WO2017131216A1 - Puce de fractionnement de biopolymère, et procédé de fractionnement de biopolymère et procédé d'analyse de biopolymère l'utilisant - Google Patents

Puce de fractionnement de biopolymère, et procédé de fractionnement de biopolymère et procédé d'analyse de biopolymère l'utilisant Download PDF

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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
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Prior art keywords
biopolymer
opening
chip
fractionation
wall
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PCT/JP2017/003069
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English (en)
Japanese (ja)
Inventor
博文 新宅
卓哉 藁谷
想太郎 上村
祐伴 小口
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国立大学法人京都大学
国立大学法人東京大学
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Priority to JP2017557217A priority Critical patent/JP6338262B2/ja
Publication of WO2017131216A1 publication Critical patent/WO2017131216A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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.

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Abstract

La présente invention concerne une nouvelle puce de fractionnement de biopolymère, qui peut fractionner les biopolymères tels que les acides nucléiques, même dans les liquides qui ne contiennent pas de molécules présentant une fonction de tamis moléculaire, un procédé de fractionnement de biopolymère et un procédé d'analyse de biopolymère. Cette puce de fractionnement de biopolymère est caractérisée en ce qu'elle possède un substrat, dans lequel le substrat possède une voie d'écoulement de séparation pour séparer les biopolymères dans le cytoplasme des cellules cibles et d'une ou plusieurs ouvertures ; la une ou plusieurs ouvertures ayant une première ouverture, sur la voie d'écoulement de séparation, dans laquelle un échantillon, qui comprend les cellules cibles, peut être introduit ; la première ouverture communique avec la voie d'écoulement de séparation ; la voie d'écoulement de séparation présente une paroi qui se trouve dans la voie d'écoulement et dans le sens transversal ; et la paroi présente une ouverture.
PCT/JP2017/003069 2016-01-28 2017-01-27 Puce de fractionnement de biopolymère, et procédé de fractionnement de biopolymère et procédé d'analyse de biopolymère l'utilisant WO2017131216A1 (fr)

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JP2019037159A (ja) * 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 細胞内容物の回収方法および回収装置

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JP2002503336A (ja) * 1997-05-16 2002-01-29 アルバータ リサーチ カウンシル 微量流通システムおよびその使用方法
JP2006078475A (ja) * 2004-08-09 2006-03-23 Nsk Ltd 反応器及びその製造方法
JP2008539711A (ja) * 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 個々に細胞を解析するための装置及び方法
JP2015515263A (ja) * 2012-02-29 2015-05-28 フリューダイム コーポレーション マイクロ流体工学を利用した複数の単一細胞の捕捉及び処理の方法、システム、並びにデバイス
WO2015106146A2 (fr) * 2014-01-09 2015-07-16 The Board Of Trustees Of The Leland Stanford Junior University Extraction et séparation simultanées d'arn et d'adn à partir dans des celulles uniques au moyen de techniques électrophorétiques

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JP2002503336A (ja) * 1997-05-16 2002-01-29 アルバータ リサーチ カウンシル 微量流通システムおよびその使用方法
JP2006078475A (ja) * 2004-08-09 2006-03-23 Nsk Ltd 反応器及びその製造方法
JP2008539711A (ja) * 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 個々に細胞を解析するための装置及び方法
JP2015515263A (ja) * 2012-02-29 2015-05-28 フリューダイム コーポレーション マイクロ流体工学を利用した複数の単一細胞の捕捉及び処理の方法、システム、並びにデバイス
WO2015106146A2 (fr) * 2014-01-09 2015-07-16 The Board Of Trustees Of The Leland Stanford Junior University Extraction et séparation simultanées d'arn et d'adn à partir dans des celulles uniques au moyen de techniques électrophorétiques

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019037159A (ja) * 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 細胞内容物の回収方法および回収装置
JP6994711B2 (ja) 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 細胞内容物の回収方法および回収装置

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