WO2020012816A1 - Cell analysis method - Google Patents

Cell analysis method Download PDF

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Publication number
WO2020012816A1
WO2020012816A1 PCT/JP2019/021458 JP2019021458W WO2020012816A1 WO 2020012816 A1 WO2020012816 A1 WO 2020012816A1 JP 2019021458 W JP2019021458 W JP 2019021458W WO 2020012816 A1 WO2020012816 A1 WO 2020012816A1
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liquid
analysis method
cell
cell analysis
introducing
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PCT/JP2019/021458
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French (fr)
Japanese (ja)
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駿佑 川邉
友幸 坂井
白井　正敬
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株式会社日立ハイテクノロジーズ
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present disclosure relates to a cell analysis method.
  • Patent Literature 1 gene analysis (single cell analysis) for each cell is enabled by using an array device having a plurality of holes capable of capturing cells one by one.
  • a reaction for performing gene analysis can be performed in the device by continuously administering a plurality of reagents into the array device. Biomolecules having cell information are captured by biomolecule capturing beads filled in the reaction tank, and administration of a plurality of reagents causes a reaction required for analysis.
  • Patent Literature 1 when performing single-cell analysis, biomolecules in cells are captured by beads for capturing biomolecules in a reaction tank to cause a reaction.
  • the particle size of the biomolecule capturing beads is small, and for that purpose, the pore size of the porous membrane provided for holding the biological capturing beads is reduced. Have to do it.
  • the smaller the pore size of the porous membrane the more the gas becomes clogged with the porous membrane when a plurality of reagents are sequentially administered and fed, and the more difficult it is to send the liquid. This is because when the hydrophilic porous membrane holds the liquid, the pressure required for gas to pass through the wet porous membrane increases. In single-cell analysis, multiple reagents are administered and delivered sequentially without mixing. For this reason, it is difficult to remove gas present between the time when one reagent has been sent and the time when the next reagent is administered. Therefore, it is inevitable that the gas must pass through the porous membrane when the liquid is sent. In that case, a liquid sending pump having a high ability to suck the liquid is required, and the cost increases.
  • the present disclosure has been made in view of the above points, and provides a technique capable of analyzing cell information with high efficiency without increasing cost.
  • a cell analysis method using a single cell analysis device including an analysis chip and a membrane filter laminated on the analysis chip, wherein the first analysis chip includes cells. Introducing a liquid, introducing a second liquid having a smaller surface tension than the first liquid to the analysis chip, and placing the first liquid and the second liquid on the membrane filter side.
  • a cell analysis method including: a step of sucking a reaction reagent by the liquid sending pump; and a step of taking out and analyzing the solid phase from the analysis chip.
  • a cell analysis method using a single cell analysis device including an analysis chip and a membrane filter stacked on the analysis chip, wherein the first liquid containing cells and the surface tension of the first liquid Preparing a mixed liquid by mixing with a second liquid that is smaller than the surface tension and is larger than a predetermined value that is a measure for not destroying the cells, and introducing the mixed liquid into the analysis chip; A step of aspirating the mixed liquid by a liquid sending pump connected to a channel on the membrane filter side, introducing a reaction reagent that reacts with the cells into the analysis chip, and sending the reaction reagent to the analysis chip.
  • a cell analysis method comprising the steps of: aspirating by a pump for analysis; and taking out and analyzing the solid phase from the analysis chip.
  • cell information can be analyzed with high efficiency without increasing costs. Problems, configurations, and effects other than those described above will be apparent from the following description of the embodiments.
  • FIG. 1 is a schematic diagram illustrating a single-cell analysis system according to the present disclosure. It is an enlarged view of a cell capture hole and a nucleic acid capture bead, and an enlarged view of a DNA probe.
  • 1 is an overall schematic view of a single cell analysis system. It is a flowchart which shows the operation flow of the single cell analysis system using a pipetting device.
  • FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4.
  • FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4.
  • FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4.
  • FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4.
  • FIG. 4 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4.
  • FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4.
  • FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4. It is the schematic which shows the example which implements the PCR amplification reaction of the chip for analysis.
  • cell analysis means analyzing a biomolecule (for example, nucleic acid or protein) having cell information.
  • the term “cell” used herein is not limited to cells derived from general eukaryotic cells, but also includes fungi, bacteria, viruses, and the like included in prokaryotic cells.
  • analysis refers to extracting a biomolecule from the inside of a cell to a solid phase for reaction, performing an arbitrary reaction, and obtaining information on the cell, such as its type and function. Examples of cell analysis include gene expression analysis, genomic analysis, and protein analysis.
  • gene expression analysis refers to quantitatively analyzing the expression of a gene in a cell, that is, a target nucleic acid to be tested, analyzing the expression distribution of a gene (test nucleic acid) in a sample, Means obtaining correlation data between the expression level of a specific cell and a gene (test nucleic acid).
  • the sample is not particularly limited as long as it is a biological sample for which gene expression is to be analyzed, and any sample such as a cell sample, a tissue sample, and a liquid sample can be used.
  • the organism from which the sample is derived is not particularly limited, and vertebrates (eg, mammals, birds, reptiles, fish, amphibians), invertebrates (eg, insects, nematodes, crustaceans), protists , Plants, fungi, bacteria, and viruses.
  • vertebrates eg, mammals, birds, reptiles, fish, amphibians
  • invertebrates eg, insects, nematodes, crustaceans
  • protists eg, Plants, fungi, bacteria, and viruses.
  • test nucleic acid to be a target in the present specification, messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA and DNA, and fragments thereof can be used.
  • mRNA messenger RNA
  • ncRNA non-coding RNA
  • DNA DNA, and fragments thereof can be used.
  • surfactant refers to ⁇ (the surface tension of the liquid introduced into the analysis chip) and ⁇ (the contact angle between the liquid introduced into the analysis chip and the membrane filter) in Formula 1 shown below.
  • ⁇ the surface tension of the liquid introduced into the analysis chip
  • ⁇ the contact angle between the liquid introduced into the analysis chip and the membrane filter
  • P Pressure difference generated between the liquid on the analysis chip side and the liquid on the membrane filter side by the liquid sending pump
  • k Specific coefficient ⁇ relating to the shape of the membrane filter: Surface tension ⁇ of the liquid introduced into the analysis chip
  • Contact angle D between the liquid introduced into the analysis chip and the membrane filter the diameter of the hole provided in the membrane filter
  • FIG. 1 is a schematic diagram illustrating a single-cell analysis system according to the present disclosure.
  • the single-cell analysis system includes a single-cell analysis device 11, a dispensing device 9, and a liquid-feeding pump 10.
  • the single-cell analyzer 11 is composed of an analysis chip 7, a membrane filter 8 which is provided in close contact with the analysis chip 7, and a flow path wall 6 which sandwiches and fixes these two components from above and below.
  • the analysis chip 7 is provided with a cell capturing hole 2 for individually capturing the cells 1, and a nucleic acid capturing bead 3 for capturing the nucleic acid 22 in the cell is filled below the cell capturing hole 2. I have.
  • the upper part of the analysis chip 7 is open to the atmospheric pressure.
  • the membrane filter 8 will be described by taking a porous membrane provided with holes on a cylinder as shown in FIG. 2 as an example, but a mesh filter may be used.
  • the dispensing device 9 is provided with the cell suspension 5, the reaction reagent containing the surfactant 4, and the surfactant 4.
  • the liquid sending pump 10 applies a suction pressure to the lower part of the analysis chip 7 through the flow path of the single cell analyzer 11 and sends the upper solution that is released to the atmospheric pressure to the lower part of the analysis chip 7.
  • FIG. 1 shows a state in which a cell suspension 5 containing cells 1 and a surfactant 4 are dispensed in two layers.
  • FIG. 2 is an enlarged view of the cell capture hole 2 and the nucleic acid capture bead 3 and an enlarged view of the DNA probe 21.
  • a DNA probe 21 for capturing the test nucleic acid 22 released from the cell 1 is fixed on the surface of the nucleic acid capture bead 3.
  • the method of immobilizing the DNA probe 21 on the surface of the nucleic acid capture bead 3 is performed, for example, by modifying the 5 ′ end of the DNA probe 21 with biotin and binding to streptavidin previously immobilized on the surface of the nucleic acid capture bead 3. be able to.
  • the DNA probe 21 has a poly-T sequence at the 3 'end, and captures the test nucleic acid 22 by hybridizing with the poly A sequence at the 3' end of the test nucleic acid 22 (e.g., mRNA).
  • the poly T sequence in the DNA probe 21 may be changed to a known sequence that hybridizes with the analysis target.
  • the DNA probe 21 has a primer sequence for PCR amplification at the 5 'end fixed to the nucleic acid capture bead 3.
  • the primer sequence for PCR amplification is not particularly limited as long as it is a known sequence having an appropriate length for performing nucleic acid amplification, and those skilled in the art can appropriately design such a sequence.
  • the primer sequence for PCR amplification can be, for example, 10 to 50 bases, 15 to 40 bases, 15 to 30 bases or 15 to 20 bases. By including the common primer sequence in the probe, the amplification reaction in the subsequent nucleic acid amplification step can be easily performed.
  • the DNA probe 21 having a different cell recognition sequence for each cell trapping hole 2 is fixed to the nucleic acid trapping bead 3, so that it is possible to discriminate from which cell the sequence analyzed later is derived.
  • the cell recognition sequence is a random sequence of 5 bases, 4 5, that is, 1024 cells can be identified. Therefore, the cell recognition sequence can be arbitrarily set according to the number of the cell capturing holes 2, and specifically, can be in the range of 5 to 30 bases, 5 to 20 bases, 5 to 15 bases or 5 to 10 bases.
  • FIG. 3 is a schematic diagram of the entire single cell analysis system.
  • the single cell analysis system includes, in addition to the single cell analyzer 11 shown in FIG. 1, a camera 31 for monitoring the state and remaining amount of the liquid introduced into the single cell analyzer 11 in real time, and a camera 31 for control.
  • a PC 32 is attached.
  • the analysis chip 7 was manufactured by molding 2.0 mm square polydimethylsiloxane (PDMS) to have a thickness of 100 ⁇ m.
  • the material of the analysis chip 7 may be a resin such as polypropylene or polystyrene.
  • the analysis chip 7 can be manufactured by fine processing of a silicon wafer or the like.
  • One hundred cell capture holes 2 were provided on the upper surface side of the analysis chip 7 at a pitch of 3 ⁇ m in diameter and at intervals of 125 ⁇ m.
  • Nucleic acid capture beads 3 were dispensed into the space below the cell capture holes 2 using an inkjet printer.
  • the size of the nucleic acid capturing beads 3 is 1 micrometer in diameter.
  • the membrane filter 8 has pores with a diameter of about 800 nanometers, and has been subjected to a hydrophilic treatment.
  • the diameter of the pores provided in the membrane filter 8 is, for example, 0.5 to 3.0 micrometers.
  • polycarbonate, polyvinylidene fluoride, nylon, polyethylene, or the like can be used as a material of the membrane filter 8.
  • the channel wall 6 was made of acrylic, and the analysis chip 7 and the membrane filter 8 were sandwiched between the upper and lower acrylic channel walls 6 and fixed with screws.
  • the material of the channel wall 6 can be made of polypropylene, polystyrene, polytetrafluoroethylene, or the like, in addition to acrylic.
  • streptavidin was previously immobilized on the nucleic acid capture beads 3, and a DNA probe 21 having a 5'-end modified with biotin was immobilized thereon.
  • the DNA probe 21 has a common sequence for PCR amplification of 30 bases from the 5 'end, a cell recognition sequence of 5 bases, a molecular recognition sequence consisting of a random sequence of 7 bases, an oligo sequence of 18 bases, and a VN sequence of 2 bases. did.
  • FIG. 4 is a flowchart showing an operation flow of the single cell analysis system using the dispensing device 9.
  • FIG. 5 is a schematic diagram showing each state of the single cell analysis system corresponding to the flowchart of FIG. For example, the state of (a) in the operation flow of FIG. 4 matches the state of FIG. 5- (a).
  • FIG. 5 each step of the operation flow of the single cell analysis system shown in FIG. 4 will be described.
  • (A) Dispensing of cell suspension S401
  • the cell suspension 5 containing the cells 1 to be tested is set in the dispensing device 9 and dispensed on the analysis chip 7 of the single cell analyzer 11 while being controlled by the control PC 32.
  • the amount of the cell suspension 5 to be dispensed is about 5 microliters, and the number of cells existing in the cell suspension 5 is about 80.
  • the state of the upper part of the analysis chip 7 is monitored by the camera 31 to check the state of cells being captured in the cell capturing hole 2 and the remaining amount of the solution on the upper part of the analysis chip 7. After the required amount of the cell suspension 5 has been dispensed, the flow (a) is terminated.
  • the surfactant 4 set in the dispensing device 9 is controlled by the control PC 32 while checking with the camera 31, and is additionally dispensed on the liquid surface of the cell suspension 5 above the analysis chip 7. .
  • the two liquids are dispensed gently so as not to mix as much as possible, and stirring is not performed.
  • the surfactant 4 is introduced into the cells 1 in the cell suspension 5 by introducing the surfactant 4 in a state where the tip of the pipetting pipette is attached to the cell suspension 5. Touching can prevent the cells 1 from being destroyed.
  • the reason for introducing the surfactant 4 is to prevent clogging of the analysis chip 7 with bubbles by flowing a liquid having a small surface tension (for example, smaller than pure water).
  • the surfactant 4 comes into contact with the cells 1 only by the liquid sending pump 10. After most of the liquid has been transferred. Since the surfactant 4 is supplied in a short time, the time during which the surfactant 4 contacts the cell 1 is short. Therefore, the surfactant 4 can be caused to flow through the flow path without destroying the cell membrane of the cell 1, and clogging of the analysis chip 7 with bubbles can be prevented.
  • the nucleic acid capturing The molecules in the cell 1 can be captured by the beads 3.
  • P pressure difference generated by the liquid sending pump 10 between the liquid on the analysis chip 7 side and the liquid on the membrane filter 8
  • k specific coefficient ⁇ 2 relating to the shape of the membrane filter 8: surfactant 4 and reaction reagent
  • the contact angle between the mixed liquid and the membrane filter 8 D the diameter of the hole provided in the membrane filter 8
  • the Lysis buffer (including the surfactant 4) is dispensed from the dispensing device 9 onto the analysis chip 7 through the control PC 32.
  • the surfactant 4 to be mixed with the reaction reagent may be the same type of surfactant as the surfactant 4 dispensed in the flow (b), or may be a different type of surfactant.
  • Example 1 Reagent dispensing (2nd step)
  • the flow (d) is performed again according to the operation flow in order to describe an example in which the reaction procedure includes all two steps.
  • an RT reagent for performing a reverse transcription reaction is dispensed as a second reagent.
  • the RT reagent mixed with the surfactant 4 so as to satisfy the condition of the following formula 4 is dispensed by the dispensing device 9.
  • P Pressure difference generated between the liquid on the analysis chip 7 side and the liquid on the membrane filter 8 by the liquid sending pump 10
  • k Specific coefficient ⁇ 22 relating to the shape of the membrane filter 8: Surfactant 4 and second Surface tension ⁇ of the liquid mixture with the reagent: contact angle between the liquid mixture and the membrane filter 8
  • D diameter of the holes provided in the membrane filter 8
  • the surfactant 4 to be mixed with the second reagent may be the same type of surfactant as the surfactant 4 dispensed in the flow (b) and the first flow (d), or may be a different type. It may be a surfactant.
  • FIG. 6 is a schematic view showing an example in which a PCR amplification reaction of the analysis chip 7 is performed.
  • the tube 41 contains a PCR reagent 42 necessary for the PCR reaction. After the end of the PCR amplification reaction, DNA sequence sequencing is performed, and the gene expression level of each cell captured in the cell capturing hole 2 can be analyzed.
  • the single cell analysis system of the present disclosure is present above the analysis chip 7 by the liquid sending pump 10 by introducing the surfactant 4 having a small surface tension into the single cell analysis device 11.
  • the liquid sending pump 10 by introducing the surfactant 4 having a small surface tension into the single cell analysis device 11.
  • the reaction step is required twice has been described, but the number of the reaction steps may be one or more.
  • the present disclosure has described the method of introducing the surfactant 4 into the reaction reagent as a method of preventing air bubbles from being clogged in the analysis chip 7; However, it is not always necessary to mix the surfactant 4. For example, in the case of an operation flow having only one reaction step, the surfactant 4 need not be mixed into the reaction reagent. In the case of an operation flow in which the reaction step is performed three times, the surfactant 4 does not have to be mixed with the third reaction reagent.
  • Example 2 a method will be described in which the damage to the cell 1 from the surfactant 4 can be further reduced as compared with the method shown in the first embodiment.
  • the cell analysis method according to the second embodiment differs from the first embodiment in the flow (b) (S402) among the operation flows described above.
  • the flow (b) of the second embodiment will be described.
  • a program is recorded in the control PC 32 so as to start dispensing of the prepared surfactant 4 at a timing before the cell suspension 5 is completely fed, or set by the user. You. Alternatively, the surfactant 4 may be dispensed by operating the control PC 32 while observing the remaining amount of the cell suspension 5 with the camera 31. After the completion of the feeding of the surfactant 4, the flow (d) and subsequent steps are performed in the same manner as in the first embodiment.
  • the feeding of the cell suspension 5 is started before the additional dispensing of the surfactant 4, and the surfactant 4 is additionally dispensed immediately before the feeding of the cell suspension 5 is completed. This makes it possible to shorten the time during which the cell 1 comes into contact with the surfactant 4 as much as possible.
  • Example 3 In Examples 1 and 2, the surfactant 4 was dispensed after the cell suspension 5 was dispensed.
  • the flows (a) and (b) described in the first embodiment are substituted by dispensing a liquid mixture obtained by mixing the surfactant 4 with the cell suspension 5.
  • the surfactant 4 is selected so as to satisfy the following conditional expression 5.
  • P pressure difference generated by the liquid sending pump 10 between the liquid on the analysis chip 7 side and the liquid on the membrane filter 8 side
  • k specific coefficient ⁇ 3 relating to the shape of the membrane filter 8: surfactant 4 and cell suspension
  • Surface tension ⁇ of the mixed liquid with the suspension liquid 5 contact angle between the mixed liquid and the membrane filter 8
  • D diameter of the hole provided in the membrane filter 8
  • Example 3 the surface tension of the surfactant 4 is required to be larger than a predetermined value that does not break the cells 1 in the above flow. This ⁇ 3 is determined, for example, by experiment. Further, if the mixed liquid of the surfactant 4 and the cell suspension 5 is dispensed and then sent as soon as possible, damage to the cells 1 can be reduced.
  • the surface tension of the solution to be introduced into the single cell analyzer 11 is reduced by additionally dispensing the surfactant 4 or previously mixing the surfactant 4 with the cell suspension 5 or the reaction reagent.
  • the surfactant 4 or previously mixing the surfactant 4 with the cell suspension 5 or the reaction reagent We were able to. Accordingly, it is possible to prevent the gas from being clogged in the analysis chip 7, and it is not necessary to provide the liquid supply pump 10 having a high capacity.
  • the pores provided in the membrane filter 8 and the pore diameter of the cell trapping pores 2 can be reduced, small nucleic acid trapping beads 3 can be used, and a highly efficient reaction can be performed. Further, by executing the above-described operation flow, it is possible to prevent the cell membrane from being destroyed even when the surface tension of the solution is reduced.
  • the present disclosure is not limited to the above-described embodiments, and includes various modifications.
  • the above-described embodiments have been described in detail in order to easily explain the present disclosure, and are not necessarily limited to those having all the configurations described above.
  • a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of one embodiment can be added to the configuration of another embodiment.
  • liquid dispensing may be performed by a technique using a pipette or the like.
  • the pump 10 for liquid supply is operated, and the remaining amount of the cell suspension 5 is reduced.
  • the surfactant 4 may be additionally dispensed onto the liquid surface of the cell suspension 5.

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Abstract

To highly efficiently analyze cell data without increasing cost. A cell analysis method, wherein a single cell analyzer 11 provided with an analysis chip and a membrane filter laminated on the analysis chip is used, comprises: a step for introducing a first liquid containing a cell into the analysis chip; a step for introducing a second liquid having a smaller surface tension than the first liquid into the analysis chip; a step for sucking the first and second liquids with a liquid feeding pump connected to a flow channel on the membrane filter side; a step for introducing a reaction reagent capable of reacting with the cell into the analysis chip and reacting a molecule extracted from the cell with a solid phase; a step for sucking the reaction reagent with a liquid feeding pump; and a step for taking out the solid phase from the analysis chip and analyzing the same.

Description

細胞解析方法Cell analysis method
　本開示は、細胞解析方法に関する。 The present disclosure relates to a cell analysis method.
　近年、多数の細胞から構成される生体組織のゲノム解析、遺伝子発現解析または蛋白解析を行うときに、個々の細胞のゲノム、遺伝子発現または蛋白質の違いに注目して解析する単一細胞解析の重要性が認識され始めている。従来の解析では生体組織から採取した多数の細胞を１種類のサンプルとし、それら細胞からＤＮＡおよび／またはＲＮＡを抽出して解析を行う。しかし、この方法では全細胞の平均データしか得ることができず、個々の細胞に含まれるＤＮＡおよび／またはＲＮＡの存在量が平均値から乖離していたとしても評価することができない。 In recent years, when performing genomic analysis, gene expression analysis or protein analysis of living tissue composed of many cells, the importance of single cell analysis that focuses on analysis of differences in genome, gene expression or protein of individual cells is important Sex is beginning to be recognized. In conventional analysis, many cells collected from a living tissue are used as one type of sample, and DNA and / or RNA are extracted from the cells and analyzed. However, according to this method, only average data of all cells can be obtained, and it cannot be evaluated even if the abundance of DNA and / or RNA contained in each cell deviates from the average value.
　そこで特許文献１で用いられているようなアレイデバイスを用いることで、細胞一つずつの遺伝子解析を行うことが近年すすめられている。特許文献１では細胞を一つずつ捕捉可能な孔を複数持つアレイデバイスを用いて、細胞一つずつの遺伝子解析（単一細胞解析）を可能にしている。また当該アレイデバイスでは、遺伝子解析を行うにあたって、アレイデバイス中に複数の試薬を連続的に投与することによって遺伝子解析を行うための反応をデバイス中で行うことが可能である。細胞情報を持つ生体分子は反応槽内に充填されている生体分子捕捉用ビーズによって捕捉され、複数の試薬を投与することで、解析に必要な反応が生じる。 Therefore, in recent years, it has been recommended to perform a gene analysis for each cell by using an array device as used in Patent Document 1. In Patent Literature 1, gene analysis (single cell analysis) for each cell is enabled by using an array device having a plurality of holes capable of capturing cells one by one. In the array device, when performing gene analysis, a reaction for performing gene analysis can be performed in the device by continuously administering a plurality of reagents into the array device. Biomolecules having cell information are captured by biomolecule capturing beads filled in the reaction tank, and administration of a plurality of reagents causes a reaction required for analysis.
国際公開第２０１６／０３８６７０号WO 2016/038670
　上述のとおり、特許文献１では、単一細胞解析を行う際に、細胞内の生体分子を反応槽内の生体分子捕捉用ビーズに捕捉させて反応を生じさせている。ここで、高効率な反応を行うためには、生体分子捕捉用ビーズの粒径が小さいことが望ましく、そのためには生体捕捉用ビーズを保持するために設置されている多孔質膜の孔径を小さくしなければいけない。 As described above, in Patent Literature 1, when performing single-cell analysis, biomolecules in cells are captured by beads for capturing biomolecules in a reaction tank to cause a reaction. Here, in order to perform a highly efficient reaction, it is desirable that the particle size of the biomolecule capturing beads is small, and for that purpose, the pore size of the porous membrane provided for holding the biological capturing beads is reduced. Have to do it.
　しかしながら、多孔質膜の孔径が小さくなればなるほど、複数の試薬を順番に投与および送液する際に、気体が多孔質膜に詰まり、送液が困難になる。これは親水性である多孔質膜が液体を保持した状態になると、濡れた多孔質膜を気体が通過するために必要な圧力が高くなるためである。単一細胞解析では複数の試薬を混ぜずに順番に投与および送液する。そのため、一つの試薬を送液し終えてから次の試薬を投与するまでの間に存在する気体は除去することが難しい。したがって、必然的に、液体の送液時に気体も併せて多孔質膜を通過させる必要がある。その場合、液体を吸引する能力が高い送液ポンプが必要となりコストが増大する。 However, the smaller the pore size of the porous membrane, the more the gas becomes clogged with the porous membrane when a plurality of reagents are sequentially administered and fed, and the more difficult it is to send the liquid. This is because when the hydrophilic porous membrane holds the liquid, the pressure required for gas to pass through the wet porous membrane increases. In single-cell analysis, multiple reagents are administered and delivered sequentially without mixing. For this reason, it is difficult to remove gas present between the time when one reagent has been sent and the time when the next reagent is administered. Therefore, it is inevitable that the gas must pass through the porous membrane when the liquid is sent. In that case, a liquid sending pump having a high ability to suck the liquid is required, and the cost increases.
　本開示は、上記の点に鑑みてなされたものであり、コストを増大させずに細胞情報を高効率に解析できる技術を提供する。 The present disclosure has been made in view of the above points, and provides a technique capable of analyzing cell information with high efficiency without increasing cost.
　上記課題を解決するために、解析用チップと前記解析用チップに積層された膜フィルタとを備える単一細胞解析装置を用いる細胞解析方法であって、前記解析用チップに細胞を含む第１の液体を導入するステップと、前記解析用チップに前記第１の液体よりも表面張力が小さい第２の液体を導入するステップと、前記第１の液体と前記第２の液体とを前記膜フィルタ側の流路と接続された送液用ポンプによって吸引するステップと、前記解析用チップに前記細胞と反応する反応試薬を導入し、前記細胞から抽出される分子を固相に反応させるステップと、前記反応試薬を前記送液用ポンプによって吸引するステップと、前記解析用チップの中から前記固相を取り出して解析するステップと、を含む細胞解析方法を提供する。 In order to solve the above-mentioned problem, there is provided a cell analysis method using a single cell analysis device including an analysis chip and a membrane filter laminated on the analysis chip, wherein the first analysis chip includes cells. Introducing a liquid, introducing a second liquid having a smaller surface tension than the first liquid to the analysis chip, and placing the first liquid and the second liquid on the membrane filter side. Aspirating by a liquid sending pump connected to the flow path of, introducing a reaction reagent that reacts with the cells into the analysis chip, and reacting a molecule extracted from the cells with a solid phase, There is provided a cell analysis method including: a step of sucking a reaction reagent by the liquid sending pump; and a step of taking out and analyzing the solid phase from the analysis chip.
　また、解析用チップと前記解析用チップに積層された膜フィルタとを備える単一細胞解析装置を用いる細胞解析方法であって、細胞を含む第１の液体と表面張力が前記第１の液体の表面張力よりも小さく前記細胞を破壊しない目安となる所定の値よりも大きい第２の液体とを混合して混合液体を調製するステップと、前記解析用チップに前記混合液体を導入するステップと、前記混合液体を前記膜フィルタ側の流路と接続された送液用ポンプによって吸引するステップと、前記解析用チップに前記細胞と反応する反応試薬を導入するステップと、前記反応試薬を前記送液用ポンプによって吸引するステップと、前記解析用チップの中から前記固相を取り出して解析するステップと、を含む細胞解析方法を提供する。 Also, a cell analysis method using a single cell analysis device including an analysis chip and a membrane filter stacked on the analysis chip, wherein the first liquid containing cells and the surface tension of the first liquid Preparing a mixed liquid by mixing with a second liquid that is smaller than the surface tension and is larger than a predetermined value that is a measure for not destroying the cells, and introducing the mixed liquid into the analysis chip; A step of aspirating the mixed liquid by a liquid sending pump connected to a channel on the membrane filter side, introducing a reaction reagent that reacts with the cells into the analysis chip, and sending the reaction reagent to the analysis chip. A cell analysis method comprising the steps of: aspirating by a pump for analysis; and taking out and analyzing the solid phase from the analysis chip.
　本開示によれば、コストを増大させずに細胞情報を高効率に解析できる。上記以外の課題、構成および効果は、以下の実施の形態の説明により明らかにされる。 According to the present disclosure, cell information can be analyzed with high efficiency without increasing costs. Problems, configurations, and effects other than those described above will be apparent from the following description of the embodiments.
本開示の単一細胞解析システムを表す模式図である。1 is a schematic diagram illustrating a single-cell analysis system according to the present disclosure. 細胞捕捉孔と核酸捕捉ビーズの拡大図およびＤＮＡプローブの拡大図である。It is an enlarged view of a cell capture hole and a nucleic acid capture bead, and an enlarged view of a DNA probe. 単一細胞解析システムの全体外略図である。1 is an overall schematic view of a single cell analysis system. 分注装置を用いた単一細胞解析システムの操作フローを示すフローチャートである。It is a flowchart which shows the operation flow of the single cell analysis system using a pipetting device. 図４のフローチャートと対応する単一細胞解析システムの各状態を表した概略図である。FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4. 図４のフローチャートと対応する単一細胞解析システムの各状態を表した概略図である。FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4. 図４のフローチャートと対応する単一細胞解析システムの各状態を表した概略図である。FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4. 図４のフローチャートと対応する単一細胞解析システムの各状態を表した概略図である。FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4. 図４のフローチャートと対応する単一細胞解析システムの各状態を表した概略図である。FIG. 5 is a schematic diagram illustrating each state of the single cell analysis system corresponding to the flowchart of FIG. 4. 解析用チップのＰＣＲ増幅反応を実施する例を示す概略図である。It is the schematic which shows the example which implements the PCR amplification reaction of the chip for analysis.
　以下、図面に基づいて、本開示の実施例を説明する。なお、本開示の実施例は、後述する実施例に限定されるものではなく、その技術思想の範囲において、種々の変形が可能である。また、後述する各実施例の説明に使用する各図の対応部分には同一の符号を付して示し、重複する説明を省略する。まず、本明細書において使用されるいくつかの用語について説明し、続いて実施例に係る単一細胞解析システムについて説明する。 Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. It should be noted that embodiments of the present disclosure are not limited to the embodiments described below, and various modifications are possible within the scope of the technical idea. Corresponding parts in the drawings used in the description of each embodiment described later are denoted by the same reference numerals, and redundant description will be omitted. First, some terms used in the present specification will be described, and then a single cell analysis system according to an example will be described.
　本明細書において「細胞解析」とは、細胞の情報を持った生体分子（例えば、核酸や蛋白質など）を解析することを意味する。ここでいう「細胞」とは一般的な真核細胞由来の細胞のみに限らず、原核細胞に含まれるような真菌、細菌およびウイルスなども含まれる。また、ここでいう「解析」とは、生体分子を細胞内から反応用の固相へと抽出し、任意の反応を行い、細胞の情報、例えば種類や機能といったものを得ることを指す。細胞解析の例として、遺伝子発現解析、ゲノム解析および蛋白質解析などが挙げられる。 書 In this specification, “cell analysis” means analyzing a biomolecule (for example, nucleic acid or protein) having cell information. The term “cell” used herein is not limited to cells derived from general eukaryotic cells, but also includes fungi, bacteria, viruses, and the like included in prokaryotic cells. The term “analysis” as used herein refers to extracting a biomolecule from the inside of a cell to a solid phase for reaction, performing an arbitrary reaction, and obtaining information on the cell, such as its type and function. Examples of cell analysis include gene expression analysis, genomic analysis, and protein analysis.
　本明細書において「遺伝子発現解析」とは、細胞における遺伝子、すなわちターゲットになる被検核酸の発現を定量的に分析すること、サンプルにおける遺伝子（被検核酸）の発現分布を分析すること、サンプルにおける特定の細胞と遺伝子（被検核酸）発現量との相関データを得ることを意味する。 As used herein, “gene expression analysis” refers to quantitatively analyzing the expression of a gene in a cell, that is, a target nucleic acid to be tested, analyzing the expression distribution of a gene (test nucleic acid) in a sample, Means obtaining correlation data between the expression level of a specific cell and a gene (test nucleic acid).
　サンプルは、遺伝子発現を解析しようとする生体由来サンプルであれば特に限定されるものではなく、細胞サンプル、組織サンプルおよび液体サンプルなどの任意のサンプルを用いることができる。 The sample is not particularly limited as long as it is a biological sample for which gene expression is to be analyzed, and any sample such as a cell sample, a tissue sample, and a liquid sample can be used.
　また、サンプルの由来となる生体も特に限定されるものではなく、脊椎動物（例えば、哺乳類、鳥類、爬虫類、魚類、両生類）、無脊椎動物（例えば、昆虫、線虫、甲殻類）、原生生物、植物、真菌、細菌およびウイルスなどの任意の生体に由来するサンプルを用いることができる。 Also, the organism from which the sample is derived is not particularly limited, and vertebrates (eg, mammals, birds, reptiles, fish, amphibians), invertebrates (eg, insects, nematodes, crustaceans), protists , Plants, fungi, bacteria, and viruses.
　本明細書においてターゲットとなる被検核酸としてはメッセンジャーＲＮＡ（ｍＲＮＡ）、非コードＲＮＡ（ｎｃＲＮＡ）、ｍｉｃｒｏＲＮＡおよびＤＮＡならびにそれらの断片を用いることができる。 メ ッ As the test nucleic acid to be a target in the present specification, messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA and DNA, and fragments thereof can be used.
　本明細書において「界面活性剤」とは、以下に示す式１において、γ（解析用チップに導入する液体の表面張力）およびθ（解析用チップに導入された液体と膜フィルタとの接触角）の二つを変更するための化学物質の総称であり、一般的な界面活性剤の総称である、イオン性界面活性剤、非イオン性界面活性剤など以外にも、水に混合することで上記二つのパラメータを変更することが可能な物質（例えばアルコールおよび有機溶媒）も含む。なお、上記界面活性剤の表面張力は、例えば、純水の表面張力よりも小さい。
Figure JPOXMLDOC01-appb-M000004
 ここで、
Ｐ：送液用ポンプが解析用チップ側の液体と膜フィルタ側の液体との間に生じさせる圧力差
ｋ：膜フィルタの形状に関する固有係数
γ：解析用チップに導入する液体の表面張力
θ：解析用チップに導入する液体と膜フィルタとの接触角
Ｄ：膜フィルタに設けられた孔の孔径 In the present specification, the term “surfactant” refers to γ (the surface tension of the liquid introduced into the analysis chip) and θ (the contact angle between the liquid introduced into the analysis chip and the membrane filter) in Formula 1 shown below. ) Is a general term for chemical substances to change the two, and in addition to the general term for general surfactants, such as ionic surfactants and nonionic surfactants, can be mixed with water Substances that can change the above two parameters (eg, alcohols and organic solvents) are also included. The surface tension of the surfactant is, for example, smaller than the surface tension of pure water.
Figure JPOXMLDOC01-appb-M000004
 here,
P: Pressure difference generated between the liquid on the analysis chip side and the liquid on the membrane filter side by the liquid sending pump k: Specific coefficient γ relating to the shape of the membrane filter: Surface tension θ of the liquid introduced into the analysis chip Contact angle D between the liquid introduced into the analysis chip and the membrane filter: the diameter of the hole provided in the membrane filter
＜実施例１＞
　図１は、本開示の単一細胞解析システムを表す模式図である。単一細胞解析システムは、単一細胞解析装置１１、分注装置９および送液用ポンプ１０を備える。 <Example 1>
FIG. 1 is a schematic diagram illustrating a single-cell analysis system according to the present disclosure. The single-cell analysis system includes a single-cell analysis device 11, a dispensing device 9, and a liquid-feeding pump 10.
　単一細胞解析装置１１は、解析用チップ７と、解析用チップ７と密着して設置する膜フィルタ８と、それら二部品を上下から挟み込んで固定する流路壁６で構成される。解析用チップ７には、細胞１を個別に捕捉するための細胞捕捉孔２が設けられ、細胞捕捉孔２の下部には細胞内の核酸２２を捕捉するための核酸捕捉ビーズ３が充填されている。また、解析用チップ７の上部は、大気圧開放されている。膜フィルタ８は、図２に示されるような円筒上の孔が設けられた多孔質膜を例にとって説明するが、網目状のフィルタを用いてもよい。 The single-cell analyzer 11 is composed of an analysis chip 7, a membrane filter 8 which is provided in close contact with the analysis chip 7, and a flow path wall 6 which sandwiches and fixes these two components from above and below. The analysis chip 7 is provided with a cell capturing hole 2 for individually capturing the cells 1, and a nucleic acid capturing bead 3 for capturing the nucleic acid 22 in the cell is filled below the cell capturing hole 2. I have. The upper part of the analysis chip 7 is open to the atmospheric pressure. The membrane filter 8 will be described by taking a porous membrane provided with holes on a cylinder as shown in FIG. 2 as an example, but a mesh filter may be used.
　分注装置９には、細胞懸濁液５、界面活性剤４入り反応試薬および界面活性剤４が設置されている。送液用ポンプ１０は、単一細胞解析装置１１の流路を通して、解析用チップ７の下部に引圧を印加し、大気圧開放されている上部の溶液を解析用チップ７の下部へと送液する。図１には、細胞１を含む細胞懸濁液５および界面活性剤４が二層をなすように分注された様子が示されている。 The dispensing device 9 is provided with the cell suspension 5, the reaction reagent containing the surfactant 4, and the surfactant 4. The liquid sending pump 10 applies a suction pressure to the lower part of the analysis chip 7 through the flow path of the single cell analyzer 11 and sends the upper solution that is released to the atmospheric pressure to the lower part of the analysis chip 7. Liquid. FIG. 1 shows a state in which a cell suspension 5 containing cells 1 and a surfactant 4 are dispensed in two layers.
　図２は、細胞捕捉孔２と核酸捕捉ビーズ３の拡大図およびＤＮＡプローブ２１の拡大図である。核酸捕捉ビーズ３の表面には細胞１から放出された被検核酸２２を捕捉するためのＤＮＡプローブ２１が固定されている。核酸捕捉ビーズ３の表面にＤＮＡプローブ２１を固定させる方法は、例えば、ＤＮＡプローブ２１の５’末端にビオチン修飾を行い、予め核酸捕捉ビーズ３の表面に固定させたストレプトアビジンと結合させることにより行うことができる。 FIG. 2 is an enlarged view of the cell capture hole 2 and the nucleic acid capture bead 3 and an enlarged view of the DNA probe 21. A DNA probe 21 for capturing the test nucleic acid 22 released from the cell 1 is fixed on the surface of the nucleic acid capture bead 3. The method of immobilizing the DNA probe 21 on the surface of the nucleic acid capture bead 3 is performed, for example, by modifying the 5 ′ end of the DNA probe 21 with biotin and binding to streptavidin previously immobilized on the surface of the nucleic acid capture bead 3. be able to.
　ＤＮＡプローブ２１は、３’末端にポリＴ配列を有し、被検核酸２２（例えば、ｍＲＮＡなど）の３’末端のポリＡ配列とハイブリダイズすることにより被検核酸２２を捕捉する。なお、ｍＲＮＡに代えてｍｉｃｒｏＲＮＡなどを解析したい場合には、ＤＮＡプローブ２１内のポリＴ配列を解析対象とハイブリダイズする既知配列に変更しても良い。 The DNA probe 21 has a poly-T sequence at the 3 'end, and captures the test nucleic acid 22 by hybridizing with the poly A sequence at the 3' end of the test nucleic acid 22 (e.g., mRNA). When it is desired to analyze microRNA or the like instead of mRNA, the poly T sequence in the DNA probe 21 may be changed to a known sequence that hybridizes with the analysis target.
　ＤＮＡプローブ２１は、核酸捕捉ビーズ３に固定されている５’末端にＰＣＲ増幅用プライマー配列を有する。ＰＣＲ増幅用プライマー配列は、核酸増幅を行うために適切な長さの既知配列であれば特に限定されるものではなく、当業者であればそのような配列を適宜設計することが可能である。ＰＣＲ増幅用プライマー配列は、例えば、１０～５０塩基、１５～４０塩基、１５～３０塩基または１５～２０塩基とすることができる。共通のプライマー配列をプローブに含めることによって、後の核酸増幅工程における増幅反応を簡便に実施することが可能になる。 The DNA probe 21 has a primer sequence for PCR amplification at the 5 'end fixed to the nucleic acid capture bead 3. The primer sequence for PCR amplification is not particularly limited as long as it is a known sequence having an appropriate length for performing nucleic acid amplification, and those skilled in the art can appropriately design such a sequence. The primer sequence for PCR amplification can be, for example, 10 to 50 bases, 15 to 40 bases, 15 to 30 bases or 15 to 20 bases. By including the common primer sequence in the probe, the amplification reaction in the subsequent nucleic acid amplification step can be easily performed.
　核酸捕捉ビーズ３には、細胞捕捉孔２ごとに異なる細胞認識配列を有するＤＮＡプローブ２１が固定されており、それ故、後に分析した配列がいずれの細胞由来であるかを判別することが可能となる。例えば、細胞認識配列を５塩基のランダム配列にした場合には４の５乗、すなわち１０２４の細胞を識別することが可能となる。したがって細胞認識配列は細胞捕捉孔２の数に応じて任意に設定でき、具体的には５～３０塩基、５～２０塩基、５～１５塩基または５～１０塩基の範囲とすることができる。 The DNA probe 21 having a different cell recognition sequence for each cell trapping hole 2 is fixed to the nucleic acid trapping bead 3, so that it is possible to discriminate from which cell the sequence analyzed later is derived. Become. For example, when the cell recognition sequence is a random sequence of 5 bases, 4 5, that is, 1024 cells can be identified. Therefore, the cell recognition sequence can be arbitrarily set according to the number of the cell capturing holes 2, and specifically, can be in the range of 5 to 30 bases, 5 to 20 bases, 5 to 15 bases or 5 to 10 bases.
　図３は、単一細胞解析システムの全体外略図である。単一細胞解析システムには、図１に示された単一細胞解析装置１１の他に、単一細胞解析装置１１に導入された液体の様子や残量をリアルタイムでモニタリングするカメラ３１および制御用ＰＣ３２が付属している。 FIG. 3 is a schematic diagram of the entire single cell analysis system. The single cell analysis system includes, in addition to the single cell analyzer 11 shown in FIG. 1, a camera 31 for monitoring the state and remaining amount of the liquid introduced into the single cell analyzer 11 in real time, and a camera 31 for control. A PC 32 is attached.
　解析用チップ７は２．０ミリメートル四方のポリジメチルシロキサン（ＰＤＭＳ）を成型して厚さ１００マイクロメートルで作製した。ただし解析用チップ７の素材はポリプロピレン、ポリスチレンなどの樹脂でもよい。また、解析用チップ７は、シリコンウェハ等の微細加工によって作製することが可能である。 The analysis chip 7 was manufactured by molding 2.0 mm square polydimethylsiloxane (PDMS) to have a thickness of 100 μm. However, the material of the analysis chip 7 may be a resin such as polypropylene or polystyrene. Further, the analysis chip 7 can be manufactured by fine processing of a silicon wafer or the like.
　細胞捕捉孔２は、直径３マイクロメートル、１２５マイクロメートル間隔のピッチで、解析用チップ７の上面側に百個設置した。細胞捕捉孔２の下部に存在する空間に核酸捕捉ビーズ３をインクジェットプリンタで分注した。核酸捕捉ビーズ３のサイズは直径１マイクロメートルである。膜フィルタ８は、直径約８００ナノメートルの細孔を有し、親水性処理がなされている。膜フィルタ８に設ける細孔の径は、例えば、０．５～３．０マイクロメートルとする。膜フィルタ８の素材は、ポリカーボネート、ポリフッ化ビニリデン、ナイロンおよびポリエチレン等が使用可能である。流路壁６はアクリルによって作製し、上下のアクリル製流路壁６によって解析用チップ７と膜フィルタ８とを挟み込んでネジで固定した。流路壁６の素材はアクリルの他にも、ポリプロピレン、ポリスチレンまたはポリテトラフルオロエチレン等でも作製可能である。また、核酸捕捉ビーズ３には予めストレプトアビジンが固定化されており、そこに５’末端がビオチン修飾されたＤＮＡプローブ２１を固定した。ＤＮＡプローブ２１は５’末端から３０塩基のＰＣＲ増幅用共通配列、５塩基の細胞認識配列、７塩基のランダム配列からなる分子認識配列、１８塩基のオリゴ配列および２塩基のＶＮ配列を有するようにした。 百 One hundred cell capture holes 2 were provided on the upper surface side of the analysis chip 7 at a pitch of 3 μm in diameter and at intervals of 125 μm. Nucleic acid capture beads 3 were dispensed into the space below the cell capture holes 2 using an inkjet printer. The size of the nucleic acid capturing beads 3 is 1 micrometer in diameter. The membrane filter 8 has pores with a diameter of about 800 nanometers, and has been subjected to a hydrophilic treatment. The diameter of the pores provided in the membrane filter 8 is, for example, 0.5 to 3.0 micrometers. As a material of the membrane filter 8, polycarbonate, polyvinylidene fluoride, nylon, polyethylene, or the like can be used. The channel wall 6 was made of acrylic, and the analysis chip 7 and the membrane filter 8 were sandwiched between the upper and lower acrylic channel walls 6 and fixed with screws. The material of the channel wall 6 can be made of polypropylene, polystyrene, polytetrafluoroethylene, or the like, in addition to acrylic. In addition, streptavidin was previously immobilized on the nucleic acid capture beads 3, and a DNA probe 21 having a 5'-end modified with biotin was immobilized thereon. The DNA probe 21 has a common sequence for PCR amplification of 30 bases from the 5 'end, a cell recognition sequence of 5 bases, a molecular recognition sequence consisting of a random sequence of 7 bases, an oligo sequence of 18 bases, and a VN sequence of 2 bases. did.
　図４は、分注装置９を用いた単一細胞解析システムの操作フローを示すフローチャートである。図５は、図４のフローチャートと対応する単一細胞解析システムの各状態を表した概略図である。例えば、図４の操作フローの（ａ）の状態は、図５－（ａ）図と一致している。以下に、図４に示された単一細胞解析システムの操作フローの各ステップについて説明する。 FIG. 4 is a flowchart showing an operation flow of the single cell analysis system using the dispensing device 9. FIG. 5 is a schematic diagram showing each state of the single cell analysis system corresponding to the flowchart of FIG. For example, the state of (a) in the operation flow of FIG. 4 matches the state of FIG. 5- (a). Hereinafter, each step of the operation flow of the single cell analysis system shown in FIG. 4 will be described.
（ａ）細胞懸濁液分注（Ｓ４０１）
　被検対象である細胞１を含んだ細胞懸濁液５を分注装置９にセットし、制御用ＰＣ３２によって制御しながら単一細胞解析装置１１の解析用チップ７の上部に分注する。細胞懸濁液５の分注量は５マイクロリットルほどとし、細胞懸濁液５の内部に存在する細胞数は八十個ほどを目安にする。また、解析用チップ７の上部の様子はカメラ３１でモニタリングし、細胞捕捉孔２に細胞が捕捉される様子および解析チップ７の上部の溶液の残量を確認する。必要な量の細胞懸濁液５が分注された後、フロー（ａ）を終了する。 (A) Dispensing of cell suspension (S401)
The cell suspension 5 containing the cells 1 to be tested is set in the dispensing device 9 and dispensed on the analysis chip 7 of the single cell analyzer 11 while being controlled by the control PC 32. The amount of the cell suspension 5 to be dispensed is about 5 microliters, and the number of cells existing in the cell suspension 5 is about 80. The state of the upper part of the analysis chip 7 is monitored by the camera 31 to check the state of cells being captured in the cell capturing hole 2 and the remaining amount of the solution on the upper part of the analysis chip 7. After the required amount of the cell suspension 5 has been dispensed, the flow (a) is terminated.
（ｂ）界面活性剤分注（Ｓ４０２）
　フロー（ａ）終了後、以下の式２を満たすような界面活性剤４を準備し、分注装置９にセットする。
Figure JPOXMLDOC01-appb-M000005
 ここで、
Ｐ：送液用ポンプ１０が解析用チップ７側の液体と膜フィルタ８側の液体との間に生じさせる圧力差
ｋ：膜フィルタ８の形状に関する固有係数
γ1：界面活性剤４の表面張力
θ：界面活性剤４と膜フィルタ８との接触角
Ｄ：膜フィルタ８に設けられた孔の孔径 (B) Surfactant dispensing (S402)
After the end of the flow (a), a surfactant 4 that satisfies the following equation 2 is prepared and set in the dispensing device 9.
Figure JPOXMLDOC01-appb-M000005
 here,
P: pressure difference generated by the liquid sending pump 10 between the liquid on the analysis chip 7 side and the liquid on the membrane filter 8 k: intrinsic coefficient γ 1 relating to the shape of the membrane filter 8: surface tension of the surfactant 4 θ: contact angle between surfactant 4 and membrane filter 8 D: pore diameter of pore provided in membrane filter 8
　続いて、分注装置９にセットした界面活性剤４を、カメラ３１で確認しながら制御用ＰＣ３２によって制御し、解析用チップ７の上部の細胞懸濁液５の液面上に追加分注する。この際、できる限り二液（細胞懸濁液５および界面活性剤４）が混ざらないように静かに分注し、撹拌を行わない。 Subsequently, the surfactant 4 set in the dispensing device 9 is controlled by the control PC 32 while checking with the camera 31, and is additionally dispensed on the liquid surface of the cell suspension 5 above the analysis chip 7. . At this time, the two liquids (cell suspension 5 and surfactant 4) are dispensed gently so as not to mix as much as possible, and stirring is not performed.
　例えば、細胞懸濁液５からなる第１の液層の上方に界面活性剤４からなる第２の液層が形成されるよう静かに分注を行う。界面活性剤４を分注する際は、細胞懸濁液５に分注ピペットの先端が付着した状態で界面活性剤４を導入すると、細胞懸濁液５中の細胞１に界面活性剤４が触れ、細胞１が破壊されるのを防ぐことができる。界面活性剤４を導入する理由は、表面張力が小さい（例えば、純水よりも小さい）液体を流すことにより解析用チップ７内に気泡による目詰まりが生じるのを防ぐためである。必要量の界面活性剤４が分注されればフロー（ｂ）を終了する。 For example, gently perform dispensing so that a second liquid layer made of the surfactant 4 is formed above the first liquid layer made of the cell suspension 5. When the surfactant 4 is dispensed, the surfactant 4 is introduced into the cells 1 in the cell suspension 5 by introducing the surfactant 4 in a state where the tip of the pipetting pipette is attached to the cell suspension 5. Touching can prevent the cells 1 from being destroyed. The reason for introducing the surfactant 4 is to prevent clogging of the analysis chip 7 with bubbles by flowing a liquid having a small surface tension (for example, smaller than pure water). When the required amount of the surfactant 4 is dispensed, the flow (b) ends.
（ｃ）細胞懸濁液および界面活性剤の送液（Ｓ４０３）
　フロー（ｂ）の終了後、送液用ポンプ１０を起動する。送液用ポンプ１０によって解析用チップ７の上方に存在する細胞懸濁液５は解析用チップ７の下部の流路を介して排出され始め、それに伴い細胞懸濁液５中の細胞１は細胞捕捉孔２に捕捉される。細胞懸濁液５の送液完了後、細胞懸濁液５の上方に存在していた界面活性剤４が解析用チップ７の上方に存在しなくなるまで完全に送液される。解析チップ７の上方の溶液の残量はカメラ３１で確認することが可能で、界面活性剤４が完全に送液されたことを確認されれば、送液用ポンプ１０を停止してフロー（ｃ）を終了する。 (C) Delivery of cell suspension and surfactant (S403)
After the end of the flow (b), the liquid supply pump 10 is started. The cell suspension 5 existing above the analysis chip 7 is started to be discharged by the liquid sending pump 10 through the flow path below the analysis chip 7, and the cells 1 in the cell suspension 5 are replaced with the cells. It is captured in the capturing hole 2. After the completion of the transfer of the cell suspension 5, the surfactant 4 existing above the cell suspension 5 is completely sent until it does not exist above the analysis chip 7. The remaining amount of the solution above the analysis chip 7 can be checked by the camera 31. If it is confirmed that the surfactant 4 has been completely sent, the solution sending pump 10 is stopped and the flow ( End c).
　上述のとおり、細胞懸濁液５と界面活性剤４とはできるだけ混合しないように分注されているため、細胞１に界面活性剤４が接触するのは送液ポンプ１０によって細胞懸濁液５の大部分が送液し終わった後である。そして、短い時間で界面活性剤４を送液するため細胞１に界面活性剤４が接触する時間が短い。それ故、細胞１の細胞膜を破壊することなく界面活性剤４を流路に流すことができ、解析用チップ７内に気泡による目詰まりが生じるのを防ぐことができる。また、仮に細胞１の細胞膜が破壊されて中の分子が出てしまったとしても、細胞懸濁液５の送液により個々の細胞１が細胞捕捉孔２に既に捕捉されているため、核酸捕捉ビーズ３に細胞１内の分子を捕捉させることができる。 As described above, since the cell suspension 5 and the surfactant 4 are dispensed so as not to be mixed as much as possible, the surfactant 4 comes into contact with the cells 1 only by the liquid sending pump 10. After most of the liquid has been transferred. Since the surfactant 4 is supplied in a short time, the time during which the surfactant 4 contacts the cell 1 is short. Therefore, the surfactant 4 can be caused to flow through the flow path without destroying the cell membrane of the cell 1, and clogging of the analysis chip 7 with bubbles can be prevented. Even if the cell membrane of the cell 1 is destroyed and the molecules in the cell 1 come out, since the individual cells 1 have already been captured in the cell capturing holes 2 by sending the cell suspension 5, the nucleic acid capturing The molecules in the cell 1 can be captured by the beads 3.
（ｄ）反応試薬分注（Ｓ４０４）
　フロー（ｃ）の終了後、解析用チップ７上には細胞捕捉孔２に捕捉された被検細胞１が残っている状態となる。フロー（ｄ）では遺伝子発現解析を行うための試薬を分注し、解析用チップ中で反応を行う。実施例１では細胞膜を破壊するＬｙｓｉｓバッファを第１試薬として分注した。試薬の種類はこれに限ったものではなくユーザの任意で選択して構わない。単一細胞解析装置１１に導入する全ての反応試薬には、以下の式３を満たすような界面活性剤４を混合し、予め分注装置９にセットしておく。
Figure JPOXMLDOC01-appb-M000006
 　ここで、
Ｐ：送液用ポンプ１０が解析用チップ７側の液体と膜フィルタ８側の液体との間に生じさせる圧力差
ｋ：膜フィルタ８の形状に関する固有係数
γ2：界面活性剤４と反応試薬との混合液体の表面張力
θ：混合液体と膜フィルタ８との接触角
Ｄ：膜フィルタ８に設けられた孔の孔径 (D) Dispensing of reaction reagent (S404)
After the end of the flow (c), the test cell 1 captured in the cell capturing hole 2 remains on the analysis chip 7. In the flow (d), a reagent for performing gene expression analysis is dispensed and a reaction is performed in an analysis chip. In Example 1, a Lysis buffer that disrupts the cell membrane was dispensed as the first reagent. The type of reagent is not limited to this, and may be arbitrarily selected by the user. A surfactant 4 that satisfies the following formula 3 is mixed with all the reaction reagents to be introduced into the single cell analyzer 11 and set in the dispenser 9 in advance.
Figure JPOXMLDOC01-appb-M000006
 here,
P: pressure difference generated by the liquid sending pump 10 between the liquid on the analysis chip 7 side and the liquid on the membrane filter 8 k: specific coefficient γ 2 relating to the shape of the membrane filter 8: surfactant 4 and reaction reagent The contact angle between the mixed liquid and the membrane filter 8 D: the diameter of the hole provided in the membrane filter 8
　反応試薬に表面張力が小さい界面活性剤４を混合しておくことにより、送液完了時に解析用チップ７内に気泡による目詰まりが生じることを防ぐことができる。カメラ３１でフロー（ｃ）の終了を確認後、制御用ＰＣ３２を通して、分注装置９からＬｙｓｉｓバッファ（界面活性剤４を含む）を解析用チップ７上に分注する。必要量のＬｙｓｉｓバッファが分注されればフロー（ｄ）を終了する。なお、反応試薬に混合させる界面活性剤４は、フロー（ｂ）において分注した界面活性剤４と同一種類の界面活性剤であってもよく、異なる種類の界面活性剤であってもよい。 (4) By mixing the surfactant 4 having a small surface tension with the reaction reagent, it is possible to prevent the analysis chip 7 from being clogged with bubbles when the liquid supply is completed. After confirming the end of the flow (c) by the camera 31, the Lysis buffer (including the surfactant 4) is dispensed from the dispensing device 9 onto the analysis chip 7 through the control PC 32. When the required amount of Lysis buffer is dispensed, the flow (d) ends. The surfactant 4 to be mixed with the reaction reagent may be the same type of surfactant as the surfactant 4 dispensed in the flow (b), or may be a different type of surfactant.
（ｅ）反応試薬の送液（Ｓ４０５）
　フロー（ｄ）の終了後、送液用ポンプ１０を起動する。フロー（ｄ）で分注したＬｙｓｉｓバッファによって細胞捕捉孔２に捕捉した細胞１の細胞膜を破壊し、細胞１内の被検核酸２２を細胞捕捉孔２の下部へ吸引する。被検核酸２２は、核酸捕捉ビーズ３の表面に固定されているＤＮＡプライマー２１に捕捉される。細胞１の細胞膜が破壊されたかどうかをカメラ３１でモニタリングし、細胞膜の破壊が不十分な場合は、制御用ＰＣ３２が分注装置９を制御してＬｙｓｉｓバッファの追加分注を行う。捕捉された全ての細胞１が破壊されたのを確認し、全てのＬｙｓｉｓバッファの送液が完了したら、送液用ポンプ１０を停止し、フロー（ｅ）を終了する。フロー（ｅ）を終了した後、解析用チップ７の上方には何もない状態となる。 (E) Feeding the reaction reagent (S405)
After the end of the flow (d), the liquid supply pump 10 is started. The cell membrane of the cell 1 captured in the cell capturing hole 2 is destroyed by the Lysis buffer dispensed in the flow (d), and the test nucleic acid 22 in the cell 1 is sucked into the lower part of the cell capturing hole 2. The test nucleic acid 22 is captured by the DNA primer 21 fixed on the surface of the nucleic acid capture bead 3. The camera 31 monitors whether or not the cell membrane of the cell 1 has been destroyed. If the cell membrane has not been sufficiently destroyed, the control PC 32 controls the dispensing device 9 to additionally dispense the Lysis buffer. It is confirmed that all the captured cells 1 have been destroyed, and when the liquid supply of all the Lysis buffers is completed, the liquid supply pump 10 is stopped, and the flow (e) is terminated. After the end of the flow (e), there is nothing above the analysis chip 7.
（ｄ’）反応試薬分注（２工程目）
　実施例１では、反応手順が全２工程存在する例を説明するため、操作フローにしたがってもう一度、フロー（ｄ）を行う。実施例１では第２試薬として逆転写反応を行うためのＲＴ試薬を分注する。以下に示す式４の条件を満たすように界面活性剤４を混合したＲＴ試薬を分注装置９によって分注する。
Figure JPOXMLDOC01-appb-M000007
 　ここで、
Ｐ：送液用ポンプ１０が解析用チップ７側の液体と膜フィルタ８側の液体との間に生じさせる圧力差
ｋ：膜フィルタ８の形状に関する固有係数
γ22：界面活性剤４と第２試薬との混合液体の表面張力
θ：混合液体と膜フィルタ８との接触角
Ｄ：膜フィルタ８に設けられた孔の孔径 (D ') Reagent dispensing (2nd step)
In Example 1, the flow (d) is performed again according to the operation flow in order to describe an example in which the reaction procedure includes all two steps. In Example 1, an RT reagent for performing a reverse transcription reaction is dispensed as a second reagent. The RT reagent mixed with the surfactant 4 so as to satisfy the condition of the following formula 4 is dispensed by the dispensing device 9.
Figure JPOXMLDOC01-appb-M000007
 here,
P: Pressure difference generated between the liquid on the analysis chip 7 side and the liquid on the membrane filter 8 by the liquid sending pump 10 k: Specific coefficient γ 22 relating to the shape of the membrane filter 8: Surfactant 4 and second Surface tension θ of the liquid mixture with the reagent: contact angle between the liquid mixture and the membrane filter 8 D: diameter of the holes provided in the membrane filter 8
　任意の分量（例えば、数マイクロリットル）を分注後、フロー（ｄ’）を終了する。なお、第２試薬に混合させる界面活性剤４は、フロー（ｂ）および１回目のフロー（ｄ）において分注した界面活性剤４と同一種類の界面活性剤であってもよく、異なる種類の界面活性剤であってもよい。 後 After dispensing an arbitrary amount (for example, several microliters), the flow (d ') ends. The surfactant 4 to be mixed with the second reagent may be the same type of surfactant as the surfactant 4 dispensed in the flow (b) and the first flow (d), or may be a different type. It may be a surfactant.
（ｅ’）反応試薬の送液（２工程目）
　フロー（ｄ’）の終了後、送液用ポンプ１０を起動し、界面活性剤４入りのＲＴ試薬を送液する。解析チップ７の上方のＲＴ試薬の残量をカメラ３１で確認しながら送液を行い、全ての溶液を送液するまでに、一度、送液用ポンプ１０を停止し、逆転写反応が十分行われる時間（例えば５０分）静置する。逆転写反応の終了後、再度、送液用ポンプ１０を起動し、残りのＲＴ試薬を全て送液する。全ての溶液の送液が完了した後、送液用ポンプ１０を停止し、フロー（ｅ’）を終了する。 (E ') Reaction reagent sending (second step)
After the end of the flow (d '), the liquid sending pump 10 is started, and the RT reagent containing the surfactant 4 is sent. The solution is fed while confirming the remaining amount of the RT reagent above the analysis chip 7 with the camera 31, and the pump 10 is once stopped until all the solutions are sent, so that the reverse transcription reaction is sufficiently performed. Allow to stand still (eg, 50 minutes). After the end of the reverse transcription reaction, the solution sending pump 10 is started again to send all the remaining RT reagents. After all the solutions have been sent, the solution sending pump 10 is stopped, and the flow (e ′) ends.
　以上で分注装置９を備える単一細胞解析システムを用いての操作は全て終了する。続いて、単一細胞解析装置１１から解析用チップ７を取り出し、ＰＣＲ増幅に必要な反応をチューブ４１中で行う。 で All operations using the single cell analysis system including the dispensing device 9 are completed. Subsequently, the analysis chip 7 is taken out of the single-cell analyzer 11, and a reaction necessary for PCR amplification is performed in the tube 41.
　図６は、解析用チップ７のＰＣＲ増幅反応を実施する例を示す概略図である。チューブ４１中にはＰＣＲ反応に必要なＰＣＲ用試薬４２が入っている。ＰＣＲ増幅反応終了後、ＤＮＡ配列シーケンシングを行い、細胞捕捉孔２で捕捉した一細胞ごとの遺伝子発現量を解析することが可能になる。 FIG. 6 is a schematic view showing an example in which a PCR amplification reaction of the analysis chip 7 is performed. The tube 41 contains a PCR reagent 42 necessary for the PCR reaction. After the end of the PCR amplification reaction, DNA sequence sequencing is performed, and the gene expression level of each cell captured in the cell capturing hole 2 can be analyzed.
　上記のとおり、本開示の単一細胞解析システムは、表面張力が小さい界面活性剤４を単一細胞解析装置１１に導入することにより、送液用ポンプ１０で解析用チップ７の上方に存在する液体を送液し終えた際に、解析用チップ７内に気泡の目詰まりが生じることを防ぐことができる。 As described above, the single cell analysis system of the present disclosure is present above the analysis chip 7 by the liquid sending pump 10 by introducing the surfactant 4 having a small surface tension into the single cell analysis device 11. When the liquid has been sent, clogging of bubbles in the analysis chip 7 can be prevented.
　なお、上記の説明では、反応工程が二回必要な例を説明したが、反応工程は一回でもそれ以上でもよい。本開示は、解析用チップ７内に気泡の目詰まりを防ぐ方法として反応試薬に界面活性剤４を導入する方法を説明したが、次の送液を控えてない最後の反応試薬に対しては、必ずしも界面活性剤４を混入する必要はない。例えば、反応工程が一回しかない操作フローの場合は、反応試薬に界面活性剤４を混入しなくともよい。また、反応工程が三回存在する操作フローの場合は、三回目の反応試薬に対して界面活性剤４を混入しなくともよい。 In the above description, an example in which the reaction step is required twice has been described, but the number of the reaction steps may be one or more. The present disclosure has described the method of introducing the surfactant 4 into the reaction reagent as a method of preventing air bubbles from being clogged in the analysis chip 7; However, it is not always necessary to mix the surfactant 4. For example, in the case of an operation flow having only one reaction step, the surfactant 4 need not be mixed into the reaction reagent. In the case of an operation flow in which the reaction step is performed three times, the surfactant 4 does not have to be mixed with the third reaction reagent.
＜実施例２＞
　実施例２では、実施例１に示した方法よりも細胞１が界面活性剤４から受けるダメージをさらに減少させることができる方法を説明する。実施例２の細胞解析方法は、上で説明した操作フローのうち、フロー（ｂ）（Ｓ４０２）が実施例１とは異なる。以下に、実施例２のフロー（ｂ）について説明する。 <Example 2>
In the second embodiment, a method will be described in which the damage to the cell 1 from the surfactant 4 can be further reduced as compared with the method shown in the first embodiment. The cell analysis method according to the second embodiment differs from the first embodiment in the flow (b) (S402) among the operation flows described above. Hereinafter, the flow (b) of the second embodiment will be described.
（ｂ）界面活性剤分注（Ｓ４０２）
　フロー（ａ）の終了後、上記式２を満たすような界面活性剤４を準備し、分注装置９にセットする。フロー（ａ）の終了後、解析チップ７の上方には細胞懸濁液５のみが分注されている状態である。続いて、界面活性剤４を分注する前に送液用ポンプ１０を起動し、送液を開始する。分注装置９は、細胞懸濁液５の液面のレベルが解析用チップ７の上方にあるうちに、界面活性剤４の分注を開始する。界面活性剤４を分注し終え、且つ分注した界面活性剤４の送液を終えた後、送液用ポンプ１０を停止する。この際、制御用ＰＣ３２には、細胞懸濁液５が送液し終わる前のタイミングで準備した界面活性剤４の分注を開始するようにプログラムが記録されるか、または、ユーザによって設定される。または、カメラ３１によって細胞懸濁液５の残量を観察しながら制御用ＰＣ３２を操作し、界面活性剤４を分注してもよい。界面活性剤４の送液が完了後、実施例１と同様にフロー（ｄ）以降を行う。 (B) Surfactant dispensing (S402)
After the end of the flow (a), the surfactant 4 satisfying the above formula 2 is prepared and set in the dispensing device 9. After the end of the flow (a), only the cell suspension 5 is dispensed above the analysis chip 7. Then, before dispensing the surfactant 4, the liquid sending pump 10 is started to start the liquid sending. The dispensing device 9 starts dispensing the surfactant 4 while the liquid surface level of the cell suspension 5 is above the analysis chip 7. After the dispensing of the surfactant 4 and the termination of the dispensing of the dispensed surfactant 4, the pump 10 for liquid delivery is stopped. At this time, a program is recorded in the control PC 32 so as to start dispensing of the prepared surfactant 4 at a timing before the cell suspension 5 is completely fed, or set by the user. You. Alternatively, the surfactant 4 may be dispensed by operating the control PC 32 while observing the remaining amount of the cell suspension 5 with the camera 31. After the completion of the feeding of the surfactant 4, the flow (d) and subsequent steps are performed in the same manner as in the first embodiment.
　上記のように細胞懸濁液５の送液を界面活性剤４の追加分注よりも先に開始し、細胞懸濁液５の送液が完了する直前に界面活性剤４を追加分注することで、細胞１が界面活性剤４と接触する時間をできる限り短くすることが可能である。 As described above, the feeding of the cell suspension 5 is started before the additional dispensing of the surfactant 4, and the surfactant 4 is additionally dispensed immediately before the feeding of the cell suspension 5 is completed. This makes it possible to shorten the time during which the cell 1 comes into contact with the surfactant 4 as much as possible.
＜実施例３＞
　実施例１および２では、細胞懸濁液５を分注し終えてから界面活性剤４を分注した。実施例３では、細胞懸濁液５に界面活性剤４を混合した混合液体を分注することによって、実施例１において説明したフロー（ａ）および（ｂ）を代替する。この際、界面活性剤４は、以下の式５の条件式を満たすように選ばれる。
Figure JPOXMLDOC01-appb-M000008
 　ここで、
Ｐ：送液用ポンプ１０が解析用チップ７側の液体と膜フィルタ８側の液体との間に生じさせる圧力差
ｋ：膜フィルタ８の形状に関する固有係数
γ3：界面活性剤４と細胞懸濁液５との混合液体の表面張力
θ：混合液体と膜フィルタ８との接触角
Ｄ：膜フィルタ８に設けられた孔の孔径 <Example 3>
In Examples 1 and 2, the surfactant 4 was dispensed after the cell suspension 5 was dispensed. In the third embodiment, the flows (a) and (b) described in the first embodiment are substituted by dispensing a liquid mixture obtained by mixing the surfactant 4 with the cell suspension 5. At this time, the surfactant 4 is selected so as to satisfy the following conditional expression 5.
Figure JPOXMLDOC01-appb-M000008
 here,
P: pressure difference generated by the liquid sending pump 10 between the liquid on the analysis chip 7 side and the liquid on the membrane filter 8 side k: specific coefficient γ 3 relating to the shape of the membrane filter 8: surfactant 4 and cell suspension Surface tension θ of the mixed liquid with the suspension liquid 5: contact angle between the mixed liquid and the membrane filter 8 D: diameter of the hole provided in the membrane filter 8
　実施例３では、界面活性剤４の表面張力は、上記フローにおいて細胞１を壊さない程度の所定の値よりも大きい表面張力であることが要求される。このγ3は、例えば、実験によって決定される。また、界面活性剤４と細胞懸濁液５との混合液体は、分注してからなるべく早急に送液すると細胞１へのダメージを軽減することができる。 In Example 3, the surface tension of the surfactant 4 is required to be larger than a predetermined value that does not break the cells 1 in the above flow. This γ 3 is determined, for example, by experiment. Further, if the mixed liquid of the surfactant 4 and the cell suspension 5 is dispensed and then sent as soon as possible, damage to the cells 1 can be reduced.
　上で説明した実施例１～３では、界面活性剤４を追加分注または細胞懸濁液５もしくは反応試薬に予め混合することによって、単一細胞解析装置１１に導入する溶液の表面張力を小さくすることができた。これにより、解析用チップ７内に気体の目詰まりが生じることを防ぐことができ、高い能力の送液用ポンプ１０を必要とせずに済む。また、膜フィルタ８に設ける孔および細胞捕捉孔２の孔径を小さくすることができるため、小さい核酸捕捉用ビーズ３を使用することができ、高効率な反応が実施できる。また、上述の操作フローを実行することにより、溶液の表面張力を小さくした場合であっても、細胞膜が破壊されることを防ぐことができる。 In Examples 1 to 3 described above, the surface tension of the solution to be introduced into the single cell analyzer 11 is reduced by additionally dispensing the surfactant 4 or previously mixing the surfactant 4 with the cell suspension 5 or the reaction reagent. We were able to. Accordingly, it is possible to prevent the gas from being clogged in the analysis chip 7, and it is not necessary to provide the liquid supply pump 10 having a high capacity. Further, since the pores provided in the membrane filter 8 and the pore diameter of the cell trapping pores 2 can be reduced, small nucleic acid trapping beads 3 can be used, and a highly efficient reaction can be performed. Further, by executing the above-described operation flow, it is possible to prevent the cell membrane from being destroyed even when the surface tension of the solution is reduced.
　なお、本開示は上記した実施例に限定されるものではなく、様々な変形例が含まれる。例えば、上記した実施例は本開示を分かりやすく説明するために詳細に説明したものであり、必ずしも説明した全ての構成を備えるものに限定されるものではない。また、ある実施例の構成の一部を他の実施例の構成に置き換えることが可能であり、また、ある実施例の構成に他の実施例の構成を加えることも可能である。また、各実施例の構成の一部について、他の構成の追加・削除・置換をすることが可能である。 Note that the present disclosure is not limited to the above-described embodiments, and includes various modifications. For example, the above-described embodiments have been described in detail in order to easily explain the present disclosure, and are not necessarily limited to those having all the configurations described above. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of one embodiment can be added to the configuration of another embodiment. Further, for a part of the configuration of each embodiment, it is possible to add / delete / replace another configuration.
　例えば、上記の実施例１および２では、分注装置９を使用して細胞懸濁液５、界面活性剤４および反応試薬等の各種液体を単一細胞解析デバイス１１に分注する例を説明したが、液体の分注はピペット等を使用して手技によって実行されてもよい。その場合、実施例２では、例えば、カメラ３１で解析用チップ７上の細胞懸濁液５の残量を確認しながら、送液用ポンプ１０を稼働させ、細胞懸濁液５の残量が少なくなった時点で、界面活性剤４を細胞懸濁液５の液面上に追加分注してもよい。 For example, in Examples 1 and 2 described above, an example is described in which various liquids such as the cell suspension 5, the surfactant 4, and the reaction reagent are dispensed to the single-cell analysis device 11 using the dispensing device 9. However, liquid dispensing may be performed by a technique using a pipette or the like. In this case, in the second embodiment, for example, while checking the remaining amount of the cell suspension 5 on the analysis chip 7 with the camera 31, the pump 10 for liquid supply is operated, and the remaining amount of the cell suspension 5 is reduced. When the amount becomes low, the surfactant 4 may be additionally dispensed onto the liquid surface of the cell suspension 5.
１…細胞、２…細胞捕捉用孔、３…核酸捕捉用ビーズ、４…界面活性剤、５…細胞懸濁液、６…流路壁、７…解析用チップ、８…多孔質膜、９…分注装置、１０…送液用ポンプ、２１…ＤＮＡプローブ、２２…被検核酸、３１…カメラ、３２…制御用ＰＣ、４１…チューブ、４２…ＰＣＲ用試薬 DESCRIPTION OF SYMBOLS 1 ... Cell, 2 ... Cell trapping hole, 3 ... Nucleic acid trapping beads, 4 ... Surfactant, 5 ... Cell suspension, 6 ... Channel wall, 7 ... Analysis chip, 8 ... Porous membrane, 9 ... Dispensing device, 10 ... Sending pump, 21 ... DNA probe, 22 ... Test nucleic acid, 31 ... Camera, 32 ... Control PC, 41 ... Tube, 42 ... PCR reagent

Claims (15)

  1. 　解析用チップと前記解析用チップに積層された膜フィルタとを備える単一細胞解析装置を用いる細胞解析方法であって、
    　前記解析用チップに細胞を含む第１の液体を導入するステップと、
    　前記解析用チップに前記第１の液体よりも表面張力が小さい第２の液体を導入するステップと、
    　前記第１の液体と前記第２の液体とを前記膜フィルタ側の流路と接続された送液用ポンプによって吸引するステップと、
    　前記解析用チップに前記細胞と反応する反応試薬を導入し、前記細胞から抽出される分子を固相に反応させるステップと、
    　前記反応試薬を前記送液用ポンプによって吸引するステップと、
    　前記解析用チップの中から前記固相を取り出して解析するステップと、
    　を含む細胞解析方法。     A cell analysis method using a single cell analysis device including an analysis chip and a membrane filter stacked on the analysis chip,
    Introducing a first liquid containing cells into the analysis chip;
    Introducing a second liquid having a smaller surface tension than the first liquid to the analysis chip;
    Suctioning the first liquid and the second liquid by a liquid-feeding pump connected to a flow path on the membrane filter side;
    Introducing a reaction reagent that reacts with the cells into the analysis chip, and reacting a molecule extracted from the cells with a solid phase,
    Aspirating the reaction reagent by the liquid sending pump;
    Removing and analyzing the solid phase from the analysis chip,
    A cell analysis method comprising:
  2. 　請求項１に記載の細胞解析方法であって、
    　前記第２の液体を導入するステップにおいて、前記第１の液体からなる第１の液層と前記第２の液体からなる第２の液層とが形成されるように前記第２の液体を導入する、
    　細胞解析方法。     The cell analysis method according to claim 1, wherein
    In the step of introducing the second liquid, the second liquid is introduced such that a first liquid layer made of the first liquid and a second liquid layer made of the second liquid are formed. Do
    Cell analysis method.
  3. 　請求項１に記載の細胞解析方法であって、
    　前記第２の液体を導入するステップにおいて、前記第２の液体として界面活性剤を導入する、
    　細胞解析方法。     The cell analysis method according to claim 1, wherein
    In the step of introducing the second liquid, a surfactant is introduced as the second liquid,
    Cell analysis method.
  4. 　請求項１に記載の細胞解析方法であって、
    　前記膜フィルタは多孔質膜または網目状のフィルタである、
    　細胞解析方法。     The cell analysis method according to claim 1, wherein
    The membrane filter is a porous membrane or a mesh filter.
    Cell analysis method.
  5. 　請求項１に記載の細胞解析方法であって、
    　前記第２の液体を導入するステップにおいて、前記第１の液体を吸引しながら前記第２の液体を導入する、
    　細胞解析方法。     The cell analysis method according to claim 1, wherein
    Introducing the second liquid while sucking the first liquid in the step of introducing the second liquid;
    Cell analysis method.
  6. 　請求項１に記載の細胞解析方法であって、
    　前記第２の液体を導入するステップにおいて、前記第２の液体の表面張力は純水の表面張力よりも小さい、
    　細胞解析方法。     The cell analysis method according to claim 1, wherein
    In the step of introducing the second liquid, the surface tension of the second liquid is smaller than the surface tension of pure water.
    Cell analysis method.
  7. 　請求項１に記載の細胞解析方法であって、
    　前記膜フィルタは多孔質膜であり、
    　前記第２の液体、前記多孔質膜および前記送液用ポンプは、以下の数式を満たすパラメータで特徴づけられる、
    　細胞解析方法。
    Figure JPOXMLDOC01-appb-M000001
     Ｐ：前記送液用ポンプが前記解析用チップ側の液体と前記多孔質膜側の液体との間に生じさせる圧力差
    ｋ：前記多孔質膜の形状に関する固有係数
    γ1：前記第２の液体の表面張力
    θ：前記第２の液体と前記多孔質膜との接触角
    Ｄ：前記多孔質膜に設けられた孔の孔径     The cell analysis method according to claim 1, wherein
    The membrane filter is a porous membrane,
    The second liquid, the porous membrane and the pump for sending liquid are characterized by parameters satisfying the following mathematical formulas:
    Cell analysis method.
    Figure JPOXMLDOC01-appb-M000001
     P: a pressure difference generated between the liquid on the analysis chip side and the liquid on the porous membrane side by the liquid sending pump k: an intrinsic coefficient γ 1 regarding the shape of the porous membrane: the second liquid Surface tension θ: contact angle between the second liquid and the porous membrane D: pore diameter of pores provided in the porous membrane
  8. 　請求項１に記載の細胞解析方法であって、
    　前記第２の液体を導入するステップにおいて、前記解析用チップの映像をモニタに表示しながら前記第２の液体を導入する、
    　細胞解析方法。     The cell analysis method according to claim 1, wherein
    Introducing the second liquid while displaying an image of the analysis chip on a monitor in the step of introducing the second liquid;
    Cell analysis method.
  9. 　請求項１に記載の細胞解析方法であって、
    　前記膜フィルタは０．５～３．０マイクロメートルの孔径が設けられた多孔質膜である、
    　細胞解析方法。     The cell analysis method according to claim 1, wherein
    The membrane filter is a porous membrane provided with a pore size of 0.5 to 3.0 micrometers.
    Cell analysis method.
  10. 　請求項１に記載の細胞解析方法であって、
    　前記第２の液体を導入するステップにおいて、前記第１の液体に分注ピペットの先端が付着した状態で前記第２の液体を導入する、
    　細胞解析方法。     The cell analysis method according to claim 1, wherein
    In the step of introducing the second liquid, the second liquid is introduced with a tip of a pipetting pipet attached to the first liquid,
    Cell analysis method.
  11. 　請求項１に記載の細胞解析方法であって、
    　前記解析用チップは大気圧下に置かれる、
    　細胞解析方法。     The cell analysis method according to claim 1, wherein
    The analysis chip is placed under atmospheric pressure,
    Cell analysis method.
  12. 　請求項７に記載の細胞解析方法であって、
    　前記細胞から抽出される分子を固相に反応させるステップにおいて、前記反応試薬に前記反応試薬よりも表面張力が小さい第３の液体を導入し、
    　前記第３の液体、前記多孔質膜および前記送液用ポンプは、以下の数式を満たすパラメータで特徴づけられる、
    　細胞解析方法。
    Figure JPOXMLDOC01-appb-M000002
     Ｐ：前記送液用ポンプが前記解析用チップ側の液体と前記多孔質膜側の液体との間に生じさせる圧力差
    ｋ：前記多孔質膜の形状に関する固有係数
    γ2：前記反応試薬と前記第３の液体との混合液体の表面張力
    θ：前記混合液体と前記多孔質膜との接触角
    Ｄ：前記多孔質膜に設けられた孔の孔径     It is a cell analysis method of Claim 7, Comprising:
    In the step of reacting a molecule extracted from the cells with a solid phase, introducing a third liquid having a smaller surface tension than the reaction reagent to the reaction reagent,
    The third liquid, the porous membrane, and the liquid sending pump are characterized by parameters that satisfy the following mathematical formula:
    Cell analysis method.
    Figure JPOXMLDOC01-appb-M000002
     P: a pressure difference generated between the liquid on the analysis chip side and the liquid on the porous membrane side by the liquid sending pump k: an intrinsic coefficient γ 2 relating to the shape of the porous membrane: the reaction reagent and the reaction reagent Surface tension θ of the liquid mixture with the third liquid: contact angle D between the liquid mixture and the porous film D: diameter of holes provided in the porous film
  13. 　解析用チップと前記解析用チップに積層された膜フィルタとを備える単一細胞解析装置を用いる細胞解析方法であって、
    　細胞を含む第１の液体と表面張力が前記第１の液体の表面張力よりも小さく前記細胞を破壊しない目安となる所定の値よりも大きい第２の液体とを混合して混合液体を調製するステップと、
    　前記解析用チップに前記混合液体を導入するステップと、
    　前記混合液体を前記膜フィルタ側の流路と接続された送液用ポンプによって吸引するステップと、
    　前記解析用チップに前記細胞と反応する反応試薬を導入するステップと、
    　前記反応試薬を前記送液用ポンプによって吸引するステップと、
    　前記解析用チップの中から前記固相を取り出して解析するステップと、
    　を含む細胞解析方法。     A cell analysis method using a single cell analysis device including an analysis chip and a membrane filter stacked on the analysis chip,
    A mixed liquid is prepared by mixing a first liquid containing cells and a second liquid having a surface tension smaller than the surface tension of the first liquid and larger than a predetermined value which is a measure not to destroy the cells. Steps and
    Introducing the mixed liquid into the analysis chip,
    Aspirating the mixed liquid with a liquid sending pump connected to the flow path on the membrane filter side,
    Introducing a reaction reagent that reacts with the cells in the analysis chip,
    Aspirating the reaction reagent by the liquid sending pump;
    Removing and analyzing the solid phase from the analysis chip,
    A cell analysis method comprising:
  14. 　請求項１３に記載の細胞解析方法であって、
    　前記第２の液体は界面活性剤である、
    　細胞解析方法。     The cell analysis method according to claim 13, wherein
    The second liquid is a surfactant;
    Cell analysis method.
  15. 　請求項１３に記載の細胞解析方法であって、
    　前記膜フィルタは多孔質膜であり、
    　前記混合液体、前記膜フィルタおよび前記送液用ポンプは、以下の数式を満たすパラメータで特徴づけられる、
    　細胞解析方法。
    Figure JPOXMLDOC01-appb-M000003
     Ｐ：前記送液用ポンプが前記解析用チップ側の液体と前記多孔質膜側の液体との間に生じさせる圧力差
    ｋ：前記多孔質膜の形状に関する固有係数
    γ3：前記混合液体の表面張力
    θ：前記混合液体と前記多孔質膜との接触角
    Ｄ：前記多孔質膜に設けられた孔の孔径     The cell analysis method according to claim 13, wherein
    The membrane filter is a porous membrane,
    The mixed liquid, the membrane filter and the pump for sending liquid are characterized by parameters that satisfy the following equation:
    Cell analysis method.
    Figure JPOXMLDOC01-appb-M000003
     P: pressure difference generated between the liquid on the analysis chip side and the liquid on the porous membrane side by the liquid sending pump k: intrinsic coefficient γ 3 relating to the shape of the porous membrane: surface of the mixed liquid Tension θ: contact angle between the liquid mixture and the porous membrane D: pore diameter of pores provided in the porous membrane
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