WO2018025636A1 - Biological sample analysis method and biological sample analysis device - Google Patents

Biological sample analysis method and biological sample analysis device Download PDF

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Publication number
WO2018025636A1
WO2018025636A1 PCT/JP2017/026058 JP2017026058W WO2018025636A1 WO 2018025636 A1 WO2018025636 A1 WO 2018025636A1 JP 2017026058 W JP2017026058 W JP 2017026058W WO 2018025636 A1 WO2018025636 A1 WO 2018025636A1
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biological sample
dna
fixing member
nanopore
substrate
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PCT/JP2017/026058
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French (fr)
Japanese (ja)
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匡 柴原
剛 大浦
井上　智博
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株式会社日立ハイテクノロジーズ
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Publication of WO2018025636A1 publication Critical patent/WO2018025636A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a biological sample analyzer and a biological sample analysis method for performing sequence analysis of nucleic acids such as DNA and RNA using a thin film having nano-sized pores.
  • a biological sample analyzer and a biological sample analysis method for performing sequence analysis of nucleic acids such as DNA and RNA using a thin film having nano-sized pores.
  • it relates to a technique in the case of using arrayed nanopores, that is, multi-nanopores.
  • Patent Literature 1 relating to nanopore DNA sequencing.
  • This publication describes that “the inner diameter of the nanopore was about 10 nm”, and the nanopore DNA sequencer directly measures the DNA base sequence electrically without performing an extension reaction or a fluorescent label. Is attracting attention.
  • Several methods have been proposed for the direct measurement method, one of which is a blocking current method.
  • a few nm pore (nanopore) is prepared on the thin film by a transmission electron microscope or the like, and a liquid tank filled with the electrolyte solution is provided on both sides of the thin film. When an electrode is provided in each liquid tank and a voltage is applied between these electrodes, an ionic current flows through the nanopore.
  • the ion current is proportional to the cross-sectional area of the nanopore as a first order approximation. As DNA passes through the nanopore, the DNA blocks the nanopore, reducing the effective cross-sectional area through which ions can pass, thereby reducing the ionic current. This amount of decrease is called the blocking current. Based on the magnitude of the blocking current, the difference between the single strand and double strand of DNA and the type of base are determined.
  • a method is also considered in which one end of DNA single strand or double strand is fixed to a plane parallel to the plane on which the nanopore is formed, and the speed at which the DNA passes through the nanopore is controlled by a stage driven on an axis perpendicular to the plane. It is done.
  • sample moving stage that can position the position of single-stranded DNA or double-stranded DNA at a desired location on the nanopore.
  • the base cannot be determined unless one end of DNA is fixed and the other end is blocked.
  • the movement of a labeling substance made of charged particles such as polymer ions can be arbitrarily controlled by the principle of a magnetic field or optical tweezers or by an electric field.
  • the inter-space is sub-nanomail, and it is not configured to control a base with a plurality of nanopores blocked with extremely high accuracy.
  • a method is also considered in which one end of DNA single strand or double strand is fixed to a plane parallel to the plane on which the nanopore is formed, and the speed at which the DNA passes through the nanopore is controlled by a stage driven on an axis perpendicular to the plane. It is done.
  • a fixing member (substrate or bead) to which a biological sample is fixed, a substrate having a through-hole through which the biological sample passes, and the biological sample are detected when the biological sample passes through the through-hole.
  • a biological sample analysis method using a biological sample analyzer comprising a detection member, a probe for holding the fixing member, and a drive mechanism for driving the probe, wherein the fixing member is held on the probe, and the fixing member is It is made close to the substrate, the fixing member is released from the probe, the detection member confirms that the biological sample has blocked the through hole, the fixing member is held by the probe, and the probe is pulled up by the driving mechanism.
  • a biological sample analysis method and a biological sample analyzer are provided.
  • the “nanopore” described below is a nano-sized hole provided in the thin film and penetrates the front and back of the thin film.
  • the thin film is mainly formed from an inorganic material.
  • the thin film material may include an organic substance, a polymer material, and the like.
  • the substrate or the beads to which one end of the DNA fragment is fixed are mainly formed from an inorganic material.
  • the thin film material may include an organic substance, a polymer material, and the like.
  • FIG. 1 is a diagram showing an example of the configuration of a biological sample analyzer, and shows a base sequence reading mechanism using a biological sample analysis chip.
  • a biological sample analyzer for example, a DNA analyzer
  • the partition body 101 includes a nanopore device having nanopores.
  • the two tanks 102A and 102B are filled with the electrolyte solution 103.
  • the two tanks 102A and 102B are electrically connected by an electrode 104 and a power source 105.
  • the entire flow path partitioned by the nanopore device is called a flow cell 106.
  • the DNA 111 in the liquid passes through the nanopore of the nanopore device 101 and enters one tank. Although it migrates from 107A to the opposite tank 107B, it does not pass.
  • the base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing one end of the DNA 111 introduced into the nanopore by the driving stage 109.
  • the current value is amplified by an amplifier (not shown) and recorded on a PC (Personal Computer not shown) via an ADC (not shown).
  • the nanopore device 101 includes a nanopore thin film 113 having a thickness of several nanometers on which nanopores 112 are formed.
  • the area of the very thin nanopore thin film 113 is small, and the thickness of the nanopore device has a thickness of several hundred nm for reinforcement.
  • the DNA 111 passes through the nanopore 112 by the driving stage, the flow of ions is hindered, so that the current value decreases by the cross-sectional integral of the DNA 111 in the nanopore 112.
  • the DNA 111 has been described using a sphere (bead), but hereinafter, the DNA 111 will be described using a single strand of yarn in which the DNA 111 is connected in a bead shape.
  • FIG. 2 is an example of a current change for each base species read by applying a voltage at the electrode 104. As shown in the sequence reading example 200, different current values are detected for each of the four types of bases.
  • FIG. 3 shows a biological sample analyzer 300 partially enlarged and showing a state before one end of the DNA 111 is introduced into the nanopore. It is known that a single strand of DNA is entangled in a solution because it is not chemically stable compared to a double strand.
  • the power source 105 generates a force that causes the DNA 111 to be drawn into the nanopore within a radius of several hundred nanomails around the nanopore in the solution.
  • a range in which a force for drawing DNA 111 is generated is referred to as a DNA drawing range 301.
  • the one end of the DNA 111 In order for one end of the DNA 111 to be introduced into the nanopore, the one end of the DNA 111 is brought close to the DNA pulling range 301, and when one end of the DNA 111 enters the DNA pulling range 301 by Brownian motion, it is pulled into the nanopore 112. That is, it is important how one end of the DNA 111 is brought close to the DNA drawing range 301.
  • FIG. 4 shows a case where the actual mechanical accuracy is taken into account in order to explain the problem to be solved by the present invention.
  • the parallelism of the DNA fixing substrate 108 attached to the nanopore device 101 and the probe 107 cannot be 0 degree. Therefore, when one end of the DNA 111 is brought close to the DNA pulling range 301, the nanopore device 101 and the DNA fixing substrate 108 come into contact with each other due to the difference in parallelism, and it is difficult for the DNA 111 to approach the DNA pulling range 301 of several hundred nanometers. .
  • the single-stranded DNA 111 has various shapes due to differences in entanglement and Brownian motion, even if the distance between the nanopore device 101 and the DNA fixing substrate 108 does not approach the DNA pulling range 301 of several hundred nanometers, In particular, there is a possibility that the DNA 111 is introduced into the nanopore, but it is difficult to control the waiting time, which greatly affects the throughput.
  • FIG. 5 shows a first implementation method of the biological sample analyzer 500 of the present invention.
  • An enlarged view of the circle 100A is shown on the right side of FIG.
  • the analysis method is performed according to the procedures (1) to (5).
  • (1) The DNA fixing substrate 108 in the DNA fixing solution 501 is sucked by creating a negative pressure with a pump such as a syringe pump 502.
  • (2) The sucked DNA fixing substrate 108 is brought close to the nanopore 112 using a drive mechanism.
  • (4) The separated DNA fixing substrate 108 falls on the nanopore device 101, and the distance between the DNA fixing substrate 108 and the nanopore device 101 becomes almost zero.
  • the distance 0 is not completely 0 nanometers, and the DNA fixing substrate 108 and the nanopore device 101 have surface roughness and the like, and the nanopores cannot be completely blocked.
  • the gap between the DNA fixing substrate 108 and the nanopore device 101 is not less than the area of the nanopore diameter of 10 nm and does not affect the blocking current. If it becomes completely zero and the blocking current is affected, a member may be manufactured so as to create a design gap on both ends of the DNA fixing substrate 108.
  • the specific gravity of the material of the DNA fixing substrate 108 is larger than the specific gravity of the electrolyte solution 103.
  • the DNA immobilization substrate 108 in order to bring the DNA immobilization substrate 108 closer to the nanopore device 101, the DNA immobilization substrate 108 once separated may be pressed with the probe 107. (5) Thereafter, after confirming the blocking signal, a negative pressure is generated again by a pump such as the syringe pump 502, and the DNA fixing substrate 108 is sucked. (6) The suctioned DNA immobilization substrate 108 is precisely controlled by the drive stage 109, and the base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. .
  • FIG. 6 shows a second implementation method of the biological sample analysis method 600 of the present invention.
  • An enlarged view of the nanopore device 101 is shown in the circle 100A.
  • the analysis method is performed according to the procedures (1) to (5).
  • a magnetic material (metal) 601 is attached, bonded, bonded, or applied to the surface of the DNA fixing substrate 108 opposite to the surface on which the DNA 111 is fixed.
  • the DNA immobilization substrate 108 in the DNA immobilization solution 501 is attracted by creating a magnetic force with an electromagnet 602 or the like.
  • the sucked DNA fixing substrate 108 is brought close to the nanopore 112 using a drive mechanism.
  • the magnetic force is released, and the DNA immobilization substrate 108 is separated from the probe 107.
  • the separated DNA fixing substrate 108 falls on the nanopore device 101, and the distance between the DNA fixing substrate 108 and the nanopore device 101 becomes almost zero.
  • the distance 0 is not completely 0 nanometers, and the DNA fixing substrate 108 and the nanopore device 101 have surface roughness and the like, and the nanopores cannot be completely blocked.
  • the gap between the DNA fixing substrate 108 and the nanopore device 101 is not less than the area of the nanopore diameter of 10 nm and does not affect the blocking current. Further, if it becomes completely zero and the blocking current is affected, a member may be manufactured so as to create a gap in design between both ends of the DNA fixing substrate 108.
  • the specific gravity of the material of the DNA fixing substrate 108 is larger than the specific gravity of the electrolyte solution 103.
  • one end of the DNA 111 can approach the DNA drawing range 301 as much as possible, and one end of the entangled DNA 111 is drawn into the nanopore 112, and the DNA 111 in the nanopore 112 is drawn.
  • the blockage signal (current value) reduced by the cross-sectional integral is amplified by an amplifier (not shown) and confirmed by a PC (Personal Computer not shown) via an ADC (not shown). This effect can be obtained not only in a single pore but also in a multipore system.
  • the DNA immobilization substrate 108 once separated may be pressed with the probe 107.
  • the electromagnet 602 After confirming the blocking signal, the electromagnet 602 again creates a magnetic force and attracts the DNA fixing substrate 108. (6) The suctioned DNA immobilization substrate 108 is precisely controlled by the drive stage 109, and the base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. .
  • FIG. 7 shows a third implementation method of the biological sample analysis method 700 of the present invention.
  • An enlarged view of the nanopore device 101 is shown in the circle 100A.
  • the analysis method is performed according to the procedures (1) to (5).
  • An automatic gripping mechanism 702 using a motor 701 or the like as a driving force is provided at the probe tip, and grips the DNA fixing substrate 108 in the DNA fixing solution 501.
  • the grasped DNA fixing substrate 108 is brought close to the nanopore 112 using a drive mechanism.
  • the automatic gripping mechanism 702 is opened, and the DNA immobilization substrate 108 is separated from the probe 107.
  • the separated DNA fixing substrate 108 falls on the nanopore device 101, and the distance between the DNA fixing substrate 108 and the nanopore device 101 becomes almost zero.
  • the distance 0 is not completely 0 nanometers, and the DNA fixing substrate 108 and the nanopore device 101 have surface roughness and the like, and the nanopores cannot be completely blocked.
  • the gap between the DNA fixing substrate 108 and the nanopore device 101 is not less than the area of the nanopore diameter of 10 nm and does not affect the blocking current. Further, if it becomes completely zero and the blocking current is affected, a member may be manufactured so as to create a gap in design between both ends of the DNA fixing substrate 108.
  • the specific gravity of the material of the DNA fixing substrate 108 is larger than the specific gravity of the electrolyte solution 103.
  • one end of the DNA 111 can approach the DNA drawing range 301 as much as possible, and one end of the entangled DNA 111 is drawn into the nanopore 112, and the DNA 111 in the nanopore 112 is drawn.
  • the blockage signal (current value) reduced by the cross-sectional integral is amplified by an amplifier (not shown) and confirmed by a PC (Personal Computer not shown) via an ADC (not shown). This effect can be obtained not only in a single pore but also in a multipore system.
  • the DNA fixing substrate 108 may be pressed with the probe 107.
  • the DNA holding substrate 108 is held again by the automatic holding mechanism 702.
  • the grasped DNA immobilization substrate 108 is precisely controlled by the drive stage 109, and the base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. .
  • FIG. 8 shows a fourth biological sample analysis method of the present invention.
  • spherical DNA fixing beads 801 are used in place of the DNA fixing substrate 108 on the flat plate shown in Example 1. DNA is fixed to the DNA fixing beads 801. The point that the DNA fixing beads 801 are sucked into and separated from the probe 107 using the syringe pump 502 is the same as in the first embodiment.
  • the spherical DNA fixed bead 801 probe tip 802 has a large number of openings.
  • FIG. 9 shows a fifth biological sample analysis method of the present invention.
  • spherical DNA-immobilized magnetic beads 901 are used in place of the DNA-immobilized substrate 108 on the flat plate shown in Example 2. DNA is fixed to the spherical DNA fixing magnetic beads 901.
  • the point of using the electromagnet 602 is the same as that of the second embodiment.
  • the operation of the probe 107 is the same as that of the second embodiment.
  • FIG. 10 shows a sixth biological sample analysis method of the biological sample analysis method 1000 of the present invention.
  • the sucked DNA immobilization substrate 108 is brought close to the nanopore 112 using a drive mechanism, and is brought close to the extent that the DNA immobilization substrate 108 does not contact the nanopore device 101.
  • the distance of separation is not a problem in the present invention.
  • the DNA fixing solution 501 may be grasped with tweezers and manually inserted into the flow cell opening 110.
  • the subsequent procedure follows the procedure of Example 1 to Example 3 (4), and the DNA immobilization substrate 108 falls on the nanopore device 101, and the distance between the DNA immobilization substrate 108 and the nanopore device 101 becomes almost zero.
  • One end of the DNA 111 can approach the DNA pull-in range 301 as much as possible, and one end of the entangled DNA 111 is drawn into the nanopore 112, and a blocking signal (current value) reduced by the cross-sectional integral of the DNA 111 in the nanopore 112 is obtained.
  • This effect can be obtained not only in a single pore but also in a multipore system.
  • the distance 0 is not completely 0 nanometers, and the DNA fixing substrate 108 and the nanopore device 101 have surface roughness and the like, and the nanopores cannot be completely blocked.
  • the gap between the DNA fixing substrate 108 and the nanopore device 101 is not less than the area of the nanopore diameter of 10 nm and does not affect the blocking current. If it becomes completely zero and the blocking current is affected, a member may be manufactured so as to create a design gap on both ends of the DNA fixing substrate 108.
  • the specific gravity of the material of the DNA fixing substrate 108 is larger than the specific gravity of the electrolyte solution 103.
  • the DNA fixing substrate 108 is sucked by negative pressure, magnetic force, or gripping mechanism.
  • the suctioned DNA immobilization substrate 108 is precisely controlled by the drive stage 109, and the base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. .
  • FIG. 11 shows a seventh biological sample analysis method of the biological sample analysis method 1100 of the present invention.
  • the attracted DNA-immobilized magnetic beads 901 are brought close to the nanopore 112 using a drive mechanism, so that the DNA-immobilized magnetic beads 901 are not brought into contact with the nanopore device 101. Thereafter, the magnetic force is released, and the DNA-fixed magnetic beads 901 are separated from the probe 107, but the distance of separation is not a problem in the present invention.
  • the DNA fixing magnetic beads 901 from the DNA fixing solution 501 may be manually inserted into the flow cell opening 110.
  • the subsequent procedure follows the procedure of Example 4 or (4) of Example 5, and the DNA-immobilized magnetic beads 901 drop on the nanopore device 101, and the distance between the DNA-immobilized magnetic beads 901 and the nanopore device 101 is
  • One end of the DNA 111 can approach the DNA pull-in range 301 as much as possible, and one end of the entangled DNA 111 is drawn into the nanopore 112, and the blocking signal (decrease by the cross-sectional integral of the DNA 111 in the nanopore 112 is reduced.
  • the current value is amplified by an amplifier (not shown) and confirmed by a PC (Personal Computer not shown) via an ADC (not shown). This effect can be obtained not only in a single pore but also in a multipore system.
  • the DNA-fixed magnetic beads 901 may be pressed with the probe 107.
  • an electromagnet 902 may be disposed in the lower part on the lower tank 102A side, and the magnetic beads may be pulled in the ⁇ Z direction to be closer to the nanopore device 101.
  • This method is not limited to beads, and the DNA immobilization substrate 108 with the magnetic body 601 may be pulled in the ⁇ Z direction by the electromagnet 902 so as to be closer to the nanopore device 101.
  • the magnetic force is again generated by the electromagnet 602 and the DNA-fixed magnetic beads 901 are attracted.
  • the attracted DNA-fixed magnetic beads 901 are precisely controlled by the drive stage 109, and the base sequence is determined from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. read.
  • Biological sample analyzer 100A Biological sample analyzer partially enlarged view 101: Partition with nanopore installed (nanopore device) 102A, 102B: Tank 103: Electrolyte solution 104: Electrode 105: Power source 106: Flow cell 107: Probe 108: DNA fixing substrate 109: Drive mechanism 110: Flow cell opening 111: DNA 112: Nanopore 113: Nanopore thin film 200: Array reading example 300: Biological sample analysis method 301: DNA drawing range 500: Biological sample analysis method 501 of Example 1: DNA fixing solution 502: Syringe pump 503: Syringe pump flow path 600: Biological sample analysis method 601 of Example 2: Magnetic material (metal) 602: Electromagnet 700: Biological sample analysis method 701 of Example 3 Motor 702: Automatic gripping mechanism 800: Biological sample analysis method 801 of Example 4 DNA fixing bead 802: Probe tip channel 900: Biological sample of Example 5 Analysis method 901: DNA-fixed magnetic beads 902: Electromagnet 1000: Biological sample analysis method

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Abstract

The objective of the present invention is to make it possible for bases which are blocking a plurality of nanopores to be controlled rapidly and with very high precision, and to improve throughput, reduce running costs, and shorten waiting time. This biological sample analysis method employs a biological sample analysis device provided with a fixing member to which a biological sample is fixed, a substrate having a through-hole through which the biological sample passes, and a drive mechanism which drives the fixing member, to detect the biological sample when the biological sample passes through the through-hole, wherein after it has been confirmed that the biological sample is blocking the through-hole, the fixing member is held and is pulled up by the drive mechanism.

Description

生体試料分析方法及び生体試料分析装置Biological sample analysis method and biological sample analyzer
　本発明は、ナノサイズのポアが開いた薄膜によるＤＮＡ、ＲＮＡなどの核酸の配列解析等を行う生体試料分析装置及び生体試料分析方法に関する。特に、アレイ化されたナノポア、即ちマルチナノポアを用いる場合における技術に関する。 The present invention relates to a biological sample analyzer and a biological sample analysis method for performing sequence analysis of nucleic acids such as DNA and RNA using a thin film having nano-sized pores. In particular, it relates to a technique in the case of using arrayed nanopores, that is, multi-nanopores.
　本技術分野の背景技術として、ナノポアＤＮＡシーケンスに関する特許文献１がある。この公報には、「ナノポアの内径は約１０ｎｍであった。」と記載されており、ナノポア
ＤＮＡシーケンサは、伸長反応や蛍光ラベルは行わずに、ＤＮＡの塩基配列を電気的に直接計測する手法が注目を浴びている。直接計測法にはいくつかの手法が提案されているが、その一つに封鎖電流方式がある。薄膜に透過電子顕微鏡などによって数ｎｍのポア（ナノポア）を作製し、その薄膜の両側に電解質溶液を満たした液槽を設ける。それぞれの液槽に電極を設け、これらの電極間に電圧をかけると、ナノポアを通してイオン電流が流れる。イオン電流は一次近似としてナノポアの断面積に比例する。ＤＮＡがナノポアを通過する際に、ＤＮＡがナノポアを封鎖し、イオンが通過できる有効断面積が減少するため、イオン電流が減少する。この減少量を封鎖電流と呼ぶ。封鎖電流の大きさを元に、ＤＮＡの１本鎖と２本鎖との差異や、塩基の種類を判別する。 As background art of this technical field, there is Patent Literature 1 relating to nanopore DNA sequencing. This publication describes that “the inner diameter of the nanopore was about 10 nm”, and the nanopore DNA sequencer directly measures the DNA base sequence electrically without performing an extension reaction or a fluorescent label. Is attracting attention. Several methods have been proposed for the direct measurement method, one of which is a blocking current method. A few nm pore (nanopore) is prepared on the thin film by a transmission electron microscope or the like, and a liquid tank filled with the electrolyte solution is provided on both sides of the thin film. When an electrode is provided in each liquid tank and a voltage is applied between these electrodes, an ionic current flows through the nanopore. The ion current is proportional to the cross-sectional area of the nanopore as a first order approximation. As DNA passes through the nanopore, the DNA blocks the nanopore, reducing the effective cross-sectional area through which ions can pass, thereby reducing the ionic current. This amount of decrease is called the blocking current. Based on the magnitude of the blocking current, the difference between the single strand and double strand of DNA and the type of base are determined.
　ナノポア内を通過するＤＮＡのブラウン運動が大きいことや、ナノポア内を通過するスピードが速すぎて検出器の測定スピードが追い付かないなどの課題があるため、各塩基を判別する測定精度を確保しにくい。ＤＮＡのブラウン運動や通過スピードをコントロールするために、ＤＮＡの一端を固定して運動を制御した上で測定する手法がいくつか提案されている。ＡＦＭなどで用いられるカンチレバーにＤＮＡを固定する方式（特許文献２）、ビーズに生体試料を固定する方式（特許文献３）などが開示されている。 It is difficult to ensure the measurement accuracy for distinguishing each base because the Brownian motion of DNA passing through the nanopore is large and the measurement speed of the detector cannot catch up because the speed of passing through the nanopore is too fast. . In order to control the Brownian movement and the passing speed of DNA, several methods have been proposed in which measurement is performed while the movement is controlled by fixing one end of DNA. A method of fixing DNA to a cantilever used in AFM (Patent Document 2), a method of fixing a biological sample to beads (Patent Document 3), and the like are disclosed.
　ＤＮＡの１本鎖や２本鎖の一端を、ナノポアが作成された平面と平行な面に固定し、その面に垂直な軸に駆動するステージによって、ＤＮＡのナノポア通過速度を制御する方式も考えられる。 A method is also considered in which one end of DNA single strand or double strand is fixed to a plane parallel to the plane on which the nanopore is formed, and the speed at which the DNA passes through the nanopore is controlled by a stage driven on an axis perpendicular to the plane. It is done.
　ＤＮＡの１本鎖や２本鎖の位置を、ナノポア上の所望の場所に位置付けることが可能な試料移動ステージが設けられている場合もある。 There may be a sample moving stage that can position the position of single-stranded DNA or double-stranded DNA at a desired location on the nanopore.
　また、従来は１つの薄膜に１つのナノポアを設けた、いわゆるシングルナノポアを用いるのが通例である。一方、１つの薄膜に複数のナノポアを設けた、いわゆるマルチナノポアを用いれば、薄膜あたりの測定効率を向上できると考えられる。 Also, conventionally, it is customary to use a so-called single nanopore in which one nanopore is provided in one thin film. On the other hand, it is considered that the measurement efficiency per thin film can be improved by using a so-called multi-nanopore in which a plurality of nanopores are provided in one thin film.
国際公開ＷＯ２０１２－０４３０２８号公報International Publication WO2012-043028 米国特許公開公報第２００４－０１４４６５８号公報US Patent Publication No. 2004-0144658 特開２０１１－２１１９０５号公報JP 2011-211905 A
　上述のように、ナノポアＤＮＡシーケンサのシステムには、ＤＮＡの一端を固定し、もう一端はナノポアを封鎖しない限り、塩基を判別することができない。 As described above, in the nanopore DNA sequencer system, the base cannot be determined unless one end of DNA is fixed and the other end is blocked.
　マルチポア化には、複数のナノポアを封鎖することが求められるが、特許文献２に開示された技術によれば、カンチレバーに、ＤＮＡの一端を固定し、上下動作を行う。カンチレバーの先端は非常に細く、複数のナノポアを封鎖することはできない問題があった。 For multipore formation, it is required to block a plurality of nanopores. However, according to the technique disclosed in Patent Document 2, one end of DNA is fixed to a cantilever and moved up and down. The tip of the cantilever was very thin, and there was a problem that multiple nanopores could not be blocked.
　また、特許文献３に開示された技術によれば、磁場や光ピンセットの原理により、また高分子イオンなどの荷電粒子からなる標識物質は電場により、その動きを任意に制御可能であるが、塩基間距離がサブナノメールであり、複数のナノポアを封鎖した塩基を超高精度に制御するような構成にはなっていない。 Further, according to the technique disclosed in Patent Document 3, the movement of a labeling substance made of charged particles such as polymer ions can be arbitrarily controlled by the principle of a magnetic field or optical tweezers or by an electric field. The inter-space is sub-nanomail, and it is not configured to control a base with a plurality of nanopores blocked with extremely high accuracy.
　ＤＮＡの１本鎖や２本鎖の一端を、ナノポアが作成された平面と平行な面に固定し、その面に垂直な軸に駆動するステージによって、ＤＮＡのナノポア通過速度を制御する方式も考えられる。 A method is also considered in which one end of DNA single strand or double strand is fixed to a plane parallel to the plane on which the nanopore is formed, and the speed at which the DNA passes through the nanopore is controlled by a stage driven on an axis perpendicular to the plane. It is done.
　しかしながら、このような構成の場合、ＤＮＡを固定した面を高精度に制御した場合、ＤＮＡを固定した面とナノポアデバイスが片当たりする可能性がある。この場合、ナノポアにＤＮＡが十分な長さ入らない可能性がある。 However, in the case of such a configuration, when the surface on which DNA is immobilized is controlled with high accuracy, the surface on which DNA is immobilized and the nanopore device may come into contact with each other. In this case, there is a possibility that DNA does not enter the nanopore sufficiently long.
　上記課題を解決するために、生体試料が固定された固定部材（基板またはビーズ）と、当該生体試料が通過する貫通孔を有する基板と、生体試料が貫通孔を通過時に当該生体試料を検出する検出部材と、前記固定部材を保持するプローブと、前記プローブを駆動する駆動機構を備えた、生体試料分析装置を用いた生体試料分析方法であって、プローブに固定部材を保持し、固定部材を基板に接近させ、プローブから固定部材をリリースし、検出部材にて、生体試料が貫通孔を封鎖したことを確認し、固定部材をプローブに保持し、駆動機構によってプローブを引き上げることを特徴とする生体試料分析方法，及び，生体試料分析装置を提供する。 In order to solve the above problems, a fixing member (substrate or bead) to which a biological sample is fixed, a substrate having a through-hole through which the biological sample passes, and the biological sample are detected when the biological sample passes through the through-hole. A biological sample analysis method using a biological sample analyzer, comprising a detection member, a probe for holding the fixing member, and a drive mechanism for driving the probe, wherein the fixing member is held on the probe, and the fixing member is It is made close to the substrate, the fixing member is released from the probe, the detection member confirms that the biological sample has blocked the through hole, the fixing member is held by the probe, and the probe is pulled up by the driving mechanism. A biological sample analysis method and a biological sample analyzer are provided.
　上記構成によれば、複数のナノポアを封鎖した塩基を迅速、かつ、高精度に制御することができる。したがって、スループットを向上でき、ランニングコストを削減でき、待ち時間も短縮できる。 According to the above configuration, it is possible to quickly and accurately control a base that blocks a plurality of nanopores. Therefore, throughput can be improved, running cost can be reduced, and waiting time can be shortened.
生体試料分析装置の構成と生体試料分析チップの拡大図の一例を示す図である。It is a figure which shows an example of the structure of a biological sample analyzer, and an enlarged view of a biological sample analysis chip. 生体試料分析チップによって取得される配列読取例である。It is an example of the sequence reading acquired by the biological sample analysis chip. ＤＮＡの一端をナノポアに導入前の拡大図の一例を示す図である。It is a figure which shows an example of the enlarged view before introduce | transducing one end of DNA into nanopore. ＤＮＡ固定基板とナノポア基板の平行度の違いを示す図である。It is a figure which shows the difference in the parallelism of a DNA fixed board | substrate and a nanopore board | substrate. 第１実施例における生体試料分析方法，及び，生体試料分析装置を示す図である。It is a figure which shows the biological sample analysis method and biological sample analyzer in 1st Example. 第２実施例における生体試料分析方法，及び，生体試料分析装置を示す図である。It is a figure which shows the biological sample analysis method and biological sample analyzer in 2nd Example. 第３実施例における生体試料分析方法，及び，生体試料分析装置を示す図である。It is a figure which shows the biological sample analysis method and biological sample analyzer in 3rd Example. 第４実施例における生体試料分析方法，及び，生体試料分析装置を示す図である。It is a figure which shows the biological sample analysis method and biological sample analyzer in 4th Example. 第５実施例における生体試料分析方法，及び，生体試料分析装置を示す図である。It is a figure which shows the biological sample analysis method and biological sample analyzer in 5th Example. 第６実施例における生体試料分析方法，及び，生体試料分析装置を示す図である。It is a figure which shows the biological sample analysis method and biological sample analyzer in 6th Example. 第７実施例における生体試料分析方法，及び，生体試料分析装置を示す図である。It is a figure which shows the biological sample analysis method and biological sample analyzer in 7th Example.
以下、添付図面を参照して本発明の実施例について説明する。なお、添付図面は本発明の原理に則った具体的な実施例を示しているが、これらは本発明の理解のためのものであり、決して本発明を限定的に解釈するために用いられるものではない。 Embodiments of the present invention will be described below with reference to the accompanying drawings. The accompanying drawings show specific embodiments in accordance with the principle of the present invention, but these are for the understanding of the present invention, and are never used to interpret the present invention in a limited manner. is not.
　以下で述べる「ナノポア」とは、薄膜に設けたナノサイズの孔であり、薄膜の表裏を貫通する。薄膜は主に無機材料から形成される。薄膜材料は、他に、有機物質、高分子材料などを含むこともできる。また、ＤＮＡ断片の一端が固定される基板またはビーズは、主に無機材料から形成される。薄膜材料は、他に、有機物質、高分子材料などを含むこともできる。 The “nanopore” described below is a nano-sized hole provided in the thin film and penetrates the front and back of the thin film. The thin film is mainly formed from an inorganic material. In addition, the thin film material may include an organic substance, a polymer material, and the like. In addition, the substrate or the beads to which one end of the DNA fragment is fixed are mainly formed from an inorganic material. In addition, the thin film material may include an organic substance, a polymer material, and the like.
　まず、ナノポアデバイスを用いた生体試料分析装置を説明する。図１は、生体試料分析装置の構成の一例を示す図であり、生体試料分析チップによる塩基配列読取機構を示す。 First, a biological sample analyzer using a nanopore device will be described. FIG. 1 is a diagram showing an example of the configuration of a biological sample analyzer, and shows a base sequence reading mechanism using a biological sample analysis chip.
　図１に示すように、生体試料分析装置（例えば、ＤＮＡ解析装置）１００は、仕切り体１０１により分けられた二つの槽１０２Ａ、１０２Ｂを備える。仕切り体１０１は、ナノポアを有するナノポアデバイスを備える。二つの槽１０２Ａ、１０２Ｂには、電解質溶液１０３が満たされている。二つの槽１０２Ａ、１０２Ｂは、電極１０４及び電源１０５で電気的に接続されている。このナノポアデバイスで仕切られた流路全体をフローセル１０６と呼ぶ。 As shown in FIG. 1, a biological sample analyzer (for example, a DNA analyzer) 100 includes two tanks 102A and 102B separated by a partition body 101. The partition body 101 includes a nanopore device having nanopores. The two tanks 102A and 102B are filled with the electrolyte solution 103. The two tanks 102A and 102B are electrically connected by an electrode 104 and a power source 105. The entire flow path partitioned by the nanopore device is called a flow cell 106.
　プローブ１０７に取り付けられたＤＮＡ固定基板１０８に化学的にＤＮＡの一端を固定し、駆動機構１０９によって、フローセル開口部１１０から進入すると、液中のＤＮＡ１１１は、ナノポアデバイス１０１のナノポアを通して、一方の槽１０７Ａから反対側の槽１０７Ｂに泳動するが、通り過ぎることはない。ナノポアに導入されたＤＮＡ１１１の一端を駆動ステージ１０９によって引き抜いたり、押し込んだりすることで、ＤＮＡ１１１がナノポアデバイス１０１のナノポアを通過する際に変化する電流値から塩基配列を読み取る。電流値は、アンプ（図示せず）で増幅されて、ＡＤＣ（図示せず）を介してＰＣ（Ｐｅｒｓｏｎａｌ　Ｃｏｍｐｕｔｅｒ　図示せず）に記録される。 When one end of the DNA is chemically fixed to the DNA fixing substrate 108 attached to the probe 107 and entered from the flow cell opening 110 by the driving mechanism 109, the DNA 111 in the liquid passes through the nanopore of the nanopore device 101 and enters one tank. Although it migrates from 107A to the opposite tank 107B, it does not pass. The base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing one end of the DNA 111 introduced into the nanopore by the driving stage 109. The current value is amplified by an amplifier (not shown) and recorded on a PC (Personal Computer not shown) via an ADC (not shown).
　図１の円形１００Ａ内に、ナノポアデバイス１０１の拡大図を示す。ナノポアデバイス１０１は、ナノポア１１２が形成された数ｎｍのナノポア薄膜１１３を備える。非常に薄いナノポア薄膜１１３の面積は小さく，ナノポアデバイスの厚みは補強のため、数百ｎｍの厚みを有している。ナノポアデバイス１０１の上下の槽１０２Ａ、１０２Ｂに電解質溶液１０３を満たし、ナノポアデバイス１０１を介して上下に電圧を印加すると、電解質溶液１０３中のイオン由来の、ナノポア１１２のポア径の断面積に応じた電流が検出される。ＤＮＡ１１１が駆動ステージによって、ナノポア１１２を通過すると、イオンの流れが妨げられるため、電流値は、ナノポア１１２中のＤＮＡ１１１の断面積分だけ減少する。図１の拡大図では、ＤＮＡ１１１は、球体（ビーズ）を用いて説明したが，以下，ＤＮＡ１１１が数珠状につなぎなった，糸状の1本鎖を用いて説明する。 An enlarged view of the nanopore device 101 is shown in a circle 100A of FIG. The nanopore device 101 includes a nanopore thin film 113 having a thickness of several nanometers on which nanopores 112 are formed. The area of the very thin nanopore thin film 113 is small, and the thickness of the nanopore device has a thickness of several hundred nm for reinforcement. When the upper and lower tanks 102A and 102B of the nanopore device 101 are filled with the electrolyte solution 103 and a voltage is applied vertically through the nanopore device 101, the cross-sectional area of the pore diameter of the nanopore 112 derived from ions in the electrolyte solution 103 is determined. A current is detected. When the DNA 111 passes through the nanopore 112 by the driving stage, the flow of ions is hindered, so that the current value decreases by the cross-sectional integral of the DNA 111 in the nanopore 112. In the enlarged view of FIG. 1, the DNA 111 has been described using a sphere (bead), but hereinafter, the DNA 111 will be described using a single strand of yarn in which the DNA 111 is connected in a bead shape.
　ＤＮＡ解析装置１００では、塩基種毎の電流値の変化量の違いから塩基識別を行う。図２は、電極１０４で電圧を印加することにより読み取られる塩基種ごとの電流変化の例である。配列読取例２００に示されるように、４種類の塩基ごとに異なる電流値が検出される。 In the DNA analysis apparatus 100, base identification is performed from the difference in the amount of change in the current value for each base type. FIG. 2 is an example of a current change for each base species read by applying a voltage at the electrode 104. As shown in the sequence reading example 200, different current values are detected for each of the four types of bases.
　ここで、ＤＮＡ１１１の一端がナノポアに導入する方法，及び，生体試料分析装置の詳細を述べる。図３は、生体試料分析装置３００を示しており一部、拡大し、ＤＮＡ１１１の一端がナノポアに導入される前の状態を示す。ＤＮＡの１本鎖は、２本鎖と比べ、化学的に安定している状態ではないため、溶液中で絡まっていることが知られている。電源１０５により、溶液中のナノポアを中心として、半径数百ナノメールの範囲に、ＤＮＡ１１１がナノポアに引き込まれる力が発生する。ＤＮＡ１１１を引き込む力が発生する範囲を、ＤＮＡ引込範囲３０１とする。 Here, the method of introducing one end of the DNA 111 into the nanopore and the details of the biological sample analyzer will be described. FIG. 3 shows a biological sample analyzer 300 partially enlarged and showing a state before one end of the DNA 111 is introduced into the nanopore. It is known that a single strand of DNA is entangled in a solution because it is not chemically stable compared to a double strand. The power source 105 generates a force that causes the DNA 111 to be drawn into the nanopore within a radius of several hundred nanomails around the nanopore in the solution. A range in which a force for drawing DNA 111 is generated is referred to as a DNA drawing range 301.
　ＤＮＡ１１１の一端がナノポアに導入するためには、ＤＮＡ引込範囲３０１に、ＤＮＡ１１１の一端を近づけ、ブラウン運動によって、ＤＮＡ１１１の一端がＤＮＡ引込範囲３０１に入ったとき、ナノポア１１２に引き込まれる。つまり、いかにしてＤＮＡ１１１の一端をＤＮＡ引込範囲３０１に近づけるかが重要である。 In order for one end of the DNA 111 to be introduced into the nanopore, the one end of the DNA 111 is brought close to the DNA pulling range 301, and when one end of the DNA 111 enters the DNA pulling range 301 by Brownian motion, it is pulled into the nanopore 112. That is, it is important how one end of the DNA 111 is brought close to the DNA drawing range 301.
　本発明が解決する課題を説明するために、実際のメカ精度を考慮した場合を図４に示す。ナノポアデバイス１０１とプローブ１０７に取り付けられたＤＮＡ固定基板１０８の平行度は、０度にすることはできない。そのため、ＤＮＡ１１１の一端をＤＮＡ引込範囲３０１に近づける際、平行度の違いから、ナノポアデバイス１０１とＤＮＡ固定基板１０８が接触してしまい、ＤＮＡ１１１が数百ナノメートルのＤＮＡ引込範囲３０１に近づくことが難しい。 FIG. 4 shows a case where the actual mechanical accuracy is taken into account in order to explain the problem to be solved by the present invention. The parallelism of the DNA fixing substrate 108 attached to the nanopore device 101 and the probe 107 cannot be 0 degree. Therefore, when one end of the DNA 111 is brought close to the DNA pulling range 301, the nanopore device 101 and the DNA fixing substrate 108 come into contact with each other due to the difference in parallelism, and it is difficult for the DNA 111 to approach the DNA pulling range 301 of several hundred nanometers. .
　１本鎖のＤＮＡ１１１は、絡まり方の違いやブラウン運動によって、形状が様々のため、ナノポアデバイス１０１とＤＮＡ固定基板１０８の距離が、数百ナノメートルのＤＮＡ引込範囲３０１まで近づかなくても、偶然的に、ＤＮＡ１１１がナノポア導入される可能性はあるが、待ち時間を制御することは難しく、スループットに多く影響する。 Since the single-stranded DNA 111 has various shapes due to differences in entanglement and Brownian motion, even if the distance between the nanopore device 101 and the DNA fixing substrate 108 does not approach the DNA pulling range 301 of several hundred nanometers, In particular, there is a possibility that the DNA 111 is introduced into the nanopore, but it is difficult to control the waiting time, which greatly affects the throughput.
　図５は、本発明の生体試料分析装置５００の第１の実施方法を示している。円形１００Ａ内の拡大図を図５の右側に示す。 FIG. 5 shows a first implementation method of the biological sample analyzer 500 of the present invention. An enlarged view of the circle 100A is shown on the right side of FIG.
　ナノポアデバイス１０１の拡大図を示す。分析方法は、（１）～（５）の手順で行っていく。（１）ＤＮＡ固定溶液５０１内にあるＤＮＡ固定基板１０８を、シリンジポンプ５０２などのポンプで、陰圧を作り出し、吸引する。（２）吸引したＤＮＡ固定基板１０８を、ナノポア１１２に駆動機構を用いて、近づける。（３）ＤＮＡ固定基板１０８がナノポアデバイス１０１と接触しない程度に近づけた後、陰圧を開放し、プローブ１０７から、ＤＮＡ固定基板１０８を離反する。（４）離反されたＤＮＡ固定基板１０８は、ナノポアデバイス１０１上に落下し、ＤＮＡ固定基板１０８とナノポアデバイス１０１の距離は、ほぼ０になる。ここで、距離０と言うのは、完全な０ナノメートルではなく、ＤＮＡ固定基板１０８と、ナノポアデバイス１０１には、表面粗さなどがあり、完全にナノポアを封鎖することはできない。ＤＮＡ固定基板１０８とナノポアデバイス１０１との隙間は、ナノポア径１０ｎｍの面積以上あり、封鎖電流には影響はない。仮に完全に０になってしまい、封鎖電流に影響がある場合は、ＤＮＡ固定基板１０８の両端に、設計上、隙間を作るように、部材を製作してもよい。 An enlarged view of the nanopore device 101 is shown. The analysis method is performed according to the procedures (1) to (5). (1) The DNA fixing substrate 108 in the DNA fixing solution 501 is sucked by creating a negative pressure with a pump such as a syringe pump 502. (2) The sucked DNA fixing substrate 108 is brought close to the nanopore 112 using a drive mechanism. (3) After bringing the DNA immobilization substrate 108 close to the extent that it does not contact the nanopore device 101, the negative pressure is released and the DNA immobilization substrate 108 is separated from the probe 107. (4) The separated DNA fixing substrate 108 falls on the nanopore device 101, and the distance between the DNA fixing substrate 108 and the nanopore device 101 becomes almost zero. Here, the distance 0 is not completely 0 nanometers, and the DNA fixing substrate 108 and the nanopore device 101 have surface roughness and the like, and the nanopores cannot be completely blocked. The gap between the DNA fixing substrate 108 and the nanopore device 101 is not less than the area of the nanopore diameter of 10 nm and does not affect the blocking current. If it becomes completely zero and the blocking current is affected, a member may be manufactured so as to create a design gap on both ends of the DNA fixing substrate 108.
　また、ＤＮＡ固定基板１０８の材質の比重は、電解質溶液１０３の比重よりも大きいとする。 Further, it is assumed that the specific gravity of the material of the DNA fixing substrate 108 is larger than the specific gravity of the electrolyte solution 103.
　プローブ１０７から、ＤＮＡ固定基板１０８を離反したことで、図４に示すようにＤＮＡ固定基板１０８とナノポアデバイス１０１との接触を防止することができ、ＤＮＡ１１１の一端は、ＤＮＡ引込範囲３０１（図３）に、限りなく近づくことができ、絡まっていたＤＮＡ１１１の一端がナノポア１１２に引き込まれ、ナノポア１１２中のＤＮＡ１１１の断面積分だけ減少した封鎖信号（電流値）が、アンプ（図示せず）で増幅されて、ＡＤＣ（図示せず）を介してＰＣ（Ｐｅｒｓｏｎａｌ　Ｃｏｍｐｕｔｅｒ　図示せず）で確認される。またこの効果は、シングルポアだけでなく、マルチポアシステムでも同様の効果が得られる。さらに、よりＤＮＡ固定基板１０８を、ナノポアデバイス１０１に近づけるため、一旦離反したＤＮＡ固定基板１０８をプローブ１０７で押し付けることをしてもよい。（５）その後、封鎖信号を確認した後、再び、シリンジポンプ５０２などのポンプで、陰圧を作り出し、ＤＮＡ固定基板１０８を吸引する。（６）吸引したＤＮＡ固定基板１０８を駆動ステージ１０９によって、精密制御を行い、引き抜いたり、押し込んだりすることで、ＤＮＡ１１１がナノポアデバイス１０１のナノポアを通過する際に変化する電流値から塩基配列を読み取る。 By separating the DNA immobilization substrate 108 from the probe 107, contact between the DNA immobilization substrate 108 and the nanopore device 101 can be prevented as shown in FIG. 4, and one end of the DNA 111 has a DNA pulling range 301 (FIG. 3). ), An end of the tangled DNA 111 is drawn into the nanopore 112, and a blocking signal (current value) reduced by the cross-sectional integral of the DNA 111 in the nanopore 112 is amplified by an amplifier (not shown). And confirmed by a PC (Personal Computer not shown) via an ADC (not shown). This effect can be obtained not only in a single pore but also in a multipore system. Furthermore, in order to bring the DNA immobilization substrate 108 closer to the nanopore device 101, the DNA immobilization substrate 108 once separated may be pressed with the probe 107. (5) Thereafter, after confirming the blocking signal, a negative pressure is generated again by a pump such as the syringe pump 502, and the DNA fixing substrate 108 is sucked. (6) The suctioned DNA immobilization substrate 108 is precisely controlled by the drive stage 109, and the base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. .
　（５）において、陰圧による吸引の際、平行度の違いから、駆動ステージ１０９よる精密制御できない領域が存在するが、それは、ＤＮＡ配列を読まない領域（バッファ）を設けることする。１枚の基板にＤＮＡが固定されているため、基板のみを制御できることも利点の一つである。 In (5), there is a region that cannot be precisely controlled by the drive stage 109 due to the difference in parallelism during suction by negative pressure, but this is to provide a region (buffer) that does not read the DNA sequence. One of the advantages is that only the substrate can be controlled because the DNA is fixed on one substrate.
　図６は、本発明の生体試料分析方法６００の第２の実施方法を示している。円形１００Ａ内に、ナノポアデバイス１０１の拡大図を示す。分析方法は、（１）～（５）の手順で行っていく。予め、ＤＮＡ固定基板１０８のＤＮＡ１１１が固定される面とは反対の面に、磁性体（金属）６０１を、付着、接合、接着、または塗布などを行っておく。（１）ＤＮＡ固定溶液５０１内にあるＤＮＡ固定基板１０８を、電磁石６０２などで、磁力を作り出し、吸引する。（２）吸引したＤＮＡ固定基板１０８を、ナノポア１１２に駆動機構を用いて、近づける。（３）ＤＮＡ固定基板１０８がナノポアデバイス１０１と接触しない程度に近づけた後、磁力を開放し、プローブ１０７から、ＤＮＡ固定基板１０８を離反する。（４）離反されたＤＮＡ固定基板１０８は、ナノポアデバイス１０１上に落下し、ＤＮＡ固定基板１０８とナノポアデバイス１０１の距離は、ほぼ０になる。ここで、距離０と言うのは、完全な０ナノメートルではなく、ＤＮＡ固定基板１０８と、ナノポアデバイス１０１には、表面粗さなどがあり、完全にナノポアを封鎖することはできない。ＤＮＡ固定基板１０８とナノポアデバイス１０１との隙間は、ナノポア径１０ｎｍの面積以上あり、封鎖電流には影響はない。また仮に完全に０になってしまい、封鎖電流に影響がある場合は、ＤＮＡ固定基板１０８の両端に、設計上、隙間を作るように、部材を製作してもよい。 FIG. 6 shows a second implementation method of the biological sample analysis method 600 of the present invention. An enlarged view of the nanopore device 101 is shown in the circle 100A. The analysis method is performed according to the procedures (1) to (5). In advance, a magnetic material (metal) 601 is attached, bonded, bonded, or applied to the surface of the DNA fixing substrate 108 opposite to the surface on which the DNA 111 is fixed. (1) The DNA immobilization substrate 108 in the DNA immobilization solution 501 is attracted by creating a magnetic force with an electromagnet 602 or the like. (2) The sucked DNA fixing substrate 108 is brought close to the nanopore 112 using a drive mechanism. (3) After bringing the DNA immobilization substrate 108 close to the extent that it does not contact the nanopore device 101, the magnetic force is released, and the DNA immobilization substrate 108 is separated from the probe 107. (4) The separated DNA fixing substrate 108 falls on the nanopore device 101, and the distance between the DNA fixing substrate 108 and the nanopore device 101 becomes almost zero. Here, the distance 0 is not completely 0 nanometers, and the DNA fixing substrate 108 and the nanopore device 101 have surface roughness and the like, and the nanopores cannot be completely blocked. The gap between the DNA fixing substrate 108 and the nanopore device 101 is not less than the area of the nanopore diameter of 10 nm and does not affect the blocking current. Further, if it becomes completely zero and the blocking current is affected, a member may be manufactured so as to create a gap in design between both ends of the DNA fixing substrate 108.
　また、ＤＮＡ固定基板１０８の材質の比重は、電解質溶液１０３の比重よりも大きいとする。 Further, it is assumed that the specific gravity of the material of the DNA fixing substrate 108 is larger than the specific gravity of the electrolyte solution 103.
　プローブ１０７から、ＤＮＡ固定基板１０８を離反したことで、ＤＮＡ１１１の一端は、ＤＮＡ引込範囲３０１に、限りなく近づくことができ、絡まっていたＤＮＡ１１１の一端がナノポア１１２に引き込まれ、ナノポア１１２中のＤＮＡ１１１の断面積分だけ減少した封鎖信号（電流値）が、アンプ（図示せず）で増幅されて、ＡＤＣ（図示せず）を介してＰＣ（Ｐｅｒｓｏｎａｌ　Ｃｏｍｐｕｔｅｒ　図示せず）で確認される。またこの効果は、シングルポアだけでなく、マルチポアシステムでも同様の効果が得られる。さらに、よりＤＮＡ固定基板１０８を、ナノポアデバイス１０１に近づけるため、一旦離反したＤＮＡ固定基板１０８をプローブ１０７で、押し付けることをしてもよい。（５）その後、封鎖信号を確認した後、再び、電磁石６０２で、磁力を作り出し、ＤＮＡ固定基板１０８を吸引する。（６）吸引したＤＮＡ固定基板１０８を駆動ステージ１０９によって、精密制御を行い、引き抜いたり、押し込んだりすることで、ＤＮＡ１１１がナノポアデバイス１０１のナノポアを通過する際に変化する電流値から塩基配列を読み取る。 By separating the DNA fixing substrate 108 from the probe 107, one end of the DNA 111 can approach the DNA drawing range 301 as much as possible, and one end of the entangled DNA 111 is drawn into the nanopore 112, and the DNA 111 in the nanopore 112 is drawn. The blockage signal (current value) reduced by the cross-sectional integral is amplified by an amplifier (not shown) and confirmed by a PC (Personal Computer not shown) via an ADC (not shown). This effect can be obtained not only in a single pore but also in a multipore system. Furthermore, in order to bring the DNA immobilization substrate 108 closer to the nanopore device 101, the DNA immobilization substrate 108 once separated may be pressed with the probe 107. (5) After confirming the blocking signal, the electromagnet 602 again creates a magnetic force and attracts the DNA fixing substrate 108. (6) The suctioned DNA immobilization substrate 108 is precisely controlled by the drive stage 109, and the base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. .
　（５）において、磁力による吸引の際、平行度の違いから、駆動ステージ１０９よる精密制御できない領域が存在するが、それは、ＤＮＡ配列を読まない領域（バッファ）を設けることする。１枚の基板にＤＮＡが固定されているため、基板のみを制御できることも利点の一つである。 In (5), there is a region that cannot be precisely controlled by the drive stage 109 due to the difference in parallelism when attracted by magnetic force, but this is to provide a region (buffer) where the DNA sequence is not read. One of the advantages is that only the substrate can be controlled because the DNA is fixed on one substrate.
　図７は、本発明の生体試料分析方法７００の第３の実施方法を示している。円形１００Ａ内に、ナノポアデバイス１０１の拡大図を示す。分析方法は、（１）～（５）の手順で行っていく。（１）プローブ先端に、モータ７０１などを駆動力とした、自動把持機構７０２になっており、ＤＮＡ固定溶液５０１内にあるＤＮＡ固定基板１０８を、把持する。（２）把持したＤＮＡ固定基板１０８を、ナノポア１１２に駆動機構を用いて、近づける。（３）ＤＮＡ固定基板１０８がナノポアデバイス１０１と接触しない程度に近づけた後、自動把持機構７０２を開放し、プローブ１０７から、ＤＮＡ固定基板１０８を離反する。（４）離反されたＤＮＡ固定基板１０８は、ナノポアデバイス１０１上に落下し、ＤＮＡ固定基板１０８とナノポアデバイス１０１の距離は、ほぼ０になる。ここで、距離０と言うのは、完全な０ナノメートルではなく、ＤＮＡ固定基板１０８と、ナノポアデバイス１０１には、表面粗さなどがあり、完全にナノポアを封鎖することはできない。ＤＮＡ固定基板１０８とナノポアデバイス１０１との隙間は、ナノポア径１０ｎｍの面積以上あり、封鎖電流には影響はない。また仮に完全に０になってしまい、封鎖電流に影響がある場合は、ＤＮＡ固定基板１０８の両端に、設計上、隙間を作るように、部材を製作してもよい。 FIG. 7 shows a third implementation method of the biological sample analysis method 700 of the present invention. An enlarged view of the nanopore device 101 is shown in the circle 100A. The analysis method is performed according to the procedures (1) to (5). (1) An automatic gripping mechanism 702 using a motor 701 or the like as a driving force is provided at the probe tip, and grips the DNA fixing substrate 108 in the DNA fixing solution 501. (2) The grasped DNA fixing substrate 108 is brought close to the nanopore 112 using a drive mechanism. (3) After bringing the DNA immobilization substrate 108 close to the extent that it does not contact the nanopore device 101, the automatic gripping mechanism 702 is opened, and the DNA immobilization substrate 108 is separated from the probe 107. (4) The separated DNA fixing substrate 108 falls on the nanopore device 101, and the distance between the DNA fixing substrate 108 and the nanopore device 101 becomes almost zero. Here, the distance 0 is not completely 0 nanometers, and the DNA fixing substrate 108 and the nanopore device 101 have surface roughness and the like, and the nanopores cannot be completely blocked. The gap between the DNA fixing substrate 108 and the nanopore device 101 is not less than the area of the nanopore diameter of 10 nm and does not affect the blocking current. Further, if it becomes completely zero and the blocking current is affected, a member may be manufactured so as to create a gap in design between both ends of the DNA fixing substrate 108.
　また、ＤＮＡ固定基板１０８の材質の比重は、電解質溶液１０３の比重よりも大きいとする。 Further, it is assumed that the specific gravity of the material of the DNA fixing substrate 108 is larger than the specific gravity of the electrolyte solution 103.
　プローブ１０７から、ＤＮＡ固定基板１０８を離反したことで、ＤＮＡ１１１の一端は、ＤＮＡ引込範囲３０１に、限りなく近づくことができ、絡まっていたＤＮＡ１１１の一端がナノポア１１２に引き込まれ、ナノポア１１２中のＤＮＡ１１１の断面積分だけ減少した封鎖信号（電流値）が、アンプ（図示せず）で増幅されて、ＡＤＣ（図示せず）を介してＰＣ（Ｐｅｒｓｏｎａｌ　Ｃｏｍｐｕｔｅｒ　図示せず）で確認される。またこの効果は、シングルポアだけでなく、マルチポアシステムでも同様の効果が得られる。さらに、よりＤＮＡ固定基板１０８を、ナノポアデバイス１０１に近づけるため、プローブ１０７で、ＤＮＡ固定基板１０８を押し付けることをしてもよい。（５）封鎖信号を確認した後、再び、自動把持機構７０２で、ＤＮＡ固定基板１０８を把持する。（６）把持したＤＮＡ固定基板１０８を駆動ステージ１０９によって、精密制御を行い、引き抜いたり、押し込んだりすることで、ＤＮＡ１１１がナノポアデバイス１０１のナノポアを通過する際に変化する電流値から塩基配列を読み取る。 By separating the DNA fixing substrate 108 from the probe 107, one end of the DNA 111 can approach the DNA drawing range 301 as much as possible, and one end of the entangled DNA 111 is drawn into the nanopore 112, and the DNA 111 in the nanopore 112 is drawn. The blockage signal (current value) reduced by the cross-sectional integral is amplified by an amplifier (not shown) and confirmed by a PC (Personal Computer not shown) via an ADC (not shown). This effect can be obtained not only in a single pore but also in a multipore system. Furthermore, in order to bring the DNA fixing substrate 108 closer to the nanopore device 101, the DNA fixing substrate 108 may be pressed with the probe 107. (5) After confirming the blocking signal, the DNA holding substrate 108 is held again by the automatic holding mechanism 702. (6) The grasped DNA immobilization substrate 108 is precisely controlled by the drive stage 109, and the base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. .
　（５）において、自動把持機構による把持の際、平行度の違いから、駆動ステージ１０９よる精密制御できない領域が存在するが、それは、ＤＮＡ配列を読まない領域（バッファ）を設けることする。１枚の基板にＤＮＡが固定されているため、基板のみを制御できることも利点の一つである。 In (5), when gripping by the automatic gripping mechanism, there is a region that cannot be precisely controlled by the drive stage 109 due to the difference in parallelism, and this is to provide a region (buffer) where the DNA sequence is not read. One of the advantages is that only the substrate can be controlled because the DNA is fixed on one substrate.
　図８は、本発明の第４の生体試料分析方法を示している。 FIG. 8 shows a fourth biological sample analysis method of the present invention.
　本実施例は、実施例１と類似しているため、相違点を中心に説明し、重複する部分は割愛する。本実施例では、実施例１に示す平板上のＤＮＡ固定基板１０８の代わりに、球状のＤＮＡ固定ビーズ８０１を用いている。このＤＮＡ固定ビーズ８０１にＤＮＡを固定する。シリンジポンプ５０２を用いてＤＮＡ固定ビーズ８０１をプローブ１０７に吸引したり、離反したりする点は、実施例１と同じである。なお、図８に示すように球状のＤＮＡ固定ビーズ８０１プローブ先端８０２には多数の開口部が設けられている。 Since the present embodiment is similar to the first embodiment, differences will be mainly described and overlapping portions will be omitted. In this example, spherical DNA fixing beads 801 are used in place of the DNA fixing substrate 108 on the flat plate shown in Example 1. DNA is fixed to the DNA fixing beads 801. The point that the DNA fixing beads 801 are sucked into and separated from the probe 107 using the syringe pump 502 is the same as in the first embodiment. In addition, as shown in FIG. 8, the spherical DNA fixed bead 801 probe tip 802 has a large number of openings.
　図９は、本発明の第５の生体試料分析方法を示している。 FIG. 9 shows a fifth biological sample analysis method of the present invention.
　本実施例は、実施例２と類似しているため、相違点を中心に説明し、重複する部分は割愛する。本実施例では、実施例２に示す平板上のＤＮＡ固定基板１０８の代わりに球状のＤＮＡ固定磁気ビーズ９０１を用いている。この球状のＤＮＡ固定磁気ビーズ９０１にＤＮＡを固定する。電磁石６０２を用いる点は実施例２と同じである。その他、プローブ１０７の動作なども実施例２と同じである。 Since the present embodiment is similar to the second embodiment, differences will be mainly described and overlapping portions will be omitted. In this example, spherical DNA-immobilized magnetic beads 901 are used in place of the DNA-immobilized substrate 108 on the flat plate shown in Example 2. DNA is fixed to the spherical DNA fixing magnetic beads 901. The point of using the electromagnet 602 is the same as that of the second embodiment. In addition, the operation of the probe 107 is the same as that of the second embodiment.
　図１０は、本発明の生体試料分析方法１０００の第６の生体試料分析方法を示している。 FIG. 10 shows a sixth biological sample analysis method of the biological sample analysis method 1000 of the present invention.
　実施例１の手順（２）～（３）は、吸引したＤＮＡ固定基板１０８を、ナノポア１１２に駆動機構を用いて、近づけ、ＤＮＡ固定基板１０８がナノポアデバイス１０１と接触しない程度に近づけた後、陰圧を開放し、プローブ１０７から、ＤＮＡ固定基板１０８を離反しているが、離反する距離は、本発明において、問題ではない。たとえば、ＤＮＡ固定液５０１からピンセットで把持し、手動で、フローセル開口部１１０に挿入してもよい。その後の手順は、実施例１～実施例３の（４）の手順に従い、ＤＮＡ固定基板１０８は、ナノポアデバイス１０１上に落下し、ＤＮＡ固定基板１０８とナノポアデバイス１０１の距離は、ほぼ０になり、ＤＮＡ１１１の一端は、ＤＮＡ引込範囲３０１に、限りなく近づくことができ、絡まっていたＤＮＡ１１１の一端がナノポア１１２に引き込まれ、ナノポア１１２中のＤＮＡ１１１の断面積分だけ減少した封鎖信号（電流値）が、アンプ（図示せず）で増幅されて、ＡＤＣ（図示せず）を介してＰＣ（Ｐｅｒｓｏｎａｌ　Ｃｏｍｐｕｔｅｒ　図示せず）で確認される。またこの効果は、シングルポアだけでなく、マルチポアシステムでも同様の効果が得られる。ここで、距離０と言うのは、完全な０ナノメートルではなく、ＤＮＡ固定基板１０８と、ナノポアデバイス１０１には、表面粗さなどがあり、完全にナノポアを封鎖することはできない。ＤＮＡ固定基板１０８とナノポアデバイス１０１との隙間は、ナノポア径１０ｎｍの面積以上あり、封鎖電流には影響はない。仮に完全に０になってしまい、封鎖電流に影響がある場合は、ＤＮＡ固定基板１０８の両端に、設計上、隙間を作るように、部材を製作してもよい。 In the procedures (2) to (3) of Example 1, the sucked DNA immobilization substrate 108 is brought close to the nanopore 112 using a drive mechanism, and is brought close to the extent that the DNA immobilization substrate 108 does not contact the nanopore device 101. Although the negative pressure is released and the DNA fixing substrate 108 is separated from the probe 107, the distance of separation is not a problem in the present invention. For example, the DNA fixing solution 501 may be grasped with tweezers and manually inserted into the flow cell opening 110. The subsequent procedure follows the procedure of Example 1 to Example 3 (4), and the DNA immobilization substrate 108 falls on the nanopore device 101, and the distance between the DNA immobilization substrate 108 and the nanopore device 101 becomes almost zero. One end of the DNA 111 can approach the DNA pull-in range 301 as much as possible, and one end of the entangled DNA 111 is drawn into the nanopore 112, and a blocking signal (current value) reduced by the cross-sectional integral of the DNA 111 in the nanopore 112 is obtained. Amplified by an amplifier (not shown) and confirmed by a PC (Personal Computer not shown) through an ADC (not shown). This effect can be obtained not only in a single pore but also in a multipore system. Here, the distance 0 is not completely 0 nanometers, and the DNA fixing substrate 108 and the nanopore device 101 have surface roughness and the like, and the nanopores cannot be completely blocked. The gap between the DNA fixing substrate 108 and the nanopore device 101 is not less than the area of the nanopore diameter of 10 nm and does not affect the blocking current. If it becomes completely zero and the blocking current is affected, a member may be manufactured so as to create a design gap on both ends of the DNA fixing substrate 108.
　また、ＤＮＡ固定基板１０８の材質の比重は、電解質溶液１０３の比重よりも大きいとする。（５）封鎖信号を確認した後、陰圧や磁力、把持機構によって、ＤＮＡ固定基板１０８を吸引する。（６）吸引したＤＮＡ固定基板１０８を駆動ステージ１０９によって、精密制御を行い、引き抜いたり、押し込んだりすることで、ＤＮＡ１１１がナノポアデバイス１０１のナノポアを通過する際に変化する電流値から塩基配列を読み取る。 Further, it is assumed that the specific gravity of the material of the DNA fixing substrate 108 is larger than the specific gravity of the electrolyte solution 103. (5) After confirming the blocking signal, the DNA fixing substrate 108 is sucked by negative pressure, magnetic force, or gripping mechanism. (6) The suctioned DNA immobilization substrate 108 is precisely controlled by the drive stage 109, and the base sequence is read from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. .
　（５）において、陰圧による吸引の際、平行度の違いから、駆動ステージ１０９よる精密制御できない領域が存在するが、それは、ＤＮＡ配列を読まない領域（バッファ）を設けることする。１枚の基板にＤＮＡが固定されているため、基板のみを制御できることも利点の一つである。 In (5), there is a region that cannot be precisely controlled by the drive stage 109 due to the difference in parallelism during suction by negative pressure, but this is to provide a region (buffer) that does not read the DNA sequence. One of the advantages is that only the substrate can be controlled because the DNA is fixed on one substrate.
　図１１は、本発明の生体試料分析方法１１００の第７の生体試料分析方法を示している。 FIG. 11 shows a seventh biological sample analysis method of the biological sample analysis method 1100 of the present invention.
　実施例５の手順（２）～（３）は、吸引したＤＮＡ固定磁気ビーズ９０１を、ナノポア１１２に駆動機構を用いて、近づけ、ＤＮＡ固定磁気ビーズ９０１がナノポアデバイス１０１と接触しない程度に近づけた後、磁力を開放し、プローブ１０７から、ＤＮＡ固定磁気ビーズ９０１を離反しているが、離反する距離は、本発明において、問題ではない。たとえば、ＤＮＡ固定液５０１からＤＮＡ固定磁気ビーズ９０１を、手動で、フローセル開口部１１０に挿入してもよい。その後の手順は、実施例４、または、実施例５の（４）の手順に従い、ＤＮＡ固定磁気ビーズ９０１は、ナノポアデバイス１０１上に落下し、ＤＮＡ固定磁気ビーズ９０１とナノポアデバイス１０１の距離は、ほぼ０になり、ＤＮＡ１１１の一端は、ＤＮＡ引込範囲３０１に、限りなく近づくことができ、絡まっていたＤＮＡ１１１の一端がナノポア１１２に引き込まれ、ナノポア１１２中のＤＮＡ１１１の断面積分だけ減少した封鎖信号（電流値）が、アンプ（図示せず）で増幅されて、ＡＤＣ（図示せず）を介してＰＣ（Ｐｅｒｓｏｎａｌ　Ｃｏｍｐｕｔｅｒ　図示せず）で確認される。またこの効果は、シングルポアだけでなく、マルチポアシステムでも同様の効果が得られる。さらに、よりＤＮＡ固定磁気ビーズ９０１を、ナノポアデバイス１０１に近づけるため、プローブ１０７で、ＤＮＡ固定磁気ビーズ９０１を押し付けることをしてもよい。磁気ビーズの特性を利用し、下の槽１０２Ａ側の下部に、電磁石９０２を配置し、磁気ビーズを－Ｚ方向に引っ張り、よりナノポアデバイス１０１に近づけることをしてもよい。またこの方法は，ビーズに限らず，磁性体６０１付きＤＮＡ固定基板１０８を電磁石９０２で－Ｚ方向に引っ張り、よりナノポアデバイス１０１に近づけることをしてもよい。（５）封鎖信号を確認した後、再び、電磁石６０２などで、磁力を作り出し、ＤＮＡ固定磁気ビーズ９０１を吸引する。この磁力を発生するのは、電磁石である必要はない。永久磁石でも可能である。（６）吸引したＤＮＡ固定磁気ビーズ９０１を駆動ステージ１０９によって、精密制御を行い、引き抜いたり、押し込んだりすることで、ＤＮＡ１１１がナノポアデバイス１０１のナノポアを通過する際に変化する電流値から塩基配列を読み取る。 In the procedures (2) to (3) of Example 5, the attracted DNA-immobilized magnetic beads 901 are brought close to the nanopore 112 using a drive mechanism, so that the DNA-immobilized magnetic beads 901 are not brought into contact with the nanopore device 101. Thereafter, the magnetic force is released, and the DNA-fixed magnetic beads 901 are separated from the probe 107, but the distance of separation is not a problem in the present invention. For example, the DNA fixing magnetic beads 901 from the DNA fixing solution 501 may be manually inserted into the flow cell opening 110. The subsequent procedure follows the procedure of Example 4 or (4) of Example 5, and the DNA-immobilized magnetic beads 901 drop on the nanopore device 101, and the distance between the DNA-immobilized magnetic beads 901 and the nanopore device 101 is One end of the DNA 111 can approach the DNA pull-in range 301 as much as possible, and one end of the entangled DNA 111 is drawn into the nanopore 112, and the blocking signal (decrease by the cross-sectional integral of the DNA 111 in the nanopore 112 is reduced. The current value is amplified by an amplifier (not shown) and confirmed by a PC (Personal Computer not shown) via an ADC (not shown). This effect can be obtained not only in a single pore but also in a multipore system. Furthermore, in order to bring the DNA-fixed magnetic beads 901 closer to the nanopore device 101, the DNA-fixed magnetic beads 901 may be pressed with the probe 107. By utilizing the characteristics of the magnetic beads, an electromagnet 902 may be disposed in the lower part on the lower tank 102A side, and the magnetic beads may be pulled in the −Z direction to be closer to the nanopore device 101. This method is not limited to beads, and the DNA immobilization substrate 108 with the magnetic body 601 may be pulled in the −Z direction by the electromagnet 902 so as to be closer to the nanopore device 101. (5) After confirming the blocking signal, the magnetic force is again generated by the electromagnet 602 and the DNA-fixed magnetic beads 901 are attracted. It is not necessary for the electromagnet to generate this magnetic force. Permanent magnets are also possible. (6) The attracted DNA-fixed magnetic beads 901 are precisely controlled by the drive stage 109, and the base sequence is determined from the current value that changes when the DNA 111 passes through the nanopore of the nanopore device 101 by pulling out or pushing in. read.
　（５）において、磁力による吸引の際、平行度の違いから、駆動ステージ１０９よる精密制御できない領域が存在するが、それは、ＤＮＡ配列を読まない領域（バッファ）を設けることする。 In (5), there is a region that cannot be precisely controlled by the drive stage 109 due to the difference in parallelism when attracted by magnetic force, but this is to provide a region (buffer) where the DNA sequence is not read.
１００：生体試料分析装置
１００Ａ：生体試料分析装置一部拡大図
１０１：ナノポアを有するが設置された仕切り体（ナノポアデバイス）
１０２Ａ、１０２Ｂ：槽
１０３：電解質溶液
１０４：電極
１０５：電源
１０６：フローセル
１０７：プローブ
１０８：ＤＮＡ固定基板
１０９：駆動機構
１１０：フローセル開口部
１１１：ＤＮＡ
１１２：ナノポア
１１３：ナノポア薄膜
２００：配列読取例
３００：生体試料分析方法
３０１：ＤＮＡ引込範囲
５００：実施例１の生体試料分析方法
５０１：ＤＮＡ固定溶液
５０２：シリンジポンプ
５０３：シリンジポンプ流路
６００：実施例２の生体試料分析方法
６０１：磁性体（金属）
６０２：電磁石
７００：実施例３の生体試料分析方法
７０１：モータ
７０２：自動把持機構
８００：実施例４の生体試料分析方法
８０１：ＤＮＡ固定ビーズ
８０２：プローブ先端流路
９００：実施例５の生体試料分析方法
９０１：ＤＮＡ固定磁気ビーズ
９０２：電磁石
１０００：実施例６の生体試料分析方法
１１００：実施例７の生体試料分析方法 100: Biological sample analyzer 100A: Biological sample analyzer partially enlarged view 101: Partition with nanopore installed (nanopore device)
102A, 102B: Tank 103: Electrolyte solution 104: Electrode 105: Power source 106: Flow cell 107: Probe 108: DNA fixing substrate 109: Drive mechanism 110: Flow cell opening 111: DNA
112: Nanopore 113: Nanopore thin film 200: Array reading example 300: Biological sample analysis method 301: DNA drawing range 500: Biological sample analysis method 501 of Example 1: DNA fixing solution 502: Syringe pump 503: Syringe pump flow path 600: Biological sample analysis method 601 of Example 2: Magnetic material (metal)
602: Electromagnet 700: Biological sample analysis method 701 of Example 3 Motor 702: Automatic gripping mechanism 800: Biological sample analysis method 801 of Example 4 DNA fixing bead 802: Probe tip channel 900: Biological sample of Example 5 Analysis method 901: DNA-fixed magnetic beads 902: Electromagnet 1000: Biological sample analysis method of Example 6 1100: Biological sample analysis method of Example 7

Claims (10)

  1. 　生体試料が固定された固定部材と、当該生体試料が通過する貫通孔を有する基板と、前記固定部材を駆動する駆動機構を備えた、生体試料分析装置を用い、生体試料が貫通孔を通過する時に生体試料を検出する生体試料分析方法であって、
    　(a) 生体試料が貫通孔を封鎖したことを確認した後に固定部材を保持し、
    　(b) 駆動機構によって固定部材を引き上げることを特徴とする生体試料分析方法。     The biological sample passes through the through-hole using a biological sample analyzer that includes a fixing member to which the biological sample is fixed, a substrate having a through-hole through which the biological sample passes, and a drive mechanism that drives the fixing member. A biological sample analysis method that sometimes detects a biological sample,
    (a) holding the fixing member after confirming that the biological sample has blocked the through-hole,
    (b) A biological sample analysis method, wherein the fixing member is pulled up by a drive mechanism.
  2. 　請求項１において、
    　前記(a)の工程の前に、
    (c) プローブに固定部材を保持し、
    (d) 固定部材を基板に接近させ、
    (e) プローブから固定部材をリリースすること
    　を特徴とする生体試料分析方法。     In claim 1,
    Before the step (a),
    (c) Hold the fixing member on the probe,
    (d) The fixing member is brought close to the substrate,
    (e) A biological sample analysis method comprising releasing a fixing member from a probe.
  3. 　請求項１において、
    　前記(a)の工程の前に、
    　固定部材を生体試料が通過する貫通孔を有する基板が挿入されている槽に落下させることを特徴とする生体試料分析方法。     In claim 1,
    Before the step (a),
    A biological sample analysis method comprising dropping a fixing member into a tank in which a substrate having a through hole through which a biological sample passes is inserted.
  4. 　請求項１から３のいずれか１項において、
    　生体試料は核酸であり、
    　基板は、電解質溶液を収容した槽内に、挿入されており、
    　生体試料が貫通孔を通過する時に封鎖される面積に応じて核酸の塩基配列を読み取ることを特徴とする生体試料分析方法。     In any one of Claim 1 to 3,
    The biological sample is a nucleic acid,
    The substrate is inserted into a tank containing the electrolyte solution,
    A method for analyzing a biological sample, comprising: reading a base sequence of a nucleic acid according to an area sealed when the biological sample passes through a through-hole.
  5. 　請求項１から３のいずれか１項において、
    　プローブへの固定部材の保持は、陰圧で行うことを特徴とする生体試料分析方法。     In any one of Claim 1 to 3,
    A biological sample analysis method characterized in that holding of a fixing member to a probe is performed under negative pressure.
  6. 　請求項１から３のいずれか１項において、
    　プローブへの固定部材の保持は、磁力で行うことを特徴とする生体試料分析方法。     In any one of Claim 1 to 3,
    A biological sample analysis method, wherein holding of a fixing member to a probe is performed by magnetic force.
  7. 　請求項１から３のいずれか１項において、
    　プローブへの固定部材の保持は、機械的な把持により行うことを特徴とする生体試料分析方法。     In any one of Claim 1 to 3,
    A biological sample analysis method, wherein holding of a fixing member to a probe is performed by mechanical gripping.
  8. 　請求項１から３のいずれか１項において、
    　固定部材は、平板状の基板であることを特徴とする生体試料分析方法。     In any one of Claim 1 to 3,
    The biological sample analysis method, wherein the fixing member is a flat substrate.
  9. 　請求項１から３のいずれか１項において、
    　固定部材は、球状ビーズであることを特徴とする生体試料分析方法。     In any one of Claim 1 to 3,
    The biological sample analysis method, wherein the fixing member is a spherical bead.
  10. 　生体試料が固定された固定部材と、当該生体試料が通過する貫通孔を有する基板と、前記固定部材を駆動する駆動機構と、生体試料が貫通孔を通過する時に生体試料を検出する検出機構とを備えた、生体試料分析装置において、
    　生体試料が貫通孔を封鎖したことを確認した後に固定部材を保持し、駆動機構によって固定部材を引き上げることを特徴とする生体試料分析装置。     A fixing member to which the biological sample is fixed, a substrate having a through-hole through which the biological sample passes, a driving mechanism for driving the fixing member, and a detection mechanism for detecting the biological sample when the biological sample passes through the through-hole. A biological sample analyzer comprising:
    A biological sample analyzer characterized by holding a fixing member after confirming that the biological sample has blocked the through-hole, and pulling up the fixing member by a driving mechanism.
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