WO2004018664A1 - 核酸分析チップと核酸分析装置 - Google Patents
核酸分析チップと核酸分析装置 Download PDFInfo
- Publication number
- WO2004018664A1 WO2004018664A1 PCT/JP2003/010762 JP0310762W WO2004018664A1 WO 2004018664 A1 WO2004018664 A1 WO 2004018664A1 JP 0310762 W JP0310762 W JP 0310762W WO 2004018664 A1 WO2004018664 A1 WO 2004018664A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- analysis chip
- hole
- acid analysis
- culture
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
Definitions
- the invention of this application relates to a nucleic acid analysis chip and a nucleic acid analyzer using the same. More specifically, the invention of this application relates to the field of biotechnology research using microorganisms and cells. The present invention relates to a new nucleic acid analysis chip capable of analyzing nucleic acid components and an analyzer using the same. Background art
- the micro-chamber realized by the inventor of this application performs measurement while completely controlling the environment on a cell basis.
- this is a culture technology that makes it possible, it is necessary to quantitatively measure the amount of expressed mRNA in cells in order to measure changes in cells such as response to drugs.
- no nucleic acid analysis technology optimized for the system has been developed.
- an object of the invention of this application is to provide a new technical means for analyzing a nucleic acid component of each cell cultured in a cell culture micro-champer based on the above background.
- the invention of this application directly measures quantitative analysis of nucleic acid components of cells in each container in the cell culture micro-chamber developed by the inventors of the present application as described above.
- a nucleic acid analysis chip having means for performing the above and a nucleic acid analyzer using the same.
- the invention of the present application is, firstly, that a plurality of holes capable of completely enclosing a single cell are arranged as a culture vessel on a substrate, and an aqueous solution is left in each of these holes. It has an upper region that can be filled with a liquid having a lower specific gravity than the aqueous solution without mixing the upper part of the hole with the aqueous solution.
- a nucleic acid analysis chip for performing analysis.
- the invention of the present application provides a nucleic acid analyzer using a nucleic acid analysis chip, which comprises means for optically measuring each container in the microchamber, optical means, There is a means for heating the solution, a means for introducing the reaction solution into each container in the microchamber, a means for preventing the reaction solution in each container from diffusing and evaporating.
- FIG. 1 is a schematic diagram showing an example of the basic configuration of the nucleic acid analysis chip of the present invention.
- FIG. 2 is a schematic diagram showing an example of a device configuration for observing the nucleic acid analysis chip shown in FIG. 1 and locally heating the focused light.
- the symbols in FIG. 1 indicate the following.
- a nucleic acid analysis chip is capable of recovering a nucleic acid component from a hole after the introduction of a reaction solution.
- Various forms such as a sealing material, for example, a semi-permeable membrane interposed between the and the upper region are adopted.
- a semi-permeable membrane 107 is sealed on the upper surface of the substrate 101 so that the culture solution can be exchanged, and that the cells 103 do not escape from the container or contain impurities such as bacteria.
- the cells can be continuously cultured in the same manner.
- a lighter liquid for example liquid paraffin or silicone oil, is introduced into the upper region 105.
- each container 102 is isolated by a liquid such as liquid paraffin.
- the semi-permeable membrane 107 on the upper surface of the container 102 is broken using a micropipet 104 to introduce a PCR reaction solution.
- a fluorescent dye that can optically measure the PCR reaction for example, Taq Man Probe (Amersham, USA), and nucleic acid quantitative measurement using the fluorescence energy transfer method Becomes possible.
- the amount of fluorescence it is possible to measure the fluorescence collected by the objective lens 106.
- it is possible to perform heating necessary for the PCR reaction by irradiating infrared light through the objective lens 106.
- nucleic acid components recovered from each container 102 by micropipette 104 can be analyzed comprehensively and quantitatively using capillary electrophoresis. is there.
- the PCR reaction can be performed by an existing heating / cooling device such as a thermal cycler.
- FIG. 2 shows an example of the configuration of an apparatus of the cell culture microchamber 100 for heating the vessel 102 and introducing focused light for causing a PCR reaction.
- cells in nucleic acid analysis chip 100 It has a microscope observation system for observing changes in the sample and the amount of fluorescence, and has an infrared focused light irradiation system for heating the aqueous solution in the container at the same time.
- the nucleic acid analysis chip 100 is arranged on the optical path of the microscopic observation optical system.
- the microscopic observation optical system has the following configuration.
- the light emitted from the light source 201 is adjusted to a specific wavelength by the fill-in filter 202, collected by the condenser lens 203, and irradiated to the nucleic acid analysis chip 100.
- the irradiated light is used for observation with an objective lens 205 as transmitted light.
- the transmitted light image inside the nucleic acid analysis chip 100 passes through the filter 212 by the mirror 211, is guided to the camera 213, and forms an image on the light receiving surface of the camera. Therefore, it is desirable that the material of the chip 100 is a material that is optically transparent to the light having the wavelength selected by the filter 202.
- glass such as borosilicate glass or quartz glass, resin or plastic such as polystyrene, or a solid substrate such as a silicon substrate is used.
- a silicon substrate it is desirable to use light having a wavelength of 900 nm or more for observation.
- the excitation light for fluorescence observation emitted from the light source 208 is guided to the objective lens 205 by the dichroic mirror 210 after the wavelength is selected by the filter 209. Used as excitation light for fluorescence observation inside the nucleic acid analysis chip 100.
- the fluorescence emitted from the chip 100 is collected again by the objective lens 205, and only the fluorescence and transmitted light after the excitation light is turned on by the filter 212 can be observed with the camera 211. You.
- the combination of the filters 202, 209, and 212 only the transmitted light can be observed with the camera 21 or only the fluorescent light, or the transmitted light image and The fluorescent image can be observed at the same time.
- This laser light is focused by the objective lens 205, and can locally heat the nucleic acid analysis chip 100.
- Focus point In the case of moving, by moving the movable dichroic mirror, it is possible to move the focusing position of the laser light in the nucleic acid analysis chip 100.
- the wavelength of this laser beam is preferably a wavelength that has water absorption but has no photochemical action and has little absorption to the substrate. For example, in the case of light having a wavelength of 150 nm, laser light is effectively absorbed by water, and the reaction solution locally generates heat in the container in which the light is absorbed.
- the image data obtained by the camera is analyzed by the image processing / analysis device 214, and the movable dichroic mirror 206 and the nucleic acid analysis chip 100 are mounted on the basis of various analysis results.
- a stage movement motor 215 that can freely move in the X-Y direction can be driven.
- the measurement is performed using one camera.
- a change in the fluorescence intensity at at least one different wavelength is measured by adding a dichroic mirror. Quantitative analysis by the energy transfer method can be performed.
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Optical Measuring Cells (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/525,876 US20050272039A1 (en) | 2002-08-26 | 2003-08-26 | Nucleic acid analysis chip and nucleic acid analyzer |
EP03792844A EP1541678A4 (en) | 2002-08-26 | 2003-08-26 | NUCLEIC ACID ANALYSIS CHIP AND NUCLEIC ACID ANALYZER |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2002245900A JP4171775B2 (ja) | 2002-08-26 | 2002-08-26 | 核酸分析装置 |
JP2002-245900 | 2002-08-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2004018664A1 true WO2004018664A1 (ja) | 2004-03-04 |
Family
ID=31944204
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2003/010762 WO2004018664A1 (ja) | 2002-08-26 | 2003-08-26 | 核酸分析チップと核酸分析装置 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20050272039A1 (ja) |
EP (1) | EP1541678A4 (ja) |
JP (1) | JP4171775B2 (ja) |
CN (1) | CN1678737A (ja) |
WO (1) | WO2004018664A1 (ja) |
Families Citing this family (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4795840B2 (ja) * | 2006-04-24 | 2011-10-19 | 富士通株式会社 | 小胞体反応チップ、小胞体収納方法、反応液回収方法、及び、小胞体回収方法 |
JP4864525B2 (ja) * | 2006-04-26 | 2012-02-01 | 富士通株式会社 | 物質導入装置及び物質導入方法 |
DE102007031244B3 (de) * | 2007-07-05 | 2009-01-02 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Vorrichtung und Verfahren zur Durchführung statischer und dynamischer Streulichtmessungen in kleinen Volumina |
KR101362905B1 (ko) * | 2007-11-30 | 2014-02-19 | (주)바이오니아 | 마이크로 챔버 플레이트, 그 제조방법 |
WO2009157211A1 (ja) * | 2008-06-26 | 2009-12-30 | 国立大学法人東京大学 | 培養細胞の活性計測方法、マイクロチャンバ、マイクロチャンバアレイ、培養容器および培養細胞の活性計測装置 |
JP4574707B2 (ja) * | 2008-10-20 | 2010-11-04 | 一般社団法人オンチップ・セロミクス・コンソーシアム | 細胞分取及び培養方法とその装置 |
WO2011056643A2 (en) * | 2009-10-27 | 2011-05-12 | The Regents Of The University Of Michigan | Microfluidic platform for discrete cell assay |
KR101356076B1 (ko) | 2010-07-23 | 2014-01-28 | (주)바이오니아 | 시료 내장 마이크로 챔버 플레이트 및 분석용 마이크로 챔버 프레이트의 제조 방법, 분석용 마이크로 챔버 플레이트 및 시료 내장 마이크로 챔버 플레이트 제조 장치 셋 |
JP5771962B2 (ja) * | 2010-12-08 | 2015-09-02 | 大日本印刷株式会社 | マイクロ流路チップ及びその製造方法 |
CN102435544A (zh) * | 2011-11-04 | 2012-05-02 | 深圳市普康电子有限公司 | 血细胞分析仪计数池预热机构 |
WO2013177560A1 (en) | 2012-05-25 | 2013-11-28 | The Regents Of The University Of California | Microfluidic systems for particle trapping and separation |
US9708654B2 (en) | 2012-06-15 | 2017-07-18 | Board Of Regents, The University Of Texas System | High throughput sequencing of multiple transcripts |
KR102019973B1 (ko) | 2013-11-20 | 2019-11-04 | (주)바이오니아 | 마이크로 챔버 플레이트 |
CN103894248B (zh) * | 2014-04-09 | 2015-09-16 | 国家纳米科学中心 | 一种单细胞分析用微流控芯片和***及单细胞分析方法 |
US10513733B2 (en) | 2015-03-23 | 2019-12-24 | Board Of Regents, The University Of Texas System | High throughout sequencing of paired VH and VL transcripts from B cells secreting antigen-specific antibodies |
US10549277B2 (en) | 2015-10-14 | 2020-02-04 | The Regents Of The University Of California | Integrated microfluidic platform for selective extraction of single-cell mRNA |
US9862941B2 (en) * | 2015-10-14 | 2018-01-09 | Pioneer Hi-Bred International, Inc. | Single cell microfluidic device |
WO2018071448A1 (en) | 2016-10-11 | 2018-04-19 | The Regents Of The University Of California | Systems and methods to encapsulate and preserve organic matter for analysis |
US11517901B2 (en) | 2017-06-09 | 2022-12-06 | The Regents Of The University Of California | High-efficiency particle encapsulation in droplets with particle spacing and downstream droplet sorting |
WO2018227210A1 (en) | 2017-06-09 | 2018-12-13 | The Regents Of The University Of California | High-efficiency encapsulation in droplets based on hydrodynamic vortices control |
WO2019075409A1 (en) | 2017-10-12 | 2019-04-18 | The Regents Of The University Of California | ISOLATION AND IDENTIFICATION WITHOUT MICROFLUIDIC LABEL OF CELLS USING FLUORESCENCE LIFE IMAGING (FLIM) |
US11745179B2 (en) | 2017-10-20 | 2023-09-05 | The Regents Of The University Of California | Microfluidic systems and methods for lipoplex-mediated cell transfection |
US11499127B2 (en) | 2017-10-20 | 2022-11-15 | The Regents Of The University Of California | Multi-layered microfluidic systems for in vitro large-scale perfused capillary networks |
WO2019084538A1 (en) | 2017-10-27 | 2019-05-02 | Board Of Regents, The University Of Texas System | TUMOR SPECIFIC ANTIBODIES, T CELL RECEPTORS AND METHODS OF IDENTIFICATION THEREOF |
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JP2001108684A (ja) * | 1999-10-05 | 2001-04-20 | Hitachi Ltd | Dna検査方法及びdna検査装置 |
JP2002027984A (ja) * | 2000-07-17 | 2002-01-29 | Mitsubishi Chemicals Corp | マイクロリアクタチップ,化学反応試験方法及びマイクロリアクタチップ用薄膜部材 |
WO2002042411A1 (fr) * | 2000-11-22 | 2002-05-30 | Japan Science And Technology Corporation | Appareil destine a l'observation microscopique de la culture durable d'une cellule unique |
EP1239279A1 (en) * | 2000-01-17 | 2002-09-11 | Yokogawa Electric Corporation | Biochip reader |
JP2003009860A (ja) * | 2001-06-27 | 2003-01-14 | Fuji Photo Film Co Ltd | 区画培養基板及びそれを用いたdnaチップ |
JP2003083965A (ja) * | 2001-09-10 | 2003-03-19 | Adgene Co Ltd | 蛋白・核酸解析用チップ |
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JP2662215B2 (ja) * | 1986-11-19 | 1997-10-08 | 株式会社日立製作所 | 細胞保持装置 |
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US6093370A (en) * | 1998-06-11 | 2000-07-25 | Hitachi, Ltd. | Polynucleotide separation method and apparatus therefor |
US6653124B1 (en) * | 2000-11-10 | 2003-11-25 | Cytoplex Biosciences Inc. | Array-based microenvironment for cell culturing, cell monitoring and drug-target validation |
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2002
- 2002-08-26 JP JP2002245900A patent/JP4171775B2/ja not_active Expired - Fee Related
-
2003
- 2003-08-26 WO PCT/JP2003/010762 patent/WO2004018664A1/ja active Application Filing
- 2003-08-26 US US10/525,876 patent/US20050272039A1/en not_active Abandoned
- 2003-08-26 CN CNA038201356A patent/CN1678737A/zh active Pending
- 2003-08-26 EP EP03792844A patent/EP1541678A4/en not_active Withdrawn
Patent Citations (6)
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JP2001108684A (ja) * | 1999-10-05 | 2001-04-20 | Hitachi Ltd | Dna検査方法及びdna検査装置 |
EP1239279A1 (en) * | 2000-01-17 | 2002-09-11 | Yokogawa Electric Corporation | Biochip reader |
JP2002027984A (ja) * | 2000-07-17 | 2002-01-29 | Mitsubishi Chemicals Corp | マイクロリアクタチップ,化学反応試験方法及びマイクロリアクタチップ用薄膜部材 |
WO2002042411A1 (fr) * | 2000-11-22 | 2002-05-30 | Japan Science And Technology Corporation | Appareil destine a l'observation microscopique de la culture durable d'une cellule unique |
JP2003009860A (ja) * | 2001-06-27 | 2003-01-14 | Fuji Photo Film Co Ltd | 区画培養基板及びそれを用いたdnaチップ |
JP2003083965A (ja) * | 2001-09-10 | 2003-03-19 | Adgene Co Ltd | 蛋白・核酸解析用チップ |
Non-Patent Citations (1)
Title |
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See also references of EP1541678A4 * |
Also Published As
Publication number | Publication date |
---|---|
EP1541678A1 (en) | 2005-06-15 |
US20050272039A1 (en) | 2005-12-08 |
EP1541678A4 (en) | 2008-06-18 |
JP2004081084A (ja) | 2004-03-18 |
CN1678737A (zh) | 2005-10-05 |
JP4171775B2 (ja) | 2008-10-29 |
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