電圧は、例えば、後述する本発明のチップを用いた分画方法における放出工程および分離工程の説明における電圧の説明を援用できる。前記細胞質の生体高分子の分離方法は、例えば、その他に、前記壁の周囲、具体的には、壁の一方から他方に向かう流れを生じさせ、この流れにより、前記細胞質の生体高分子を分離する方法があげられる。 The separation step is a step of separating the released cytoplasmic biopolymer. In the separation step, the released cytoplasmic biopolymer is separated from the remainder of the target cell after separation of the cytoplasmic biopolymer, that is, the remaining biopolymer. The method for separating the cytoplasmic biopolymer is not particularly limited. For example, in the state where the remaining biopolymer is trapped in the opening of the wall, the cytoplasmic biopolymer is removed from the remaining biopolymer. The method can be separated, and specific examples include a known electrical separation method such as electrophoresis. Examples of the electrical separation method include a method of arranging a pair of electrodes so as to sandwich the opening of the wall and applying a voltage to the pair of electrodes. For the voltage applied to the pair of electrodes, for example, the description of the voltage in the description of the discharge step and the separation step in the fractionation method using the chip of the present invention described later can be used. In the method for separating the cytoplasmic biopolymer, for example, a flow around the wall, specifically, from one side of the wall to the other is generated, and the cytoplasmic biopolymer is separated by this flow. How to do.

　本発明において、前記トラップ工程と前記放出工程の順序は、特に制限されず、例えば、前記トラップ工程後に、前記放出工程を実施してもよいし、前記放出工程後に前記トラップ工程を実施してもよい。前記放出工程において、前記電気的な破壊方法により前記ターゲット細胞の細胞膜を破壊する場合、前記ターゲット細胞がトラップされる前記壁の開口の周囲の電流密度を増加させることができ、前記開口を有する壁を有さない分離用流路（例えば、前記特許文献１の分離用流路）と比較して、より低い電圧で、前記ターゲット細胞の細胞膜を破壊できることから、前記トラップ工程後に前記放出工程を実施することが好ましい。 In the present invention, the order of the trapping step and the discharging step is not particularly limited. For example, the discharging step may be performed after the trapping step, or the trapping step may be performed after the discharging step. Good. In the discharging step, when the cell membrane of the target cell is destroyed by the electrical destruction method, the current density around the opening of the wall where the target cell is trapped can be increased, and the wall having the opening Since the cell membrane of the target cell can be destroyed at a lower voltage compared to a separation channel (for example, the separation channel disclosed in Patent Document 1) that does not have a cell, the release step is performed after the trap step. It is preferable to do.

　本発明の分画方法は、例えば、前記本発明のチップを用いて実施することもできる。この場合、本発明の分画方法は、例えば、前記第１の開口部から前記ターゲット細胞を含む試料を導入する導入工程、前記ターゲット細胞を、前記壁の開口にトラップするトラップ工程、前記ターゲット細胞から生体高分子を放出させる放出工程、および前記壁を基準として前記ターゲット細胞がトラップされる側とは逆方向の前記分離用流路に、前記ターゲット細胞の細胞質の生体高分子を分離する分離工程を含む。前記ターゲット細胞を前記壁の開口の上流流路側にトラップする場合、前記分離工程では、例えば、前記細胞質の生体高分子を前記下流流路に分離する。また、前記ターゲット細胞を前記壁の開口の下流流路側にトラップする場合、前記分離工程では、例えば、前記細胞質の生体高分子を前記上流流路に分離する。以下、前記壁の開口の上流流路側にトラップし、前記分離用流路に電圧を印加することにより、前記放出工程および前記分離工程を実施する場合を例にあげて説明するが、本発明は、これに何ら限定されない。 The fractionation method of the present invention can be carried out, for example, using the chip of the present invention. In this case, the fractionation method of the present invention includes, for example, an introducing step of introducing a sample containing the target cell from the first opening, a trapping step of trapping the target cell in the opening of the wall, the target cell And a separation step of separating the cytoplasmic biopolymer of the target cell into the separation channel in a direction opposite to the side on which the target cell is trapped with respect to the wall. including. When trapping the target cells on the upstream channel side of the opening of the wall, in the separation step, for example, the cytoplasmic biopolymer is separated into the downstream channel. When the target cell is trapped on the downstream channel side of the opening of the wall, in the separation step, for example, the cytoplasmic biopolymer is separated into the upstream channel. Hereinafter, the case where the discharge step and the separation step are performed by trapping on the upstream channel side of the opening of the wall and applying a voltage to the separation channel will be described as an example. This is not a limitation.

　前記本発明のチップを用いて、本発明の分画方法を実施する場合、本発明の分画方法は、さらに、前記分離用流路に前記分離液を供給する工程を含んでもよい。前記分離液は、例えば、前述の説明を援用できる。 When performing the fractionation method of the present invention using the chip of the present invention, the fractionation method of the present invention may further include a step of supplying the separation liquid to the separation channel. For example, the above description can be used for the separation liquid.

　前記導入工程は、前記第１の開口部から前記ターゲット細胞を含む試料を導入する工程である。前記試料の導入方法は、特に制限されず、例えば、公知の分注手段等を用いることができる。前記導入工程において、導入される試料の体積は、例えば、前述の説明を援用できる。 The introduction step is a step of introducing a sample containing the target cells from the first opening. The method for introducing the sample is not particularly limited, and for example, a known dispensing means can be used. In the introduction step, the above description can be used for the volume of the sample to be introduced, for example.

　前記トラップ工程は、前記ターゲット細胞を、前記壁の開口にトラップする工程である。前記試料が圧等を加えずに前記第１の開口部から前記分離用流路に導入される場合、前記ターゲット細胞は、例えば、前記試料または前記分離液の流れにより、前記分離用流路内を前記壁の方向に移動し、前記壁の開口にトラップされる。また、前記試料が圧等を加えることにより、前記第１の開口部から前記分離用流路に導入される場合、前記ターゲット細胞は、例えば、前記第１の開口部から前記下流流路方向（例えば、前記第２の開口部方向）への電気浸透流を生じさせることにより、前記分離用流路内を前記壁の方向に移動し、前記壁の開口にトラップされる。前記電気浸透流は、例えば、前記第１の開口部の電極と前記下流流路側の電極（例えば、前記下流流路の電極、前記第２の開口部の電極等）とに電圧を印加することにより生じる。また、前記チップが前記第３の開口部を有する場合、例えば、前記第１の開口部から前記第３の開口部への前記試料もしくは前記分離液の流れ、または前記電気浸透流等により、前記ターゲット細胞は、前記分離用流路内を前記壁の方向に移動し、前記壁の開口にトラップされてもよい。 The trapping step is a step of trapping the target cell at the opening of the wall. When the sample is introduced into the separation channel from the first opening without applying pressure or the like, the target cell is, for example, in the separation channel by the flow of the sample or the separation liquid. Is moved in the direction of the wall and trapped in the opening of the wall. In addition, when the sample is introduced into the separation channel from the first opening by applying pressure or the like, the target cell is, for example, from the first opening to the downstream channel direction ( For example, by generating an electroosmotic flow in the direction of the second opening), the separation channel is moved in the direction of the wall and trapped in the opening of the wall. The electroosmotic flow applies a voltage to, for example, the electrode of the first opening and the electrode on the downstream channel side (for example, the electrode of the downstream channel, the electrode of the second opening, etc.). Caused by. Further, when the chip has the third opening, for example, by the flow of the sample or the separation liquid from the first opening to the third opening, the electroosmotic flow, or the like, The target cell may move in the direction of the wall in the separation channel and be trapped in the opening of the wall.

　前記放出工程は、例えば、前記分離用流路に電圧を印加することにより、前記ターゲット細胞から生体高分子を放出させる工程である。また、前記分離工程は、例えば、前記下流流路側（例えば、前記第２の開口部側）に、前記ターゲット細胞の細胞質の生体高分子を分離する工程である。具体的に、前記放出工程は、例えば、前記第１の開口部の電極および前記下流流路側の電極（例えば、前記下流流路の電極、前記第２の開口部の電極等）に電圧を印加することにより実施できる。また、前記分離工程は、前記第１の開口部の電極および前記下流流路側の電極に電圧を印加することにより実施できる。前記分離用流路への電圧印加は、例えば、電圧印加手段により実施できる。前記電圧印加手段は、例えば、前述の説明を援用できる。前記第１の開口部の電極および前記下流流路側の電極に印加する電圧は、特に制限されず、前記ターゲット細胞の細胞膜を破砕できる電圧であればよく、例えば、前記分離液の種類に応じて、適宜設定できる。具体的に、前記第１の開口部の電極の電圧（Ｖ１）と、前記下流流路側の電極の電圧（Ｖ２）との差の絶対値（｜Ｖ１－Ｖ２｜）が、例えば、５０～１０００Ｖの範囲である。具体例として、Ｖ１とＶ２との組合せは、特に制限されず、Ｖ２が０Ｖの場合、Ｖ１は、例えば、－５０～－１０００Ｖであり、Ｖ１が０Ｖの場合、Ｖ２は、例えば、５０～１０００Ｖである。前記チップが前記第３の開口部を有する場合、前記第３の開口部の電極に電圧を印加してもよい。前記第３の開口部の電極の電圧は、例えば、前記調整用流路内の液体中の陰イオンが、前記分離用流路へ流入することにより、前記分離用流路から前記調整用流路への陰イオンの流出を抑制可能な範囲で、適宜設定できる。前記第１の開口部の電極の電圧および前記下流流路側の電極の電圧が前記具体例の範囲の場合、前記第３の開口部の電極の電圧は、例えば、－５０～－１０００Ｖに設定できる。前記電極への電圧の印加時間は、例えば、前記電圧印加後の電流値が平衡状態に達する時間であり、具体例として、１０～５００秒に設定できる。 The releasing step is a step of releasing the biopolymer from the target cell, for example, by applying a voltage to the separation channel. The separation step is a step of separating the cytoplasmic biopolymer of the target cell, for example, on the downstream flow channel side (for example, the second opening side). Specifically, in the discharging step, for example, a voltage is applied to the electrode of the first opening and the electrode on the downstream channel side (for example, the electrode of the downstream channel, the electrode of the second opening, etc.) Can be implemented. Further, the separation step can be performed by applying a voltage to the electrode of the first opening and the electrode on the downstream flow path side. The voltage application to the separation channel can be performed by, for example, a voltage application unit. The above-mentioned explanation can be used for the voltage application means, for example. The voltage applied to the electrode of the first opening and the electrode on the downstream flow path side is not particularly limited as long as it is a voltage that can disrupt the cell membrane of the target cell. For example, depending on the type of the separation liquid Can be set as appropriate. Specifically, the absolute value (| V 1 −V 2 |) of the difference between the voltage (V 1 ) of the electrode of the first opening and the voltage (V 2 ) of the electrode on the downstream channel side is, for example, , 50 to 1000V. As a specific example, the combination of V 1 and V 2 is not particularly limited. When V 2 is 0 V, V 1 is, for example, −50 to −1000 V, and when V 1 is 0 V, V 2 is For example, it is 50 to 1000V. When the chip has the third opening, a voltage may be applied to the electrode of the third opening. The voltage of the electrode of the third opening is, for example, that the anion in the liquid in the adjustment channel flows from the separation channel into the adjustment channel by flowing into the separation channel. As long as the outflow of anions to the can be suppressed, it can be set as appropriate. When the voltage of the electrode of the first opening and the voltage of the electrode on the downstream flow path side are in the range of the specific example, the voltage of the electrode of the third opening can be set to −50 to −1000 V, for example. . The voltage application time to the electrode is, for example, a time for the current value after the voltage application to reach an equilibrium state, and can be set to 10 to 500 seconds as a specific example.

　本発明の分画方法は、さらに、前記細胞質の生体高分子を精製する工程を含むことが好ましい。前記精製工程では、例えば、前記細胞質の生体高分子に含まれる複数の生体高分子のうち、いずれか１つまたは２つ以上の生体高分子を精製する。本発明の分画方法は、前記精製工程を含むことにより、例えば、前記ターゲット細胞の細胞膜の破砕により、放出されたタンパク質と、前記細胞質の核酸とを分離でき、例えば、より純度の高い前記細胞質の核酸を分画できる。前記細胞質の核酸の精製方法は、特に制限されず、公知のタンパク質と核酸との分画方法が利用でき、例えば、等速電気泳動法等により実施できる。 The fractionation method of the present invention preferably further includes a step of purifying the cytoplasmic biopolymer. In the purification step, for example, any one or two or more of the biopolymers contained in the cytoplasmic biopolymer are purified. By including the purification step, the fractionation method of the present invention can separate the released protein and the cytoplasmic nucleic acid, for example, by disrupting the cell membrane of the target cell. For example, the cytoplasm having higher purity can be separated. Can be fractionated. The method for purifying the cytoplasmic nucleic acid is not particularly limited, and a known fractionation method of protein and nucleic acid can be used. For example, it can be performed by isotachophoresis or the like.

　本発明の分画方法は、さらに、前記分離工程において、分離された細胞質の生体高分子を、所定位置まで輸送してもよい。前記所定位置は、例えば、前記本発明のチップにおける第１の開口部、上流流路、下流流路、第２の開口部等があげられる。前記輸送は、例えば、前記分離方法と同様にして実施できる。 The fractionation method of the present invention may further transport the separated cytoplasmic biopolymer to a predetermined position in the separation step. Examples of the predetermined position include a first opening, an upstream flow channel, a downstream flow channel, and a second opening in the chip of the present invention. The transport can be performed, for example, in the same manner as the separation method.

　本発明の分画方法は、例えば、さらに、前記細胞質の生体高分子および前記残部の生体高分子の少なくとも一方を回収する回収工程を含んでもよい。前記細胞質の生体高分子および前記残部の生体高分子の回収は、例えば、マイクロマニピュレータ、マイクロピペット等の吸引手段等を用いて、実施できる。本発明の分画方法が前記本発明のチップを用い、前記細胞質の生体高分子を前記下流流路側に分離する場合、前記細胞質の生体高分子および前記残部の生体高分子は、例えば、前記第１の開口部から回収できる。また、前記本発明のチップが前記第２の開口部を有する場合、前記細胞質の生体高分子および前記残部の生体高分子は、例えば、それぞれ、前記第２の開口部および前記第１の開口部から回収できる。本発明の分画方法は、例えば、前記残部の生体高分子のみを回収してもよい。前記残部の生体高分子は、例えば、前記吸引手段を用いて、前記第１の開口部から回収できる。また、前記本発明のチップが前記捕獲部を有する場合、例えば、前記捕獲部を回収することにより、前記捕獲部に捕獲された前記細胞質の生体高分子および前記残部の生体高分子の少なくとも一方の生体高分子を回収できる。 The fractionation method of the present invention may further include, for example, a recovery step of recovering at least one of the cytoplasmic biopolymer and the remaining biopolymer. The cytoplasmic biopolymer and the remaining biopolymer can be collected using, for example, a suction means such as a micromanipulator or a micropipette. When the fractionation method of the present invention uses the chip of the present invention to separate the cytoplasmic biopolymer to the downstream flow channel side, the cytoplasmic biopolymer and the remaining biopolymer are, for example, the first It can collect | recover from one opening part. When the chip of the present invention has the second opening, the cytoplasmic biopolymer and the remaining biopolymer are, for example, the second opening and the first opening, respectively. Can be recovered from. In the fractionation method of the present invention, for example, only the remaining biopolymer may be recovered. The remaining biopolymer can be recovered from the first opening using, for example, the suction means. In addition, when the chip of the present invention includes the capture unit, for example, by collecting the capture unit, at least one of the cytoplasmic biopolymer captured by the capture unit and the remaining biopolymer Biopolymers can be recovered.

　前記生体高分子が核酸である場合、本発明の分画方法は、例えば、前記分離された細胞質の核酸および前記残部の核酸の少なくとも一方の核酸を増幅する増幅工程を含んでもよい。また、前記核酸がＲＮＡの場合、本発明の分画方法は、前記増幅工程に先立ち、前記ＲＮＡからｃＤＮＡを合成する逆転写工程を含んでもよい。本発明の分画方法は、このような工程を含むことにより、分離した細胞質の核酸および他の核酸から核酸分析方法に供するための核酸試料を作製することができる。前記核酸試料は、例えば、ｃＤＮＡライブラリということもできる。前記核酸の増幅および逆転写は、例えば、公知の核酸増幅方法および核酸の逆転写方法により実施できる。 When the biopolymer is a nucleic acid, the fractionation method of the present invention may include, for example, an amplification step of amplifying at least one of the separated cytoplasmic nucleic acid and the remaining nucleic acid. When the nucleic acid is RNA, the fractionation method of the present invention may include a reverse transcription step of synthesizing cDNA from the RNA prior to the amplification step. By including such steps, the fractionation method of the present invention can prepare a nucleic acid sample for use in the nucleic acid analysis method from the separated cytoplasmic nucleic acid and other nucleic acids. The nucleic acid sample can also be referred to as, for example, a cDNA library. The amplification and reverse transcription of the nucleic acid can be performed, for example, by a known nucleic acid amplification method and nucleic acid reverse transcription method.

　本発明の分画方法は、さらに、前記分画された細胞質の生体高分子および細胞質の生体高分子の分離後のターゲット細胞の残部が含む生体高分子の分画状態を保持する保持工程を含んでもよい。これにより、前記分画された細胞質の生体高分子および残部の生体高分子が再混合することを防止できる。前記分画状態の保持は、例えば、前記分画された細胞質の生体高分子および残部の生体高分子の再混合を防止できる程度に、前記細胞質の生体高分子および前記残部の生体高分子の少なくとも一方の生体高分子の移動を抑制することを意味する。前記分画状態の保持は、例えば、前記分画された細胞質の生体高分子および残部の生体高分子間の液体をゲル化、固体化等することにより実施できる。前記ゲル化により前記分画状態を保持する場合、例えば、ＰＥＧ－ＤＡ（Poly(ethylene glycol) diacrylate）、Pluronic（登録商標） F127、gelatin methacrylateの刺激応答性のゲル化剤を、前記分画された細胞質の生体高分子および残部の生体高分子間の液体と混合または置換させ、刺激を加えることにより分画状態を保持できる。また、前記液体移動制御部を有する本発明のチップを使用し、本発明の分画方法を実施する場合、前記分画状態の保持は、例えば、前記液体移動制御部により実施できる。具体例として、前記下流流路に前記液体移動制御部を有するチップを用い、前記下流流路側に前記細胞質の生体高分子を分離する場合、前記液体移動制御部として、前記下流流路から前記上流流路方向へ前記分離用流路内の液体の移動を抑制する液体移動制御部を用いることで、前記分離工程において、前記細胞質の生体高分子を、前記液体移動制御部より下流側に輸送することにより、前記分画された細胞質の生体高分子および細胞質の生体高分子の分離後のターゲット細胞の残部が含む生体高分子の分画状態を保持することができる。 The fractionation method of the present invention further includes a holding step of retaining the fractionated state of the biopolymer contained in the fractionated cytoplasmic biopolymer and the remainder of the target cell after separation of the cytoplasmic biopolymer. But you can. Thereby, it is possible to prevent the fractionated cytoplasmic biopolymer and the remaining biopolymer from being mixed again. The maintenance of the fractionated state is, for example, at least of the cytoplasmic biopolymer and the remaining biopolymer to such an extent that re-mixing of the fractionated cytoplasmic biopolymer and the remaining biopolymer can be prevented. It means to suppress the movement of one biopolymer. The fractionation state can be maintained, for example, by gelling or solidifying the liquid between the fractionated cytoplasmic biopolymer and the remaining biopolymer. When the fractionation state is maintained by the gelation, for example, the fractionated responsive gelling agent such as PEG-DA (Poly (ethylene glycol) diacrylate), Pluronic (registered trademark) F127, gelatin methacrylate is separated. The fractional state can be maintained by mixing or substituting the liquid between the cytoplasmic biopolymer and the remaining biopolymer and applying a stimulus. In addition, when the chip of the present invention having the liquid movement control unit is used and the fractionation method of the present invention is performed, the fractionation state can be maintained, for example, by the liquid movement control unit. As a specific example, when a chip having the liquid movement control unit is used in the downstream flow channel and the cytoplasmic biopolymer is separated on the downstream flow channel side, the liquid movement control unit is used as the liquid movement control unit. By using a liquid movement control unit that suppresses the movement of the liquid in the separation channel in the flow path direction, the cytoplasmic biopolymer is transported downstream from the liquid movement control unit in the separation step. Thereby, the fractionated state of the biopolymer contained in the fractionated cytoplasmic biopolymer and the remainder of the target cell after separation of the cytoplasmic biopolymer can be maintained.

＜生体高分子の分析方法＞
　本発明の生体高分子の分析方法は、前述のように、ターゲット細胞から細胞質の生体高分子を分画する分画工程、および前記細胞質の生体高分子および前記細胞質の生体高分子の分画後のターゲット細胞の残部が含む生体高分子の少なくとも一方を分析する分析工程を含み、前記分画工程が、前記本発明の生体高分子の分画方法により実施されることを特徴とする。本発明の分析方法は、前記分画工程が、前記本発明の分画方法により実施されることが特徴であり、その他の工程および条件は、特に制限されない。本発明の分析方法によれば、例えば、１個のターゲット細胞から、精度よく細胞質の生体高分子を分画し、前記細胞質の生体高分子および前記残部の生体高分子を分析できる。本発明の分析方法は、前記本発明の分画方法の説明を援用できる。本発明において、分析は、例えば、定性分析および定量分析のいずれの意味も含む。 <Method for analyzing biopolymers>
As described above, the method for analyzing a biopolymer of the present invention includes a fractionation step of fractionating a cytoplasmic biopolymer from a target cell, and after fractionation of the cytoplasmic biopolymer and the cytoplasmic biopolymer. An analysis step of analyzing at least one of the biopolymers contained in the remainder of the target cells, wherein the fractionation step is performed by the biopolymer fractionation method of the present invention. The analysis method of the present invention is characterized in that the fractionation step is performed by the fractionation method of the present invention, and other steps and conditions are not particularly limited. According to the analysis method of the present invention, for example, a cytoplasmic biopolymer can be accurately fractionated from one target cell, and the cytoplasmic biopolymer and the remaining biopolymer can be analyzed. The description of the fractionation method of the present invention can be used for the analysis method of the present invention. In the present invention, the analysis includes, for example, any meaning of qualitative analysis and quantitative analysis.

　本発明の分析方法は、例えば、前記本発明のチップを用いて前記分画工程を実施する場合、さらに、前記ターゲット細胞を含む試料を調製する工程を含んでもよい。前記試料の調製方法は、特に制限されず、前記ターゲット細胞の種類に応じて、適宜決定できる。１個のターゲット細胞を含む試料を調製する場合、前記試料は、例えば、フローサイトメーターを用い、所望の１個のターゲット細胞を分離することにより、調製できる。また、前記試料は、例えば、マイクロマニピュレータ、マイクロピペット等の吸引手段を用い、所望の１個のターゲット細胞を分離することにより、調製できる。 For example, when the fractionation step is performed using the chip of the present invention, the analysis method of the present invention may further include a step of preparing a sample containing the target cells. The method for preparing the sample is not particularly limited, and can be appropriately determined according to the type of the target cell. When preparing a sample containing one target cell, the sample can be prepared, for example, by separating a desired single target cell using a flow cytometer. Moreover, the said sample can be prepared by isolate | separating a desired one target cell using suction means, such as a micromanipulator and a micropipette, for example.

　前記分画工程は、例えば、前記生体高分子の分画方法の説明を援用できる。 For the fractionation step, for example, the description of the biopolymer fractionation method can be cited.

　前記分析工程では、前記細胞質の生体高分子および前記細胞質の生体高分子の分画（分離）後のターゲット細胞の残部が含む生体高分子の少なくとも一方を分析する。前記分析工程では、例えば、前記細胞質の生体高分子および前記残部の生体高分子のいずれかを分析してもよいし、両者を分析してもよい。前記残部の生体高分子は、例えば、前記分画工程において、分画された生体高分子以外の生体高分子であり、具体的には、核の生体高分子等があげられる。前記分画工程において、前記細胞質の生体高分子の一部の核酸を分画した場合、前記残部の生体高分子は、例えば、分画しなかった他の細胞質の生体高分子を含んでもよい。 In the analysis step, at least one of the cytoplasmic biopolymer and the biopolymer contained in the remainder of the target cell after fractionation (separation) of the cytoplasmic biopolymer is analyzed. In the analysis step, for example, either the cytoplasmic biopolymer or the remaining biopolymer may be analyzed, or both may be analyzed. The remaining biopolymer is, for example, a biopolymer other than the biopolymer fractionated in the fractionation step, and specifically includes a nuclear biopolymer. In the fractionation step, when a part of the nucleic acid of the cytoplasmic biopolymer is fractionated, the remaining biopolymer may contain, for example, another cytoplasmic biopolymer that has not been fractionated.

　前記細胞質の生体高分子および前記残部の生体高分子の分析方法は、特に制限されず、前記分析の対象および目的に応じて適宜決定できる。具体例として、前記生体高分子が核酸であり、分析対象の核酸の有無を分析する場合、例えば、前記分析対象の核酸にハイブリダイズするプローブを用い、融解曲線法等により分析できる。前記分析対象の核酸の発現量を分析する場合、例えば、ＰＣＲ、ｑＲＴ－ＰＣＲ等により分析できる。また、複数の分析対象の核酸の発現パターンを分析する場合、例えば、ＲＮＡ－Ｓｅｑ等のトランスクリプトーム解析、ＤＮＡマイクロアレイ解析等により分析できる。また、前記生体高分子が糖であり、糖の分子量、構造等を分析する場合、液体クロマトグラフ－飛行時間型質量分析計（ＬＣ－ＴＯＦ－ＭＳ）により分析できる。前記生体高分子がタンパク質であり、分析対象のタンパク質有無、量等を分析する場合、ウエスタンプロット（western blotting）、extended ligation assay、proximity ligation assay等により分析できる。 The method of analyzing the cytoplasmic biopolymer and the remaining biopolymer is not particularly limited, and can be appropriately determined according to the analysis target and purpose. As a specific example, when the biopolymer is a nucleic acid and the presence / absence of the nucleic acid to be analyzed is analyzed, for example, a probe that hybridizes to the nucleic acid to be analyzed can be used and analyzed by a melting curve method or the like. When analyzing the expression level of the nucleic acid to be analyzed, for example, it can be analyzed by PCR, qRT-PCR or the like. In addition, when analyzing the expression patterns of a plurality of nucleic acids to be analyzed, it can be analyzed by, for example, transcriptome analysis such as RNA-Seq, DNA microarray analysis or the like. Further, when the biopolymer is a sugar and the molecular weight, structure, etc. of the sugar are analyzed, it can be analyzed by a liquid chromatograph-time-of-flight mass spectrometer (LC-TOF-MS). When the biopolymer is a protein and the presence / absence, amount, etc. of the protein to be analyzed are analyzed, it can be analyzed by Western plotting, extended ligation assay, proximity ligation assay, etc.

　本発明の分析方法は、さらに、分析された細胞質の生体高分子および残部の生体高分子の少なくとも一方を回収する工程を含んでもよい。前記分析された細胞質の生体高分子および前記残部の生体高分子の回収方法は、特に制限されず、例えば、前記本発明の分画方法における回収工程の説明を援用できる。 The analysis method of the present invention may further include a step of collecting at least one of the analyzed cytoplasmic biopolymer and the remaining biopolymer. The method for recovering the analyzed cytoplasmic biopolymer and the remaining biopolymer is not particularly limited, and for example, the description of the recovery step in the fractionation method of the present invention can be cited.

　本発明の分析方法は、例えば、さらに、前記本発明の分画方法における増幅工程、逆転写工程、および／または保持工程を含んでもよい。前記増幅工程、前記逆転写工程、および前記保持工程は、前記本発明の分画方法の説明を援用できる。 The analysis method of the present invention may further include, for example, an amplification step, a reverse transcription step, and / or a holding step in the fractionation method of the present invention. The description of the fractionation method of the present invention can be used for the amplification step, the reverse transcription step, and the holding step.

　つぎに、本発明のチップおよび分画方法について、図面を参照し、例をあげて詳細に説明する。ただし、本発明は、以下の例に限定されない。なお、各図において、同一部分には、同一符号を付しており、特に示さない限り、各形態の記載を援用できる。また、図面は、説明の便宜上、各部の構造は適宜簡略化して示す場合があり、各部の大きさ、その比率等は、実際とは異なり、模式的に示す場合がある。 Next, the chip and fractionation method of the present invention will be described in detail with reference to the drawings and examples. However, the present invention is not limited to the following examples. In each figure, the same numerals are given to the same portion, and description of each form can be used unless otherwise indicated. In the drawings, for convenience of explanation, the structure of each part may be simplified as appropriate, and the size, the ratio, and the like of each part may be schematically shown, unlike actual ones.

（実施形態１）
　図１は、本発明のチップの一例を示す概略図であり、（Ａ）は、上面図、（Ｂ）は、前記（Ａ）のＩ－Ｉ方向からみた断面図、（Ｃ）は、前記（Ａ）のII－II方向からみた断面図である。図１に示すように、チップ１０は、上基板１ａと下基板１ｂとからなる基板１を有する。上基板１ａは、２つの貫通孔１２および１３と、下表面における凹部１４、１５ａ、１５ｂ、１５ｃ、１６とを有し、これらは、上基板１ａと下基板１ｂとの積層により、それぞれ、第１の開口部１２、第２の開口部１３、上流流路１４、トラップ口１５ａ、バイパス口１５ｂ、１５ｃ、下流流路１６を構成している。また、上基板１ａは、下表面における凹部１５ａ、１５ｂ、１５ｃの間の２つの凸部１７ａ、１７ｂを有し、これらは、上基板１ａと下基板１ｂとの積層により、壁１７ａ、１７ｂを構成している。第１の開口部１２と第２の開口部１３とは、上流流路１４、開口１５、および下流流路１６を含む分離用流路１１で連通する。 (Embodiment 1)
FIG. 1 is a schematic view showing an example of the chip of the present invention, (A) is a top view, (B) is a cross-sectional view taken along the II direction of (A), and (C) is the above-mentioned FIG. It is sectional drawing seen from the II-II direction of (A). As shown in FIG. 1, the chip 10 has a substrate 1 composed of an upper substrate 1a and a lower substrate 1b. The upper substrate 1a has two through holes 12 and 13 and concave portions 14, 15a, 15b, 15c, 16 on the lower surface, which are respectively formed by stacking the upper substrate 1a and the lower substrate 1b. 1 opening part 12, 2nd opening part 13, upstream flow path 14, trap port 15a, bypass ports 15b and 15c, and downstream flow path 16 are comprised. Further, the upper substrate 1a has two convex portions 17a, 17b between the concave portions 15a, 15b, 15c on the lower surface, and these are formed by laminating the upper substrate 1a and the lower substrate 1b to form the walls 17a, 17b. It is composed. The first opening 12 and the second opening 13 communicate with each other in the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16.

　チップ１０の大きさは、特に制限されず、以下の条件が例示できる。
第１の開口部１２
　　　直径　３～１０ｍｍ（例えば、５ｍｍ）
　　　容積　５～５００μＬ、５～６０μＬ（例えば、５０、１０μＬ）
第２の開口部１３
　　　直径　３～１０ｍｍ（例えば、５ｍｍ）
　　　容積　５～５００μＬ、５～６０μＬ（例えば、５０、１０μＬ）
上流流路１４
　　　長さ　１０～５０００μｍ（例えば、２００μｍ）
　　　幅　　２０～５００μｍ（例えば、５０μｍ）
　　　深さ　１５～４０μｍ（例えば、２５μｍ）
トラップ口１５ａ
　　　長さ　１～２０μｍ（例えば、１０μｍ）
　　　幅　　１～１０μｍ（例えば、３μｍ）
　　　深さ　０．１～４０μｍ、１５～４０μｍ（例えば、２５μｍ）
バイパス口１５ｂ、１５ｃ
　　　長さ　１～２００μｍ（例えば、５μｍ）
　　　幅　　０．１～３μｍ（例えば、３μｍ）
　　　深さ　１～４０μｍ（例えば、２５μｍ）
下流流路１６
　　　長さ　５０００～２００００μｍ（例えば、２００００μｍ）
　　　幅　　１０～３００μｍ（例えば、５０μｍ）
　　　深さ　５～４０μｍ（例えば、２５μｍ） The size of the chip 10 is not particularly limited, and the following conditions can be exemplified.
First opening 12
Diameter 3-10mm (eg 5mm)
Volume 5 to 500 μL, 5 to 60 μL (for example, 50, 10 μL)
Second opening 13
Diameter 3-10mm (eg 5mm)
Volume 5 to 500 μL, 5 to 60 μL (for example, 50, 10 μL)
Upstream flow path 14
Length 10-5000μm (for example, 200μm)
Width 20-500μm (for example, 50μm)
Depth 15-40μm (for example, 25μm)
Trap mouth 15a
Length 1-20μm (for example, 10μm)
Width 1 ~ 10μm (eg 3μm)
Depth 0.1-40μm, 15-40μm (for example, 25μm)
Bypass ports 15b, 15c
Length 1 ~ 200μm (eg 5μm)
Width 0.1-3μm (for example, 3μm)
Depth 1-40μm (for example, 25μm)
Downstream channel 16
Length 5000-20000μm (eg 20000μm)
Width 10 to 300 μm (for example, 50 μm)
Depth 5-40μm (for example, 25μm)

　まず、チップ１０の第１の開口部１２に前記分離液を導入することで、分離用流路１１に前記分離液を充填する。また、これにより第１の開口部１２から第２の開口部１３への分離液の流れを生じさせる。そして、前記試料を第１の開口部１２に導入する。前記試料は、前記分離液の流れにより第２の開口部１３方向へ移動し、前記試料中のターゲット細胞が、トラップ口１５ａにトラップされる。前記トラップ後、例えば、第１の開口部１２の溶液を前記分離液と置換してもよい。 First, the separation liquid 11 is filled into the separation channel 11 by introducing the separation liquid into the first opening 12 of the chip 10. This also causes the separation liquid to flow from the first opening 12 to the second opening 13. Then, the sample is introduced into the first opening 12. The sample moves in the direction of the second opening 13 by the flow of the separation liquid, and target cells in the sample are trapped in the trap port 15a. After the trap, for example, the solution in the first opening 12 may be replaced with the separation liquid.

　つぎに、チップ１０は、例えば、電圧印加手段を備える分画装置を使用することで、生体高分子を分離できる。この際、電圧を印加する電極系は、前記分画装置が備えてもよいし、チップ１０が備えてもよい。前者の場合、前記分画装置の電極系を、チップ１０の第１の開口部１２および第２の開口部１３に挿入すればよい。 Next, the chip 10 can separate the biopolymer by using, for example, a fractionation device including a voltage application unit. At this time, an electrode system for applying a voltage may be provided in the fractionation device or the chip 10. In the former case, the electrode system of the fractionation device may be inserted into the first opening 12 and the second opening 13 of the chip 10.

　そして、前記分画装置の前記電圧印加手段により前記電圧を印加することにより、前記ターゲット細胞の細胞膜を破砕する。そして、前記ターゲットから生体高分子を放出させ、前記第２の開口部１３側に、前記ターゲット細胞の細胞質の生体高分子を分離する。他方、核の生体高分子等の分離されなかった残部の生体高分子は、トラップ口１５ａにトラップされる。そして、第２の開口部１３から前記細胞質の生体高分子を、第１の開口部１２から前記残部の生体高分子を回収する。 Then, the cell membrane of the target cell is disrupted by applying the voltage by the voltage applying means of the fractionation device. Then, the biopolymer is released from the target, and the cytoplasmic biopolymer of the target cell is separated on the second opening 13 side. On the other hand, the remaining biopolymer that has not been separated, such as nuclear biopolymer, is trapped in the trap port 15a. Then, the cytoplasmic biopolymer is recovered from the second opening 13, and the remaining biopolymer is recovered from the first opening 12.

（実施形態２）
　図２は、本発明のチップの一例を示す概略図であり、（Ａ）は、上面図、（Ｂ）は、前記（Ａ）の（Ｂ）で示す二点鎖線で囲った領域の拡大図（上面図）、（Ｃ）は、前記（Ｂ）のＩ－Ｉ方向からみた断面図、（Ｄ）は、前記（Ａ）の（Ｄ）で示す二点鎖線で囲った領域の拡大図（上面図）である。図２に示すように、本実施形態のチップ２０は、さらに、調整用流路２１、第３の開口部２２、および接続流路２３を含み、壁１７の開口１５が１つであること以外、図１に示したチップと同様の構成を有する。調整用流路２１は、下流流路１６の壁１７の近傍で下流流路１６と連通する。また、接続流路２３は、一端が、上流流路１４の壁１７近傍で上流流路１４と連通し、他端が、調整用流路２１と連通する。 (Embodiment 2)
2A and 2B are schematic views showing an example of the chip of the present invention, where FIG. 2A is a top view, and FIG. 2B is an enlarged view of a region surrounded by a two-dot chain line shown in FIG. (Top view), (C) is a cross-sectional view as viewed from the II direction of (B), and (D) is an enlarged view of a region surrounded by a two-dot chain line (D) of (A). FIG. As shown in FIG. 2, the chip 20 of this embodiment further includes an adjustment channel 21, a third opening 22, and a connection channel 23, except that the wall 17 has one opening 15. 1 has the same configuration as the chip shown in FIG. The adjustment channel 21 communicates with the downstream channel 16 in the vicinity of the wall 17 of the downstream channel 16. Further, one end of the connection channel 23 communicates with the upstream channel 14 near the wall 17 of the upstream channel 14, and the other end communicates with the adjustment channel 21.

　チップ２０の大きさは、特に制限されず、以下の条件が例示できる。
第１の開口部１２
　　　直径　３～１０ｍｍ（例えば、５ｍｍ）
　　　容積　５～５００μＬ、５～６０μＬ（例えば、５０、１０μＬ）
第２の開口部１３
　　　直径　３～１０ｍｍ（例えば、５ｍｍ）
　　　容積　５～５００μＬ、５～６０μＬ（例えば、５０、１０μＬ）
上流流路１４
　　　長さ　１０～５０００μｍ（例えば、２００μｍ）
　　　幅　　２０～５００μｍ（例えば、５０μｍ）
　　　深さ　１５～４０μｍ（例えば、２５μｍ）
開口１５
　　　長さ　１～２０μｍ（例えば、１０μｍ）
　　　幅　　１～１０μｍ（例えば、３μｍ）
　　　深さ　０．１～４０μｍ、１５～４０μｍ（例えば、２５μｍ）
下流流路１６
　　　長さ　５０００～２００００μｍ（例えば、２００００μｍ）
　　　幅　　１０～３００μｍ（例えば、５０μｍ）
　　　深さ　５～４０μｍ（例えば、２５μｍ）
調整用流路２１
　　　長さ　３０００～４００００μｍ（例えば、１１０００μｍ）
　　　幅　　２０～５００μｍ（例えば、５０μｍ）
　　　深さ　５～４０μｍ（例えば、２５μｍ）
第３の開口部２２
　　　直径　３～１０ｍｍ（例えば、５ｍｍ）
　　　容積　１０～１００μＬ（例えば、６０μＬ）
接続流路２３
　　　長さ　２０～２００００μｍ（例えば、１００００μｍ）
　　　幅　　２０～３００μｍ（例えば、２５μｍ）
　　　深さ　５～４０μｍ（例えば、２５μｍ） The size of the chip 20 is not particularly limited, and the following conditions can be exemplified.
First opening 12
Diameter 3-10mm (eg 5mm)
Volume 5 to 500 μL, 5 to 60 μL (for example, 50, 10 μL)
Second opening 13
Diameter 3-10mm (eg 5mm)
Volume 5 to 500 μL, 5 to 60 μL (for example, 50, 10 μL)
Upstream flow path 14
Length 10-5000μm (for example, 200μm)
Width 20-500μm (for example, 50μm)
Depth 15-40μm (for example, 25μm)
Opening 15
Length 1-20μm (for example, 10μm)
Width 1 ~ 10μm (eg 3μm)
Depth 0.1-40μm, 15-40μm (for example, 25μm)
Downstream channel 16
Length 5000-20000μm (eg 20000μm)
Width 10 to 300 μm (for example, 50 μm)
Depth 5-40μm (for example, 25μm)
Adjustment flow path 21
Length 3000-40000μm (for example, 11000μm)
Width 20-500μm (for example, 50μm)
Depth 5-40μm (for example, 25μm)
Third opening 22
Diameter 3-10mm (eg 5mm)
Volume 10-100μL (for example, 60μL)
Connection channel 23
Length 20 to 20000 μm (for example, 10,000 μm)
Width 20-300μm (for example, 25μm)
Depth 5-40μm (for example, 25μm)

　まず、チップ２０の第１の開口部１２および第２の開口部１３に前記分離液を導入し、第３の開口部２２から吸引することで、分離用流路１１、調整用流路２１、接続流路２３に前記分離液を充填する。また、これにより、第１の開口部１２から第３の開口部２２への分離液の流れを生じさせる。そして、前記試料を第１の開口部１２に導入する。前記試料は、前記分離液の流れにより第３の開口部２２方向へ移動し、前記試料中のターゲット細胞が、開口１５にトラップされる。前記試料が複数のターゲット細胞を含む場合、トラップされなかった細胞は、接続流路２３を介して、調整用流路２１へと移動する。そして、第３の開口部２２に前記分離液を導入し、第１の開口部１２から第３の開口部２２への流れを緩和する。 First, the separation liquid 11 is introduced into the first opening 12 and the second opening 13 of the chip 20 and sucked from the third opening 22, whereby the separation channel 11, the adjustment channel 21, The connection channel 23 is filled with the separation liquid. This also causes the separation liquid to flow from the first opening 12 to the third opening 22. Then, the sample is introduced into the first opening 12. The sample moves toward the third opening 22 by the flow of the separation liquid, and target cells in the sample are trapped in the opening 15. When the sample includes a plurality of target cells, the cells that have not been trapped move to the adjustment channel 21 via the connection channel 23. Then, the separation liquid is introduced into the third opening 22 to relieve the flow from the first opening 12 to the third opening 22.

　つぎに、チップ２０は、例えば、電圧印加手段を備える分画装置を使用することで、生体高分子を分離できる。この際、電圧を印加する電極系は、前記分画装置が備えてもよいし、チップ２０が備えてもよい。前者の場合、前記分画装置の電極系を、チップ２０の第１の開口部１２および第２の開口部１３に挿入すればよい。 Next, the chip 20 can separate the biopolymer by using, for example, a fractionation device including a voltage applying unit. At this time, an electrode system for applying a voltage may be provided in the fractionation device or the chip 20. In the former case, the electrode system of the fractionation device may be inserted into the first opening 12 and the second opening 13 of the chip 20.

　そして、前記分画装置の前記電圧印加手段により前記電圧を印加することにより、前記ターゲット細胞の細胞膜を破砕する。そして、前記ターゲットから生体高分子を放出させ、前記第２の開口部１３側に、前記ターゲット細胞の細胞質の生体高分子を分離する。他方、核の生体高分子等の分離されなかった残部の生体高分子は、開口１５にトラップされる。また、前記細胞質の生体高分子の調整用流路２１への導入を抑制する場合、第３の開口部２２に電極を配置し、前記電圧印加手段により電圧を印加してもよい。 Then, the cell membrane of the target cell is disrupted by applying the voltage by the voltage applying means of the fractionation device. Then, the biopolymer is released from the target, and the cytoplasmic biopolymer of the target cell is separated on the second opening 13 side. On the other hand, the remaining biopolymer that has not been separated, such as nuclear biopolymer, is trapped in the opening 15. Moreover, when suppressing introduction | transduction to the flow path 21 for adjustment of the said cytoplasmic biopolymer, an electrode may be arrange | positioned to the 3rd opening part 22, and a voltage may be applied by the said voltage application means.

（実施形態３）
　図３は、本発明のチップの一例を示す上面図である。図３に示すように、チップ３０は、分離用流路１１と、第１の開口部１２と、吸引吐出部３１と、捕獲部３２とを有する。分離用流路１１は、上流流路１４、開口１７および下流流路１６を有し、壁１７は、壁１７ａおよび１７ｂと、開口１５とを有する。また、下流流路１６には、捕獲部３２が配置され、また、下流流路１６の第１の開口部１２と反対方向の端には、吸引吐出部３１が接続されている。第１の開口部１２は、上流流路１４、開口１５、および下流流路１６を含む分離用流路１１と連通する。 (Embodiment 3)
FIG. 3 is a top view showing an example of the chip of the present invention. As shown in FIG. 3, the chip 30 includes a separation channel 11, a first opening 12, a suction / discharge unit 31, and a capturing unit 32. The separation channel 11 has an upstream channel 14, an opening 17, and a downstream channel 16, and the wall 17 has walls 17 a and 17 b and an opening 15. In addition, a capture unit 32 is disposed in the downstream flow channel 16, and a suction / discharge unit 31 is connected to an end of the downstream flow channel 16 in the direction opposite to the first opening 12. The first opening 12 communicates with the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16.

　チップ３０の大きさは、特に制限されず、以下の条件が例示できる。また、チップ３０において、捕獲部３２は、下流流路１６において、開口１５から０～２００００μｍの位置に配置されている。
第１の開口部１２
　　　直径　３～１０ｍｍ（例えば、５ｍｍ）
　　　容積　５～５００μＬ、５～６０μＬ（例えば、５０、１０μＬ）
上流流路１４
　　　長さ　１０～５０００μｍ（例えば、２００μｍ）
　　　幅　　２０～５００μｍ（例えば、５０μｍ）
　　　深さ　１５～４０μｍ（例えば、２５μｍ）
開口１５
　　　長さ　１～２０μｍ（例えば、１０μｍ）
　　　幅　　１～１０μｍ（例えば、３μｍ）
　　　深さ　０．１～４０μｍ、１５～４０μｍ（例えば、２５μｍ）
下流流路１６
　　　長さ　５０００～２００００μｍ（例えば、２００００μｍ）
　　　幅　　１０～３００μｍ（例えば、５０μｍ）
　　　深さ　５～４０μｍ（例えば、２５μｍ） The size of the chip 30 is not particularly limited, and the following conditions can be exemplified. Further, in the chip 30, the capture part 32 is arranged at a position of 0 to 20000 μm from the opening 15 in the downstream flow path 16.
First opening 12
Diameter 3-10mm (eg 5mm)
Volume 5 to 500 μL, 5 to 60 μL (for example, 50, 10 μL)
Upstream flow path 14
Length 10-5000μm (for example, 200μm)
Width 20-500μm (for example, 50μm)
Depth 15-40μm (for example, 25μm)
Opening 15
Length 1-20μm (for example, 10μm)
Width 1 ~ 10μm (eg 3μm)
Depth 0.1-40μm, 15-40μm (for example, 25μm)
Downstream channel 16
Length 5000-20000μm (eg 20000μm)
Width 10 to 300 μm (for example, 50 μm)
Depth 5-40μm (for example, 25μm)

　まず、チップ３０の第１の開口部１２に前記試料を導入する。導入された試料は、分離用流路１１において、上流流路１４から開口１５を介して、下流流路１６方向に移動する。この際に、前記試料中のターゲット細胞が、開口１５にトラップされる。 First, the sample is introduced into the first opening 12 of the chip 30. The introduced sample moves from the upstream flow path 14 to the downstream flow path 16 through the opening 15 in the separation flow path 11. At this time, target cells in the sample are trapped in the opening 15.

　つぎに、チップ３０は、例えば、第１の開口部１２から界面活性剤を含む溶液を導入することにより、開口１５にトラップされたターゲット細胞の細胞膜を破壊でき、これにより、ターゲットから生体高分子を放出させる。そして、吸引吐出部３１により吸引することにより、下流流路１６側に前記ターゲット細胞の細胞質の生体高分子を分離することで、下流流路１６に配置された捕獲部３２に前記細胞質の生体高分子が捕獲される。他方、核の生体高分子等の残部の生体高分子は、開口１５にトラップされる。そして、例えば、前記吸引手段を用い、第１の開口部１２から、吸引することで、まず、開口１５にトラップされていた残部の生体高分子を回収する。さらに、例えば、前記吸引手段を用い、第１の開口部１２から、吸引することで、まず、捕獲部３２にトラップされていた細胞質の生体高分子を回収する。 Next, the chip 30 can destroy the cell membrane of the target cell trapped in the opening 15 by introducing, for example, a solution containing a surfactant from the first opening 12, and thereby the biopolymer from the target. To release. Then, by sucking with the suction / discharge unit 31, the cytoplasmic biopolymer of the target cell is separated on the downstream channel 16 side, so that the cytoplasmic living body height is transferred to the capturing unit 32 arranged in the downstream channel 16. Molecules are captured. On the other hand, the remaining biopolymers such as nuclear biopolymers are trapped in the openings 15. For example, the remaining biopolymer trapped in the opening 15 is first recovered by sucking from the first opening 12 using the suction means. Further, for example, the cytoplasmic biopolymer trapped in the capture unit 32 is first recovered by suction from the first opening 12 using the suction means.

（変形例１）
　図４は、本発明のチップの一例を示す上面図である。図４に示すように、変形例１のチップ４０は、吸引吐出部３１として、吸引吐出部３１ａおよび３１ｂを有する。また、チップ４０において、吸引吐出部３１ａおよび３１ｂは、下流流路１６に接続されている。また、下流流路１６の第１の開口部１２と反対方向の端には、吸引吐出部３１ａが接続されている。そして、吸引吐出部３１ａと下流流路１６との接続部より開口１５側において、吸引吐出部３１ｂが接続されている。吸引吐出部３１ａと下流流路１６との接続部および吸引吐出部３１ｂと下流流路１６との接続部の間に、捕獲部３２が配置されている。この点を除き、変形例１のチップ４０は、前記実施形態３のチップ３０と同様の構成を有し、その説明を援用できる。 (Modification 1)
FIG. 4 is a top view showing an example of the chip of the present invention. As shown in FIG. 4, the chip 40 of the first modification has suction / discharge portions 31 a and 31 b as the suction / discharge portion 31. Further, in the chip 40, the suction / discharge portions 31 a and 31 b are connected to the downstream flow path 16. A suction / discharge part 31 a is connected to the end of the downstream flow path 16 in the direction opposite to the first opening 12. The suction / discharge part 31b is connected to the opening 15 side of the connection part between the suction / discharge part 31a and the downstream flow path 16. A capture unit 32 is disposed between a connection portion between the suction / discharge portion 31 a and the downstream flow channel 16 and a connection portion between the suction / discharge portion 31 b and the downstream flow channel 16. Except for this point, the chip 40 of the first modification has the same configuration as the chip 30 of the third embodiment, and the description thereof can be used.

　まず、チップ４０の第１の開口部１２に前記試料を導入する。導入された試料は、分離用流路１１において、上流流路１４から開口１５を介して、下流流路１６方向に移動する。この際に、前記試料中のターゲット細胞が、開口１５にトラップされる。 First, the sample is introduced into the first opening 12 of the chip 40. The introduced sample moves from the upstream flow path 14 to the downstream flow path 16 through the opening 15 in the separation flow path 11. At this time, target cells in the sample are trapped in the opening 15.

　つぎに、チップ４０は、例えば、第１の開口部１２から界面活性剤を含む溶液を導入することにより、開口１５にトラップされたターゲット細胞の細胞膜を破壊でき、これにより、ターゲットから生体高分子を放出させる。そして、吸引吐出部３１ａにより吸引することにより、下流流路１６側に前記ターゲット細胞の細胞質の生体高分子を分離することで、下流流路１６に配置された捕獲部３２に前記細胞質の生体高分子が捕獲される。他方、核の生体高分子等の残部の生体高分子は、開口１５にトラップされる。つぎに、例えば、吸引吐出部３１ｂにより吐出することで、下流流路１６から第１の開口部１２への流れを生じさせることにより、開口１５にトラップされた前記残部の生体高分子を開口１５から第１の開口部１２へ移動させる。そして、例えば、前記吸引手段を用い、第１の開口部１２から、吸引することで、前記残部の生体高分子を回収する。さらに、吸引吐出部３１ａにより吐出することで、下流流路１６から第１の開口部１２への流れを生じさせることにより、捕獲部３２にトラップされた前記細胞質の生体高分子を捕獲部３２から第１の開口部１２へ移動させる。そして、例えば、前記吸引手段を用い、第１の開口部１２から、吸引することで、前記細胞質の生体高分子を回収する。 Next, the chip 40 can destroy the cell membrane of the target cell trapped in the opening 15 by introducing, for example, a solution containing a surfactant from the first opening 12, and thereby the biopolymer from the target. To release. Then, the cytoplasmic living body high polymer is separated into the capturing part 32 disposed in the downstream flow path 16 by separating the cytoplasmic biopolymer of the target cell on the downstream flow path 16 side by being sucked by the suction / discharge section 31a. Molecules are captured. On the other hand, the remaining biopolymers such as nuclear biopolymers are trapped in the openings 15. Next, for example, the remaining biopolymer trapped in the opening 15 is opened by generating a flow from the downstream flow path 16 to the first opening 12 by being discharged by the suction discharge section 31b. To the first opening 12. Then, for example, the remaining biopolymer is recovered by sucking from the first opening 12 using the suction means. Furthermore, the cytoplasmic biopolymer trapped in the capture unit 32 is discharged from the capture unit 32 by generating a flow from the downstream flow path 16 to the first opening 12 by being discharged by the suction / discharge unit 31a. Move to the first opening 12. Then, for example, the cytoplasmic biopolymer is recovered by sucking from the first opening 12 using the suction means.

（実施形態４）
　図５は、本発明のチップの一例を示す上面図である。図５に示すように、チップ５０は、分離用流路１１と、第１の開口部１２と、電極３３とを有する。分離用流路１１は、上流流路１４、壁１７および下流流路１６を有し、壁１７は、壁１７ａおよび１７ｂと、開口１５とを有する。また、下流流路１６の第１の開口部１２と反対方向の端には、電極３３が配置されている。第１の開口部１２は、上流流路１４、開口１５、および下流流路１６を含む分離用流路１１と連通する。チップ５０の大きさは、特に制限されず、例えば、チップ３０の大きさの説明を援用できる。 (Embodiment 4)
FIG. 5 is a top view showing an example of the chip of the present invention. As shown in FIG. 5, the chip 50 includes a separation channel 11, a first opening 12, and an electrode 33. The separation channel 11 has an upstream channel 14, a wall 17, and a downstream channel 16, and the wall 17 has walls 17 a and 17 b and an opening 15. In addition, an electrode 33 is disposed at the end of the downstream flow path 16 in the direction opposite to the first opening 12. The first opening 12 communicates with the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16. The size of the chip 50 is not particularly limited, and for example, description of the size of the chip 30 can be used.

　まず、チップ５０の第１の開口部１２に前記試料を導入する。導入された試料は、分離用流路１１において、上流流路１４から開口１５を介して、下流流路１６方向に移動する。この際に、前記試料中のターゲット細胞が、開口１５にトラップされる。 First, the sample is introduced into the first opening 12 of the chip 50. The introduced sample moves from the upstream flow path 14 to the downstream flow path 16 through the opening 15 in the separation flow path 11. At this time, target cells in the sample are trapped in the opening 15.

　つぎに、チップ５０は、例えば、電圧印加手段を備える分画装置を使用することで、生体高分子を分離できる。この際、電圧を印加する電極系は、前記分画装置が備えてもよいし、チップ５０が備えてもよい。前者の場合、前記分画装置の電極系を、チップ５０の第１の開口部１２に挿入すればよい。 Next, the chip 50 can separate the biopolymer by using, for example, a fractionation device including a voltage application unit. At this time, the electrode system for applying the voltage may be provided in the fractionation device or the chip 50. In the former case, the electrode system of the fractionation device may be inserted into the first opening 12 of the chip 50.

　そして、前記分画装置の前記電圧印加手段により前記電圧を印加することにより、前記ターゲット細胞の細胞膜を破砕する。そして、前記ターゲットから生体高分子を放出させ電極３３側に、前記ターゲット細胞の細胞質の生体高分子を分離する。他方、核の生体高分子等の分離されなかった残部の生体高分子は、開口１５にトラップされる。そして、例えば、前記吸引手段を用い、第１の開口部１２から、吸引することで、まず、開口１５にトラップされていた残部の生体高分子を回収する。さらに、再度、例えば、前記吸引手段を用い、第１の開口部１２から、吸引することで、前記細胞質の生体高分子を回収する。 Then, the cell membrane of the target cell is disrupted by applying the voltage by the voltage applying means of the fractionation device. Then, the biopolymer is released from the target, and the cytoplasmic biopolymer of the target cell is separated on the electrode 33 side. On the other hand, the remaining biopolymer that has not been separated, such as nuclear biopolymer, is trapped in the opening 15. For example, the remaining biopolymer trapped in the opening 15 is first recovered by sucking from the first opening 12 using the suction means. Furthermore, for example, the cytoplasmic biopolymer is recovered by sucking from the first opening 12 again using the suction means, for example.

（実施形態５）
　図６は、本発明のチップの一例を示す上面図である。図６に示すように、チップ６０は、分離用流路１１と、第１の開口部１２と、吸引吐出部３１と、液体移動制御部３４と、バイパス流路３５と、第２の液体移動制御部３６ａおよび３６ｂとを有する。分離用流路１１は、上流流路１４、壁１７および下流流路１６を有し、壁１７は、壁１７ａおよび１７ｂと、開口１５とを有する。また、下流流路１６の第１の開口部１２と反対方向の端には、吸引吐出部３１が接続されている。液体移動制御部３４は、開口１５と、上流流路１４およびバイパス流路３５の接続部との間に配置されている。そして、第２の液体移動制御部３６ａは、バイパス流路３５と上流流路１４との接続部（連通部）に隣接するように配置されている。第２の液体移動制御部３６ｂは、バイパス流路３５と下流流路１６との接続部に隣接するように配置されている。上流流路１４および下流流路１６は、バイパス流路３５に連通されている。また、第１の開口部１２は、上流流路１４、開口１５、および下流流路１６を含む分離用流路１１と連通する。チップ６０の大きさは、特に制限されず、例えば、チップ３０の大きさの説明を援用できる。 (Embodiment 5)
FIG. 6 is a top view showing an example of the chip of the present invention. As shown in FIG. 6, the chip 60 includes a separation channel 11, a first opening 12, a suction / discharge unit 31, a liquid movement control unit 34, a bypass channel 35, and a second liquid movement. And controllers 36a and 36b. The separation channel 11 has an upstream channel 14, a wall 17, and a downstream channel 16, and the wall 17 has walls 17 a and 17 b and an opening 15. A suction / discharge unit 31 is connected to an end of the downstream flow path 16 in the direction opposite to the first opening 12. The liquid movement control unit 34 is disposed between the opening 15 and the connection portion between the upstream flow path 14 and the bypass flow path 35. The second liquid movement control unit 36a is disposed so as to be adjacent to a connection part (communication part) between the bypass flow path 35 and the upstream flow path 14. The second liquid movement control unit 36 b is disposed so as to be adjacent to the connection portion between the bypass channel 35 and the downstream channel 16. The upstream flow path 14 and the downstream flow path 16 are communicated with the bypass flow path 35. Further, the first opening 12 communicates with the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16. The size of the chip 60 is not particularly limited, and for example, description of the size of the chip 30 can be used.

　まず、チップ６０において、液体移動制御部３４を液体が通過できないようにする。また、第２の液体移動制御部３６ａおよび３６ｂを液体が通過できるようにする。つぎに、第１の開口部１２に前記試料を導入する。そして、吸引吐出部３１で吸引することにより、バイパス流路３５を介して下流流路１６に、前記試料中のターゲット細胞が導入される。 First, in the chip 60, the liquid is prevented from passing through the liquid movement control unit 34. Further, the liquid is allowed to pass through the second liquid movement control units 36a and 36b. Next, the sample is introduced into the first opening 12. Then, the target cells in the sample are introduced into the downstream flow path 16 via the bypass flow path 35 by being sucked by the suction / discharge section 31.

　つぎに、液体移動制御部３４を液体が通過できるようにし、また、第２の液体移動制御部３６ａおよび３６ｂを液体が通過できないようにする。そして、吸引吐出部３１により吐出することで、下流流路１６から第１の開口部１２への流れを生じさせる。この際、前記ターゲット細胞が、開口１５にトラップされる。 Next, the liquid is allowed to pass through the liquid movement control unit 34, and the liquid is prevented from passing through the second liquid movement control units 36a and 36b. And it discharges by the suction discharge part 31, and the flow from the downstream flow path 16 to the 1st opening part 12 is produced. At this time, the target cell is trapped in the opening 15.

　つぎに、チップ６０は、例えば、第１の開口部１２から界面活性剤を含む溶液を導入することにより、開口１５にトラップされたターゲット細胞の細胞膜を破壊でき、これにより、ターゲットから生体高分子を放出させる。そして、吸引吐出部３１により吐出することにより、上流流路１４側に前記ターゲット細胞の細胞質の生体高分子を分離し、さらに、第１の開口部１２へ移動させる。そして、例えば、前記吸引手段を用い、第１の開口部１２から、吸引することで、前記細胞質の生体高分子を回収する。さらに、吸引吐出部３１により吸引することにより、開口１５にトラップされた前記残部の生体高分子を開口１５から下流流路１６に移動させる。そして、液体移動制御部３４を液体が通過できないようにし、また、第２の液体移動制御部３６ａおよび３６ｂを液体が通過できるようにする。その後、吸引吐出部３１により吐出することで、前記残部の生体高分子は、バイパス流路３５および上流流路１４を介して、第１の開口部１２へ移動する。そして、例えば、前記吸引手段を用い、第１の開口部１２から、吸引することで、前記残部の生体高分子を回収する。 Next, the chip 60 can destroy the cell membrane of the target cell trapped in the opening 15 by, for example, introducing a solution containing a surfactant from the first opening 12, and thereby the biopolymer from the target. To release. Then, by discharging by the suction / discharge unit 31, the biopolymer in the cytoplasm of the target cell is separated to the upstream flow path 14 side, and further moved to the first opening 12. Then, for example, the cytoplasmic biopolymer is recovered by sucking from the first opening 12 using the suction means. Further, the remaining biopolymer trapped in the opening 15 is moved from the opening 15 to the downstream flow path 16 by being sucked by the suction and discharge section 31. Then, the liquid is prevented from passing through the liquid movement control unit 34, and the liquid is allowed to pass through the second liquid movement control units 36a and 36b. Thereafter, the remaining biopolymer moves to the first opening 12 via the bypass channel 35 and the upstream channel 14 by being discharged by the suction / discharge unit 31. Then, for example, the remaining biopolymer is recovered by sucking from the first opening 12 using the suction means.

（実施形態６）
　図７は、本発明のチップの一例を示す上面図である。図７に示すように、チップ７０は、分離用流路１１と、第１の開口部１２と、第２の開口部１３と、液体移動制御部３４とを有する。分離用流路１１は、上流流路１４、壁１７および下流流路１６を有し、壁１７は、壁１７ａおよび１７ｂと、開口１５とを有する。また、下流流路１６には、液体移動制御部３４が配置されている。第１の開口部１２および第２の開口部１３は、上流流路１４、開口１５、および下流流路１６を含む分離用流路１１と連通する。液体移動制御部３４は、例えば、前記マイクロバルブである。 (Embodiment 6)
FIG. 7 is a top view showing an example of the chip of the present invention. As shown in FIG. 7, the chip 70 includes a separation channel 11, a first opening 12, a second opening 13, and a liquid movement control unit 34. The separation channel 11 has an upstream channel 14, a wall 17, and a downstream channel 16, and the wall 17 has walls 17 a and 17 b and an opening 15. A liquid movement control unit 34 is disposed in the downstream flow path 16. The first opening 12 and the second opening 13 communicate with the separation channel 11 including the upstream channel 14, the opening 15, and the downstream channel 16. The liquid movement control unit 34 is, for example, the microvalve.

　チップ７０の大きさは、特に制限されず、以下の条件が例示できる。また、チップ７０において、液体移動制御部３４は、下流流路１６において、開口１５から０～２００００μｍの位置に配置されている。
第１の開口部１２
　　　直径　３～１０ｍｍ（例えば、５ｍｍ）
　　　容積　５～５００μＬ、５～６０μＬ（例えば、５０、１０μＬ）
第２の開口部１３
　　　直径　３～１０ｍｍ（例えば、５ｍｍ）
　　　容積　５～６０μＬ（例えば、１０μＬ）
上流流路１４
　　　長さ　１０～５０００μｍ（例えば、２００μｍ）
　　　幅　　２０～５００μｍ（例えば、５０μｍ）
　　　深さ　１５～４０μｍ（例えば、２５μｍ）
開口１５
　　　長さ　１～２０μｍ（例えば、１０μｍ）
　　　幅　　１～１０μｍ（例えば、３μｍ）
　　　深さ　０．１～４０μｍ、１５～４０μｍ（例えば、２５μｍ）
下流流路１６
　　　長さ　５０００～２００００μｍ（例えば、２００００μｍ）
　　　幅　　１０～３００μｍ（例えば、５０μｍ）
　　　深さ　５～４０μｍ（例えば、２５μｍ） The size of the chip 70 is not particularly limited, and the following conditions can be exemplified. Further, in the chip 70, the liquid movement control unit 34 is disposed at a position of 0 to 20000 μm from the opening 15 in the downstream flow path 16.
First opening 12
Diameter 3-10mm (eg 5mm)
Volume 5 to 500 μL, 5 to 60 μL (for example, 50, 10 μL)
Second opening 13
Diameter 3-10mm (eg 5mm)
Volume 5-60μL (for example, 10μL)
Upstream flow path 14
Length 10-5000μm (for example, 200μm)
Width 20-500μm (for example, 50μm)
Depth 15-40μm (for example, 25μm)
Opening 15
Length 1-20μm (for example, 10μm)
Width 1 ~ 10μm (eg 3μm)
Depth 0.1-40μm, 15-40μm (for example, 25μm)
Downstream channel 16
Length 5000-20000μm (eg 20000μm)
Width 10 to 300 μm (for example, 50 μm)
Depth 5-40μm (for example, 25μm)

　まず、液体移動制御部３４を解放し、通液可能とする。つぎに、チップ７０の第１の開口部１２に前記試料を導入する。導入された試料は、分離用流路１１において、上流流路１４から開口１５を介して、下流流路１６方向に移動する。この際に、前記試料中のターゲット細胞が、開口１５にトラップされる。 First, the liquid movement control unit 34 is released to enable liquid passage. Next, the sample is introduced into the first opening 12 of the chip 70. The introduced sample moves from the upstream flow path 14 to the downstream flow path 16 through the opening 15 in the separation flow path 11. At this time, target cells in the sample are trapped in the opening 15.

　つぎに、チップ７０は、例えば、電圧印加手段を備える分画装置を使用することで、生体高分子を分離できる。この際、電圧を印加する電極系は、前記分画装置が備えてもよいし、チップ７０が備えてもよい。前者の場合、前記分画装置の電極系を、チップ７０の第１の開口部１２および第２の開口部１３に挿入すればよい。また、チップ７０は、第１の開口部１２から界面活性剤を含む溶液を導入することにより、開口１５にトラップされたターゲット細胞の細胞膜を破壊してもよい。 Next, the chip 70 can separate the biopolymer by using, for example, a fractionation device including a voltage applying unit. At this time, an electrode system for applying a voltage may be provided in the fractionation device or the chip 70. In the former case, the electrode system of the fractionation device may be inserted into the first opening 12 and the second opening 13 of the chip 70. In addition, the chip 70 may destroy the cell membrane of the target cell trapped in the opening 15 by introducing a solution containing a surfactant from the first opening 12.

　つぎに、分離用流路１１に電圧を印加することにより、前記細胞質の生体高分子を、下流流路１６において、液体移動制御部３４と第２の開口部１３との間に分離する。さらに、前記分離後、液体移動制御部３４を閉塞し、通液できないようにする。そして、例えば、前記吸引手段を用い、第２の開口部１３から前記細胞質の生体高分子を、第１の開口部１２から前記残部の生体高分子を回収する。 Next, by applying a voltage to the separation channel 11, the cytoplasmic biopolymer is separated between the liquid movement control unit 34 and the second opening 13 in the downstream channel 16. Further, after the separation, the liquid movement control unit 34 is closed so that liquid cannot pass therethrough. For example, the cytoplasmic biopolymer is recovered from the second opening 13 and the remaining biopolymer is recovered from the first opening 12 using the suction means.

　以下、実施例を用いて本発明を詳細に説明するが、本発明は実施例に記載された態様に限定されるものではない。 Hereinafter, the present invention will be described in detail using examples, but the present invention is not limited to the modes described in the examples.

［実施例１］
　本発明のチップを作製し、核酸を分画できることを確認した。 [Example 1]
The chip of the present invention was produced, and it was confirmed that the nucleic acid could be fractionated.

（１）チップ
　図１に示すチップ１０を作製した。チップ１０の各部位の大きさは、以下の通りとした。
第１の開口部１２
　　　直径　５ｍｍの円柱形、容積　１０μＬ
第２の開口部１３
　　　直径　５ｍｍの円柱形、容積　１０μＬ
上流流路１４
　　　長さ　４６００μｍ、幅　５０μｍ、深さ　２５μｍ
トラップ口１５ａ
　　　長さ　５μｍ、幅　３μｍ、深さ　２５μｍ
バイパス口１５ｂ、１５ｃ
　　　長さ　５μｍ、幅　３μｍ、深さ　２５μｍ
下流流路１６
　　　長さ　２００００μｍ、幅　５０μｍ、深さ　２５μｍ (1) Chip A chip 10 shown in FIG. 1 was produced. The size of each part of the chip 10 was as follows.
First opening 12
Cylindrical shape with a diameter of 5 mm, volume 10 μL
Second opening 13
Cylindrical shape with a diameter of 5 mm, volume 10 μL
Upstream flow path 14
Length 4600μm, width 50μm, depth 25μm
Trap mouth 15a
Length 5μm, width 3μm, depth 25μm
Bypass ports 15b, 15c
Length 5μm, width 3μm, depth 25μm
Downstream channel 16
Length 20000 μm, width 50 μm, depth 25 μm

　液状のＰＤＭＳ（Sylgard 184, Dow Corning社製）をマイクロ流路の鋳型に流し入れ、脱気後、１５０℃のオーブンにおいて３０分加熱することでＰＤＭＳを固形化させた。前記固形化したＰＤＭＳを、カミソリを用いて切り出した後、分離用流路１１の両端に第１の開口部１２および第２の開口部１３を、パンチを用いて作製し、流路構造体を得た。得られた流路構造体をガラス基板にプラズマ接合を利用して封止した。 Liquid PDMS (Sylgard 184, manufactured by Dow Corning) was poured into a microchannel mold, degassed, and heated in an oven at 150 ° C. for 30 minutes to solidify PDMS. After the solidified PDMS is cut out using a razor, the first opening 12 and the second opening 13 are formed at both ends of the separation channel 11 using a punch, and a channel structure is formed. Obtained. The obtained flow path structure was sealed to a glass substrate using plasma bonding.

（２）試料
　浮遊細胞であるＫ５６２細胞（医薬基盤研究所から入手）を３７℃、５％ＣＯ２の条件下で培養した。培養液は、１０％ＦＢＳおよび１％ペニシリン／ストレプトマイシンを含むＲＰＭＩ－１６４０培養液（SigmaAldrich社製）を使用した。前記培養後、前記細胞を回収し、ピペットにより撹拌することにより一細胞化した。つぎに、１０００ｒｐｍで３分遠心後、上清を除去し、細胞分散液に分散した。前記細胞分散液の組成は、５０ｍｍｏｌ／Ｌ　Ｔｒｉｓ、２５ｍｍｏｌ／Ｌ　ＨＥＰＥＳ、１７５ｍｍｏｌ／Ｌ　スクロースとし、ｐＨは、８．３とした。前記分散後の細胞は、後述するチップ１０の第１の導入口１２への導入に先立ち、前記細胞分散液で、さらに、２０倍以上に希釈し、希釈後の細胞懸濁液を試料として使用した。 (2) K562 cells as a sample suspension cells (obtained from National Institute of Biomedical Innovation) to 37 ° C., and cultured under the conditions of 5% CO 2. As the culture solution, RPMI-1640 culture solution (Sigma Aldrich) containing 10% FBS and 1% penicillin / streptomycin was used. After the culture, the cells were collected and made into one cell by stirring with a pipette. Next, after centrifugation at 1000 rpm for 3 minutes, the supernatant was removed and dispersed in a cell dispersion. The composition of the cell dispersion was 50 mmol / L Tris, 25 mmol / L HEPES, and 175 mmol / L sucrose, and the pH was 8.3. Prior to introduction into the first inlet 12 of the chip 10 to be described later, the dispersed cells are further diluted 20 times or more with the cell dispersion, and the diluted cell suspension is used as a sample. did.

（３）核酸分画
　チップ１０の第１の開口部１２に１０μＬの分離液１を導入した。前記分離液１の組成は、５０ｍｍｏｌ／Ｌ　Ｔｒｉｓおよび２５ｍｍｏｌ／Ｌ　ＨＣｌとし、ｐＨは、８．２とした。つぎに、第２の開口部１３から１０μＬの分離液１を除去し、さらに、第１の開口部１２に１０μＬの分離液１を導入することにより、第１の開口部１２から第２の開口部１３への分離液の流れを生じさせた。さらに、Ｋ５６２細胞を１個含む試料を第１の開口部１２の内部に導入した。前記細胞は、前記分離液の流れにより、第２の開口部１３方向に移動し、トラップ口１５ａにトラップされた。前記細胞が、トラップ口１５ａにトラップされたことは、光学顕微鏡（ＩＸ７３、オリンパス社製）により確認した。そして、第１の開口部１２内の液を１０μＬの分離液２に置換し、第２の開口部１３に１０μＬの分離液１を導入した。前記分離液２の組成は、５０ｍｍｏｌ／Ｌ　Ｔｒｉｓおよび２５ｍｍｏｌ／Ｌ　ＨＥＰＥＳとし、ｐＨは、８．２とした。 (3) Nucleic acid fractionation 10 μL of the separation liquid 1 was introduced into the first opening 12 of the chip 10. The composition of the separation liquid 1 was 50 mmol / L Tris and 25 mmol / L HCl, and the pH was 8.2. Next, 10 μL of the separation liquid 1 is removed from the second opening 13, and 10 μL of the separation liquid 1 is further introduced into the first opening 12, whereby the second opening is formed from the first opening 12. A flow of the separated liquid to the part 13 was generated. Further, a sample containing one K562 cell was introduced into the first opening 12. The cells moved toward the second opening 13 by the flow of the separation liquid and were trapped in the trap port 15a. It was confirmed with an optical microscope (IX73, manufactured by Olympus) that the cells were trapped in the trap port 15a. Then, the liquid in the first opening 12 was replaced with 10 μL of the separation liquid 2, and 10 μL of the separation liquid 1 was introduced into the second opening 13. The composition of the separation liquid 2 was 50 mmol / L Tris and 25 mmol / L HEPES, and the pH was 8.2.

　第１の開口部１２および第２の開口部１３に白金線電極（直径０．８ｍｍ）を挿入後、第１の開口部１２の電極に－１５０Ｖ、第２の開口部１３の電極に０Ｖの電圧を印加した。前記電圧印加時にあわせ、第１の開口部１２および第２の開口部１３から供給される電流を計測した。そして、計測した電流値が一定値となるまで、電圧を印加することにより、前記細胞質の生体高分子を分離した。前記分離後、前記光学顕微鏡により、細胞膜を破砕された細胞（ターゲット細胞の残部）がトラップ口１５ａにトラップされていることを確認した。 After inserting a platinum wire electrode (diameter 0.8 mm) into the first opening 12 and the second opening 13, -150V is applied to the electrode of the first opening 12, and 0V is applied to the electrode of the second opening 13. A voltage was applied. The current supplied from the first opening 12 and the second opening 13 was measured in accordance with the voltage application. The cytoplasmic biopolymer was separated by applying a voltage until the measured current value became a constant value. After the separation, it was confirmed by the optical microscope that cells (the remainder of the target cells) whose cell membrane was crushed were trapped in the trap port 15a.

　前記分画後、チップ１０内の分離液１を、第２の開口部１３から全量回収した。前記回収した分離液１について、逆転写キット（TaqMan RNA-to-Ct 1-Step Kit、Thermo Fisher Scientific社製）、ＲＴ－ＰＣＲキット（gene expression assay、Thermo Fisher Scientific社製）およびサーマルサイクラー（LightCycler96、Roche社製）を用い、添付のプロトコルに基づき実施した。プライマーは、ＧＡＰＤＨプライマー（Hs02758991_g1、Applied Biosystems社製）を用いた。具体的に、９μＬの前記回収した分離液１を含む、合計２０μＬの反応液について、４８℃１５分の条件で逆転写を行い、さらに、９５℃１５分の条件でインキュベート後、９５℃１５秒、６０℃１分を１サイクルとし、５０回、前記サイクルを実施した。そして、６０℃でインキュベート中に蛍光強度を測定し、ＧＡＰＤＨの発現量を測定した。この結果、ＧＡＰＤＨの発現が確認されたことから、細胞質中に存在するＧＡＰＤＨ　ｍＲＮＡが分画できていることが確認できた。以上のことから、本発明のチップにより、細胞質の核酸を分画できることが分かった。 After the fractionation, the entire amount of the separation liquid 1 in the chip 10 was recovered from the second opening 13. For the collected separation solution 1, reverse transcription kit (TaqMan RNA-to-Ct 1-Step Kit, Thermo Fisher Fisher Scientific), RT-PCR kit (gene expression assay, Thermo Fisher Scientific) and thermal cycler (LightCycler96) And manufactured by Roche) based on the attached protocol. As the primer, a GAPDH primer (Hs02758991_g1, Applied Biosystems) was used. Specifically, a total of 20 μL of the reaction solution containing 9 μL of the recovered separation solution 1 was subjected to reverse transcription at 48 ° C. for 15 minutes, and further incubated at 95 ° C. for 15 minutes, and then 95 ° C. for 15 seconds. The cycle was carried out 50 times at 60 ° C. for 1 minute. And fluorescence intensity was measured during incubation at 60 degreeC, and the expression level of GAPDH was measured. As a result, since the expression of GAPDH was confirmed, it was confirmed that GAPDH mRNA present in the cytoplasm was fractionated. From the above, it was found that cytoplasmic nucleic acid can be fractionated by the chip of the present invention.

［実施例２］
　本発明のチップを作製し、核酸を分画できることを確認した。 [Example 2]
The chip of the present invention was produced, and it was confirmed that the nucleic acid could be fractionated.

（１）チップ
　図２に示すチップ２０を作製した。チップ２０の各部位の大きさは、以下の通りとした。そして、実施例１（１）と同様にして、チップ２０を作製した。調整用流路２１は、壁１７から３０μｍ下流側で、下流流路１６と連通させた。
第１の開口部１２
　　　直径　５ｍｍの逆円錐形、容積　１０μＬ
第２の開口部１３
　　　直径　５ｍｍの逆円錐形、容積　１０μＬ
上流流路１４
　　　長さ　４６００μｍ、幅　５０μｍ、深さ　２５μｍ
開口１５
　　　長さ　５μｍ、幅　３μｍ、深さ　２５μｍ
下流流路１６
　　　長さ　２００００μｍ、幅　５０μｍ、深さ　２５μｍ
調整用流路２１
　　　長さ　１１００μｍ、幅　５０μｍ、深さ　２５μｍ
第３の開口部２２
　　　直径　５ｍｍの円柱形、容積　６０μＬ
接続流路２３
　　　長さ　７３１０μｍ、幅　２５μｍ、深さ　２５μｍ (1) Chip A chip 20 shown in FIG. 2 was produced. The size of each part of the chip 20 was as follows. And the chip | tip 20 was produced like Example 1 (1). The adjustment channel 21 was communicated with the downstream channel 16 on the downstream side of the wall 17 by 30 μm.
First opening 12
5mm diameter inverted cone, volume 10μL
Second opening 13
5mm diameter inverted cone, volume 10μL
Upstream flow path 14
Length 4600μm, width 50μm, depth 25μm
Opening 15
Length 5μm, width 3μm, depth 25μm
Downstream channel 16
Length 20000 μm, width 50 μm, depth 25 μm
Adjustment flow path 21
Length 1100μm, width 50μm, depth 25μm
Third opening 22
5mm diameter cylindrical shape, volume 60μL
Connection channel 23
Length 7310μm, width 25μm, depth 25μm

（２）試料
　前記実施例１（２）と同様に、Ｋ５６２細胞を培養、回収および遠心後、上清を除去し、前記細胞分散液に分散した。 (2) Sample In the same manner as in Example 1 (2), K562 cells were cultured, collected and centrifuged, and then the supernatant was removed and dispersed in the cell dispersion.

（３）核酸分画
　チップ２０には、第１の開口部１２に１０μＬの分離液１を第２の開口部１３に１０μＬの分離液１を導入した。なお、本実施例の分離液１には、SYBR（登録商標）Green II（Thermo Fisher Scientific社製）を添加したものを使用した。つぎに、第３の開口部２２から吸引することにより第１の開口部１２から第３の開口部２２への分離液の流れを生じさせた。さらに、第１の開口部１２内の液を１０μＬの分離液２に置換し、第２の開口部１３内の液を１０μＬの分離液１に置換した。さらに、Ｋ５６２細胞を複数個含む試料を第１の開口部１２の内部に導入した。前記細胞は、分離液の流れにより、第３の開口部２２方向に移動し、１個のＫ５６２細胞は、開口１５にトラップされ、他の細胞は、接続流路２３を介して、調整用流路２１へ移動した。前記１個のＫ５６２細胞が、開口１５にトラップされたことは、前記光学顕微鏡により確認した。そして、第３の開口部２２に６９μＬの分離液２を導入した。 (3) Nucleic Acid Fraction Into the chip 20, 10 μL of the separation liquid 1 was introduced into the first opening 12 and 10 μL of the separation liquid 1 was introduced into the second opening 13. In addition, what added SYBR (trademark) Green II (made by Thermo Fisher Scientific) was used for the separation liquid 1 of a present Example. Next, a flow of the separation liquid from the first opening 12 to the third opening 22 was generated by suction from the third opening 22. Further, the liquid in the first opening 12 was replaced with 10 μL of the separation liquid 2, and the liquid in the second opening 13 was replaced with 10 μL of the separation liquid 1. Further, a sample containing a plurality of K562 cells was introduced into the first opening 12. The cells move in the direction of the third opening 22 due to the flow of the separation liquid, one K562 cell is trapped in the opening 15, and the other cells flow through the connection flow path 23 to adjust flow. Moved to Road 21. It was confirmed with the optical microscope that the single K562 cell was trapped in the opening 15. Then, 69 μL of the separation liquid 2 was introduced into the third opening 22.

　第１の開口部１２、第２の開口部１３および第３の開口部２２に白金線電極（直径０．８ｍｍ）を挿入後、第１の開口部１２の電極に－１５０Ｖ、第２の開口部１３の電極に０Ｖ、第３の開口部２２の電極に－１３０Ｖの電圧を印加した。前記電圧印加時にあわせ、第１の開口部１２および第３の開口部２２から供給される電流を計測した。そして、蛍光顕微鏡の観察下、計測した電流値が一定値となるまで、電圧を印加した。 After a platinum wire electrode (diameter 0.8 mm) is inserted into the first opening 12, the second opening 13, and the third opening 22, -150V is applied to the electrode of the first opening 12, and the second opening A voltage of 0 V was applied to the electrode of the portion 13, and a voltage of −130 V was applied to the electrode of the third opening 22. In accordance with the voltage application, the current supplied from the first opening 12 and the third opening 22 was measured. And voltage was applied until the measured electric current value became a fixed value under observation of the fluorescence microscope.

　この結果を図８に示す。図８は、電圧印加中の核酸の分離を示す写真である。図８において、（Ａ）は、電圧印加開始時の開口１５および下流流路１６の写真であり、（Ｂ）は、電圧印加後５秒の開口１５および下流流路１６の写真であり、（Ｃ）は、電圧印加後１１．５秒の下流流路１６の写真である。図８（Ａ）において矢印で示すように、電圧印加開始時において、Ｋ５６２細胞は、開口１５にトラップされていた。また、図８（Ｂ）において矢印で示すように、開口１５にトラップされたＫ５６２細胞は、蛍光強度が低下し、細胞質の核酸が分離された。さらに、図８（Ｃ）において矢印で示すように、前記細胞質の核酸が、下流流路１６に分離された。 This result is shown in FIG. FIG. 8 is a photograph showing separation of nucleic acids during voltage application. 8A is a photograph of the opening 15 and the downstream flow path 16 at the start of voltage application, and FIG. 8B is a photograph of the opening 15 and the downstream flow path 16 5 seconds after the voltage application. C) is a photograph of the downstream flow path 16 11.5 seconds after voltage application. As indicated by an arrow in FIG. 8A, the K562 cells were trapped in the opening 15 at the start of voltage application. In addition, as indicated by an arrow in FIG. 8B, the fluorescence intensity of the K562 cells trapped in the opening 15 was reduced, and cytoplasmic nucleic acids were separated. Further, as indicated by an arrow in FIG. 8C, the cytoplasmic nucleic acid was separated into the downstream flow path 16.

　また、チップ２０において、開口１５の幅を、２～５μｍとしたチップ、開口１５の長さを、８～１４μｍとしたチップ、または接続流路２３の長さを、４．９～１０ｍｍとしたチップを用いた以外は、同様にして、電圧を印加し、前記蛍光顕微鏡で観察した。この結果、これらのチップにおいても、前記細胞質の核酸を分離できることがわかった。以上のことから、本発明のチップによれば、核酸を分離できることがわかった。 Further, in the chip 20, a chip having a width of the opening 15 of 2 to 5 μm, a chip having a length of the opening 15 of 8 to 14 μm, or a length of the connection channel 23 is 4.9 to 10 mm. A voltage was applied in the same manner except that the chip was used, and observation was performed with the fluorescence microscope. As a result, it was found that the cytoplasmic nucleic acid can be separated also in these chips. From the above, it was found that according to the chip of the present invention, nucleic acids can be separated.

［実施例３］
　本発明のチップを作製し、核酸を分画後、残部の核酸（以下、「他の核酸」ともいう）を回収できることを確認した。 [Example 3]
After producing the chip of the present invention and fractionating the nucleic acid, it was confirmed that the remaining nucleic acid (hereinafter also referred to as “other nucleic acid”) could be recovered.

　Hoechst 33258（Sigma Aldrich社製）を含む分離液１を用いた以外は、前記実施例２と同様にして、電圧を印加し、前記細胞質の核酸を分画した。前記分画後、第２の開口部１３からチップ２０内の溶液を回収することにより、前記細胞質の核酸を回収した。つぎに、前記蛍光顕微鏡の観察下、マイクロピペットを用いて第１の開口部１２からチップ２０内の溶液を吸引することにより、前記他の核酸を回収した。 The cytoplasmic nucleic acid was fractionated by applying a voltage in the same manner as in Example 2 except that the separation solution 1 containing Hoechst® 33258 (manufactured by Sigma-Aldrich) was used. After the fractionation, the cytoplasmic nucleic acid was recovered by recovering the solution in the chip 20 from the second opening 13. Next, under the observation of the fluorescence microscope, the other nucleic acid was recovered by sucking the solution in the chip 20 from the first opening 12 using a micropipette.

　この結果を図９に示す。図９は、前記他の核酸の回収を示す写真である。図９において、（Ａ）は、吸引前の開口１５の写真を示し、（Ｂ）は、吸引後の開口１５の写真を示す。図９に示すように、第１の開口部１２から吸引することにより、開口１５にトラップされた前記他の核酸が回収された。これらのことから、本発明のチップにより、核酸を分画後、他の核酸を回収できることがわかった。 The result is shown in FIG. FIG. 9 is a photograph showing the recovery of the other nucleic acid. In FIG. 9, (A) shows a photograph of the opening 15 before suction, and (B) shows a photograph of the opening 15 after suction. As shown in FIG. 9, the other nucleic acid trapped in the opening 15 was collected by aspiration from the first opening 12. From these facts, it was found that the nucleic acid was fractionated and other nucleic acids could be recovered using the chip of the present invention.

［実施例４］
　異なる開口１５を有するチップを用いて、核酸を分画できることを確認した。 [Example 4]
It was confirmed that nucleic acids can be fractionated using chips having different openings 15.

（１）チップ
　前記実施例２（１）のチップ２０において、開口１５の側面を、下流流路１６から上流流路１４にかけてテーパー状に形成した以外は同様にしてチップを作製した。開口１５の大きさは、以下の通りとした。
開口１５
　　　長さ　４２μｍ、上流流路１４側の幅　３μｍ、下流流路１６側の幅　５０μｍ、深さ　２５μｍ (1) Chip A chip was manufactured in the same manner as in the chip 20 of Example 2 (1) except that the side surface of the opening 15 was tapered from the downstream flow path 16 to the upstream flow path 14. The size of the opening 15 was as follows.
Opening 15
Length 42 μm, upstream flow path 14 side width 3 μm, downstream flow path 16 side width 50 μm, depth 25 μm

（２）核酸分画
　チップ２０に代えて、前記（１）のチップを用いた以外は、前記実施例２（２）および（３）と同様にして、前記蛍光顕微鏡の観察下、電圧を印加し、前記細胞質の核酸を分画した。 (2) Nucleic acid fractionation A voltage was applied under the observation of the fluorescence microscope in the same manner as in Examples 2 (2) and (3) except that the chip of (1) was used instead of the chip 20. The cytoplasmic nucleic acid was fractionated.

　この結果を図１０に示す。図１０は、電圧印加中の核酸の分離を示す写真である。図１０において、（Ａ）は、電圧印加開始時の開口１５の写真であり、（Ｂ）は、電圧印加後の下流流路１６の写真である。図１０（Ａ）において矢印で示すように、電圧印加開始時において、Ｋ５６２細胞は、開口１５にトラップされていた。また、図１０（Ｂ）において矢印で示すように、電圧印加後、前記細胞質の核酸が、下流流路１６に分離された。さらに、前記実施例３と同様にして、第２の開口部１３から前記細胞質の核酸を、第１の開口部１２から前記残部の核酸を回収した。これらのことから、異なる開口１５を有するチップを用いて、核酸を分画できることおよび前記核酸を回収できることがわかった。 The result is shown in FIG. FIG. 10 is a photograph showing separation of nucleic acids during voltage application. In FIG. 10, (A) is a photograph of the opening 15 at the start of voltage application, and (B) is a photograph of the downstream flow path 16 after voltage application. As indicated by the arrows in FIG. 10A, the K562 cells were trapped in the opening 15 at the start of voltage application. 10B, the cytoplasmic nucleic acid was separated into the downstream channel 16 after voltage application. Further, in the same manner as in Example 3, the cytoplasmic nucleic acid was recovered from the second opening 13 and the remaining nucleic acid was recovered from the first opening 12. From these facts, it was found that the nucleic acid can be fractionated and the nucleic acid can be recovered using a chip having different openings 15.

［実施例５］
　本発明のチップを用いて、ミトコンドリアの核酸以外の細胞質の核酸を分画できることおよび分離した核酸を解析できることを確認した。 [Example 5]
Using the chip of the present invention, it was confirmed that cytoplasmic nucleic acids other than mitochondrial nucleic acids can be fractionated and that separated nucleic acids can be analyzed.

（１）核酸分画
　MitoTrackerGreen FM（Thermo Fisher Scientific社製）を含む分離液１を用いた以外は、前記実施例２と同様にして、前記蛍光顕微鏡の観察下、電圧を印加した。そして、前記電圧印加中、前記ターゲット細胞周囲において、ミトコンドリアの存在を示す蛍光強度を、経時的に測定した。そして、各時間の蛍光強度について、電圧印加開始時（０秒）における前記ターゲット細胞以外の領域の分離用流路１１の蛍光強度で割ることにより、補正後の蛍光強度を算出した。また、コントロールは、前記ターゲット細胞以外の領域において、前記ミトコンドリアの存在を示す蛍光強度を、経時的に測定した以外は、同様にして、補正後の蛍光強度を算出した。 (1) Nucleic acid fraction A voltage was applied under the observation of the fluorescence microscope in the same manner as in Example 2 except that the separation liquid 1 containing MitoTrackerGreen FM (manufactured by Thermo Fisher Scientific) was used. Then, during the voltage application, the fluorescence intensity indicating the presence of mitochondria was measured over time around the target cell. Then, the corrected fluorescence intensity was calculated by dividing the fluorescence intensity at each time by the fluorescence intensity of the separation channel 11 in the region other than the target cells at the start of voltage application (0 seconds). In addition, the control calculated the fluorescence intensity after correction in the same manner except that the fluorescence intensity indicating the presence of the mitochondria was measured over time in a region other than the target cell.

　この結果を図１１に示す。図１１は、補正後の蛍光強度を示すグラフである。図１１に示すように、補正後の蛍光強度は一定であり、ミトコンドリアは、開口１５にトラップされていた。これらのことから、ミトコンドリアの核酸以外の細胞質の核酸を分画できること、すなわち、細胞質の核酸のうち特定の核酸のみを分画できることがわかった。 The result is shown in FIG. FIG. 11 is a graph showing the fluorescence intensity after correction. As shown in FIG. 11, the corrected fluorescence intensity was constant, and mitochondria were trapped in the opening 15. From these, it was found that cytoplasmic nucleic acids other than mitochondrial nucleic acids can be fractionated, that is, only specific nucleic acids can be fractionated among cytoplasmic nucleic acids.

（２）核酸分析
　前記核酸分画後、前記実施例３と同様にして、前記ミトコンドリアの核酸以外の細胞質の核酸および前記他の核酸を回収した。つぎに、前記ミトコンドリアの核酸以外の細胞質の核酸については、前記実施例１（３）と同様にして、インキュベート中の蛍光強度を測定した。また、前記他の核酸は、ｑＰＣＲキット（TaqMan Copy Number Assay、Thermo Fisher Scientific社製）および前記サーマルサイクラーを用い、添付のプロトコルに基づき実施した。プライマーは、前記ＧＡＰＤＨプライマーを用いた。具体的に、９μＬの前記他の核酸を含む溶液を含む、合計２０μＬの反応液について、９５℃１５分の条件でインキュベート後、９５℃１５秒、６０℃１分を１サイクルとし、５０回、前記サイクルを実施した。そして、６０℃でインキュベート中に蛍光強度を測定した。 (2) Nucleic Acid Analysis After the nucleic acid fractionation, cytoplasmic nucleic acids other than the mitochondrial nucleic acids and the other nucleic acids were recovered in the same manner as in Example 3. Next, for the cytoplasmic nucleic acid other than the mitochondrial nucleic acid, the fluorescence intensity during the incubation was measured in the same manner as in Example 1 (3). Further, the other nucleic acids were carried out using a qPCR kit (TaqMan Copy Number Assay, manufactured by Thermo Fisher Scientific) and the thermal cycler according to the attached protocol. The GAPDH primer was used as the primer. Specifically, a total of 20 μL of the reaction solution containing 9 μL of the other nucleic acid-containing solution was incubated at 95 ° C. for 15 minutes, then 95 ° C. for 15 seconds and 60 ° C. for 1 minute, 50 times, The cycle was carried out. Then, the fluorescence intensity was measured during incubation at 60 ° C.

　この結果を図１２に示す。図１２は、蛍光強度を示すグラフである。図１２において、横軸は、サイクル数を示し、縦軸は、蛍光強度を示す。図１２に示すように、いずれの核酸を用いた場合においても、サイクル数依存的に蛍光強度が増加し、前記ミトコンドリアの核酸以外の細胞質の核酸中には、ＧＡＰＤＨ　ｍＲＮＡが、前記他の核酸には、ＧＡＰＤＨのゲノムＤＮＡが含まれていることが確認された。以上のことから、本発明のチップを用いて、ミトコンドリアの核酸以外の細胞質の核酸を分画できることおよび分画した核酸を解析できることがわかった。 The result is shown in FIG. FIG. 12 is a graph showing fluorescence intensity. In FIG. 12, the horizontal axis indicates the number of cycles, and the vertical axis indicates the fluorescence intensity. As shown in FIG. 12, in any case of using any nucleic acid, the fluorescence intensity increases depending on the number of cycles, and in the cytoplasmic nucleic acid other than the mitochondrial nucleic acid, GAPDH mRNA is different from the other nucleic acid. Was confirmed to contain GAPDH genomic DNA. From the above, it was found that cytoplasmic nucleic acids other than mitochondrial nucleic acids can be fractionated and fractionated nucleic acids can be analyzed using the chip of the present invention.

［実施例６］
　本発明のチップを用いて、核酸を分画できることを確認した。 [Example 6]
It was confirmed that nucleic acids could be fractionated using the chip of the present invention.

（１）試料
　ＢＪ細胞（ＡＴＣＣから入手、ＣＲＬ－２５２２）を３７℃、５％ＣＯ２の条件下で培養した。培養液は、１０％ＦＢＳおよび１％ペニシリン／ストレプトマイシンを含むＤＭＥＭ培養液（SigmaAldrich社製）を使用した。前記培養後、ＴｒｙｐＬＥ（Thermo Fisher Scientific社製）を用い、前記細胞を回収した以外は、前記実施例１（１）と同様にして、前記細胞分散液に分散した。 (1) (obtained from ATCC, CRL-2522) sample BJ cells 37 ° C., and cultured under the conditions of 5% CO 2. As the culture solution, a DMEM culture solution (Sigma Aldrich) containing 10% FBS and 1% penicillin / streptomycin was used. After the culturing, the cells were dispersed in the cell dispersion in the same manner as in Example 1 (1) except that the cells were collected using TrypLE (manufactured by Thermo Fisher Scientific).

（２）核酸分画
　そして、前記実施例３と同様にして、前記細胞質の核酸と前記他の核酸とを回収した。これらの結果から、本発明のチップを用いて、核酸を分画できることを確認した。 (2) Nucleic acid fractionation In the same manner as in Example 3, the cytoplasmic nucleic acid and the other nucleic acids were recovered. From these results, it was confirmed that the nucleic acid could be fractionated using the chip of the present invention.

［実施例７］
　本発明のチップを用いて、ユーグレナの核酸を分画できることを確認した。 [Example 7]
It was confirmed that Euglena nucleic acid can be fractionated using the chip of the present invention.

（１）試料
　ユーグレナ（Euglena gracilis Klebs NIES-48、ユーグレナ社より入手）を、継代後、２９℃、連続光下で、４日間培養し、ＯＤ値（６８０ｎｍ）が１．５～２．０になるように調整した。ユーグレナの培地としては、Koren-Hutner培地（ｐＨ３．５）を用いた。前記培養後、培地を回収し、純水（UltraPure(TM) DNase/RNase-Free Distilled Water、Life Technologies社製）を用いて、２００倍に希釈し、分散することにより、試料を調製した。 (1) Sample Euglena (Euglena gracilis Klebs NIES-48, obtained from Euglena) was cultured for 4 days at 29 ° C. under continuous light after passage. The OD value (680 nm) was 1.5 to 2.0. It was adjusted to become. As the Euglena medium, Koren-Hutner medium (pH 3.5) was used. After the culture, the medium was collected, diluted to 200 times with pure water (UltraPure ™ DNase / RNase-Free Distilled Water, manufactured by Life Technologies), and dispersed to prepare a sample.

　チップは、前記実施例２のチップを使用した。まず、チップ２０には、第１の開口部１２に２０μＬの分離液３を、第２の開口部１３に２０μＬの分離液２を導入した。前記分離液３の組成は、３００ｍｍｏｌ／Ｌ　Ｔｒｉｓおよび１５０ｍｍｏｌ／Ｌ　ＨＣｌとし、ｐＨは、８．２とした。なお、本実施例の分離液３には、SYBR（登録商標）Green IIを添加したものを使用した。つぎに、第３の開口部２２から吸引することにより第１の開口部１２から第３の開口部２２への分離液の流れを生じさせた。つぎに、第１の開口部１２に２０μＬの分離液２を、第２の開口部１３に２０μＬの分離液３を導入した。さらに、１μＬのユーグレナを含む試料を第１の開口部１２の内部に導入した。前記細胞は、前記分離液の流れにより、第３の開口部２２方向に移動し、開口１５にトラップされた。ユーグレナが、開口１５にトラップされたことは、前記光学顕微鏡により確認した。そして、第３の開口部２２に５０μＬの分離液２を導入した。 The chip used in Example 2 was used. First, 20 μL of the separation liquid 3 was introduced into the first opening 12 and 20 μL of the separation liquid 2 was introduced into the second opening 13 into the chip 20. The composition of the separation liquid 3 was 300 mmol / L Tris and 150 mmol / L HCl, and the pH was 8.2. In addition, what added SYBR (trademark) Green (TM) II was used for the separation liquid 3 of a present Example. Next, a flow of the separation liquid from the first opening 12 to the third opening 22 was generated by suction from the third opening 22. Next, 20 μL of the separation liquid 2 was introduced into the first opening 12, and 20 μL of the separation liquid 3 was introduced into the second opening 13. Further, a sample containing 1 μL of Euglena was introduced into the first opening 12. The cells moved toward the third opening 22 by the flow of the separation liquid and were trapped in the opening 15. It was confirmed by the optical microscope that Euglena was trapped in the opening 15. Then, 50 μL of the separation liquid 2 was introduced into the third opening 22.

　第１の開口部１２、第２の開口部１３および第３の開口部２２に白金線電極（直径０．８ｍｍ）を挿入後、第１の開口部１２の電極に－３００Ｖ、第２の開口部１３の電極に０Ｖ、第３の開口部２２の電極－２６０Ｖの電圧を印加した。前記電圧印加時にあわせ、第１の開口部１２および第３の開口部２２から供給される電流を計測した。そして、前記蛍光顕微鏡の観察下、計測した電流値が一定値となるまで、電圧を印加した。また、コントロールは、前記ユーグレナを導入しなかった以外は、同様にして、前記蛍光顕微鏡の観察下、電圧を印加した。 After a platinum wire electrode (diameter 0.8 mm) is inserted into the first opening 12, the second opening 13, and the third opening 22, -300V is applied to the electrode of the first opening 12, and the second opening The voltage of 0 V and the voltage of the electrode of the third opening 22 -260 V were applied to the electrode of the portion 13. In accordance with the voltage application, the current supplied from the first opening 12 and the third opening 22 was measured. Then, under the observation of the fluorescence microscope, a voltage was applied until the measured current value became a constant value. Further, as a control, a voltage was applied in the same manner under the observation of the fluorescence microscope except that the Euglena was not introduced.

　この結果を図１３に示す。図１３は、電圧印加中の核酸の分離を示す写真である。図１３において、（Ａ）は、電圧印加開始時の開口１５の写真であり、（Ｂ）は、電圧印加中の開口１５の写真であり、（Ｃ）は、電圧印加中の下流流路１６の写真であり、（Ｄ）は、電圧印加後の開口１５の写真であり、（Ｅ）は、コントロールにおける電圧印加中の下流流路１６の写真である。図１３（Ａ）において矢印で示すように、電圧印加開始時において、ユーグレナは、開口１５にトラップされていた。また、図１３（Ｂ）において矢印で示すように、電圧印加中に葉緑体の核酸が分離された。さらに、図１３（Ｃ）において矢印で示すように、前記葉緑体の核酸が、下流流路１６に分離された。そして、図１３（Ｄ）において矢印で示すように、電圧印加後、前記他の核酸は、開口１５にトラップされていた。他方、図１３（Ｅ）に示すように、コントロールでは、前記葉緑体の核酸の分離は観察されなかった。これらのことから、本発明のチップを用いて、ユーグレナの核酸を分画できることがわかった。 The result is shown in FIG. FIG. 13 is a photograph showing separation of nucleic acids during voltage application. In FIG. 13, (A) is a photograph of the opening 15 at the start of voltage application, (B) is a photograph of the opening 15 during voltage application, and (C) is the downstream flow path 16 during voltage application. (D) is a photograph of the opening 15 after voltage application, and (E) is a photograph of the downstream flow path 16 during voltage application in the control. As shown by the arrow in FIG. 13A, the Euglena was trapped in the opening 15 at the start of voltage application. In addition, as indicated by arrows in FIG. 13B, chloroplast nucleic acids were separated during voltage application. Further, as shown by the arrow in FIG. 13C, the chloroplast nucleic acid was separated into the downstream flow path 16. Then, as indicated by an arrow in FIG. 13D, the other nucleic acid was trapped in the opening 15 after voltage application. On the other hand, as shown in FIG. 13 (E), separation of the chloroplast nucleic acid was not observed in the control. From these results, it was found that Euglena nucleic acid can be fractionated using the chip of the present invention.

［実施例８］
　本発明のチップを用いて分画した核酸からライブラリを調製後、次世代シークエンシング技術により解析することで、細胞質の核酸と、細胞質以外の核酸を精度よく分画できていることを確認した。 [Example 8]
After preparing a library from nucleic acids fractionated using the chip of the present invention, it was confirmed that the cytoplasmic nucleic acid and the non-cytoplasmic nucleic acid could be fractionated with high precision by analyzing with a next-generation sequencing technique.

　前記ＢＪ細胞に代えて、前記Ｋ５６２用いた以外は、前記実施例３と同様にして、前記細胞質の核酸と前記他の核酸とを分画後、前記細胞質の核酸と前記他の核酸とを回収した。ｃＤＮＡ作成キット(SMART-Seq（登録商標） v4 Ultra（登録商標） Low Input RNA Kit for Sequencing、Clontech社製)およびサーマルサイクラー（S1000, Bio-rad社製）を用いて、添付のプロトコルに基づき得られた各核酸を含む溶液からｃＤＮＡを調製した。 The cytoplasmic nucleic acid and the other nucleic acid are recovered after fractionating the cytoplasmic nucleic acid and the other nucleic acid in the same manner as in Example 3 except that the K562 is used instead of the BJ cell. did. Using a cDNA generation kit (SMART-Seq (registered trademark) v4 Ultra (registered trademark) Low Input RNA Kit for Sequencing (Clontech) and a thermal cycler (S1000, Bio-rad) based on the attached protocol CDNA was prepared from the obtained solution containing each nucleic acid.

　具体的には、１μＬの前記ｃＤＮＡ作成キットのバッファーと、９．５μＬの回収した溶液とを含む、合計１０．５μＬの反応液を調製した。つぎに、前記反応液について、室温（約２５℃）、５分の条件で反応を行った。前記反応後、１０．５μＬの前記反応液に、２μＬのプライマー溶液（3’ SMART-Seq CDS Primer II A, Clontech社製）を添加後、７２℃で３分インキュベートした。さらに、前記インキュベート後の反応液と、７．５μＬの前記ｃＤＮＡ作成キットのMaster mixとを含む合計２０μＬの逆転写反応液を調製した。前記逆転写反応液について、４２℃９０分の条件でインキュベート後、さらに７０℃１０分の条件で逆転写を行った。そして、逆転写後の逆転写反応液と、３０μＬの前記ｃＤＮＡ作成キットのPCR Master Mixとを含む合計５０μＬの増幅反応液を調製した。さらに、前記増幅反応液について、９５℃１分の条件でインキュベート後、さらに、９８℃１０秒、６５℃３０秒、６８℃３分を１サイクルとし、１８回、前記サイクルを実施して増幅反応を行った。前記サイクル後、さらに、７２℃１０分の条件でインキュベートして前記増幅反応を終了した。 Specifically, a total of 10.5 μL of a reaction solution containing 1 μL of the buffer for the cDNA preparation kit and 9.5 μL of the collected solution was prepared. Next, the reaction solution was reacted at room temperature (about 25 ° C.) for 5 minutes. After the reaction, 2 μL of a primer solution (3 ′ SMART-Seq CDS Primer II A, manufactured by Clontech) was added to 10.5 μL of the reaction solution, followed by incubation at 72 ° C. for 3 minutes. Furthermore, a total of 20 μL of the reverse transcription reaction solution containing the reaction solution after the incubation and 7.5 μL of Master mix of the cDNA preparation kit was prepared. The reverse transcription reaction solution was incubated at 42 ° C. for 90 minutes, and then reverse transcription was performed at 70 ° C. for 10 minutes. Then, a total of 50 μL of the amplification reaction solution containing the reverse transcription reaction solution after reverse transcription and 30 μL of the PCR Master Mix of the cDNA preparation kit was prepared. Furthermore, after incubating the amplification reaction solution at 95 ° C. for 1 minute, the amplification reaction was carried out 18 times, further comprising 98 ° C. for 10 seconds, 65 ° C. for 30 seconds, and 68 ° C. for 3 minutes. Went. After the cycle, the amplification reaction was completed by further incubation at 72 ° C. for 10 minutes.

　つぎに、AMPure XP Kit（Beckman Coulter社製）を用いて、添付のプロトコルに基づき、前記５０μＬの増幅反応液から増幅ｃＤＮＡを精製し、１７μＬのｃＤＮＡ溶液を得た。さらに、TruSeq ChIP Sample Prep Kit（Illumina社製）を用いて、添付のプロトコルに基づき、ライブラリを調製した。そして、前記調製後のライブラリについて、HiSeq（Illumina社製)を用いて、Paired End、１００塩基／１リード、５００万リードペアの条件でシークエンス解析を行った。出力データについては、ベースコール、フィルタリング、およびIndex配列による振り分けを行い、リード配列および各塩基のクオリティデータを含むＦＡＳＴＱ形式のデータファイルを得た。前記データファイルはＳＴＡＲ（A. Dobin et al., “STAR: ultrafast universal RNA-seq aligner”, Bioinformatics, 2012, vol.29, No.1, pp.15-21）を用いてマッピングを行い、Ｃｕｆｆｌｉｎｋｓ（C. Trapnell et al., “Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.”, Nat. Protoc., 2012, Vol.7, No.3, pp562-578）により遺伝子発現解析を行った。前記遺伝子発現解析では、第２の開口部１３から回収して作成したライブラリ（細胞質の核酸に由来するライブラリ（細胞質のライブラリ））と、第１の開口部１２から回収して作成したライブラリ（その他の核酸に由来するライブラリ（核のライブラリ））とについて、各ライブラリが含む配列の種類（エキソンまたはイントロン）および各ライブラリの由来（常染色体由来またはミトコンドリアＤＮＡ）について検討した（ｎ＝２）。 Next, the amplified cDNA was purified from the 50 μL amplification reaction solution using AMPure® XP® Kit (manufactured by Beckman® Coulter) based on the attached protocol to obtain a 17 μL cDNA solution. Furthermore, a library was prepared using TruSeq プ ロ ト コ ル ChIP Sample Prep Kit (manufactured by Illumina) based on the attached protocol. The prepared library was subjected to sequence analysis using HiSeq (manufactured by Illumina) under the conditions of Paired End, 100 bases / 1 read, and 5 million read pairs. The output data was sorted by base call, filtering, and index sequence to obtain a FASTQ format data file containing the read sequence and the quality data of each base. The data file is mapped using STAR (A. Dobin et al., "STAR: ultrafast universal RNA-seq aligner", Bioinformatics, 2012, vol.29, No.1, pp.15-21), Cufflinks (C. Trapnell et al., “Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.”, Nat. Protoc., 2012, Vol.7, No.3, pp562-578) Went. In the gene expression analysis, a library created by collecting from the second opening 13 (library derived from cytoplasmic nucleic acid (cytoplasmic library)) and a library collected by creating from the first opening 12 (others) And the library (nucleus library) derived from each nucleic acid were examined for the type of sequence (exon or intron) contained in each library and the origin (autosomal or mitochondrial DNA) of each library (n = 2).

　各ライブラリが含む配列の種類の結果を図１４に示す。図１４は、各ライブラリが含む配列の種類を示すグラフである。図１４において、（Ａ）は、前記細胞質のライブラリの結果であり、（Ｂ）は、核のライブラリの結果を示す。図１４（Ａ）に示すように、前記細胞質のライブラリでは、イントロンを含むＲＮＡはほとんど存在せず、成熟したＲＮＡが大半を占めた。他方、図１４（Ｂ）に示すように、前記核のライブラリでは、イントロンを含むＲＮＡが半数を占め、多数のスプライシング前の未成熟ＲＮＡが存在した。 FIG. 14 shows the result of the types of sequences included in each library. FIG. 14 is a graph showing the types of sequences included in each library. 14A shows the results of the cytoplasmic library, and FIG. 14B shows the results of the nuclear library. As shown in FIG. 14 (A), in the cytoplasmic library, RNA containing introns hardly existed, and mature RNA occupied the majority. On the other hand, as shown in FIG. 14 (B), in the nuclear library, RNA containing introns accounted for half, and many immature RNAs before splicing existed.

　つぎに、各ライブラリの由来の結果を図１５に示す。図１５は、各ライブラリの由来（染色体名）を示すグラフである。図１５において、横軸は、各ライブラリを構成するｃＤＮＡの由来（染色体名）を示す。各ｃＤＮＡの由来において、各バーは、左から、前記細胞質のライブラリ、前記核のライブラリ、前記細胞質のライブラリ、前記核のライブラリを示す。図１５に示すように、前記細胞質のライブラリおよび前記核のライブラリは、いずれも、常染色体に由来するｃＤＮＡを含んだ。他方、前記細胞質のライブラリは、ミトコンドリアＤＮＡ由来のｃＤＮＡを含むのに対し、前記核のライブラリは、前記細胞質のライブラリをほとんど含まなかった。 Next, the results from each library are shown in FIG. FIG. 15 is a graph showing the origin (chromosome name) of each library. In FIG. 15, the horizontal axis indicates the origin (chromosome name) of the cDNA constituting each library. In the origin of each cDNA, each bar indicates the cytoplasmic library, the nuclear library, the cytoplasmic library, and the nuclear library from the left. As shown in FIG. 15, the cytoplasmic library and the nuclear library both contained autosomal cDNA. On the other hand, the cytoplasmic library contained cDNA derived from mitochondrial DNA, whereas the nuclear library contained almost no cytoplasmic library.

　一般的に、イントロンを含む未成熟ＲＮＡは、核に存在し、細胞質にはほとんど存在しないことが知られている。また、ミトコンドリアＤＮＡ由来のＲＮＡは、ほとんどが細胞質に存在し、核にはほとんど存在しないことが知られている。上述の結果は、いずれもこれらの知見と合致することから、本発明のチップおよび分画方法により、極めて精度（純度）よく、細胞質の核酸と、その他の核酸（例えば、核の核酸）とを分画できることが分かった。また、本発明のチップおよび分画方法を用いることにより、極めて精度よく分画され、且つ細胞質の核酸を豊富に含むサンプルが得られることから、本発明のチップおよび分画方法により、次世代シークエンシングに利用可能な品質を有するサンプルを調製できることが分かった。 Generally, it is known that immature RNA containing introns exists in the nucleus and hardly exists in the cytoplasm. It is known that most RNA derived from mitochondrial DNA is present in the cytoplasm and hardly present in the nucleus. Since the above results are consistent with these findings, cytochip nucleic acids and other nucleic acids (for example, nuclear nucleic acids) can be obtained with extremely high precision (purity) by the chip and fractionation method of the present invention. I found that it can be fractionated. In addition, by using the chip and the fractionation method of the present invention, a sample that is fractionated with extremely high accuracy and rich in cytoplasmic nucleic acids can be obtained. It has been found that samples with quality available for singing can be prepared.

［実施例９］
　本発明のチップを用いて核酸を分画後、前記チップ内で核酸の増幅およびライブラリの作製が可能であることを確認した。 [Example 9]
After fractionating nucleic acids using the chip of the present invention, it was confirmed that nucleic acid amplification and library preparation were possible within the chip.

　図２に示すチップ２０として、各部位の大きさが以下の通りのチップを用いた以外は、前記実施例３と同様にして、前記細胞質の核酸と前記他の核酸とを分画した。つぎに、ＰＥＧ－ＤＡ溶液（Poly(ethylene glycol) diacrylate（MW575）、１％　2,2-Dimethoxy-2-phenylacetophenoneを含む）を、第３の開口部２２からチップ２０の分離用流路１１に導入後、分離用流路１１に紫外線を照射することにより、ＰＥＧ－ＤＡをゲル化した。さらにｃＤＮＡライブラリ作成キット（REPLI-g WTA Single Cell Kit、Qiagen社製）を用い、第１の開口部１２および第２の開口部１３において、ｃＤＮＡライブラリを調製した。
第１の開口部１２
　　　直径　５ｍｍの逆円錐形、容積　３５μＬ
第２の開口部１３
　　　直径　５ｍｍの逆円錐形、容積　３５μＬ
上流流路１４
　　　長さ　４６００μｍ、幅　５０μｍ、深さ　２５μｍ
開口１５
　　　長さ　５μｍ、幅　３μｍ、深さ　２５μｍ
下流流路１６
　　　長さ　２００００μｍ、幅　５０μｍ、深さ　２５μｍ
調整用流路２１
　　　長さ　１１００μｍ、幅　５０μｍ、深さ　２５μｍ
第３の開口部２２
　　　直径　５ｍｍの円柱形、容積　１００μＬ
接続流路２３
　　　長さ　７３１０μｍ、幅　２５μｍ、深さ　２５μｍ The cytoplasmic nucleic acid and the other nucleic acids were fractionated in the same manner as in Example 3 except that a chip having the following size was used as the chip 20 shown in FIG. Next, a PEG-DA solution (containing Poly (ethylene glycol) diacrylate (MW575), 1% 2,2-Dimethoxy-2-phenylacetophenone) is transferred from the third opening 22 to the separation channel 11 of the chip 20. After the introduction, PEG-DA was gelled by irradiating the separation channel 11 with ultraviolet rays. Further, a cDNA library was prepared in the first opening 12 and the second opening 13 using a cDNA library preparation kit (REPLI-g WTA Single Cell Kit, manufactured by Qiagen).
First opening 12
5mm diameter inverted cone, 35μL capacity
Second opening 13
5mm diameter inverted cone, 35μL capacity
Upstream flow path 14
Length 4600μm, width 50μm, depth 25μm
Opening 15
Length 5μm, width 3μm, depth 25μm
Downstream channel 16
Length 20000 μm, width 50 μm, depth 25 μm
Adjustment flow path 21
Length 1100μm, width 50μm, depth 25μm
Third opening 22
Cylindrical shape with a diameter of 5 mm, volume of 100 μL
Connection channel 23
Length 7310μm, width 25μm, depth 25μm

　具体的には、第１の開口部１２および第２の開口部１３に、それぞれ、４μＬの前記キットの溶解溶液を加え、２４℃５分、９５℃３分の条件でインキュベートした。つぎに、第１の開口部１２および第２の開口部１３に、それぞれ、２μＬのDNA wipeout bufferを加え、４２℃１０分の条件でインキュベートした。さらに、ＲＮＡの逆転写を行なうため、オリゴｄＴプライマーを含む６μＬの反応液を、第１の開口部１２および第２の開口部１３に、それぞれに加え、４２℃６０分の条件でインキュベート後、９５℃３分の条件で加熱した。そして、ライゲーション反応溶液を、第１の開口部１２および第２の開口部１３に、それぞれ、１０μＬ加えて２４℃３０分の条件で反応させた後、９５℃５分の条件で反応を停止した。前記反応停止後、３０μＬの増幅反応溶液を加えて３０℃２時間の条件でインキュベートし、ｃＤＮＡライブラリを得た。 Specifically, 4 μL of the lysis solution of the kit was added to each of the first opening 12 and the second opening 13 and incubated under conditions of 24 ° C. for 5 minutes and 95 ° C. for 3 minutes. Next, 2 μL of DNA wipeout buffer was added to each of the first opening 12 and the second opening 13 and incubated at 42 ° C. for 10 minutes. Further, in order to perform reverse transcription of RNA, 6 μL of a reaction solution containing an oligo dT primer was added to each of the first opening 12 and the second opening 13 and incubated at 42 ° C. for 60 minutes. Heating was performed at 95 ° C. for 3 minutes. And after adding 10 microliters of ligation reaction solutions to the 1st opening part 12 and the 2nd opening part 13, respectively, and making it react on the conditions for 24 degreeC for 30 minutes, reaction was stopped on the conditions for 95 degreeC for 5 minutes . After the reaction was stopped, 30 μL of amplification reaction solution was added and incubated at 30 ° C. for 2 hours to obtain a cDNA library.

　第１の開口部１２および第２の開口部１３で得られたｃＤＮＡライブラリについて、その収量を分子分光分析計（Qubit（登録商標）fluorometer、Thermo Fisher Scientific社製）および核酸定量キット（Qubit（登録商標）dsDNA HS Assay kit、Thermo Fisher Scientific社製）により測定した。 With respect to the cDNA libraries obtained in the first opening 12 and the second opening 13, the yield was measured for a molecular spectrometer (Qubit (registered trademark) fluorometer, Thermo Fisher Fisher Scientific) and a nucleic acid quantification kit (Qubit (registered)). (Trademark) dsDNA-HS-Assay-kit, Thermo-Fisher-Scientific).

　この結果を図１６に示す。図１６は、ｃＤＮＡライブラリの収量を示すグラフであり、横軸は、ライブラリの種類を示し、縦軸は、収量を示す。図１６に示すように、第１の開口部１２および第２の開口部１３のいずれで得られたｃＤＮＡライブラリも、前記ｃＤＮＡライブラリを用いて、さらなる分析を行なうのに十分な収量であった。また、第１の開口部１２および第２の開口部１３の両者で得られたｃＤＮＡライブラリの収量の合計は、一般的に１細胞から得られるｃＤＮＡライブラリの収量と同等以上であった。これらのことから、本発明のチップを用いて核酸を分画後、前記チップ内で細胞質の核酸およびその他の核酸の増幅が可能であり、さらに、前記チップ内で、前記細胞質の核酸およびその他の核酸に由来するライブラリの作製が可能であることがわかった。 The results are shown in FIG. FIG. 16 is a graph showing the yield of cDNA library. The horizontal axis represents the type of library, and the vertical axis represents the yield. As shown in FIG. 16, the cDNA library obtained from both the first opening 12 and the second opening 13 had a sufficient yield for further analysis using the cDNA library. Further, the total yield of the cDNA library obtained from both the first opening 12 and the second opening 13 was generally equal to or higher than the yield of the cDNA library obtained from one cell. From these facts, it is possible to amplify cytoplasmic nucleic acids and other nucleic acids in the chip after fractionating the nucleic acids using the chip of the present invention. It was found that a library derived from nucleic acid can be prepared.

［実施例１０］
　本発明のチップにおいて、前記ターゲット細胞が前記壁の開口にトラップされた状態で、前記ターゲット細胞の細胞膜を破壊でき、かつ前記ターゲット細胞の核膜の破壊をより抑制できる、壁の開口の径（ｗ１）と、壁の開口とターゲット細胞の核膜との最短距離（ｄ）との関係式を算出した。 [Example 10]
In the chip of the present invention, in the state where the target cell is trapped in the opening of the wall, the cell membrane of the target cell can be destroyed, and the destruction of the nuclear membrane of the target cell can be further suppressed. The relational expression between w 1 ) and the shortest distance (d) between the opening of the wall and the nuclear membrane of the target cell was calculated.

　シミュレーションソフト（COMSOL、COMSOL Inc.社製）を用い、下記の条件を設定し、壁の開口から距離ｄｏの位置にある核膜の電場（電流密度、Ｅｎｕｃ）の壁の開口の電場（電流密度、Ｅｏｒｉｆｉｃｅ）に対する比をシミュレーションした。
・ターゲット細胞：核膜と細胞膜とが同心球（（ｗｔ－ｗｎ）／２＝ｄ）
・分離用流路の壁の性質：絶縁
・壁の開口の電流密度：一様な電流密度（電場）
・分離用流路内の溶媒：水 Simulation software (COMSOL, COMSOL Inc. Co.) was used, set the following conditions, the electric field (current density, E nuc) of the nuclear membrane at the position of the opening of the wall distance d o of the walls of the opening of the field ( The ratio to the current density (E orifice ) was simulated.
Target cell: the nuclear membrane and the cell membrane are concentric spheres ((w t −w n ) / 2 = d)
-Wall characteristics of separation channel: Insulation-Current density of wall opening: Uniform current density (electric field)
-Solvent in the separation channel: water

　壁の開口から出てくる電気力線は、壁の開口端から放射状に広がるため、二次元の場合は円筒状に、三次元の場合は、球面状に広がる。このため、壁の開口から距離ｄｏの位置にある核膜での電気力線の密度は、壁の開口から出てくる電気力線の総数を円筒の表面積（三次元の場合は球面の表面積）で割ると算出できる。また、電場の強さは、電気力線の密度に比例する。したがって、壁の開口の電場（Ｅｏｒｉｆｉｃｅ）と、核膜の電場（Ｅｎｕｃ）とは、下記式（４）により近似できると考えられた。
　　　Ｅｎｕｃ＝Ｅｏｒｉｆｉｃｅ×ｗ１／（πｄ＋ｗ１）　　　・・・（４）
　　　　Ｅｎｕｃ：核膜の電場
　　　　Ｅｏｒｉｆｉｃｅ：壁の開口の電場
　　　　ｗ１：壁の開口の径
　　　　π：円周率
　　　　ｄ：壁の開口と、ターゲット細胞の核膜との最短距離 Since the electric lines of force that emerge from the opening of the wall spread radially from the opening end of the wall, they expand in a cylindrical shape in the two-dimensional case and in a spherical shape in the three-dimensional case. Therefore, the density of electric lines of force in the nuclear membrane in the opening of the wall at a distance d o, if the total number of electric flux lines coming out of the opening in the wall of the cylindrical surface area (three-dimensional spherical surface area ) Divided by The strength of the electric field is proportional to the density of the electric field lines. Therefore, it was considered that the electric field (E orifice ) of the wall opening and the electric field (E nuc ) of the nuclear membrane can be approximated by the following equation (4).
E nuc = E orifice × w 1 / (πd + w 1) ··· (4)
E nuc: field of nuclear envelope E Orifice: field wall of the opening w 1: diameter of the wall of the opening [pi: pi d: minimum distance between the opening of the wall, and the nuclear membrane of the target cell

　つぎに、前記シミュレーション結果が、前記近似式により近似できるかを、前記シミュレーション結果と前記近似式とを比較することにより確認した。これらの結果を図１７に示す。図１７は、前記シミュレーション結果および前記近似式を示すグラフである。図１７において、横軸は、壁の開口から距離ｄｏを示し、縦軸は、壁の開口から距離ｄｏの位置にある核膜の電場（Ｅｎｕｃ）の壁の開口の電場（Ｅｏｒｉｆｉｃｅ）に対する比（Ｅｎｕｃ／Ｅｏｒｉｆｉｃｅ）を示す。また、図１７において、図中の記号は、前記シミュレーション結果を示し、実線は、前記式（４）の近似式を示し、図中の矢印で示す数字は、壁の開口の径（ｗ１）を示す。図１７に示すように、前記式（４）の近似式は、前記シミュレーション結果を高い相関で近似できることが分かった。 Next, it was confirmed by comparing the simulation result and the approximate expression whether the simulation result can be approximated by the approximate expression. These results are shown in FIG. FIG. 17 is a graph showing the simulation result and the approximate expression. 17, the horizontal axis represents the distance d o from the opening of the wall, and the vertical axis represents the electric field of the wall of the opening of the electric field of the nuclear membrane in the opening of the wall at a distance d o (E nuc) (E orifice ) represents the ratio (E nuc / E orifice) for. In FIG. 17, the symbol in the figure indicates the simulation result, the solid line indicates the approximate expression of the expression (4), and the number indicated by the arrow in the figure indicates the diameter (w 1 ) of the wall opening. Indicates. As shown in FIG. 17, it was found that the approximate expression of the expression (4) can approximate the simulation result with high correlation.

　本発明者らは、壁の開口の電場および核膜の電場が、それぞれ、下記式（５）および（６）を満たすことにより、前記ターゲット細胞が前記壁の開口にトラップされた状態で、前記ターゲット細胞の細胞膜を破壊でき、かつ前記ターゲット細胞の核膜の破壊をより抑制できるとの知見を得ている。
　　　Ｅｏｒｉｆｉｃｅ≧１００（ｋＶ／ｍ）・・・（５）
　　　Ｅｎｕｃ≦５０（ｋＶ／ｍ）・・・・（６）
　　　　Ｅｎｕｃ：核膜の電場
　　　　Ｅｏｒｉｆｉｃｅ：壁の開口の電場 The present inventors have satisfied that the target cell is trapped in the opening of the wall by satisfying the following formulas (5) and (6), respectively, in the electric field of the wall opening and the nuclear membrane: It has been found that the cell membrane of the target cell can be destroyed, and the destruction of the nuclear membrane of the target cell can be further suppressed.
E orientation ≧ 100 (kV / m) (5)
E nuc ≦ 50 (kV / m ) ···· (6)
E nuc: field of nuclear envelope E Orifice: field wall opening

　そこで、前記式（４）～（６）に基づき、前記壁の開口の断面形状が矩形および円形である場合の壁の開口の径（ｗ１）と、壁の開口とターゲット細胞の核膜との最短距離（ｄ）との関係式を算出した。 Therefore, based on the equations (4) to (6), the diameter (w 1 ) of the wall opening when the cross-sectional shape of the wall opening is rectangular and circular, the wall opening, and the nuclear membrane of the target cell The relational expression with the shortest distance (d) was calculated.

　この結果、前記壁の開口の断面形状が長方形等の矩形である場合、前記壁の開口の径（ｗ１）は、下記式（２）を満たすことで、前記ターゲット細胞の細胞膜を破壊でき、かつ前記ターゲット細胞の核膜の破壊を抑制できることが分かった。また、前記壁の開口の断面形状が円形である場合、前記壁の開口の径（ｗ１）は、下記式（３）を満たすことで、前記ターゲット細胞の細胞膜を破壊でき、かつ前記ターゲット細胞の核膜の破壊をより抑制できることが分かった。
　　　ｗ１≦πｄ　　　　・・・（２）
　　　ｗ１ ２≦８ｄ２　　　・・・（３）
　　　　ｗ１：壁の開口の径
　　　　π：円周率
　　　　ｄ：壁の開口と、ターゲット細胞の核膜との最短距離 As a result, when the cross-sectional shape of the opening of the wall is a rectangle such as a rectangle, the diameter (w 1 ) of the opening of the wall can satisfy the following formula (2), thereby destroying the cell membrane of the target cell, And it was found that the destruction of the nuclear membrane of the target cell can be suppressed. Moreover, when the cross-sectional shape of the opening of the wall is circular, the diameter (w 1 ) of the opening of the wall satisfies the following formula (3), so that the cell membrane of the target cell can be destroyed, and the target cell It was found that the destruction of the nuclear membrane can be further suppressed.
w 1 ≦ πd ··· (2)
w 1 2 ≦ 8d 2 (3)
w 1 : Diameter of the opening of the wall π: Circumference d: Shortest distance between the opening of the wall and the nuclear membrane of the target cell

　以上のことから、本発明のチップにおいて、壁の開口の径（ｗ１）と、壁の開口とターゲット細胞の核膜との最短距離（ｄ）との関係を、前記式（２）または（３）とすることにより、前記ターゲット細胞が前記壁の開口にトラップされた状態で、前記ターゲット細胞の細胞膜を破壊でき、かつ前記ターゲット細胞の核膜の破壊を抑制できることが分かった。 From the above, in the chip of the present invention, the relationship between the diameter (w 1 ) of the opening of the wall and the shortest distance (d) between the opening of the wall and the nuclear membrane of the target cell is expressed by the above formula (2) or ( 3), it was found that the cell membrane of the target cell can be destroyed while the target cell is trapped in the opening of the wall, and the destruction of the nuclear membrane of the target cell can be suppressed.

　以上、実施形態および実施例を参照して本発明を説明したが、本発明は上記実施形態および実施例に限定されるものではない。本発明の構成や詳細には、本発明のスコープ内で当業者が理解し得る様々な変更をすることができる。 As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

　この出願は、２０１６年１月２８日に出願された日本出願特願２０１６－０１４７０８および２０１６年９月９日に出願された日本出願特願２０１６－１７７１６３を基礎とする優先権を主張し、その開示のすべてをここに取り込む。 This application claims priority based on Japanese Patent Application No. 2016-014708 filed on Jan. 28, 2016 and Japanese Application No. 2016-177163 filed on Sep. 9, 2016. The entire disclosure is incorporated herein.

　以上のように、本発明のチップによれば、前記分子篩機能を有する分子を含まない液体系においても、核酸等の生体高分子を分画できる。また、本発明のチップによれば、前記残部の生体高分子を、前記壁の開口にトラップした状態で、前記細胞質の生体高分子を分離できるため、例えば、次世代シークエンシングに利用可能な品質を有するサンプルを調製できる。さらに、本発明のチップは、例えば、１個のターゲット細胞を前記分離用流路に導入した際に、前記壁の開口で、前記ターゲット細胞を精度よくトラップできる。このため、本発明のチップによれば、例えば、１個のターゲット細胞から細胞質の生体高分子を分離できる。また、本発明のチップは、前記分離用流路が、前記開口を有する壁をその流路内に有する。このため、本発明のチップは、例えば、前記分離用流路に電圧を印加した際に、前記開口を有する壁を有さない分離用流路（例えば、前記特許文献１の分離用流路）と比較して、前記ターゲット細胞がトラップされる前記壁の開口の周囲の電流密度を増加させることができる。このため、本発明のチップによれば、例えば、前記特許文献１の分離用流路において、前記ターゲット細胞の細胞膜を破砕する際の電圧と比較し、より低い電圧（例えば、１／１０以下の電圧）で、前記ターゲット細胞の細胞膜を破砕できる。また、本発明のチップは、例えば、より低い電圧で使用できることから、電圧印加時のジュール熱の発生を低減でき、前記ジュール熱による生体高分子の変性等の影響を低減できる。さらに、本発明のチップは、例えば、より低い電圧で使用できることから、生体高分子の分離に使用する分離液として、電解質を含む高伝導性溶液が使用できる。このため、本発明は、臨床分野、医療分野、生命科学分野等において極めて有用である。 As described above, according to the chip of the present invention, a biopolymer such as a nucleic acid can be fractionated even in a liquid system that does not contain a molecule having the molecular sieve function. In addition, according to the chip of the present invention, the cytoplasmic biopolymer can be separated in a state where the remaining biopolymer is trapped in the opening of the wall. For example, the quality that can be used for next-generation sequencing Can be prepared. Furthermore, the chip of the present invention can trap the target cells with high accuracy at the opening of the wall when, for example, one target cell is introduced into the separation channel. Therefore, according to the chip of the present invention, for example, a cytoplasmic biopolymer can be separated from one target cell. In the chip of the present invention, the separation channel has a wall having the opening in the channel. For this reason, the chip of the present invention has, for example, a separation channel that does not have a wall having the opening when a voltage is applied to the separation channel (for example, the separation channel in Patent Document 1). Compared with, the current density around the wall opening where the target cells are trapped can be increased. For this reason, according to the chip of the present invention, for example, in the separation channel of Patent Document 1, a lower voltage (for example, 1/10 or less) compared to the voltage at the time of crushing the cell membrane of the target cell. Voltage) can disrupt the cell membrane of the target cell. Moreover, since the chip of the present invention can be used at a lower voltage, for example, generation of Joule heat at the time of voltage application can be reduced, and the influence of denaturation of the biopolymer due to the Joule heat can be reduced. Furthermore, since the chip of the present invention can be used at a lower voltage, for example, a highly conductive solution containing an electrolyte can be used as a separation liquid used for biopolymer separation. For this reason, the present invention is extremely useful in the clinical field, medical field, life science field, and the like.

１　　　　　　　　　　　　　　　　　　基板
１ａ　　　　　　　　　　　　　　　　　上基板
１ｂ　　　　　　　　　　　　　　　　　下基板
１０、２０、３０、４０、５０、６０、７０　生体高分子分画用チップ
１１　　　　　　　　　　　　　　　　　分離用流路
１２　　　　　　　　　　　　　　　　　第１の開口部
１３　　　　　　　　　　　　　　　　　第２の開口部
１４　　　　　　　　　　　　　　　　　上流流路
１５　　　　　　　　　　　　　　　　　開口
１５ａ　　　　　　　　　　　　　　　　トラップ口
１５ｂ、１５ｃ　　　　　　　　　　　　バイパス口
１６　　　　　　　　　　　　　　　　　下流流路
１７、１７ａ、１７ｂ　　　　　　　　　壁
２１　　　　　　　　　　　　　　　　　調整用流路
２２　　　　　　　　　　　　　　　　　第３の開口部
２３　　　　　　　　　　　　　　　　　接続流路
３１、３１ａ、３１ｂ　　　　　　　　　吸引吐出部
３２　　　　　　　　　　　　　　　　　捕獲部
３３　　　　　　　　　　　　　　　　　電極
３４　　　　　　　　　　　　　　　　　液体移動制御部
３５　　　　　　　　　　　　　　　　　バイパス流路
３６ａ、３６ｂ　　　　　　　　　　　　第２の液体移動制御部 DESCRIPTION OF SYMBOLS 1 Substrate 1a Upper substrate 1b Lower substrate 10, 20, 30, 40, 50, 60, 70 Biopolymer fractionation chip 11 Separation flow path 12 First opening 13 Second opening 14 Upstream flow path 15 Opening 15a Trap port 15b, 15c Bypass port 16 Downstream flow path 17, 17a, 17b Wall 21 Adjusting flow path 22 Third opening 23 Connection flow path 31, 31a, 31b Suction discharge section 32 Capture section 33 Electrode 34 Liquid movement Control unit 35 Bypass channels 36a, 36b Second liquid movement control unit

Claims (40)

基板を有し、
前記基板は、ターゲット細胞が有する細胞質の生体高分子を分離する分離用流路と、１以上の開口部とを有し、
前記１以上の開口部は、前記分離用流路に前記ターゲット細胞を含む試料を導入可能な第１の開口部を有し、
前記第１の開口部は、前記分離用流路に連通され、
前記分離用流路は、その流路内であり、且つ断面方向に壁を有し、
前記壁は、開口を有することを特徴とする、生体高分子分画用チップ。 Having a substrate,
The substrate has a separation channel for separating cytoplasmic biopolymers of target cells, and one or more openings.
The one or more openings have a first opening into which a sample containing the target cells can be introduced into the separation channel,
The first opening communicates with the separation channel;
The separation channel is in the channel and has a wall in the cross-sectional direction,
The chip for biopolymer fractionation, wherein the wall has an opening. 前記壁が、２以上の開口を有する、請求項１記載の生体高分子分画用チップ。 The biopolymer fractionation chip according to claim 1, wherein the wall has two or more openings. 前記壁の開口の径（ｗ１）が、前記ターゲット細胞の径（ｗｔ）より小さい、請求項１または２記載の生体高分子分画用チップ。 The biopolymer fractionation chip according to claim 1 or 2, wherein a diameter (w 1 ) of the opening of the wall is smaller than a diameter (w t ) of the target cell. 前記壁の開口の径（ｗ１）と前記ターゲット細胞の径（ｗｔ）との比（ｗ１：ｗｔ）が、１：２以上である、請求項１から３のいずれか一項に記載の生体高分子分画用チップ。 The ratio (w 1 : w t ) of the diameter (w 1 ) of the opening of the wall and the diameter (w t ) of the target cell is 1: 2 or more, according to any one of claims 1 to 3. The chip for biopolymer fraction described. 前記壁の開口の径（ｗ１）が、前記細胞質の生体高分子の分離後のターゲット細胞の残部の径（ｗｎ）より小さい、請求項１から４のいずれか一項に記載の生体高分子分画用チップ。 The living body height according to any one of claims 1 to 4, wherein the diameter (w 1 ) of the opening of the wall is smaller than the diameter (w n ) of the remainder of the target cell after separation of the cytoplasmic biopolymer. Chip for molecular fractionation. 前記ターゲット細胞の径（ｗｔ）と前記分離用流路の径（ｗ２）との比（ｗｔ：ｗ２）が、１：１～１：１００の範囲である、請求項１から５のいずれか一項に記載の生体高分子分画用チップ。 The ratio (w t : w 2 ) between the diameter (w t ) of the target cell and the diameter (w 2 ) of the separation channel is in the range of 1: 1 to 1: 100. The biopolymer fractionation chip according to any one of the above. 前記壁の開口の断面積（Ｓ１）と前記分離用流路の断面積（Ｓ２）との比（Ｓ１：Ｓ２）が、１：２以上である、請求項１から６のいずれか一項に記載の生体高分子分画用チップ。 The ratio (S 1 : S 2 ) between the cross-sectional area (S 1 ) of the opening of the wall and the cross-sectional area (S 2 ) of the separation channel is 1: 2 or more, The biopolymer fractionation chip according to claim 1. 前記第１の開口部が、前記基板の外側面から内側面に向かってテーパー状である、請求項１から７のいずれか一項に記載の生体高分子分画用チップ。 The biopolymer fractionation chip according to any one of claims 1 to 7, wherein the first opening has a tapered shape from the outer surface to the inner surface of the substrate. 前記生体高分子分画用チップが、さらに、前記分離用流路内の液体を吸引および／または前記分離用流路内へ液体を吐出可能な吸引吐出部を１以上有し、
前記１以上の吸引吐出部は、前記壁と、前記壁を基準として前記第１の開口部とは逆方向の端部との間で、前記分離用流路に接続するように配置される、請求項１から８のいずれか一項に記載の生体高分子分画用チップ。 The biopolymer fractionation chip further has one or more suction / discharge sections capable of sucking the liquid in the separation channel and / or discharging the liquid into the separation channel,
The one or more suction / discharge sections are arranged so as to be connected to the separation channel between the wall and an end portion in a direction opposite to the first opening with respect to the wall. The chip for biopolymer fractionation according to any one of claims 1 to 8. さらに、前記生体高分子分画用チップが、前記生体高分子の捕獲部を有し、
前記捕獲部は、前記分離用流路内において、前記壁と、前記吸引吐出部の接続部との間に配置される、請求項９記載の生体高分子分画用チップ。 Furthermore, the biopolymer fractionation chip has a capture section for the biopolymer,
The biopolymer fractionation chip according to claim 9, wherein the capture unit is disposed between the wall and the connection part of the suction / discharge unit in the separation channel. 前記生体高分子分画用チップが、２以上の吸引吐出部を有し、
前記捕獲部が、前記分離用流路内において、前記各吸引吐出部の接続部間に配置される、請求項１０記載の生体高分子分画用チップ。 The biopolymer fractionating chip has two or more suction / discharge sections,
The biopolymer fractionation chip according to claim 10, wherein the capture unit is disposed between the connection portions of the suction and discharge units in the separation channel. 前記生体高分子分画用チップが、さらに、電極を有し、
前記電極は、前記壁と、前記壁を基準として前記第１の開口部とは逆方向の端部との間で、前記分離用流路内に配置される、請求項１から１１のいずれか一項に記載の生体高分子分画用チップ。 The biopolymer fractionation chip further has an electrode,
12. The electrode according to claim 1, wherein the electrode is disposed in the separation channel between the wall and an end in a direction opposite to the first opening with respect to the wall. The chip for biopolymer fraction according to one item. さらに、前記１以上の開口部が、前記細胞質の生体高分子を回収可能な第２の開口部を有し、
前記第２の開口部が、前記分離用流路に連通され、
前記壁は、前記分離用流路において、前記第１の開口部と前記第２の開口部との間に配置される、請求項１から１１のいずれか一項に記載の生体高分子分画用チップ。 Further, the one or more openings have a second opening capable of recovering the cytoplasmic biopolymer,
The second opening communicates with the separation channel;
The biopolymer fraction according to any one of claims 1 to 11, wherein the wall is disposed between the first opening and the second opening in the separation channel. For chips. さらに、前記生体高分子分画用チップが、前記壁と前記第２の開口部との間の液体の移動を制御する液体移動制御部を有し、
前記液体移動制御部が、前記分離用流路において、前記壁と前記第２の開口部との間に配置される、請求項１３記載の生体高分子分画用チップ。 Furthermore, the biopolymer fractionation chip has a liquid movement control unit that controls movement of the liquid between the wall and the second opening,
The biopolymer fractionation chip according to claim 13, wherein the liquid movement control unit is disposed between the wall and the second opening in the separation channel. さらに、前記ターゲット細胞の移動を調整する調整用流路と、第３の開口部とを有し、
前記第３の開口部と前記分離用流路とが、前記調整用流路に連通され、
前記分離用流路は、前記分離用流路の前記壁から前記第２の開口部側において、前記調整用流路に連通される、請求項１３または１４記載の生体高分子分画用チップ。 Furthermore, the flow path for adjustment for adjusting the movement of the target cell, and a third opening,
The third opening and the separation channel are communicated with the adjustment channel,
The biopolymer fractionation chip according to claim 13 or 14, wherein the separation channel communicates with the adjustment channel on the second opening side from the wall of the separation channel. さらに、前記分離用流路および前記調整用流路を連通する接続流路を有し、
前記接続流路は、前記分離用流路の前記壁から前記第１の開口部側において、前記分離用流路に連通される、請求項１５記載の生体高分子分画用チップ。 Furthermore, it has a connection flow path that communicates the separation flow path and the adjustment flow path,
The biopolymer fractionation chip according to claim 15, wherein the connection channel communicates with the separation channel on the first opening side from the wall of the separation channel. 前記接続流路の断面積および長さが、前記壁の開口が前記ターゲット細胞をトラップしていない状態において、前記壁の開口を流れる前記試料の流量（Ｆ１）と、前記接続流路を流れる前記試料の流量（Ｆｃ）との比（Ｆ１：Ｆｃ）が、１：１～２０：１の範囲となる断面積および長さを満たす、請求項１６記載の生体高分子分画用チップ。 The cross-sectional area and the length of the connection channel are such that the flow rate (F 1 ) of the sample flowing through the wall opening and the connection channel in the state where the wall opening does not trap the target cells. The biopolymer fraction according to claim 16, wherein a ratio (F 1 : F c ) to the flow rate (F c ) of the sample satisfies a cross-sectional area and a length in a range of 1: 1 to 20: 1. Chip. さらに、電極系を有し、
前記電極系が、１以上の電極を有し、
前記１以上の電極が、前記第１の開口部内、前記分離用流路における前記壁と前記第１の開口部との間、前記第２の開口部内、および前記分離用流路における前記壁と前記第２開口部との間からなる群から選択された少なくとも１つに位置するように配置されている、請求項１３から１７のいずれか一項に記載の生体高分子分画用チップ。 Furthermore, it has an electrode system,
The electrode system comprises one or more electrodes;
The one or more electrodes are in the first opening, between the wall and the first opening in the separation channel, in the second opening, and the wall in the separation channel. The biopolymer fractionation chip according to any one of claims 13 to 17, which is disposed so as to be located in at least one selected from the group consisting of the second opening and the second opening. 前記生体高分子分画用チップが、請求項１５から１７のいずれか一項に記載の生体高分子分画用チップであり、
前記１以上の電極が、さらに、前記第３の開口部内および前記調整用流路内の少なくとも一方に位置するように配置されている、請求項１８記載の生体高分子分画用チップ。 The biopolymer fractionation chip is the biopolymer fractionation chip according to any one of claims 15 to 17,
19. The biopolymer fractionation chip according to claim 18, wherein the one or more electrodes are further disposed so as to be located in at least one of the third opening and the adjustment channel. 前記生体高分子が、核酸、糖、タンパク質、および脂質からなる群から選択された少なくとも１つである、請求項１から１９のいずれか一項に記載の生体高分子分画用チップ。 The biopolymer fractionation chip according to any one of claims 1 to 19, wherein the biopolymer is at least one selected from the group consisting of nucleic acids, sugars, proteins, and lipids. 前記細胞質の生体高分子が、細胞質基質の生体高分子および細胞小器官の生体高分子の少なくとも一方である、請求項１から２０のいずれか一項に記載の生体高分子分画用チップ。 The biopolymer fractionation chip according to any one of claims 1 to 20, wherein the cytoplasmic biopolymer is at least one of a biopolymer of a cytoplasmic matrix and a biopolymer of an organelle. 前記細胞小器官の生体高分子が、葉緑体の核酸、ミトコンドリアの核酸、およびリポソームの核酸からなる群から選択された少なくとも１つである、請求項２１記載の生体高分子分画用チップ。 The biopolymer fractionation chip according to claim 21, wherein the biopolymer of the organelle is at least one selected from the group consisting of chloroplast nucleic acid, mitochondrial nucleic acid, and liposome nucleic acid. ターゲット細胞を、開口を有する壁の開口にトラップするトラップ工程、
前記ターゲット細胞の細胞膜を破壊することにより、前記ターゲット細胞から細胞質の生体高分子を放出させる放出工程、および
放出された細胞質の生体高分子を分離する分離工程を含むことを特徴とする、生体高分子の分画方法。 A trapping process for trapping target cells in a wall opening having an opening;
Characterized in that it comprises a release step of releasing a cytoplasmic biopolymer from the target cell by destroying a cell membrane of the target cell, and a separation step of separating the released cytoplasmic biopolymer. Molecular fractionation method. 前記放出工程において、前記ターゲット細胞の細胞膜を電気的または化学的に破壊する、請求項２３記載の生体高分子の分画方法。 The biopolymer fractionation method according to claim 23, wherein the cell membrane of the target cell is electrically or chemically destroyed in the releasing step. 前記放出工程において、前記ターゲット細胞の細胞膜を電気的に破壊することにより、前記ターゲット細胞から細胞質の生体高分子を放出させ、
前記分離工程において、電気的分離方法により、前記放出された細胞質の生体高分子を分離する、請求項２３または２４記載の生体高分子の分画方法。 In the release step, the cell membrane of the target cell is electrically destroyed to release a cytoplasmic biopolymer from the target cell,
25. The biopolymer fractionation method according to claim 23 or 24, wherein in the separation step, the released cytoplasmic biopolymer is separated by an electrical separation method. さらに、前記分離工程において、分離された細胞質の生体高分子および前記細胞質の生体高分子の分離後のターゲット細胞の残部が含む生体高分子の少なくとも一方を回収する回収工程を含む、請求項２３から２５のいずれか一項に記載の生体高分子の分画方法。 Furthermore, the separation step includes a recovery step of recovering at least one of the separated cytoplasmic biopolymer and the biopolymer contained in the remainder of the target cell after separation of the cytoplasmic biopolymer. 25. The method for fractionating a biopolymer according to any one of 25. 前記壁が、２以上の開口を有する、請求項２３から２６のいずれか一項に記載の生体高分子の分画方法。 The biopolymer fractionation method according to any one of claims 23 to 26, wherein the wall has two or more openings. 前記壁の開口の径（ｗ１）が、前記ターゲット細胞の径（ｗｔ）より小さい、請求項２３から２７のいずれか一項に記載の生体高分子の分画方法。 The biopolymer fractionation method according to any one of claims 23 to 27, wherein a diameter (w 1 ) of the wall opening is smaller than a diameter (w t ) of the target cell. 前記壁の開口の径（ｗ１）と前記ターゲット細胞の径（ｗｔ）との比（ｗ１：ｗｔ）が、１：２以上である、請求項２３から２８のいずれか一項に記載の生体高分子の分画方法。 The ratio (w 1 : w t ) between the diameter (w 1 ) of the opening of the wall and the diameter (w t ) of the target cell is 1: 2 or more, according to any one of claims 23 to 28. The biopolymer fractionation method as described. 前記壁の開口の径（ｗ１）が、前記細胞質の生体高分子の分離後のターゲット細胞の残部の径（ｗｎ）より小さい、請求項２３から２９のいずれか一項に記載の生体高分子の分画方法。 30. The living body height according to any one of claims 23 to 29, wherein a diameter (w 1 ) of the opening of the wall is smaller than a diameter (w n ) of the remaining portion of the target cell after separation of the cytoplasmic biopolymer. Molecular fractionation method. 請求項１から２２のいずれか一項に記載の生体高分子分画用チップを用い、
前記第１の開口部から前記ターゲット細胞を含む試料を導入する導入工程、
前記ターゲット細胞を、前記壁の開口にトラップするトラップ工程、
前記分離用流路に電圧を印加することにより、前記ターゲット細胞から生体高分子を放出させる放出工程、および
前記壁と、前記壁を基準として前記第１の開口部方向と逆方向の端部との間の前記分離用流路に、前記ターゲット細胞の細胞質の生体高分子を分離する分離工程を含む、請求項２３から３０のいずれか一項に記載の生体高分子の分画方法。 Using the biopolymer fractionation chip according to any one of claims 1 to 22,
An introducing step of introducing a sample containing the target cells from the first opening;
A trapping step for trapping the target cell in the opening of the wall;
A release step of releasing a biopolymer from the target cell by applying a voltage to the separation channel; and the wall; and an end portion in a direction opposite to the first opening direction with respect to the wall. 31. The biopolymer fractionation method according to any one of claims 23 to 30, further comprising a separation step of separating the cytoplasmic biopolymer of the target cell in the separation channel between the two. 前記生体高分子分画用チップが、請求項１３から２２のいずれか一項に記載の生体高分子分画用チップであり、
さらに、前記第２の開口部から、前記細胞質の生体高分子を回収する工程を含む、請求項３１記載の生体高分子の分画方法。 The biopolymer fractionation chip is the biopolymer fractionation chip according to any one of claims 13 to 22,
32. The biopolymer fractionation method according to claim 31, further comprising the step of recovering the cytoplasmic biopolymer from the second opening. さらに、前記第１の開口部から、前記細胞質の生体高分子の分離後のターゲット細胞の残部が含む生体高分子を回収する工程を含む、請求項３１または３２記載の生体高分子の分画方法。 The biopolymer fractionation method according to claim 31 or 32, further comprising a step of recovering the biopolymer contained in the remainder of the target cell after separation of the cytoplasmic biopolymer from the first opening. . さらに、前記ターゲット細胞を含む試料を調製する工程を含む、請求項３１から３３のいずれか一項に記載の生体高分子の分画方法。 The biopolymer fractionation method according to any one of claims 31 to 33, further comprising a step of preparing a sample containing the target cells. 前記細胞質の生体高分子が、細胞質基質の生体高分子および細胞小器官の生体高分子の少なくとも一方である、請求項２３から３４のいずれか一項に記載の生体高分子の分画方法。 35. The biopolymer fractionation method according to any one of claims 23 to 34, wherein the cytoplasmic biopolymer is at least one of a biopolymer of a cytoplasmic matrix and a biopolymer of an organelle. 前記細胞小器官の生体高分子が、葉緑体の核酸、ミトコンドリアの核酸、およびリポソームの核酸からなる群から選択された少なくとも１つである、請求項３５記載の生体高分子の分画方法。 36. The method for fractionating a biopolymer according to claim 35, wherein the biopolymer of the organelle is at least one selected from the group consisting of chloroplast nucleic acid, mitochondrial nucleic acid, and liposome nucleic acid. ターゲット細胞から細胞質の生体高分子を分画する分画工程、および
前記細胞質の生体高分子および前記細胞質の生体高分子の分画後のターゲット細胞の残部が含む生体高分子の少なくとも一方を分析する分析工程を含み、
前記分画工程が、請求項２３から３６のいずか一項に記載の生体高分子の分画方法により実施されることを特徴とする、生体高分子の分析方法。 Fractionation step of fractionating cytoplasmic biopolymer from the target cell, and analyzing at least one of the cytoplasmic biopolymer and the remainder of the target cell after fractionation of the cytoplasmic biopolymer Including analysis steps,
37. A biopolymer analysis method, wherein the fractionation step is performed by the biopolymer fractionation method according to any one of claims 23 to 36. 前記分析された細胞質の生体高分子および細胞質の生体高分子の分画後のターゲット細胞の残部が含む生体高分子の少なくとも一方を回収する工程を含む、請求項３７記載の生体高分子の分析方法。 38. The method for analyzing a biopolymer according to claim 37, comprising the step of recovering at least one of the analyzed cytoplasmic biopolymer and the remainder of the target cell after fractionation of the cytoplasmic biopolymer. . 前記分画された細胞質の生体高分子および細胞質の生体高分子の分画後のターゲット細胞の残部が含む生体高分子の分画状態を保持する保持工程を含む、請求項３７または３８記載の生体高分子の分析方法。 39. The living body according to claim 37 or 38, comprising a holding step of maintaining the fractionated state of the biopolymer contained in the fractionated cytoplasmic biopolymer and the remainder of the target cell after fractionation of the cytoplasmic biopolymer. Polymer analysis method. 前記生体高分子が、核酸であり、
前記分画された細胞質の生体高分子および細胞質の生体高分子の分画後のターゲット細胞の残部が含む生体高分子の少なくとも一方を増幅する増幅工程を含む、請求項３７から３９のいずれか一項に記載の生体高分子の分析方法。 The biopolymer is a nucleic acid;
40. The method according to any one of claims 37 to 39, comprising an amplification step of amplifying at least one of the fractionated cytoplasmic biopolymer and the remainder of the target cell after fractionation of the cytoplasmic biopolymer. The method for analyzing a biopolymer according to Item.
PCT/JP2017/003069 2016-01-28 2017-01-27 Biopolymer fractionation chip, and biopolymer fractionation method and biopolymer analysis method using same Ceased WO2017131216A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2017557217A JP6338262B2 (en) 2016-01-28 2017-01-27 Biopolymer fractionation chip, biopolymer fractionation method using the same, and biopolymer analysis method

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2016014708 2016-01-28
JP2016-014708 2016-01-28
JP2016-177163 2016-09-09
JP2016177163 2016-09-09

Publications (1)

Publication Number Publication Date
WO2017131216A1 true WO2017131216A1 (en) 2017-08-03

Family

ID=59398267

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2017/003069 Ceased WO2017131216A1 (en) 2016-01-28 2017-01-27 Biopolymer fractionation chip, and biopolymer fractionation method and biopolymer analysis method using same

Country Status (2)

Country Link
JP (1) JP6338262B2 (en)
WO (1) WO2017131216A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019037159A (en) * 2017-08-24 2019-03-14 国立大学法人北陸先端科学技術大学院大学 Method and apparatus for recovering cell contents

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002503336A (en) * 1997-05-16 2002-01-29 アルバータ リサーチ カウンシル Micro-distribution system and method of using the same
JP2006078475A (en) * 2004-08-09 2006-03-23 Nsk Ltd Reactor and production method thereof
JP2008539711A (en) * 2005-05-03 2008-11-20 オックスフォード・ジーン・テクノロジー・アイピー・リミテッド Apparatus and method for analyzing individual cells
JP2015515263A (en) * 2012-02-29 2015-05-28 フリューダイム コーポレーション Methods, systems, and devices for capturing and processing multiple single cells using microfluidics
WO2015106146A2 (en) * 2014-01-09 2015-07-16 The Board Of Trustees Of The Leland Stanford Junior University Simultaneous extraction and separation of rna and dna from single cells using electrophoretic techniques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002503336A (en) * 1997-05-16 2002-01-29 アルバータ リサーチ カウンシル Micro-distribution system and method of using the same
JP2006078475A (en) * 2004-08-09 2006-03-23 Nsk Ltd Reactor and production method thereof
JP2008539711A (en) * 2005-05-03 2008-11-20 オックスフォード・ジーン・テクノロジー・アイピー・リミテッド Apparatus and method for analyzing individual cells
JP2015515263A (en) * 2012-02-29 2015-05-28 フリューダイム コーポレーション Methods, systems, and devices for capturing and processing multiple single cells using microfluidics
WO2015106146A2 (en) * 2014-01-09 2015-07-16 The Board Of Trustees Of The Leland Stanford Junior University Simultaneous extraction and separation of rna and dna from single cells using electrophoretic techniques

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019037159A (en) * 2017-08-24 2019-03-14 国立大学法人北陸先端科学技術大学院大学 Method and apparatus for recovering cell contents
JP6994711B2 (en) 2017-08-24 2022-01-14 国立大学法人北陸先端科学技術大学院大学 Cell content recovery method and recovery device

Also Published As

Publication number Publication date
JPWO2017131216A1 (en) 2018-02-01
JP6338262B2 (en) 2018-06-06

Similar Documents

Publication Publication Date Title
Mazutis et al. Droplet-based microfluidic systems for high-throughput single DNA molecule isothermal amplification and analysis
US10427160B2 (en) Rapid and continuous analyte processing in droplet microfluidic devices
Yuan et al. An automated microwell platform for large-scale single cell RNA-seq
JP7046795B2 (en) Intracellular delivery of biomolecules mediated by surfaces with pores
CN113767176A (en) Imaging system hardware
US20180334706A1 (en) System and method for capturing and analyzing cells
CN113286893A (en) Method of generating an array
CN114423524B (en) Platform for deterministic assembly of microfluidic droplets
US20240182885A1 (en) Capillary barriers for staged loading of microfluidic devices
Qu et al. Droplet electroporation in microfluidics for efficient cell transformation with or without cell wall removal
US20210346888A1 (en) Monodispersed Particle-Triggered Droplet Formation from Stable Jets
CN113773958B (en) Cell analysis device and cell analysis method using the cell analysis device
JP2019505761A (en) Digital microfluidic system for single cell isolation and analyte characterization
CN116083530A (en) Isotachophoresis for nucleic acid purification
Quevedo et al. Electrokinetic characterization of synthetic protein nanoparticles
Bu et al. High-performance gel-free and label-free size fractionation of extracellular vesicles with two-dimensional electrophoresis in a microfluidic artificial sieve
JP6338262B2 (en) Biopolymer fractionation chip, biopolymer fractionation method using the same, and biopolymer analysis method
Zimny et al. Hydrogel droplet single-cell processing: DNA purification, handling, release, and on-chip linearization
US20240401022A1 (en) Devices, methods, and systems for extracting genomic dna from cell samples with tunable size selection
CN109289954B (en) Array PDMS-paper-based composite microfluidic chip for single cell analysis and control method thereof
Parimalam et al. Targeted permeabilization of the cell wall and extraction of charged molecules from single cells in intact plant clusters using a focused electric field
Boutonnet et al. Check for updates Chapter 2
Zhang et al. Microfluidic Technology for Single-Cell Manipulation
US11299727B2 (en) System for analyzing single cell
EP4373955A1 (en) Methods and devices for isolating rna using epitachophoresis

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: 17744446

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017557217

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17744446

Country of ref document: EP

Kind code of ref document: A1