JP2004000217A - Trapping and releasing device of dna using flow passage and method for trapping and releasing dna - Google Patents

Trapping and releasing device of dna using flow passage and method for trapping and releasing dna Download PDF

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JP2004000217A
JP2004000217A JP2003124520A JP2003124520A JP2004000217A JP 2004000217 A JP2004000217 A JP 2004000217A JP 2003124520 A JP2003124520 A JP 2003124520A JP 2003124520 A JP2003124520 A JP 2003124520A JP 2004000217 A JP2004000217 A JP 2004000217A
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dna
electric field
molecule
liquid
charged
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JP4234486B2 (en
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Zen Takamura
高村　禅
Tetsuya Hayama
葉山　哲也
Jun Kikuchi
菊地　純
Yasuhiro Horiike
堀池　靖浩
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0663—Stretching or orienting elongated molecules or particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive and simple principle capable of trapping a DNA or resembling materials selectively from flowing liquid containing proteins and various reagents, recovering or concentrating the DNA by using a simple mechanism formed on a chip, and releasing the trapped DNA by a simple operation for using it in a next treatment, and a device for the same. <P>SOLUTION: By flowing liquid containing the DNA through a flow passage having parts with partially wide width and parts with partially narrow width by a pressure difference, and impressing an electric field in a reverse direction for flowing the DNA against the pressure difference flow, it becomes possible to trap the DNA selectively in the vicinity of the narrow width part. By adjusting the strength of electric field or pressure, flowing DNA's can be trapped one after another, and the recovery or concentration of the DNA can be realized. The trapped DNA is discharged at a time to an entrance side or exit side by strengthening or weakening either one of the electric field or pressure, and thereby easily released and recovered for using it in the next treatment. <P>COPYRIGHT: (C)2004,JPO

Description

【０００１】
【発明の属する技術分野】
本発明は、健康状態の判断や、感染症の早期発見、早期治療、及び治療中の検査を行うために、血液等に含まれる、白血球、病原菌、ウイルスのＤＮＡのみを、分離、抽出、濃縮し、ＤＮＡの情報に基づく診断を行うための分離手段ならびにその装置に関する。特に必要な機能、構造の一部が一つの板状のチップに集積されており、必要な検体が微量ですみ、携帯性、即時性、使い捨て、安価などを特徴とする微小流体力学やｍｉｃｒｏ−ＴＡＳ、Ｌａｂ−ｏｎ−ａ−Ｃｈｉｐといわれる分野に関する。
【０００２】
【従来の技術】
人体から取り出した、組織や血液等に含まれるＤＮＡを分析する技術は非常に重要である。白血球や体細胞からは本人の遺伝子情報が判り、生活習慣病や遺伝病の診断、オーダーメード医療に重要である。また、血液等に含まれる病原菌やウイルスのＤＮＡを取り出して特定することにより、感染症の診断ができる。
【０００３】
精製されたＤＮＡを特定する方法としては、ＤＮＡチップや、ＰＣＲ法、ＬＡＭＰ法を用いて特定配列を持ったＤＮＡだけを増幅するような手法と組みあわせることにより、高感度で選択性の高い検出が原理的に可能である。このような検出方を用いるには、採取した細胞やウイルスが含まれているであろう液体から、ＤＮＡに有害な物質を除外し、細胞膜やウイルス壁を破砕してＤＮＡを取り出し、その後の反応や検出を阻害するような物質を除外し、ＰＣＲやＬＡＭＰ法に適当なＤＮＡ濃度、塩濃度、ｐＨを調整する前処理が必要である。前処理は試薬の混合、反応、ＤＮＡの回収処理の繰り返しからなる。この回収処理の形態としては、ＤＮＡの濃縮、分離、ＤＮＡのみを残した溶液の交換、溶液の交換によるＤＮＡの洗浄などがある。即ち、介在物が存在する溶液から、できるだけ選択的にＤＮＡのみを一箇所に集め、他の介在物と分けて回収することが必要であり、またＤＮＡを１まとめにしたまま、溶液内を移動する、あるいは逆に溶液の方を移動することにより、溶液の交換や洗浄が実現できれば、より簡単に前処理を実現できる。このようなＤＮＡの回収を行う方法としては、遠心分離法、磁気ビーズを用いてＤＮＡを絡め取った磁気ビーズを磁石により移動する方法等がある。
【０００４】
一方で、現在ウイルス性の病気の診断には、ウイルスに免疫が応対し、産出された抗体を検出する方法がとられている。これは、血液中のウイルスが余りに微量なため、検出が困難であるからである。しかし、この方法には、感染から免疫が応答し抗体ができるまでの期間、診断ができないという欠点がある。また、Ｂ型肝炎など、ウイルスが除去されても抗体だけ残ったり、また重症患者では、新たな感染があっても、抗体を産出できず、誤診される欠点があった。もし、血液中の微量なウイルス由来のＤＮＡを濃縮することができれば、これら抗原の直接検出でき、早期発見、早期治療に大きく貢献する。現在、石英など特定の材料の表面にＤＮＡを固定し洗い流す方法等を用いて濃縮が試みられているが、この要求レベルを満たすようなものはまだない。
【０００５】
【発明が解決しようとする課題】
ＤＮＡの回収処理で用いられる、遠心分離は、手による上澄みや中間層、沈殿物の取り分け作業に依存しており、微量なサンプルに適用困難である。近年、ｍｉｃｒｏＴＡＳ、Ｌａｂ−ｏｎ−ａ−Ｃｈｉｐという技術分野が立ち上がりつつあり、従来の分析技術や化学合成法を、一つのチップの上に集積化することで、システムの小型化、必要な試薬や検体量の縮小化、低コスト化、高速化、高機能化を実現している。この技術を用いると、一滴の血液、数個の細胞やウイルスからＤＮＡ分析が可能と期待される。しかしながらこれを実現するには、遠心分離に代わり、チップの中で実現できるＤＮＡの回収技術が必要である。磁気ビーズに絡め取る方法は、チップ上でも実現可能であるが、磁場の移動は煩雑であり、また集めたＤＮＡを、次の処理が行えるように再び溶液中に解放すること（即ちリリース）も、単純な緩衝液ではリリースされないなど、一般性に欠ける。診断を目的とした、ＤＮＡの前処理では、前に述べたように抗原である血中ウイルスを検出できるレベルの濃縮が可能なものはまだない。細胞や細菌レベルでは、いろいろな手法が開発されているが、煩雑で、完全チップ化に不向きであったり、感度が犠牲になったりする。また、ＤＮＡの濃縮や回収を行わず、細胞が含まれている溶液に混ぜるだけで前処理が完了するような薬品のキットが開発されているが、精製する方法に比較すると、感度や精度は悪い。
【０００６】
これらの要求に答えるべく、チップ上に形成した簡単な機構を用いて、流路を流れるたんぱく質やさまざまな試薬類を含む液体から、ＤＮＡあるいはそれに類するものを選択的に捕捉保持（即ちトラップ）し、ＤＮＡの回収、或いはＤＮＡの濃縮を行い、またトラップされたＤＮＡを簡単な操作で、リリースし、次の処理に用いることができるような、安価で単純な原理・装置はまだない。
【０００７】
【課題を解決するための手段】
図１は課題を解決する手段の説明である。流路１０１の中にＤＮＡ１０２を含む溶液を流す。溶液には、圧力差と電界を同時に印加する。即ち、溶液中のＤＮＡ１０２には、圧力流れによる力の方向１０３で示される方向の力と、電界による力の方向１０４で示される方向の力が、図１に示されるように互いに逆向きになるように同時に働かせる。流路１０１には、一つ以上の流路幅の広い部分１０５と、一つ以上の流路幅の狭い部分１０６が存在する。流路１０１の具体的な形状やその向きは後述するように様々であるが、電界と圧力流れの強さを調節することによりＤＮＡ１０２を、選択的に、幅が狭い部分の近辺に、トラップすることができる。また電界や圧力の強さを調節することにより、流れてきたＤＮＡを次々にトラップすることができる。この時トラップされるもの以外の溶液中の物質は、電界あるいは圧力流れにより流れ去るので、ＤＮＡの回収や濃縮が実現できる。また、ＤＮＡはトラップされたまま、溶液は流れているので、溶液の交換や洗浄にもちいることができる。狭い部分は、トラップするＤＮＡの大きさにより、０．０１ミクロンから、５０ミクロン、広い部分は、狭い部分に比べて断面積で２倍以上である。本発明のトラップ能力は、ＤＮＡのサイズにより異なる。流路径や形状を変えたり、電圧や圧力を変えることにより、トラップできるＤＮＡのサイズを変化させることが可能である。
【０００８】
トラップされたＤＮＡは、電界もしくは圧力のどちらかを強める、或いは弱めることにより、入り口側、あるいは出口側に放出される。これにより、簡単にリリース、回収でき、次の処理に用いることができる。
【０００９】
【発明の実施の形態】
図２に本発明に基づきＤＮＡをトラップ、リリースする最も基本的な形態を示す。トラップ機構２０１は本発明による、広い部分と狭い部分を持った流路である。入り口（リザーバ）２０２はＤＮＡの入り口でありここにＤＮＡが含まれた液体を入れる。液体は流路２０４、トラップ機構２０１を通り、出口（ウエステ）２０３へ流れる。入り口２０２と出口２０３のどちらかまたは両方から圧力源につながった管をもちいて圧力を加える。また入り口２０２と出口２０３には電極などをもちいて同時に電界を加える。圧力により入り口２０２から出口２０３へ、液を搬送し、同時に入り口２０２に正、出口２０２に負の電圧を加える。これにより入り口２０２の液のうちＤＮＡだけがトラップ機構２０１にトラップされる。入り口２０２の液が大部分出口２０３に流れたあと、電界を強める、或いは圧力を弱めるまたは切る或いは逆転することにより。入り口２０２側に濃縮されたＤＮＡを取り出すことができる。あるいは圧力を強める、または電界を弱めるか切るか反転することにより、出口２０３側に濃縮されたＤＮＡを取り出すことができる。
【００１０】
図３の上の図は、本発明の形態であり、図２に加え、入り口３０１側に、濃縮されたＤＮＡ回収流路３０２と、電極を加えるリザーバを兼ねるＤＮＡ回収口３０３が設けられている。入り口３０１からＤＮＡを圧力によって、トラップ機構２０１へ流し、ＤＮＡ回収口３０３に正、出口２０３に負の電圧を印加する。十分トラップされたあと、圧力を弱めることにより、濃縮されたＤＮＡを流路２０４を経由して、ＤＮＡ回収流路３０２、ＤＮＡ回収口３０３に回収することができる。回収路が、２０３出口側にあっても同様に回収できる。
【００１１】
図３の下図は、本発明の利用形態であり、ＤＮＡ回収流路３０２の先に、検出部３０４が取り付けられている。検出部３０４はＰＣＲやＬＡＭＰチャンバーや、ＤＮＡチップが考えられる。また、各種電気泳動カラムであってもよい。電気泳動を行う場合は、電気泳動開始時に、プラグ状にサンプルを開始点につめる必要がある。通常、十字路などを用いてこれを実現するが、本発明によりリリースされたＤＮＡはプラグ状に固まってリリースされるため、この作業を省略してもよい。また、検出部３０４は、検出以外の後処理を行う要素に置き換えられる。
【００１２】
図１８は、図３下段の構成に、液体供給装置１８０１を付加したものである。圧力により入り口２０２から出口２０３へ、ＤＮＡが含まれた液を搬送し、同時に入り口２０２に正、出口２０２に負の電圧を加える。これにより入り口２０２の液のうちＤＮＡだけがトラップ機構２０１にトラップされる。十分ＤＮＡがトラップされた後、ＤＮＡが圧力流れから受ける力と、電界から受ける力を保ったまま、入り口２０２からの液の供給を中止し、液体供給装置１８０１から、交換液リザーバ１８０２に格納されている交換液を供給することにより、ＤＮＡをトラップしたまま、ＤＮＡの周囲の液を交換できる。また、交換液を必要な時間連続して流すことにより、交換液によるＤＮＡの処理や、ＤＮＡの洗浄を行うことができる。その後、電界を強める、或いは圧力を弱めるまたは切る或いは逆転することにより、液交換し、処理あるいは洗浄されたＤＮＡを回収し、同様に次の処理に移すことが可能である。
【００１３】
図４は、本発明トラップ機構を構成する流路１０１の流路幅の狭い部分と流路幅の広い部分を実現するさまざまな形態であり、上段の図は、単純に幅の異なる流路を連結することに実現される。幅は、チップの水平方向でもよいし、深さ方向でも、その両方でもよい。中段の図は、流路の幅を変える代わりに、流路壁４０１内にビーズなど障害物４０２の詰め物をすることにより、トラップ機構を構成する。図４の下段の図は、多孔質の膜や樹脂等の多孔質物質４０３を流路壁４０１内に形成することによりこれを実現した例である。光硬化樹脂などを用いることにより簡単に実現できる。
【００１４】
図５は、本トラップ機構を複数並列に接続するための形態であり、スループットやトラップ量を向上することが可能である。上段の２つは、平面上に複数集積化する方法の形態であり、本トラップ機構は非常に微小なため、集積化により１００００倍程度の能力向上は容易に得られる。また、下段は、板状のものにあけられた孔とそれに隣接する空間によりトラップ機構を実現するものであり、さらに高い集積度が簡単に得られる。
【００１５】
図６は、トラップできるサイズの異なるトラップ機構６０１を直列に接続路６０２を介して、あるいは介さずに直接、直列に接続した形態であり、これにより異なるサイズのＤＮＡを異なるトラップ機構にトラップすることができる。これにより簡単にサイズによる分離が実現でき、分析やＤＮＡのフィルタリングに応用できる。接続路６０１に回収路６０３を接続した場合は、サイズごとに異なるＤＮＡを分けて回収したり、後処理を行うことが可能である。
【００１６】
図７は、本機構を用いたＤＮＡ分析装置の構成である。入り口７０１に注入された微量な血液、血清、大腸菌のような検体を含む液は、前処理部７０２により、酵素の非活性化処理、アルカリや酵素を用いた細胞壁やウイルス壁の破砕が行われる、次に既に述べた様々な形態の本発明を利用したＤＮＡトラップ機構７０３に注入され、破砕された細胞壁や淡白質、イオン類からＤＮＡのみを濃縮し、回収する。リリースされたＤＮＡは検出部７０４により選択的検出される。検出部７０４はＰＣＲ法やＬＡＭＰ法や電気泳動カラムなどが考えられる。これにより、血液や細胞、菌類、或いは、血清中に薄まったウイルス中のＤＮＡを濃縮して、チップ上で、高感度に検出可能である。
【００１７】
【実施例】
〔第一の実施例〕
図８は、本発明を用いてＤＮＡをトラップした実施例のひとつである。ＹＯＹＯ１で染色したＤＮＡ８０１は、蛍光顕微鏡で１分子観察が可能である。ここではＴ４−ＤＮＡ（大きさ１６０ｋＢＰ）あるいはλ−ＤＮＡ（大きさ４８ｋＢＰ）を用いた。ＤＮＡは、０．５ＴＢＥバッファーに、メルカプトエタノール、グルコースオキシダーゼ、カタラーゼ、グルコースと一緒に含まれており、溶存酸素によって観察中にＤＮＡが切れるのを抑えてある。また電気浸透流をおさえるために、ポリビニルピロリドンが含まれている。電源８０２により電圧を印加する。圧力計８０３は、シリンジ８０４を用いて加えた圧力を計測する。白金線８０５を用いて入り口と出口に電圧を印加する。８０６はガラス板であり、８０７はシリコンゴムであり、両者は入り口と出口を密閉するために用いられる。８０８は、石英製のチップであり、図１のようなクサビ型が連続したようなトラップ機構が、図２のように配置されている。クサビの最も細い部分は０．６ミクロンであり、太い部分は５ミクロン、クサビの周期は５０ミクロン、繰り返し回数は、８回である。深さは０．５ミクロンである。８０９はレンズであり、ＤＮＡの様子を拡大観察する。８１０はダイクロイックミラー、８１１はミラー、８１３は励起光（４９０ｎｍ）であり、蛍光観察のための光学系である。ＤＮＡからの蛍光を８１４、高感度ＣＣＤカメラ８１２によって観察する。
【００１８】
図９は、まず圧力だけをかけてＤＮＡを泳動させた例であり、図は圧力差およそ４０Ｐａ以下のものであるが、速度に差は見られたが、このように小さな圧力差であっても、大きいＤＮＡ（Ｔ４）も、小さいＤＮＡ（そのフラグメント）もトラップされなかった。圧力差が大きくなるにつれ、ＤＮＡは益々容易にこの流路を通過した。これはクサビの方向を逆にしても同様であった。これは、後述されるＤＮＡのトラップの後のリリースに利用できる。
【００１９】
図１０は、電場のみをかけてＤＮＡを泳動した例である。この図はもっとも電圧が低い（０．１Ｖ以下）の例であるが、ＤＮＡは簡単にこの流路を通過した。より大きい、電圧差では、更に容易にこの流路を通過した。これはクサビの方向を逆にしても同様であった。これは、後述するトラップされたＤＮＡのリリースに利用できる。
【００２０】
図１１は、クサビの方向に対して、電界がＤＮＡに及ぼす力が、電界による力の方向１１０１になるように電圧を６Ｖ印加し、圧力流れによる力の方向１１０２に圧力を５ｋＰａ印加した場合の例である。このとき、Ｔ４　ＤＮＡは、図中ＤＮＡ１０２で表示された位置にトラップされ、長時間１０分ほどたっても動かなかった。また、トラップされたＤＮＡは電界を切ると圧力流れによる力の方向１１０２の方向に直ちに、リリースされた。
【００２１】
図１２は、図１１におけるＴ４−ＤＮＡのトラップが起こる電圧と圧力の条件範囲である。１２０１はトラップの起こる範囲（斜線部）であり、この範囲の中の条件で、Ｔ４ＤＮＡが確実にトラップされた。およそ３ｋＰａ以上の領域で、圧力流とつりあう電圧の付近でトラップが見られる。トラップが起こる電圧の範囲は、圧力が増加するにつれ、広くなった。また、Ｔ４−ＤＮＡよりも小さいＤＮＡは、この条件のすこし内側で、リリースされ、トラップにはサイズ依存性があることが示された。
【００２２】
図１３は、図１１において、圧力と電圧の向きを両方とも反転した場合のトラップがおきる範囲である。図１２と同様に、圧力が増加するにつれ、電圧のトラップ範囲はひろくなった。
【００２３】
図１２及び図１３中の実線は、ＤＮＡに及ぼす、圧力流れからの力と、電界からの力が、つりあう条件である。このことから、本トラップは、ＤＮＡに及ぼす力において、圧力による力と電界による力が、つりあう条件の３０％から１７０％の範囲で起こり、形状とＤＮＡ分子によって決まるある閾値よりも強い力を印加する必要があることが判る。
【００２４】
図１４は、図１２において、トラップ領域の下側における、大、小ＤＮＡの移動を示したものである。この図から、このような形状において、圧力と電場を両方逆向きに印加した場合は、泳動速度に著しいサイズ依存性がみられた。トラップ領域の上側においても同様な著しいサイズ依存性がみられた。これは、ゲルやキャピラリーを用いた電気泳動法に代わる、ＤＮＡサイズ分離法である。
【００２５】
図１５、図１６は圧力場、電場のみの場合において、図１４と同様なプロットを行ったものであるが、圧力場、電場のみの場合はサイズによる泳動速度の差はほとんどないことが判る。
【００２６】
クサビ型の、最も狭い部分と、広い部分の大きさを変えて、トラップを行った。広い部分は、トラップされているＤＮＡの大きさからは十分無限大とおもわれる１００ミクロンを越えても問題なくトラップできることが判った。狭い部分を変化させると、同じ圧力、電場においても、トラップできるＤＮＡのサイズが変化し、０．６ミクロンでは、１０００ｂｐ以上のＤＮＡがトラップされ、０．３ミクロンでは５００ｂｐ以上のＤＮＡがトラップされた。また、５０ミクロンのものでは、ＤＮＡのトラップは見られなかった。この結果より、小さいＤＮＡをトラップするには、最も狭い部分のサイズが小さい方が有利で、また狭い部分の大きさや電圧や圧力の大きさを最適化することにより、特定の大きさ以上のＤＮＡをトラップできることがわかる。また、ＤＮＡをトラップするのに有効な狭い部分の幅は、ＤＮＡの大きさから考えて、０．０１ミクロンから５０ミクロンの間と考えられる。
【００２７】
〔第二の実施例〕
次に第２の実施例を示す。第一の実施例と同様に図８に示した蛍光観察装置を用いる。ここで、泳動チップ８０８は図１８に示すように、入り口３０１と出口２０３の他に、ＤＮＡ回収流路３０２、ＤＮＡ回収口３０３と、液体供給装置１８０１、交換液リザーバー１８０２を付加したものを用いる。トラップ機構２０１は、第１の実施例と同様にクサビの最も細い部分は０．６ミクロンであり、太い部分は５ミクロン、クサビの周期は５０ミクロン、繰り返し回数は、８回である。深さは０．５ミクロンである。０．５ＴＢＥバッファーに、メルカプトエタノール、グルコースオキシダーゼ、カタラーゼ、グルコースを付与し溶存酸素によって観察中にＤＮＡが切れるのを抑え、また電気浸透流をおさえるために、ポリビニルピロリドンを付与した、緩衝液を用意する。これを緩衝液Ａとする。まず流路全体を緩衝液Ａで満たす。実施例一と同様に調製したＤＮＡ溶液に、表面にＣＯＯＨ基を付与したポリスチレンビーズを混入した溶液を、入り口３０１に入れる。交換液リザーバ１８０２には、緩衝液Ａを入れる。入り口３０１には、圧力を印加するシリンジと白金電極を実施例一と同様に接続する。と出口２０３には、白金電極を接続する。交換液リザーバ１８０２と、ＤＮＡ回収口３０３には、それぞれ、圧力を印加するシリンジを接続する。この４つの接続は実施例一と同様にシリコーンゴムでそれぞれ密閉されている。
【００２８】
まず、入り口３０１に８ｋＰａの圧縮の圧力を加えると同時に、出口２０３と入り口３０１の間に１０Ｖの電圧を、出口２０３側が負になるように加える。この時、液体供給装置１８０１、ＤＮＡ回収流路にはＤＮＡが流れないように、交換液リザーバ１８０２、ＤＮＡ回収口３０３に印加する圧力を調整する。入り口３０１にいれた、ＤＮＡと溶液とポリスチレンビーズは、次々とトラップ機構２０１に流れ込み、ＤＮＡはトラップされ、濃縮されるが、ビーズは出口２０３へ流れ去った。十分ＤＮＡがトラップされた後、交換液リザーバへ印加する圧力を増加し、入り口３０１に印加する圧力を弱めると、入り口３０１からのＤＮＡとポリスチレンビーズの供給が止まり、トラップされたＤＮＡは交換液リザーバーからの液体にさらされた。これにより、トラップ機構に存在するポリスチレンビーズは完全に出口２０３へ流れ去り、ＤＮＡは洗浄された。次に、印加している電界と圧力を同時に０にし、ＤＮＡ回収口３０３に引っ張りの圧力を加える。この時、入り口３０１と交換液リザーバ１８０２から液体が流出しないような引っ張りの圧力を加える。トラップされたＤＮＡはＤＮＡ回収流路３０２を通って、ＤＮＡ回収口に回収された。以上により、ＤＮＡとポリスチレンビーズの混合物から、ＤＮＡのみを濃縮して抽出し、洗浄し、回収することができた。
【００２９】
図１７はトラップ力の説明である。１７０１は電場による力であり、ＤＮＡが電場から受ける力は壁面からの距離によらず一定である。これに対して、１７０２は圧力流による力であり、ＤＮＡにが圧力流からうける力は、壁面付近が小さく、中央が大きい。従って、最も狭い部分近辺では、壁面付近では電場による力が強くなり、中央部１７０３では非常に強い圧力による力ができる。ここを流れる粒子状のものは、壁面からすり抜けることが可能であるが、ＤＮＡのような長い分子は、すり抜ける過程で、逃げ出そうとするＤＮＡ１７０４のように長い分子のどこかが中央の流れに引きずられトラップ部に戻される。これによって、長い分子のみトラップされる。
【００３０】
トラップ中のＤＮＡを詳細に観察すると、図１７で示すように運動しながらトラップされるＤＮＡが観察されこのトラップ力の説明が裏付けられる。
【００３１】
【発明の効果】
以上に述べたとおり、本発明によるＤＮＡトラップ機構により、液体よりＤＮＡ、或いはＤＮＡを含む長い分子のみをトラップし、必要に応じてリリースすることが可能である。これにより、チップ上で、ＤＮＡを取り出すための前処理の溶液からＤＮＡのみを回収する機構、ＤＮＡを残してバッファーや溶液を交換する作業、あるいは、非常に薄まったＤＮＡを濃縮しＰＣＲやＬＡＭＰ法、ＤＮＡチップなどの検出感度を上げたり、チップ上で扱いやすい液量に調整したりすることが容易になる。これにより、血液中の白血球や、ウイルス、病原体のＤＮＡの分析や診断が容易になり、また、抗体検出ではなく抗原を検出することにより病気の診断がより早く、正確になる効果がある。また、トラップ機構や泳動速度のサイズ依存性を利用した、新しい、ＤＮＡ分離法や、ＤＮＡフィルタリング法、スクリーニング法も容易に構成できる。また、ＤＮＡを利用した蛋白質の合成や、ＤＮＡを用いた分析法、ＤＮＡを用いたデバイスの開発において、溶液中のＤＮＡのみを一時的に固定できることは、溶液の交換や、ＤＮＡのマニュピレーション、ＤＮＡの観察を容易にし、あらゆる波及効果が期待できる。本発明は、ＤＮＡに特化して説明を行ったが、同様の特性をもつ、ＲＮＡや長鎖線状分子に応用できることは容易に推測できる。
【図面の簡単な説明】
【図１】本発明によるトラッピング機構の図
【図２】本発明を利用する形態の基本
【図３】本発明の利用形態の応用例
【図４】本発明のトラッピング機構の構成例
【図５】本発明のトラッピング機構を複数配置して効果をあげる方法
【図６】効果の異なる本発明を直列に接続して分離を行う方法
【図７】本発明を利用して血液等を分析する構成
【図８】本発明を実施するための装置構成例
【図９】実施例における圧力場のみでのＤＮＡの泳動速度
【図１０】実施例における電場のみでのＤＮＡの泳動速度
【図１１】実施例における圧力場と電場を両方かけた場合のＤＮＡのトラッピング
【図１２】実施例におけるトラッピングが起こる電圧と圧力の範囲
【図１３】実施例における逆接続でのトラッピングが起こる電圧と圧力の範囲
【図１４】実施例における圧力場と電場を両方かけた場合のＤＮＡの移動距離と時間の関係
【図１５】実施例における圧力場のみの場合のＤＮＡの移動距離と時間の関係
【図１６】実施例における電場のみの場合のＤＮＡの移動距離と時間の関係
【図１７】現在のトラッピング現象の説明図
【図１８】本発明に液体供給装置を付加した応用例
【符号の説明】
１０１　流路
１０２　ＤＮＡ
１０３　圧力流れによる力の方向
１０４　電界による力の方向
１０５　流路幅の広い部分
１０６　流路幅の狭い部分
２０１　本発明によるトラッピング機構本体
２０２　入り口（リザーバ）
２０３　出口（ウエステ）
２０４　流路
３０１　入り口（リザーバ）
３０２　ＤＮＡ回収流路
３０３　ＤＮＡ回収口
３０４　ＤＮＡ検出機構
４０１　流路壁
４０２　ビーズなど障害物
４０３　多孔質物質
５０１　入り口
５０２　出口
６０１　効果の異なるＤＮＡトラップ機構本体
６０２　接続路（必要なときのみ）
６０３　分離したＤＮＡ回収路（必要に応じて）
７０１　血液などサンプル入り口
７０２　細胞壁やウイルス壁を壊す前処理部
７０３　本発明によるトラップ機構による濃縮器
７０４　特定のＤＮＡの検出部（ＰＣＲチャンバーなど）
８０１　ＹＯＹＯ１で染色したＤＮＡ溶液
８０２　電源
８０３　圧力計
８０４　シリンジ
８０５　白金線
８０６　ガラス板
８０７　シリコンラバー
８０８　泳動チップ
８０９　対物レンズ
８１０　ダイクロイックミラー
８１１　ミラー
８１２　ＣＣＤカメラ
８１３　励起光
８１４　蛍光
１１０１　電界による力の方向
１１０２　圧力流による力の方向
１２０１　トラップの起こる範囲（斜線部）
１７０１　電場による力
１７０２　圧力流による力
１７０３　中央部
１７０４　逃げ出そうとするＤＮＡ
１８０１　液体供給装置
１８０２　液体供給リザーバ[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention separates, extracts, and concentrates only leukocytes, pathogens, and viral DNAs contained in blood and the like to determine health conditions and to perform early detection of infectious diseases, early treatment, and tests during treatment. Further, the present invention relates to a separation means for performing a diagnosis based on DNA information and an apparatus therefor. In particular, a part of the necessary functions and structures are integrated on a single plate-shaped chip, and the required sample is small, and microfluidics and micro- TAS, a field called Lab-on-a-Chip.
[0002]
[Prior art]
Techniques for analyzing DNA contained in tissues, blood, and the like extracted from the human body are very important. Leukocytes and somatic cells provide information on the individual's genetic information, which is important for diagnosis of lifestyle-related diseases and genetic diseases, and for personalized medicine. In addition, by extracting and specifying DNA of pathogenic bacteria and viruses contained in blood and the like, an infectious disease can be diagnosed.
[0003]
As a method for specifying purified DNA, a method that amplifies only DNA having a specific sequence using a DNA chip, a PCR method, or a LAMP method to detect DNA with high sensitivity and high selectivity is used. Is possible in principle. To use such a detection method, substances that are harmful to DNA are excluded from the liquid that may contain the collected cells or virus, and the DNA is extracted by crushing the cell membrane and virus wall, and the subsequent reaction is performed. It is necessary to perform a pretreatment for adjusting a DNA concentration, a salt concentration, and a pH suitable for the PCR and the LAMP method by excluding a substance that inhibits the detection or the detection. The pretreatment consists of repeating the mixing of the reagents, the reaction, and the DNA recovery process. Examples of the form of the recovery treatment include concentration and separation of DNA, exchange of a solution in which only DNA is left, and washing of DNA by exchange of a solution. That is, it is necessary to collect only the DNA from the solution containing the inclusions as selectively as possible at one place and collect it separately from the other inclusions. If the replacement or cleaning of the solution can be realized by moving the solution or conversely, the pretreatment can be realized more easily. As a method for recovering such DNA, there are a centrifugal separation method, a method in which magnetic beads with DNA entangled using magnetic beads are moved by a magnet, and the like.
[0004]
On the other hand, for the diagnosis of viral diseases, a method is currently used in which immunity is responded to a virus and the produced antibody is detected. This is because the virus in the blood is so small that it is difficult to detect it. However, this method has a disadvantage that diagnosis cannot be performed during the period from infection to the time when immunity responds and antibodies are formed. In addition, even if the virus is removed, such as hepatitis B, only the antibody remains, and in severely ill patients, even if there is a new infection, the antibody cannot be produced, resulting in misdiagnosis. If a small amount of virus-derived DNA in the blood can be concentrated, these antigens can be directly detected, which greatly contributes to early detection and early treatment. At present, concentration is attempted using a method in which DNA is fixed on the surface of a specific material such as quartz and washed away, but there is still no material that satisfies this required level.
[0005]
[Problems to be solved by the invention]
Centrifugation, which is used in DNA recovery processing, depends on the work of manually separating the supernatant, the intermediate layer, and the precipitate, and is difficult to apply to a minute amount of sample. In recent years, the technical fields of microTAS and Lab-on-a-Chip are starting up, and by integrating conventional analysis techniques and chemical synthesis methods on a single chip, the system can be reduced in size, necessary reagents and It realizes reduction of sample volume, lower cost, higher speed, and higher functionality. Using this technique, it is expected that DNA analysis from a drop of blood, several cells or viruses is possible. However, to achieve this, instead of centrifugation, a DNA recovery technique that can be realized in a chip is required. The method of entanglement with magnetic beads can be realized on a chip, but the movement of a magnetic field is complicated, and the collected DNA may be released again into a solution (ie, release) so that the next processing can be performed. Lack of generality, such as not being released with a simple buffer. In pretreatment of DNA for the purpose of diagnosis, as described above, there is still no method capable of concentrating at a level capable of detecting a virus in the blood as an antigen. At the cell and bacterial level, various techniques have been developed, but they are cumbersome, unsuitable for complete chip formation, or sacrificing sensitivity. Also, drug kits have been developed in which the pretreatment can be completed simply by mixing the cells with the solution containing the cells without concentrating or recovering the DNA, but the sensitivity and precision are higher than those of the purification method. bad.
[0006]
To meet these demands, DNA or similar substances are selectively captured and retained (ie, trapped) from liquids containing proteins and various reagents flowing through the flow channel using a simple mechanism formed on the chip. There is not yet an inexpensive and simple principle or apparatus that can recover DNA or concentrate DNA, release the trapped DNA by a simple operation, and use it for the next processing.
[0007]
[Means for Solving the Problems]
FIG. 1 is an illustration of means for solving the problem. A solution containing DNA 102 is caused to flow through the channel 101. A pressure difference and an electric field are simultaneously applied to the solution. That is, in the DNA 102 in the solution, the force in the direction indicated by the force flow direction 103 and the force in the direction indicated by the electric field force 104 are opposite to each other as shown in FIG. Work at the same time. The flow channel 101 has one or more wide channel width portions 105 and one or more narrow flow channel width portions 106. Although the specific shape and the direction of the flow channel 101 are various as described later, the DNA 102 is selectively trapped in the vicinity of the narrow portion by adjusting the intensity of the electric field and the pressure flow. be able to. By adjusting the strength of the electric field and the pressure, the flowing DNA can be trapped one after another. Substances in the solution other than those trapped at this time flow off due to the electric field or the pressure flow, so that recovery and concentration of DNA can be realized. Further, since the solution is flowing while the DNA is trapped, the solution can be used for exchange and washing. The narrow portion has a cross-sectional area of 0.01 to 50 microns, and the wide portion has twice or more the cross-sectional area of the narrow portion, depending on the size of the DNA to be trapped. The trapping ability of the present invention depends on the size of DNA. The size of the DNA that can be trapped can be changed by changing the diameter or shape of the flow path or changing the voltage or pressure.
[0008]
The trapped DNA is released to the entrance side or the exit side by increasing or decreasing either the electric field or the pressure. Thereby, it can be easily released and collected, and can be used for the next processing.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
FIG. 2 shows the most basic form for trapping and releasing DNA according to the present invention. The trap mechanism 201 is a channel having a wide portion and a narrow portion according to the present invention. An entrance (reservoir) 202 is an entrance for DNA, into which a liquid containing DNA is put. The liquid flows to the outlet (wester) 203 through the flow path 204 and the trap mechanism 201. Pressure is applied using a tube connected to a pressure source from one or both of the inlet 202 and outlet 203. An electric field is simultaneously applied to the entrance 202 and the exit 203 using electrodes or the like. The liquid is transported from the inlet 202 to the outlet 203 by pressure, and at the same time, a positive voltage is applied to the inlet 202 and a negative voltage is applied to the outlet 202. As a result, only the DNA of the liquid at the entrance 202 is trapped by the trap mechanism 201. After the liquid at the inlet 202 has mostly flowed to the outlet 203, by increasing the electric field, or reducing, turning off or reversing the pressure. The concentrated DNA can be taken out at the entrance 202 side. Alternatively, by increasing the pressure, or weakening, turning off, or inverting the electric field, the concentrated DNA can be taken out at the outlet 203 side.
[0010]
The upper part of FIG. 3 shows an embodiment of the present invention. In addition to FIG. 2, a concentrated DNA recovery channel 302 and a DNA recovery port 303 also serving as a reservoir for adding an electrode are provided on the entrance 301 side. . The DNA flows from the entrance 301 to the trap mechanism 201 by pressure, and a positive voltage is applied to the DNA recovery port 303 and a negative voltage is applied to the exit 203. After sufficient trapping, by reducing the pressure, the concentrated DNA can be recovered to the DNA recovery channel 302 and the DNA recovery port 303 via the channel 204. Even if the collection path is on the exit 203 side, the collection can be performed similarly.
[0011]
The lower diagram of FIG. 3 shows a use form of the present invention, in which a detection unit 304 is attached at the end of the DNA recovery channel 302. The detection unit 304 may be a PCR or LAMP chamber, or a DNA chip. Moreover, various electrophoresis columns may be used. When performing electrophoresis, it is necessary to pack a sample at the starting point in a plug shape at the start of electrophoresis. Usually, this is achieved using a crossroad or the like, but since the DNA released according to the present invention is released in the form of a plug, it may be omitted. The detection unit 304 is replaced with an element that performs post-processing other than detection.
[0012]
FIG. 18 is obtained by adding a liquid supply device 1801 to the configuration in the lower part of FIG. The liquid containing DNA is conveyed from the inlet 202 to the outlet 203 by pressure, and at the same time, a positive voltage is applied to the inlet 202 and a negative voltage is applied to the outlet 202. As a result, only the DNA of the liquid at the entrance 202 is trapped by the trap mechanism 201. After the DNA has been sufficiently trapped, the supply of liquid from the inlet 202 is stopped while the DNA receives the force from the pressure flow and the force from the electric field, and is stored in the exchange liquid reservoir 1802 from the liquid supply device 1801. By supplying the exchange solution, the solution around the DNA can be exchanged while the DNA is trapped. Further, by continuously flowing the exchange solution for a required time, the treatment of DNA with the exchange solution and the washing of DNA can be performed. Thereafter, by increasing the electric field, or decreasing, turning off, or reversing the pressure, the solution can be exchanged, the treated or washed DNA can be collected, and the DNA can be similarly transferred to the next treatment.
[0013]
FIGS. 4A and 4B show various forms for realizing a narrow portion and a wide portion of the flow path 101 of the flow path 101 constituting the trap mechanism of the present invention. The upper diagram simply shows flow paths having different widths. It is realized by connecting. The width may be in the horizontal direction of the chip, in the depth direction, or both. In the middle diagram, instead of changing the width of the flow path, a trap mechanism is configured by filling the flow path wall 401 with an obstacle 402 such as a bead. The lower part of FIG. 4 shows an example in which this is realized by forming a porous substance 403 such as a porous film or a resin in the flow path wall 401. It can be easily realized by using a photocurable resin or the like.
[0014]
FIG. 5 shows a mode for connecting a plurality of the trap mechanisms in parallel, and it is possible to improve the throughput and the trap amount. The upper two are forms of a method of integrating a plurality of elements on a plane. Since the trap mechanism is very small, a capacity improvement of about 10,000 times can be easily obtained by integration. Further, the lower stage realizes a trapping mechanism by a hole formed in a plate-like material and a space adjacent thereto, and a higher degree of integration can be easily obtained.
[0015]
FIG. 6 shows a configuration in which trap mechanisms 601 of different sizes that can be trapped are connected in series via a connection path 602 directly or directly, without using a connection path, thereby trapping DNAs of different sizes into different trap mechanisms. Can be. As a result, separation by size can be easily realized and applied to analysis and filtering of DNA. When the collection path 603 is connected to the connection path 601, it is possible to collect and collect different DNAs for each size or to perform post-processing.
[0016]
FIG. 7 shows the configuration of a DNA analyzer using this mechanism. The liquid containing a trace amount of blood, serum, or a sample such as Escherichia coli injected into the entrance 701 is subjected to a deactivation treatment of an enzyme and a crush of a cell wall or a virus wall using an alkali or an enzyme by a pretreatment unit 702. Next, the DNA is injected into the DNA trap mechanism 703 utilizing various forms of the present invention described above, and the DNA is concentrated and recovered only from the cell walls, white matter, and ions that are crushed. The released DNA is selectively detected by the detection unit 704. The detection unit 704 may be a PCR method, a LAMP method, an electrophoresis column, or the like. Thus, DNA in blood, cells, fungi, or virus diluted in serum can be concentrated and detected on a chip with high sensitivity.
[0017]
【Example】
[First embodiment]
FIG. 8 shows an example of trapping DNA using the present invention. DNA 801 stained with YOYO1 can be observed for one molecule with a fluorescence microscope. Here, T4-DNA (size 160 kBP) or λ-DNA (size 48 kBP) was used. DNA is contained in 0.5 TBE buffer together with mercaptoethanol, glucose oxidase, catalase, and glucose to prevent DNA from being cut during observation due to dissolved oxygen. Further, polyvinylpyrrolidone is contained to suppress the electroosmotic flow. A voltage is applied by the power supply 802. The pressure gauge 803 measures the pressure applied using the syringe 804. A voltage is applied to the entrance and the exit using a platinum wire 805. 806 is a glass plate and 807 is a silicone rubber, both of which are used to seal the entrance and the exit. Reference numeral 808 denotes a quartz chip, in which a trapping mechanism such as a continuous wedge type as shown in FIG. 1 is arranged as shown in FIG. The thinnest part of the wedge is 0.6 microns, the thick part is 5 microns, the period of the wedge is 50 microns, and the number of repetitions is eight. The depth is 0.5 microns. Reference numeral 809 denotes a lens for observing the state of DNA in an enlarged manner. 810 is a dichroic mirror, 811 is a mirror, 813 is excitation light (490 nm), and is an optical system for fluorescence observation. The fluorescence from the DNA is observed at 814 by a high-sensitivity CCD camera 812.
[0018]
FIG. 9 shows an example in which DNA was first electrophoresed by applying only pressure. In the figure, although the pressure difference was about 40 Pa or less, there was a difference in speed. Neither large DNA (T4) nor small DNA (fragment thereof) was trapped. As the pressure difference increased, the DNA passed more easily through this channel. This was the same even if the wedge direction was reversed. This can be used for subsequent release of the trap of DNA described below.
[0019]
FIG. 10 shows an example in which DNA is migrated by applying only an electric field. In this figure, the voltage is the lowest (0.1 V or less), but DNA easily passed through this flow path. The larger, the voltage difference, passed through this channel more easily. This was the same even if the wedge direction was reversed. This can be used for the release of the trapped DNA described below.
[0020]
FIG. 11 shows a case where a voltage of 6 V is applied so that the force exerted on the DNA by the electric field is in the direction 1101 of the force of the electric field with respect to the direction of the wedge, and the pressure is 5 kPa in the direction 1102 of the force of the pressure flow. It is an example. At this time, T4 DNA was trapped at the position indicated by DNA 102 in the figure, and did not move even after a long time of about 10 minutes. Also, the trapped DNA was released immediately in the direction of force flow 1102 due to the pressure flow when the electric field was turned off.
[0021]
FIG. 12 shows the condition range of the voltage and pressure at which trapping of T4-DNA in FIG. 11 occurs. Reference numeral 1201 denotes a range in which trapping occurs (shaded area), and T4 DNA was reliably trapped under the conditions in this range. In the region above about 3 kPa, traps are seen near the voltage that balances the pressure flow. The range of voltages at which trapping occurred widened as pressure increased. Also, DNA smaller than T4-DNA was released slightly inside this condition, indicating that the trap is size dependent.
[0022]
FIG. 13 shows a range in which traps occur when the directions of the pressure and the voltage are both reversed in FIG. As in FIG. 12, as the pressure increased, the voltage trap range became wider.
[0023]
The solid line in FIG. 12 and FIG. 13 shows the condition where the force exerted on the DNA from the pressure flow and the force from the electric field balance. For this reason, in this trap, a force due to pressure and a force due to electric field occur in the range of 30% to 170% of the equilibrium condition, and a force higher than a certain threshold determined by the shape and the DNA molecule is applied to the DNA. You need to do it.
[0024]
FIG. 14 shows the movement of large and small DNAs below the trap region in FIG. From this figure, in such a shape, when both the pressure and the electric field were applied in opposite directions, a remarkable size dependency was observed in the migration speed. Similar remarkable size dependence was observed above the trap region. This is a DNA size separation method that replaces the electrophoresis method using a gel or a capillary.
[0025]
FIGS. 15 and 16 show plots similar to those in FIG. 14 in the case of only the pressure field and the electric field, but it can be seen that there is almost no difference in migration speed depending on the size in the case of only the pressure field and the electric field.
[0026]
The trap was performed while changing the size of the narrowest part and the wide part of the wedge type. It has been found that a wide portion can be trapped without any problem even if it exceeds 100 microns, which is considered to be sufficiently infinite from the size of the trapped DNA. When the narrow portion was changed, the size of the trappable DNA was changed even at the same pressure and electric field. At 0.6 micron, DNA of 1000 bp or more was trapped, and at 0.3 micron, DNA of 500 bp or more was trapped. . No DNA trap was observed in the case of 50 microns. From this result, in order to trap small DNA, it is advantageous that the size of the narrowest part is smaller, and by optimizing the size of the narrow part, the voltage and the pressure, the DNA of a specific size or more can be trapped. Can be trapped. In addition, the width of the narrow portion effective for trapping DNA is considered to be between 0.01 μm and 50 μm in view of the size of DNA.
[0027]
[Second embodiment]
Next, a second embodiment will be described. The fluorescence observation apparatus shown in FIG. 8 is used as in the first embodiment. Here, as shown in FIG. 18, the electrophoresis chip 808 has a DNA recovery channel 302, a DNA recovery port 303, a liquid supply device 1801, and an exchange liquid reservoir 1802 in addition to the entrance 301 and the exit 203. . In the trap mechanism 201, as in the first embodiment, the thinnest part of the wedge is 0.6 microns, the thick part is 5 microns, the period of the wedge is 50 microns, and the number of repetitions is eight. The depth is 0.5 microns. To 0.5 TBE buffer, a buffer solution prepared by adding mercaptoethanol, glucose oxidase, catalase, and glucose to suppress DNA breakage during observation due to dissolved oxygen, and to add polyvinylpyrrolidone to suppress electroosmotic flow is prepared. I do. This is designated as buffer A. First, the entire flow path is filled with the buffer solution A. A solution obtained by mixing polystyrene beads having a surface with a COOH group on the DNA solution prepared in the same manner as in Example 1 is put into the entrance 301. The buffer solution A is put in the exchange solution reservoir 1802. A syringe for applying pressure and a platinum electrode are connected to the entrance 301 in the same manner as in the first embodiment. And an outlet 203, a platinum electrode is connected. A syringe for applying pressure is connected to each of the exchange solution reservoir 1802 and the DNA recovery port 303. These four connections are sealed with silicone rubber as in the first embodiment.
[0028]
First, a compression pressure of 8 kPa is applied to the entrance 301, and at the same time, a voltage of 10 V is applied between the exit 203 and the entrance 301 so that the exit 203 side becomes negative. At this time, the pressure applied to the exchange liquid reservoir 1802 and the DNA recovery port 303 is adjusted so that DNA does not flow in the liquid supply device 1801 and the DNA recovery channel. The DNA, the solution, and the polystyrene beads at the entrance 301 flowed one after another into the trap mechanism 201, where the DNA was trapped and concentrated, but the beads flowed out to the exit 203. After the DNA is sufficiently trapped, the pressure applied to the exchange liquid reservoir is increased and the pressure applied to the inlet 301 is reduced, and the supply of DNA and polystyrene beads from the inlet 301 stops, and the trapped DNA is removed from the exchange liquid reservoir. Exposure to liquid from. Thereby, the polystyrene beads existing in the trap mechanism completely flowed to the outlet 203, and the DNA was washed. Next, the applied electric field and pressure are simultaneously reduced to 0, and a pulling pressure is applied to the DNA recovery port 303. At this time, a pulling pressure is applied so that the liquid does not flow out from the inlet 301 and the exchange liquid reservoir 1802. The trapped DNA passed through the DNA recovery channel 302 and was recovered at the DNA recovery port. As described above, from the mixture of DNA and polystyrene beads, only DNA was concentrated and extracted, washed, and recovered.
[0029]
FIG. 17 illustrates the trapping force. Reference numeral 1701 denotes a force by an electric field, and the force received by the DNA from the electric field is constant regardless of the distance from the wall surface. On the other hand, reference numeral 1702 denotes a force due to the pressure flow, and the force applied to the DNA from the pressure flow is small near the wall surface and large at the center. Therefore, in the vicinity of the narrowest part, the force due to the electric field becomes strong near the wall surface, and a very strong force is generated in the central portion 1703. Particles flowing here can slip through the wall, but long molecules such as DNA are dragged by the central flow during the process of slipping away, such as DNA 1704, which tries to escape. Returned to the trap section. As a result, only long molecules are trapped.
[0030]
When the DNA in the trap is observed in detail, the trapped DNA is observed while moving as shown in FIG. 17, which supports the explanation of the trapping force.
[0031]
【The invention's effect】
As described above, the DNA trapping mechanism according to the present invention can trap only DNA or a long molecule containing DNA from a liquid and release it as needed. This makes it possible to recover only the DNA from the pretreatment solution for removing the DNA on the chip, replace the buffer and the solution while leaving the DNA, or concentrate the extremely diluted DNA and use the PCR or LAMP method. In addition, it becomes easy to increase the detection sensitivity of a DNA chip or the like, or to adjust the amount of liquid to be easily handled on the chip. This facilitates the analysis and diagnosis of leukocytes, viruses, and DNA of pathogens in blood, and has the effect of diagnosing diseases faster and more accurately by detecting antigens rather than antibodies. Further, a new DNA separation method, a DNA filtering method, and a screening method utilizing the size dependence of the trapping mechanism and the migration speed can be easily configured. In addition, in the synthesis of proteins using DNA, the analysis method using DNA, and the development of devices using DNA, the ability to temporarily fix only DNA in a solution requires the exchange of solutions, manipulation of DNA, DNA Can be easily observed and all ripple effects can be expected. Although the present invention has been described specifically for DNA, it can be easily assumed that the present invention can be applied to RNA and long-chain linear molecules having similar characteristics.
[Brief description of the drawings]
FIG. 1 is a diagram of a trapping mechanism according to the present invention.
FIG. 2 is a diagram illustrating the basics of an embodiment utilizing the present invention.
FIG. 3 is an application example of a usage form of the present invention.
FIG. 4 is a configuration example of a trapping mechanism of the present invention.
FIG. 5 shows a method of increasing the effect by arranging a plurality of trapping mechanisms according to the present invention.
FIG. 6 is a diagram illustrating a method of connecting the present invention having different effects in series to perform separation.
FIG. 7 shows a configuration for analyzing blood and the like using the present invention.
FIG. 8 is an example of an apparatus configuration for implementing the present invention.
FIG. 9 shows the migration speed of DNA only in a pressure field in Examples.
FIG. 10 shows the migration rate of DNA in an electric field only in Examples.
FIG. 11 shows trapping of DNA when both a pressure field and an electric field are applied in Example.
FIG. 12 shows a range of voltage and pressure at which trapping occurs in the embodiment.
FIG. 13 shows the range of voltage and pressure at which trapping in reverse connection occurs in the embodiment.
FIG. 14 shows a relationship between a moving distance of DNA and a time when both a pressure field and an electric field are applied in the embodiment.
FIG. 15 shows a relationship between a moving distance of DNA and a time when only a pressure field is used in the embodiment.
FIG. 16 shows the relationship between the moving distance of DNA and time in the case of only an electric field in the embodiment.
FIG. 17 is an explanatory diagram of a current trapping phenomenon.
FIG. 18 is an application example in which a liquid supply device is added to the present invention.
[Explanation of symbols]
101 channel
102 DNA
103 Force direction due to pressure flow
104 Direction of force by electric field
105 Wide channel width
106 Narrow part of channel
201 Trapping Mechanism Main Body According to the Present Invention
202 entrance (reservoir)
Exit 203 (Wester)
204 channel
301 entrance (reservoir)
302 DNA recovery channel
303 DNA recovery port
304 DNA detection mechanism
401 Channel wall
402 Obstacles such as beads
403 porous material
501 entrance
Exit 502
601 DNA trap mechanism with different effects
602 connection path (only when necessary)
603 Separated DNA recovery path (if necessary)
701 blood sample inlet
702 Pretreatment unit that breaks cell wall and virus wall
703 Concentrator with trap mechanism according to the present invention
704 Detection part of specific DNA (PCR chamber etc.)
801 DNA solution stained with YOYO1
802 power supply
803 pressure gauge
804 syringe
805 platinum wire
806 glass plate
807 Silicon rubber
808 electrophoresis chip
809 Objective lens
810 dichroic mirror
811 mirror
812 CCD camera
813 Excitation light
814 fluorescence
1101 Direction of force by electric field
1102 Direction of force due to pressure flow
1201 Range where a trap occurs (shaded area)
1701 Force by electric field
1702 Force due to pressure flow
1703 Central part
1704 DNA trying to escape
1801 Liquid supply device
1802 Liquid supply reservoir

Claims (13)

液体を流すことができる流路において、少なくとも一つ以上の広い部分と狭い部分をもつ流路の中に、ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体を、圧力流による力と電界による力を同時に逆向きに印加して流し、広さの変化している部分の近くに当該線状分子の動きを一時的に制限（トラップ）し、その後必要に応じて、電界または圧力の大きさをトラップ条件からはずすことによって、一旦トラップした当該分子を速やかにあるいは計画的にリリースできることを特徴とするＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース装置。In a flow path through which a liquid can flow, in a flow path having at least one wide portion and a narrow portion, a liquid containing DNA or a linear molecule having an electric charge is applied by a force due to a pressure flow and an electric field. Simultaneously apply a force in the opposite direction and let it flow, temporarily restricting (trapping) the movement of the linear molecule in the vicinity of the changing area, and then, if necessary, the magnitude of the electric field or pressure Characterized in that the trapped molecule can be released quickly or systematically by removing the trapped molecule from the trap condition, thereby releasing a trapped / released molecule of a linear molecule having DNA or electric charge. 請求項１における広い部分と狭い部分をもつ流路が、幅或いは深さ或いはその両方、或いは直径が連続的に変化するクサビ型、或いはその繰り返しであることを特徴とするＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース装置。2. A line having a DNA or a charge, wherein the flow path having a wide portion and a narrow portion according to claim 1 is a wedge type in which a width, a depth, or both, or a diameter continuously changes, or a repetition thereof. -Like molecule trap and release device. 請求項２において、幅の狭い部分の幅が、０．０１ミクロンから５０ミクロンであることを特徴とするＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース装置。3. The trap and release device according to claim 2, wherein the width of the narrow portion is 0.01 to 50 microns. 請求項１から３のトラップ・リリース装置を、単独、或いは並列接続、或いは直列に接続し、液体の中に含まれるＤＮＡ或いは電荷をもつ線状の分子を、濃縮、又は分離、又は当該分子のみを残した溶液の交換、又は溶液の交換による当該分子の洗浄、又は当該分子の収集、又は観察や反応を目的とした当該分子の固定、又はサイズかトラップ力の違いによる当該分子の分離を行う、ＤＮＡ分析、ＤＮＡ精製、又はＤＮＡ処理装置。The trap / release device according to any one of claims 1 to 3, which is connected alone, in parallel, or connected in series, to concentrate or separate DNA or a linear molecule having a charge contained in a liquid, or to separate only the molecule. Exchange of the solution that left behind, washing of the molecule by exchanging the solution, collection of the molecule, fixation of the molecule for the purpose of observation or reaction, or separation of the molecule by difference in size or trapping force , DNA analysis, DNA purification, or DNA processing equipment. 請求項４において、血液、又は細胞、又は細菌、又はウイルスを分析する目的で、アルカリや酸、酵素などを用いて、細胞壁を破砕する前処理部と、ＰＣＲ法やＬＡＭＰ法や電気泳動法を用いた選択的検出部を組み合わせた、分析・診断装置。In claim 4, for the purpose of analyzing blood, cells, bacteria, or viruses, a pretreatment unit for crushing the cell wall using an alkali, acid, enzyme, or the like, and a PCR method, a LAMP method, or an electrophoresis method. An analysis / diagnosis device combining the used selective detection units. 請求項１において、トラップ条件を少しはずした、電圧や圧力の範囲で、ＤＮＡ或いは電荷をもつ線状の分子がサイズに大きく依存して泳動することを利用した、当該分子の分離装置。2. An apparatus according to claim 1, wherein the trapping conditions are slightly removed, and DNA or charged linear molecules migrate largely depending on the size within the range of voltage and pressure. ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体を流すことができる流路で、かつ、当該流路が少なくとも一つ以上の狭い部分と当該狭い部分に連結された当該狭い部分の断面積の２倍以上の断面積を有する広い部分を有する流路において、当該流路内において当該液体を移動させる圧力差を当該液体に印加することと、当該圧力差により当該液体が移動する方向と反対の方向に当該ＤＮＡ或いは電荷をもつ線状の分子を移動させる電界を当該液体に印加することを特徴とする当該ＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース方法。A flow path through which a liquid containing DNA or a linear molecule having an electric charge can flow, and the flow path has at least one or more narrow portions and a cross-sectional area of the narrow portion connected to the narrow portion. In a flow path having a wide portion having a cross-sectional area of twice or more, applying a pressure difference for moving the liquid in the flow path to the liquid, and opposing a direction in which the liquid moves due to the pressure difference. A method for trapping and releasing said DNA or charged linear molecules, wherein an electric field for moving said DNA or charged linear molecules in the direction is applied to said liquid. ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体を流すことができる流路において、当該流路が少なくとも一つ以上の狭い部分と当該狭い部分に連結された当該狭い部分の断面積の２倍以上の断面積を有する広い部分を有し、かつ、当該流路に連結する当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体とは異なる液体を当該流路に供給する液体供給装置を有し、当該流路内において当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体、あるいは当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体とは異なる液体を移動させる圧力差を当該液体に印加することと、当該圧力差により当該液体が移動する方向と反対の方向に当該ＤＮＡ或いは電荷をもつ線状の分子を移動させる電界を当該液体に印加することを特徴とする当該ＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース装置。In a flow path through which a liquid containing DNA or a linear molecule having a charge can flow, the flow path has at least one or more narrow portions and twice the cross-sectional area of the narrow portion connected to the narrow portion. A liquid supply device having a wide portion having the above-mentioned cross-sectional area and supplying a liquid different from the liquid containing the DNA or the linear molecule having an electric charge connected to the flow path to the flow path. Then, a pressure difference for moving a liquid containing the DNA or the linear molecule having the charge or a liquid different from the liquid containing the DNA or the linear molecule having the charge in the flow path is applied to the liquid. And D. applying said electric field to said liquid to move said DNA or linear molecules having charges in a direction opposite to the direction in which said liquid moves due to said pressure difference. Trap release device of the linear molecules with A or charge. 請求項７に記載のＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース方法において、当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体に印加する当該圧力差と当該電界により、当該ＤＮＡ或いは電荷をもつ線状の分子を当該流路内に停留させた後、当該流路内に当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体とは異なる液体を供給し、当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体とは異なる液体に当該流路内において当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体とは異なる液体を移動させる圧力差を印加し、かつ、当該圧力差により当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体とは異なる液体が移動する方向と反対の方向に当該ＤＮＡ或いは電荷をもつ線状の分子を移動させる電界を当該液体に印加することを特徴とする当該ＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース方法。8. The method for trapping and releasing DNA or charged linear molecules according to claim 7, wherein the DNA or the charged linear molecules are subjected to the pressure difference and the electric field applied to a liquid containing the DNA or charged linear molecules, whereby the DNA or the charged electric molecules are charged. After the charged linear molecules are retained in the flow path, a liquid different from the liquid containing the DNA or the charged linear molecules is supplied into the flow path, and the DNA or the charges are transferred. Applying a pressure difference that moves the DNA or a liquid different from the liquid containing the linear molecules having electric charges in the flow channel to a liquid different from the liquid containing the linear molecules having the pressure; and Due to the difference, an electric field that moves the DNA or the charged linear molecule in a direction opposite to the direction in which the liquid different from the liquid containing the DNA or the charged linear molecule is applied is applied. The DNA or trap release method of linear molecules having a charge and applying the liquid. 請求項７に記載のＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース方法において、当該流路内に流した当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体について、当該ＤＮＡ或いは電荷をもつ線状の分子が当該流路内に停留する圧力差と電界強度を求め、当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体に対して求めた当該圧力差と当該電界強度のうち電界強度のみを求めた当該電界強度より低い値で印加することを特徴とする当該ＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース方法。8. The method for trapping and releasing a DNA or a charged linear molecule according to claim 7, wherein the DNA or the charged liquid containing the DNA or the charged linear molecule that has flowed in the flow path is removed. The pressure difference and the electric field strength at which the linear molecules of the molecule stay in the flow path are obtained, and the electric field is calculated from the pressure difference and the electric field strength obtained for the liquid containing the DNA or the linear molecule having the electric charge. A method for trapping and releasing said DNA or charged linear molecules, wherein the voltage is applied at a value lower than the electric field intensity for which only the intensity has been obtained. 請求項７に記載のＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース方法において、当該流路内に流した当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体について、当該ＤＮＡ或いは電荷をもつ線状の分子が当該流路内に停留する圧力差と電界強度を求め、当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体に対して求めた当該圧力差と当該電界強度のうち電界強度のみを求めた当該電界強度より高い値で印加することを特徴とする当該ＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース方法。8. The method for trapping and releasing a DNA or a charged linear molecule according to claim 7, wherein the DNA or the charged liquid containing the DNA or the charged linear molecule that has flowed in the flow path is removed. The pressure difference and the electric field strength at which the linear molecules of the molecule stay in the flow path are obtained, and the electric field is calculated from the pressure difference and the electric field strength obtained for the liquid containing the DNA or the linear molecule having the electric charge. A method for trapping and releasing said DNA or charged linear molecules, wherein said method is applied at a value higher than said electric field intensity for which only the intensity is obtained. 請求項７、９、１０、１１に記載のＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース方法において、当該圧力差が当該ＤＮＡあるいは電荷をもつ線状の分子に及ぼす力と当該電界強度が当該ＤＮＡあるいは電荷をもつ線状分子に及ぼす力がつりあう圧力差と電界強度を共に０．３から１．７倍した圧力差と電界強度を当該流路内に流した当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体に印加することを特徴とする当該ＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース方法。12. The method for trapping and releasing DNA or charged linear molecules according to claim 7, 9, 10 or 11, wherein the force exerted by the pressure difference on the DNA or charged linear molecules and the electric field intensity are reduced. A line having the DNA or charge, in which a pressure difference and an electric field strength of 0.3 to 1.7 times both the pressure difference and the electric field strength at which the force exerted on the DNA or the charged linear molecule is balanced flow into the flow path. A method for trapping and releasing DNA or charged linear molecules, wherein the method is applied to a liquid containing the molecules. 請求項８に記載のＤＮＡ或いは電荷をもつ線状の分子のトラップ・リリース装置において、当該圧力差が当該ＤＮＡあるいは電荷をもつ線状の分子に及ぼす力と当該電界強度が当該ＤＮＡあるいは電荷をもつ線状分子に及ぼす力がつりあう圧力差と電界強度を共に０．３から１．７倍した圧力差と電界強度を当該流路内に流した当該ＤＮＡ或いは電荷をもつ線状の分子が含まれる液体に印加することを特徴とする装置。9. The trap or release device for DNA or charged linear molecules according to claim 8, wherein the force exerted by the pressure difference on the DNA or charged linear molecules and the electric field intensity have the DNA or charged. Includes linear molecules with the DNA or charge that have caused the pressure difference and the electric field strength to balance the force exerted on the linear molecules by 0.3 to 1.7 times, and the electric field strength to flow in the flow path. Apparatus characterized by applying to a liquid.
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