JP3833110B2 - Free flow electrophoresis - Google Patents

Free flow electrophoresis Download PDF

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JP3833110B2
JP3833110B2 JP2001371626A JP2001371626A JP3833110B2 JP 3833110 B2 JP3833110 B2 JP 3833110B2 JP 2001371626 A JP2001371626 A JP 2001371626A JP 2001371626 A JP2001371626 A JP 2001371626A JP 3833110 B2 JP3833110 B2 JP 3833110B2
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buffer
electrophoresis
sample
flow
buffer solution
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JP2003172725A (en
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信芳 田島
悦夫 篠原
博史 川崎
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Olympus Corp
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Olympus Corp
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【０００１】
【発明の属する技術分野】
本発明は、液体試料に電圧を印加することによって該液体に含まれる溶質を分離するフリーフロー電気泳動法に関するものである。
【０００２】
【従来の技術】
フリーフロー電気泳動とは、電気泳動層に試料と指示液を連続的に注入しながら電気泳動によって分離及び分取を行なうことができる電気泳動方法であって、２枚の板に挟まれて構成された非常に厚さの薄い液体流路に一定流速で支持液を流して流出口への支持液の層状の流れを作り、該流路への液体注入口近傍に設けた試料注入口から試料を注入し下流に試料液を流すとともに、流路の両端に設けた電極に電圧を印加することによって、試料に含まれる物質が電気泳動の原理によって当該物質によって定まる泳動方向（例えば、正負いずれかの電極方向）及び速度で泳動分離しながら所定の流出口に流出する原理を利用するものである。
【０００３】
例えば、特願第2001-210560号には、複数の緩衝液を自然拡散を抑えつつ流しながら電気泳動を行うことを可能とする構成を有するフリーフロー電気泳動素子が開示されている。この構成によって等速電気泳動や等電点電気泳動等の様々な分離法が実施可能となり、分離効率を向上することが可能であると記載されている。また、同特許出願には高分子を含む泳動緩衝液と高分子を含まない緩衝液を同時に流しながら電気泳動を行うことを特徴とするフリーフロー電気泳動法が開示されている。この方法によって、分子ふるい効果を有する泳動が可能となり、それによってサイズ分離効果を有するフリーフロー電気泳動が可能となると記載されている。
【０００４】
【発明が解決しようとする課題】
上記先行技術は、上述のような利点を有するものの、複数の緩衝液を用いた具体的なフリーフロー電気泳動方法の具体例としては、高分子を含む緩衝液と高分子を含まない緩衝液の組み合わせの一例のみの記載であり、他に具体例が記載されていない。高分子を含んだ緩衝液の利用はサイズ分離効果の利点を有するものの、粘度の高い緩衝液を薄い液体流路に流すことになり、フリーフロー電気泳動素子に圧力がかかってフリーフロー電気泳動素子の耐久性が落ちたり、フリーフロー電気泳動素子や配管が高分子によって汚染されるため装置メンテナンスを頻繁に行う必要が生じたり、分取液に高分子が混ざって分離した成分の純度が落ちるという問題点を有し、サイズ分離効果を必要とせず電場中における移動度の差で分離できる試料の分離には適していない。
【０００５】
したがって本発明は、上記問題を解決すべくなされたもので、サイズ分離効果を与えない場合にも電場中における移動度の差で分離できる試料を分離する電気泳動分離において、複数の緩衝液を流すことで分離能を向上することが可能なフリーフロー電気泳動法を提供することにある。
【０００６】
【課題を解決するための手段及び作用効果】
本発明のフリーフロー電気泳動方法は、電気泳動槽に緩衝液と試料とを連続的に注入しながら試料の分離・分取を行うフリーフロー電気泳動法において、少なくとも３種の泳動緩衝液を用い、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くするとともに、陽電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液を用いることを特徴とする（請求項１）。
【０００７】
本発明において、フリーフロー電気泳動とは、上述した通り、電気泳動槽に試料と支持液を連続的に注入しながら電気泳動による分離及び分取を行う電気泳動法である。分離操作は予め緩衝液注入口から一定流速で支持液を流して複数の流出口への液の流れを作り、試料注入口から試料を注入する。泳動槽内部は狭い空間特有の物理的現象によって強い層流が実現されているため、異なる液体を連続的に流すと液体が拡散しにくく、ほぼ直線的に流出口に向かって流れ出る。泳動槽の両端に形成した１対の電極間に電圧を印加すると、試料に含まれる物質は電気泳動の原理によって当該物質によって定まる泳動方向（例えば、正負いずれかの電極側）に泳動しながら流出口に流出する。物質によって泳動の方向及び速度が異なるため、試料に含まれる物質は分離しながら流出口に流出する。従って、複数の流出口の中から特定の分取口より液を取り出すことにより試料の分取が行える。
【０００８】
「緩衝液」とは、本発明では電気泳動に用いる「泳動液」のことを指す。
溶液に酸または塩基を加えた場合あるいは希釈した場合のｐＨの変化を緩める作用、すなわち緩衝作用をもつ溶液がそれに当たる。
弱酸と強塩基、弱塩基と強酸、弱酸と弱塩基の混合液が代表的緩衝液であるが、その組成・濃度・ｐＨは特に限定されず、一般的に電気泳動に用いられる組成・濃度・ｐＨを用いることができる。
【０００９】
電極近域とは、液体流路の流れ方向かつ電極に沿った液体流路内の領域をいい、試料流通部域とは試料を流し、電気泳動原理によって試料を電気泳動分離する領域をいう。
本発明で用いる電気泳動素子は、本発明の目的を達成できる限り特に限定されないが、先願である特願第2001-210560号に開示されている電気泳動素子を好適に用いることができる。これについては後述する。
本発明に関する実施の形態は、後記する参考例１および実施例１〜３にて具体的に説明する。
【００１０】
参考例１中では、請求項１における「試料流通部域に流す泳動緩衝液」及び「両電極近域に流す泳動緩衝液」は、それぞれ「緩衝液１」及び「緩衝液２」が該当する。
参考例を含む本発明によれば、以下の作用効果が得られる。
本発明のフリーフロー電気泳動法は、両電極近域に電気伝導度の高い緩衝液が、試料流通部域に電気伝導度の低い緩衝液が流れた状態でフリーフロー電気泳動が行われる。電極間に印加した電圧は、電気伝導度の低い緩衝液に大きな割合でかかる。このため、試料が流れる近域に効果的に高電圧を印加することが可能となり、分離能が向上する。分離速度を速くできるので、使用するフリーフロー電気泳動素子を小型化し、廉価に製造することを可能となり、将来的には使い捨てフリーフロー電気泳動素子を可能とする。また、両電極近域には電圧のかかる割合が低いためジュール熱による発熱も抑えられ、電極における気泡の発生を抑制することができる。
【００１１】
したがって参考例を含む本発明は、両電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くすることで、分離能を向上することが可能なフリーフロー電気泳動法を提供することができる。
以下に本発明の好ましい実施態様について説明する。
（１）第1の好ましい実施の態様（請求項１に対応）
第1の好ましい実施態様は、参考例を含む本発明のフリーフロー電気泳動法（請求項１記載）において、すくなくとも３種の泳動緩衝液を用い、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、陽電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液を用いることを特徴とする。
本発明の好ましい第1の実施の態様は、実施例１の実施の態様が対応する。
【００１２】
（対応する実施例）
この発明に関する実施の形態は、第１の実施の形態が対応する。本実施例１中、「試料流通部域に流す泳動緩衝液」は、「緩衝液１」が該当し、「陽電極近域に流す泳動緩衝液」は、「緩衝液２」が該当し、「陰電極近域に流す泳動緩衝液」は、「緩衝液３」が該当する。本実施例中「分離する試料」は、「キシレンシアノールＦＦとブロモフェノールブルーを混合した水溶液」が該当する。
【００１３】
（作用効果）
本発明の第1の好ましい実施の態様によれば、フリーフロー電気泳動法は、陰電極近域に電気伝導度が高く且つ分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液が、陽電極近域に電気伝導度が高く且つ分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液が、試料流通部域に電気伝導度が低い緩衝液が流れた状態でフリーフロー電気泳動が行われる。従って、電極間に印加した電圧は、電気伝導度の低い緩衝液に大きな割合でかかる。このため、試料が流れる近域に効果的に高電圧を印加することが可能となり、本発明で目的とする分離能の向上が達成できる。さらに、分離速度を速くできるので、使用するフリーフロー電気泳動素子を小型化し、廉価に製造することを可能となり、将来的には使い捨てフリーフロー電気泳動素子を可能とする。
【００１４】
また、陰電極近域に流す泳動緩衝液に含まれる陰イオンは被分離試料成分よりも移動度が小さく、陽電極近域に流す泳動緩衝液に含まれる陰イオンは被分離試料成分よりも移動度が大きい。このため、本発明の第1の好ましい実施の態様における電気泳動では等速電気泳動的な分離が実現され、試料は先行イオンと終末イオンの間に挟まれて分離しながら収束する。このため分離した成分が広がらず高濃度の状態で回収することが可能となる。ここで等速電気泳動とは、移動度の大きなイオンを含む緩衝液と移動度の小さなイオンを含む緩衝液とを用いて、分離されたゾーンの全てが同じ速度で移動するような状態を作って分離させる電気泳動法で、試料は二つの緩衝液に挟まれて分離し収束する挙動を示す。
【００１５】
したがって本発明の第1の好ましい実施の態様は、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くし、且つ陽電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液を用いることで、分離能を向上し、且つ高濃度の分取液を得ることが可能なフリーフロー電気泳動法を提供することができる。
（２）本発明の好ましい第2の実施の態様（請求項２に対応）
第２の好ましい実施態様は、本発明のフリーフロー電気泳動法の第1の好ましい実施の態様（請求項１）において、試料流通部域に流す泳動緩衝液に、陰電極近域に流す泳動緩衝液を希釈した液を用いることを特徴とする。
【００１６】
（対応する実施例）
本発明の第2の好ましい実施の態様は、実施例２の実施の態様が対応する。本実施例２中、「試料流通部域に流す泳動緩衝液」は、「緩衝液１」が該当し、「陰電極近域に流す泳動緩衝液」は、「緩衝液３」が該当する。
（作用効果）
本発明のフリーフロー電気泳動法は、陰電極近域に電気伝導度が高く且つ分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液が、陽電極近域に電気伝導度が高く且つ分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液が、試料流通部域に電気伝導度が低い緩衝液が流れた状態でフリーフロー電気泳動が行われる。電極間に印加した電圧は、電気伝導度の低い緩衝液に大きな割合でかかる。このため、試料が流れる近域に効果的に高電圧を印加することが可能となり、本発明の目的である分離能の向上が達成できる。さらに、分離速度を速くできるので、使用するフリーフロー電気泳動素子を小型化し、廉価に製造することを可能となり、将来的には使い捨てフリーフロー電気泳動素子を可能とする。
【００１７】
また、陰電極近域に流す泳動緩衝液に含まれる陰イオンは試料よりも移動度が小さく、陽電極近域に流す泳動緩衝液に含まれる陰イオンは試料よりも移動度が大きい。このため、本発明の第2の好ましい実施の態様では電気泳動は等速電気泳動的な分離が実現され、第1の好ましい実施の態様と同様に試料は先行イオンと終末イオンの間に挟まれて分離しながら収束する。このため分離した成分が広がらず高濃度の状態で回収することが可能となる。
【００１８】
また、試料流通部域に流す泳動緩衝液は陰電極近域に流す泳動緩衝液を希釈した組成であり、含まれる陰イオンが等しいため、収束に要する時間を短縮することが可能となる。また、試料流通部域に流す泳動緩衝液は陰電極近域に流す泳動緩衝液を希釈して調製でき、緩衝液の準備が簡便となる。
【００１９】
したがって本発明の第2の好ましい実施の態様は、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くし、陽電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液を用い、試料流通部域に流す泳動緩衝液に陰電極近域に流す泳動緩衝液を希釈した液を用いることで、分離能を向上し、且つ効率的に高濃度の分取液を得ることが可能なフリーフロー電気泳動法を提供することができる。
【００２０】
（３）本発明の第3の好ましい実施の態様（請求項３に対応）
本発明の第3の好ましい実施の態様は、本発明の第１の好ましい実施の態様のフリーフロー電気泳動法に加えて、陽電極近域に流す泳動緩衝液と陰電極側に流す泳動緩衝液の電気伝導度が等しいことを特徴とする。
【００２１】
（対応する実施例）
本発明の第３の好ましい実施の態様は、実施例３の実施の態様が対応する。本実施例３中、「陽電極近域に流す泳動緩衝液」は、「緩衝液２」が該当し、「陰電極近域に流す泳動緩衝液」は、「緩衝液３」が該当する。
（作用効果）
本発明の第3の好ましい実施の態様のフリーフロー電気泳動法は、陰電極近域に電気伝導度が高く且つ分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液が、陽電極近域に電気伝導度が高く且つ分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液が、試料流通部域に電気伝導度が低い緩衝液が流れた状態でフリーフロー電気泳動が行われる。電極間に印加した電圧は、電気伝導度の低い緩衝液に大きな割合でかかる。このため、試料が流れる近域に効果的に高電圧を印加することが可能となり、本発明の目的とする分離能の向上が達成できる。分離速度を速くできるので、使用するフリーフロー電気泳動素子を小型化し、廉価に製造することを可能となり、将来的には使い捨てフリーフロー電気泳動素子を可能とする。
【００２２】
また、陰電極近域に流す泳動緩衝液に含まれる陰イオンは試料よりも移動度が小さく、陽電極近域に流す泳動緩衝液に含まれる陰イオンは分離する試料成分よりも移動度が大きい。このため、本発明の第3の好ましい実施の態様における電気泳動は等速電気泳動的な分離が実現され、本発明の第１の好ましい実施の態様と同様に被分離試料成分は先行イオンと終末イオンの間に挟まれて分離しながら収束する。このため分離した成分が広がらず高濃度の状態で回収することが可能となる。
また、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液は電気伝導度が等しいため両電極で生じるジュール熱による発熱が等しく、両電極で生じる気泡の出方が偏らずに安定するため、長時間の安定な分離が可能となる。
【００２３】
したがって本発明の第3の好ましい実施の態様では、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くし、陽電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液を用い、陽電極近域に流す泳動緩衝液と陰電極側に流す泳動緩衝液との電気伝導度を等しくすることで、分離能を向上し、高濃度の分取液が得られ、且つ長時間の安定した使用が可能なフリーフロー電気泳動法を提供することができる。
【００２４】
（実施例）
本発明のフリーフロー電気泳動方法で好適に用いることができるフリーフロー電気泳動素子について説明する。本フリーフロー電気泳動素子としては特願第２０００-210560号の図1乃至図4にに示すものと実質的に同じものを用いることができる。但し、本発明のフリーフロー電気泳動方法に用いることができるフリーフロー電気泳動素子はこのものに限定されない。
【００２５】
図１はこの発明で用いるフリーフロー電気泳動素子１の上面図である。フリーフロー電気泳動素子１は、泳動槽構成基板２と注入口構成基板３との組み合わせで構成される。図１の線Ａの部分の断面図を図２に、泳動槽構成基板２の上面図を図３に、注入口構成基板３の裏面図を図４に示す。このフリーフロー電気泳動素子１は、幅広の溝からなる泳動槽８と細長い溝からなる２つの電極室９と、泳動槽８と電極室９とを区切る隔壁１０を構成した泳動槽構成基板２と、複数の緩衝液注入口４ａ〜４ｕと複数の試料注入口５ａ〜５ｅと複数の流出口６ａ〜５ｕと１対の電極７ａ，７ｂを構成した注入口構成基板３とを張り合わせて構成される。電極７は注入口構成基板３の泳動槽構成基板２と対向する面上に薄膜で構成される。隔壁１０は対向する注入口構成基板３との間に空隙を有して構成される。複数の緩衝液注入口４ａ〜４ｕと複数の流出口６ａ〜６ｕは同数で互いにそれぞれ対応し且つ流れに対して垂直方向に見て同間隔で構成される。
【００２６】
泳動槽構成基板２及び注入口構成基板３の素材はホウケイ酸ガラス、電極７は白金薄膜からなる。泳動槽８と電極室９は公知のウェットエッチング法にて深さ３０マイクロメートルに加工した。隔壁１０は対向する注入口構成基板３との間に５マイクロメートルの空隙ができるように加工した。緩衝液注入口４と試料注入口５と流出口６は超音波加工によって加工した。電極７はスパッタリング法によって成膜した。泳動槽構成基板２及び注入口構成基板３の接合は、図示しないアモルファスシリコン薄膜を介して陽極接合法によって接合した。
【００２７】
本フリーフロー電気泳動素子では、緩衝液注入口と流出口とが同数互いにそれぞれ対応し且つ流れに対して垂直方向に見て同間隔で構成されているので、狭い空間である泳動槽に緩衝液を連続的に流した場合、流れが強い層流となるため液体同志が拡散しにくい状態が生じ、それぞれの緩衝液注入口から異なる緩衝液を流した場合、それと対応する流出口に拡散することなく流れ出る。なお、等速泳動及び等電点電気泳動においては、緩衝液注入口は間隔を小さくして密に設けることが好ましい。
次に、該フリーフロー電気泳動素子１を用いたフリーフロー電気泳動法を示す。
【００２８】
まず、本発明のフリーフロー電気泳動素子１の機能について説明する。フリーフロー電気泳動とは、電気泳動槽に試料と支持液を連続的に注入しながら電気泳動による分離及び分取を行う電気泳動法である。該電気泳動槽は、本発明の電気泳動槽８がそれに当たる。分離操作は予め複数の緩衝液注入口４から一定流速で支持液を流して複数の流出口６への液の流れを作り、複数ある試料注入口５の少なくとも一つから試料を注入する。泳動槽内部は狭い空間特有の物理的現象によって強い層流が実現されているため、異なる液体を連続的に流すと液体が拡散しにくく、ほぼ直線的に流出口に向かって流れ出る。１対の電極７間に電圧を印加すると試料に含まれる物質は、電気泳動の原理によって正負いずれかの電極側に泳動しながら流出口６に流出する。物質によって泳動の方向及び速度が異なるため、試料に含まれる物質は分離しながら流出口６に流出する。従って、複数の流出口６の中から特定の分取口より液を取り出すことにより試料の分取が行える。電圧の印加に伴い電極７から発生した気泡は隔壁１０によって泳動槽８側に入ることなく電極室を流れ流出口６に向かう。
【００２９】
（参考例１）（請求項１に該当）
緩衝液１として３ｍＭトリスヒドロキシメチルアミノメタン−３ｍＭ塩酸緩衝液を、緩衝液２として１０ｍＭトリスヒドロキシメチルアミノメタン−１０ｍＭ塩酸緩衝液を、試料としてキシレンシアノールＦＦとブロモフェノールブルーを混合した水溶液を用い、図1と図2で説明したフリーフロー電気泳動素子１にてフリーフロー電気泳動を行った。フリーフロー電気泳動素子としては、図１乃至図２に示すものを用いた。フリーフロー電気泳動素子１は電極７ａを電圧印加電源の陰極に、電極７ｂを電圧印加電源の陽極に接続した。緩衝液は、陰極近域の緩衝液注入口４ａ〜４ｅと陽極近域の緩衝液注入口４ｑ〜４ｕに緩衝液２を、試料流通部域の緩衝液注入口４ｆ〜４ｐに緩衝液１をポンプで流した。試料は試料注入口５ｂからポンプで注入した。フリーフロー電気泳動は、緩衝液１と２及び試料を注入しながら電極７に電圧を印加して行った。分取液は流出口６ａ〜５ｕからの流出液を回収して得た。数ｋＶの電圧を印加しフリーフロー電気泳動を行うと、試料中のキシレンシアノールＦＦは陰極よりの流出口に、ブロモフェノールブルーは陽極よりの流出口に分離して流出した。
【００３０】
数ｋＶの電圧を印加しフリーフロー電気泳動を行うと、試料中のキシレンシアノールＦＦは陰極よりの流出ロに、プロモフェノールブルーは陽極よりの流出口に分離して流出した。
単一の緩衝液を用いた場合、キシレンシアノールＦＦとブロモフェノールブルーの流出する流出口の間隔は、流出ロの数にして一つ分あけて流出するのに対し、本実施例では流出口にして4つ分あけた分離が得られた。このように、分離率は単一の緩衝液を用いた場合よりも優れており、分離能の高い分離が可能となった。
【００３１】
本参考例では両電極近域に緩衝液２が、試料流通部域に緩衝液１が流れた状態でフリーフロー電気泳動が行われる。緩衝液２は緩衝液１に対して濃度が高いため電気伝導度が高い。電極７に印加した電圧は、電気伝導度の低い緩衝液１に大きな割合でかかる。このため、試料が流れる近域に効果的に高電圧を印加することが可能となり、分離能が向上する。また、両電極近域には電圧のかかる割合が低いためジュール熱による発熱も抑えられ、電極における気泡の発生を抑制することができる。
【００３２】
したがって参考例１を含む本発明は、両電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くすることで、分離能を向上することが可能なフリーフロー電気泳動法を提供することができる。なお、この発明の実施例は、当然、各種変更が可能である。
例えば、緩衝液の種類は限定されず通常電気泳動に用いられる緩衝液に変更できる。緩衝液の濃度は、緩衝液１に対して緩衝液２の伝導度が高い組み合わせであれば限定されない。また、緩衝液１と緩衝液２とを同一組成の緩衝液とする必要はなく、異なる組成の緩衝液とすることも可能である。緩衝液２の注入口は限定されず、両電極から適当な幅を持って流すことができる。また、試料注入口の場所は緩衝液１が流れている場所であれば限定されない。
【００３３】
また、この発明の参考例に用いたフリーフロー電気泳動素子１は、複数の緩衝液注入口を有するものであって、安定な層流状態でフリーフロー電気泳動を行うことが可能なフリーフロー電気泳動素子であれば、その構成は特に限定されない
（実施例１）（請求項１に該当）
緩衝液１として５ｍＭトリスヒドロキシメチルアミノメタン−５ｍＭ６−アミノ−ｎ−カプロン酸緩衝液を、緩衝液２として２０ｍＭトリスヒドロキシメチルアミノメタン−２０ｍＭ塩酸緩衝液を、緩衝液３として５０ｍＭトリスヒドロキシメチルアミノメタン−５００ｍＭＣＡＰＳ緩衝液を、試料としてキシレンシアノールＦＦとブロモフェノールブルーを混合した水溶液を用い、参考例１で用いたフリーフロー電気泳動素子１にてフリーフロー電気泳動を行った。フリーフロー電気泳動素子１は電極７ａを電圧印加電源の陰極に、電極７ｂを電圧印加電源の陽極に接続した。緩衝液は、陰極近域の緩衝液注入口４ａ〜４ｅに緩衝液３を、陽極側の緩衝液注入口４ｑ〜４ｕに緩衝液２を、試料流通部域の緩衝液注入口４ｆ〜４ｐに緩衝液１をポンプで流した。試料は試料注入口５ｂからポンプで注入した。フリーフロー電気泳動は、緩衝液１と２及び試料を注入しながら電極７に電圧を印加して行った。分取液は流出口６ａ〜６ｕからの流出液を回収して得た。数ｋＶの電圧を印加しフリーフロー電気泳動を行うと、試料中のキシレンシアノールＦＦとブロモフェノールブルーは分離後に収束し、キシレンシアノールＦＦは陰極よりの流出口に、ブロモフェノールブルーは陽極よりの流出口に分離して流出した。
【００３４】
数ｋＶの電圧を印加しフリーフロー電気泳動を行うと、試料中のキシレンシアノールＦＦとブロモフェノールブルーは分離後に収束し、キシレンシアノールＦＦは陰極よりの流出口に、ブロモフェノールブルーは陽極よりの流出口に分離して流出した。
単一の緩衝液を用いた場合、キシレンシアノールＦＦとブロモフェノールブルーの流出する流出口の間隔は、流出口の数にして一つ分あけて流出するのに対し、本実施例では流出口にして４つ分あけた分離が得られた。また、参考例１の条件ではキシレンシアノールＦＦとブロモフェノールブルーはそれぞれ２つの流出口にまたがって流出したが、本条件では分離後収束してそれぞれ１つの流出口のみに流出した。このように、分離率は単一の緩衝液を用いた場合よりも優れており、分離能の高い分離が可能となった。また、分離後に収束しているため、濃度の高い状態での分離が可能となった。
【００３５】
（作用・効果）
次に、この発明の実施例の作用及び効果を説明する。
本実施例では陰電極側近域に緩衝液３が、陽電極側近域に緩衝液２が、試料流通部域に緩衝液１が流れた状態でフリーフロー電気泳動が行われる。緩衝液３及び２は緩衝液１に対して濃度が高いため電気伝導度が高い。電極７に印加した電圧は、電気伝導度の低い緩衝液１に大きな割合でかかる。このため、試料が流れる近域に効果的に高電圧を印加することが可能となり、本発明で目的とする分離能が向上する。
また、緩衝液３に含まれるＣＡＰＳは試料のキシレンシアノールＦＦ及びブロモフェノールブルーよりも移動度が小さく、緩衝液２に含まれるクロルイオンは試料のキシレンシアノールＦＦ及びブロモフェノールブルーよりも移動度が大きい。このため、本実施例における電気泳動は等速電気泳動的な分離が実現され、試料は先行イオンであるクロルイオンと終末イオンであるＣＡＰＳの間に挟まれて分離しながら収束する。このため分離した成分が広がらず高濃度の状態で回収することが可能となる。ここで等速電気泳動とは、移動度の大きなイオンを含む緩衝液と移動度の小さなイオンを含む緩衝液とを用いて、分離されたゾーンの全てが同じ速度で移動するような状態を作って分離させる電気泳動法で、試料は二つの緩衝液に挟まれて分離し収束する挙動を示す。
【００３６】
したがって本発明の第1の好ましい実施の態様では、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くし、陽電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液を用いることで、分離能を向上し、且つ高濃度の分取液を得ることが可能なフリーフロー電気泳動法を提供することができる。
なお、この発明の実施例は、当然、各種変更が可能である。
【００３７】
例えば、緩衝液２は被分離試料成分よりも移動度の大きい陰イオンを含むものであれば特に限定されない。陰イオン種としては、クロルイオンの他に酢酸などを用いることができる。また、緩衝液３は被分離試料成分よりも移動度の小さい陰イオンを含むものであれば特に限定されない。陰イオン種としてはＣＡＰＳの他に、カプロン酸、グルタミン酸、β−アラニン、フェノールなどを用いることができる。緩衝液１は、含まれる陰イオンの移動度が緩衝液２中の陰イオンの移動度よりも遅いもので、緩衝液３中に含まれる陰イオンの移動度と同じもしくは速いものを用いれば、その種類は限定されない。緩衝液１，２，３共に、陰イオンの対イオンの種類は特に限定されない。トリスヒドロキシメチルアミノメタンの他に例えば、ヒスチジン、β−アラニン、アメジオール、２−アミノ−２−メチルプロパノール、バリウムイオン、カリウムイオン等を用いることができる。また、緩衝液１の濃度は緩衝液２及び３よりも電気伝導度が低い状態とすれば特に限定されない。緩衝液２，３の注入口は限定されず、電極から適当な幅を持って流すことができる。また、試料注入口の場所は緩衝液１が流れている場所であれば限定されない。
【００３８】
なお、本実施例は陰イオンを分離対象試料成分とした場合における条件である。陽イオンを分離対象とする場合、陽電極近域に流す泳動緩衝液に分離する試料成分よりも移動度の小さな陽イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液に分離する試料成分よりも移動度の大きな陽イオンを含んだ緩衝液を用いることで、同様の効果を有するフリーフロー電気泳動法を提供することができる。
【００３９】
（実施例２）（請求項２に該当）
緩衝液１として１０ｍＭトリスヒドロキシメチルアミノメタン−１００ｍＭＣＡＰＳ緩衝液を、緩衝液２として５ｍＭトリスヒドロキシメチルアミノメタン−１０ｍＭ酢酸緩衝液を、緩衝液３として５０ｍＭトリスヒドロキシメチルアミノメタン−５００ｍＭＣＡＰＳ緩衝液を、試料としてキシレンシアノールＦＦとブロモフェノールブルーを混合した水溶液を用い、参考例１で説明したフリーフロー電気泳動素子１にてフリーフロー電気泳動を行った。フリーフロー電気泳動素子１は電極７ａを電圧印加電源の陰極に、電極７ｂを電圧印加電源の陽極に接続した。緩衝液は、陰極近域の緩衝液注入口４ａ〜４ｅに緩衝液３を、陽極近域の緩衝液注入口４ｑ〜４ｕに緩衝液２を、試料流通部域の緩衝液注入口４ｆ〜４ｐに緩衝液１をポンプで流した。試料は試料注入口５ｂからポンプで注入した。フリーフロー電気泳動は、緩衝液１と２及び試料を注入しながら電極７に電圧を印加して行った。分取液は流出口６ａ〜６ｕからの流出液を回収して得た。数ｋＶの電圧を印加しフリーフロー電気泳動を行うと、試料中のキシレンシアノールＦＦとブロモフェノールブルーは分離後に収束し、キシレンシアノールＦＦは陰極よりの流出口に、ブロモフェノールブルーは陽極よりの流出口に分離して流出した。
【００４０】
数ｋＶの電圧を印加しフリーフロー電気泳動を行うと、試料中のキシレンシアノールＦＦとブロモフェノールブルーは分離後に収束し、キシレンシアノールＦＦは陰極よりの流出口に、ブロモフェノールブルーは陽極よりの流出口に分離して流出した。参考例１同様、分離率は単一の緩衝液を用いた場合よりも優れており、分離能の高い分離が可能となった。また実施例１同様、分離後に収束しているため、濃度の高い状態での分離が可能となった。また、実施例１の条件では収束に要する時間が試料注入後から３秒であったのに対し、本実施例では１秒で収束した。このように分離後の収束が短時間生じ、効率的な分離が可能となった。
【００４１】
（作用・効果）
次に、この発明の実施例の作用及び効果を説明する。
本実施例では陰電極近域に緩衝液３が、陽電極近域に緩衝液２が、試料流通部域に緩衝液１が流れた状態でフリーフロー電気泳動が行われる。緩衝液３及び２は緩衝液１に対して濃度が高いため電気伝導度が高い。電極７に印加した電圧は、電気伝導度の低い緩衝液１に大きな割合でかかる。このため、試料が流れる近域に効果的に高電圧を印加することが可能となり、本発明で目的とする分離能の向上が達成できる。
【００４２】
また、緩衝液３に含まれるＣＡＰＳは試料のキシレンシアノールＦＦ及びブロモフェノールブルーよりも移動度が小さく、緩衝液２に含まれるクロルイオンは試料のキシレンシアノールＦＦ及びブロモフェノールブルーよりも移動度が大きい。このため、本実施例における電気泳動は実施例１と同様に等速電気泳動的な分離が実現され、試料は先行イオンであるクロルイオンと終末イオンであるＣＡＰＳの間に挟まれて分離しながら収束する。このため分離した成分が広がらず高濃度の状態で回収することが可能となる。
また、緩衝液１は緩衝液３を５倍希釈した組成であり、含まれる陰イオンが等しいため、収束に要する時間を短縮することが可能となる。また、緩衝液１は緩衝液３を希釈して調製でき、緩衝液の準備が簡便となった。
【００４３】
したがって本発明は、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くし、陽電極近域に流す泳動緩衝液に分離する試料よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液に分離する試料よりも移動度の小さな陰イオンを含んだ緩衝液を用い、試料流通部域に流す泳動緩衝液に陰電極近域に流す泳動緩衝液を希釈した液を用いることで、分離能を向上し、且つ効率的に高濃度の分取液を得ることが可能なフリーフロー電気泳動法を提供することができる。
なお、この発明の実施例は、実施例１と同様に当然、各種変更が可能である。
【００４４】
例えば、緩衝液２は試料よりも移動度の大きい陰イオンを含むものであれば特に限定されない。陰イオン種としては、クロルイオンの他に酢酸などを用いることができる。また、緩衝液３は試料よりも移動度の小さい陰イオンを含むものであれば特に限定されない。陰イオン種としてはＣＡＰＳの他に、カプロン酸、グルタミン酸、β−アラニン、フェノールなどを用いることができる。緩衝液１，２，３共に、陰イオンの対イオンの種類は特に限定されない。トリスヒドロキシメチルアミノメタンの他に例えば、ヒスチジン、β−アラニン、アメジオール、２−アミノ−２−メチルプロパノール、バリウムイオン、カリウムイオン等を用いることができる。また、緩衝液１は緩衝液３を希釈したものであればその濃度は特に限定されない。緩衝液２，３の注入口は限定されず、電極から適当な幅を持って流すことができる。また、試料注入口の場所は緩衝液１が流れている場所であれば限定されない。
【００４５】
（実施例３）（請求項３に該当）
緩衝液１として５ｍＭトリスヒドロキシメチルアミノメタン−５ｍＭ６−アミノ−ｎ−カプロン酸緩衝液を、緩衝液２として５ｍＭトリスヒドロキシメチルアミノメタン−５ｍＭ塩酸緩衝液を、緩衝液３として５０ｍＭトリスヒドロキシメチルアミノメタン−５００ｍＭ６−アミノ−ｎ−カプロン酸緩衝液を、試料としてキシレンシアノールＦＦとブロモフェノールブルーを混合した水溶液を用い、図1及び図2で説明したフリーフロー電気泳動素子１にてフリーフロー電気泳動を行った。緩衝液の調製において、緩衝液２と緩衝液３は電気伝導度が等しくなるように調製した。電気電伝導度は２５℃の条件にで伝導率測定率で測定し、調整した。本実施例における緩衝液２及び３の電気伝導度は０．２５ｍＳ／ｃｍであった。フリーフロー電気泳動素子１は電極７ａを電圧印加電源の陰極に、電極７ｂを電圧印加電源の陽極に接続した。緩衝液は、陰極近域の緩衝液注入口４ａ〜ｅに緩衝液３を、陽極側の緩衝液注入口４ｑ〜ｕに緩衝液２を、試料流通部域の緩衝液注入口４ｆ〜ｐに緩衝液１をポンプで流した。試料は試料注入口５ｂからポンプで注入した。フリーフロー電気泳動は、緩衝液１と２及び試料を注入しながら電極７に電圧を印加して行った。分取液は流出口６ａ〜ｕからの流出液を回収して得た。数ｋＶの電圧を印加しフリーフロー電気泳動を行うと、試料中のキシレンシアノールＦＦとブロモフェノールブルーは分離後に収束し、キシレンシアノールＦＦは陰極よりの流出口に、ブロモフェノールブルーは陽極よりの流出口に分離して流出した。分離率は単一の緩衝液を用いた場合よりも優れており、分離能の高い分離が可能となった。また、分離後に収束しているため、濃度の高い状態での分離が可能となった。また、収束も短時間で生じ、効率的な分離が可能となった。また、電極７における気泡の発生が安定し、長時間の安定な分離が可能となった。
【００４６】
数ｋＶの電圧を印加しフリーフロー電気泳動を行うと、試料中のキシレンシアノールＦＦとブロモフェノールブルーは分離後に収束し、キシレンシアノールＦＦは陰極よりの流出口に、ブロモフェノールブルーは陽極よりの流出口に分離して流出した。参考例１同様、分離率は単一の緩衝液を用いた場合よりも優れており、分離能の高い分離が可能となった。また実施例１同様、分離後に収束しているため、濃度の高い状態での分離が可能となった。また、実施例２同様、収束も短時間で生じ効率的な分離が可能となった。また実施例２では、緩衝液２と３の電気伝導度が等しくないため電気伝導度の低い緩衝液３側に緩衝液２側よりも高いジュール熱が発生して、緩衝液３側から生じる気泡発生量が多くなり、長時間の安定した流れが得られなかったのに対し、本実施例では緩衝液２と３の電気伝導度が等しいため、両電極側の発熱量が均一となって気泡の発生量が安定した。このため、長時間の安定な分離が可能となった。
【００４７】
（作用・効果）
次に、この発明の実施例の作用及び効果を説明する。
本実施例では陰電極近域に緩衝液３が、陽電極近域に緩衝液２が、試料流通部域に緩衝液１が流れた状態でフリーフロー電気泳動が行われる。緩衝液３及び２は緩衝液１に対して濃度が高いため電気伝導度が高い。電極７に印加した電圧は、電気伝導度の低い緩衝液１に大きな割合でかかる。このため、試料が流れる近域に効果的に高電圧を印加することが可能となり、本発明で目的とする分離能の向上が達成される。
また、緩衝液３に含まれるＣＡＰＳは試料のキシレンシアノールＦＦ及びブロモフェノールブルーよりも移動度が小さく、緩衝液２に含まれるクロルイオンは試料のキシレンシアノールＦＦ及びブロモフェノールブルーよりも移動度が大きい。このため、本実施例における電気泳動は実施例２と同様に等速電気泳動的な分離が実現され、試料は先行イオンであるクロルイオンと終末イオンであるＣＡＰＳの間に挟まれて分離しながら収束する。従って、分離した成分が広がらず高濃度の状態で回収することが可能となる。また、緩衝液１と緩衝液３は含まれる陰イオンが等しいため、収束に要する時間が短縮される。また、緩衝液２と緩衝液３は電気伝導度が等しいため両電極で生じるジュール熱による発熱が等しく、両電極で生じる気泡の出方が偏らずに安定するため、長時間の安定な分離が可能となる。
【００４８】
故に、本発明は、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くし、陽電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液として分離する試料よりも移動度の小さな陰イオンを含んだ緩衝液を用い、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液との電気伝導度を等しくすることで、分離能を向上し、高濃度の分取液が得られ、且つ長時間の安定した使用が可能なフリーフロー電気泳動法を提供することができる。
なお、この発明の実施例は、実施例１と同様に当然、各種変更が可能である。
【００４９】
例えば、緩衝液２は試料よりも移動度の大きい陰イオンを含むもので、緩衝液３と同じ電気伝導度であれば組成や濃度は特に限定されない。陰イオン種としては、クロルイオンの他に酢酸などを用いることができる。また、緩衝液３は試料よりも移動度の小さい陰イオンを含むもので、緩衝液２と同じ電気伝導度であれば組成や濃度は特に限定されない。陰イオン種としてはＣＡＰＳの他に、カプロン酸、グルタミン酸、β−アラニン、フェノールなどを用いることができる。緩衝液１は、含まれる陰イオンの移動度が緩衝液２中の陰イオンの移動度と同じもしくは遅いもので、緩衝液３中に含まれる陰イオンの移動度と同じもしくは速いものを用いれば、その種類は限定されない。緩衝液１，２，３共に、陰イオンの対イオンの種類は特に限定されない。トリスヒドロキシメチルアミノメタンの他に例えば、ヒスチジン、β−アラニン、アメジオール、２−アミノ−２−メチルプロパノール、バリウムイオン、カリウムイオン等を用いることができる。また、緩衝液１の濃度は緩衝液２及び３よりも電気伝導度が低い状態とすれば特に限定されない。緩衝液２，３の注入口は限定されず、電極から適当な幅を持って流すことができる。また、試料注入口の場所は緩衝液１が流れている場所であれば限定されない。
【００５０】
【発明の効果】
参考例を含む本願請求項１によれば、電気泳動槽に緩衝液と試料とを連続的に注入しながら試料の分離・分取を行うフリーフロー電気泳動法において、少なくとも２種以上の泳動緩衝液を用い、両電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くしているので、電極間に印加した電圧は、電気伝導度の低い緩衝液に大きな割合でかかる。このため、試料が流れる近域に効果的に高電圧を印加することが可能となり、分離能が向上する。分離速度を速くできるので、使用するフリーフロー電気泳動素子を小型化し、廉価に製造することが可能となり、将来的には使い捨てフリーフロー電気泳動素子を可能とする。また、両電極近域には電圧のかかる割合が低いためジュール熱による発熱も抑えられ、電極における気泡の発生を抑制することができる。
【００５１】
また、本願請求項１によれば、少なくとも３種の泳動緩衝液を用い、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、陽電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液を用いる。このため、請求項１に含まれる参考例で得られる効果に加えて、請求項１におけるフリーフロー電気泳動法では等速電気泳動的な分離が実現され、試料は先行イオンと終末イオンの間に挟まれて分離しながら収束する。このため分離した成分が広がらず高濃度の状態で回収することが可能となる。
【００５２】
本願請求項２によれば、請求項２記載のフリーフロー電気泳動法において、試料流通部域に流す泳動緩衝液に、陰電極近域に流す泳動緩衝液を希釈した液を用いる。このため、本願請求項２におけるフリーフロー電気泳動法では、参考例１およびそれを含む請求項１で得られる効果に加えて、試料流通部域に流す泳動緩衝液は陰電極近域に流す泳動緩衝液を希釈した組成であり、含まれる陰イオンが等しいため、収束に要する時間を短縮することが可能となる。また、試料流通部域に流す泳動緩衝液は陰電極近域に流す泳動緩衝液を希釈して調製でき、緩衝液の準備が簡便となる。
【００５３】
請求項３によれば、参考例１およびそれを含む請求項１記載のフリーフロー電気泳動法において、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液の電気伝導度が等しい。参考例１を含む請求項１で得られる効果に加えて、請求項３におけるフリーフロー電気泳動法では陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液は電気伝導度が等しいため両電極で生じるジュール熱による発熱が等しく、両電極で生じる気泡の出方が偏らずに安定するため、長時間の安定な分離が可能となる。
【図面の簡単な説明】
【図１】 本発明のフリーフロー電気泳動法で用いることができるフリーフロー電泳動素子の平面図である。
【図２】 図1に示すフリーフロー電泳動素子のA−A線での断面図である。
【図３】図１のフリーフロー電気泳動素子の泳動槽構成基板の上面図である。
【図４】図１のフリーフロー電気泳動素子の注入口構成基板３の裏面図である。
【符号の説明】
１ フリーフロー電気泳動素子
２ 泳動槽構成基板
３ 注入口構成基板
４（４ａ〜４ｕ） 緩衝液注入口
５（５ａ〜５ｅ） 試料注入口
６（６ａ〜６ｕ） 流出口
７（７ａ〜７ｂ） 電極
８ 泳動槽（電気泳動槽）
９ 電極室
１０ 隔壁[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a free flow electrophoresis method in which a solute contained in a liquid is separated by applying a voltage to the liquid sample.
[0002]
[Prior art]
Free-flow electrophoresis is an electrophoresis method that allows separation and fractionation by electrophoresis while continuously injecting a sample and an indicator solution into the electrophoresis layer, and is sandwiched between two plates. The support liquid is caused to flow through the very thin liquid flow path at a constant flow rate to create a laminar flow of the support liquid to the outlet, and the sample is supplied from the sample inlet provided near the liquid inlet to the flow path. And the sample liquid is flowed downstream, and a voltage is applied to the electrodes provided at both ends of the flow path, so that the substance contained in the sample is determined by the substance according to the electrophoresis principle (for example, positive or negative) The principle of flowing out to a predetermined outlet is carried out while performing electrophoretic separation at an electrode direction) and speed.
[0003]
For example, Japanese Patent Application No. 2001-210560 discloses a free-flow electrophoretic element having a configuration that enables electrophoresis while flowing a plurality of buffer solutions while suppressing natural diffusion. It is described that with this configuration, various separation methods such as isotachophoresis and isoelectric focusing can be performed, and the separation efficiency can be improved. The patent application discloses a free-flow electrophoresis method in which electrophoresis is performed while simultaneously flowing an electrophoresis buffer containing a polymer and a buffer solution not containing a polymer. It is described that this method enables electrophoresis having a molecular sieving effect, thereby enabling free-flow electrophoresis having a size separation effect.
[0004]
[Problems to be solved by the invention]
Although the above prior art has the advantages as described above, specific examples of the free flow electrophoresis method using a plurality of buffer solutions include a buffer solution containing a polymer and a buffer solution not containing a polymer. The description is only an example of the combination, and no other specific example is described. Although the use of a buffer solution containing a polymer has the advantage of a size separation effect, a high-viscosity buffer solution is caused to flow through a thin liquid channel, and pressure is applied to the free-flow electrophoresis device. The durability of the product decreases, the free flow electrophoretic elements and piping are contaminated with polymer, so it is necessary to perform frequent maintenance of the device, and the purity of the components separated by mixing the polymer with the separation liquid decreases. It has problems and is not suitable for separation of a sample that does not require a size separation effect and can be separated by a difference in mobility in an electric field.
[0005]
Therefore, the present invention has been made to solve the above problem, and in the electrophoretic separation for separating the sample that can be separated by the difference in mobility in the electric field even when the size separation effect is not given, a plurality of buffer solutions are passed. It is an object of the present invention to provide a free flow electrophoresis method capable of improving the resolution.
[0006]
[Means for solving the problems and effects]
The free flow electrophoresis method of the present invention is a free flow electrophoresis method that separates and sorts a sample while continuously injecting a buffer and a sample into an electrophoresis tank. 3 types Using the running buffer Electrophoresis buffer flowing in the vicinity of the positive electrode and the negative electrode Running buffer and running buffer running through the sample flow area Electrical conductivity Use different electrophoresis buffers Close range Lower the conductivity of the electrophoresis buffer that flows in the sample flow area compared to the electrophoresis buffer that flows in In addition, using a buffer solution containing an anion having a higher mobility than the sample component to be separated as the running buffer flowing in the vicinity of the positive electrode, the mobility is higher than that of the sample component to be separated as the running buffer flowing in the vicinity of the negative electrode. Use a buffer containing a small amount of anion (Claim 1).
[0007]
In the present invention, free flow electrophoresis is an electrophoresis method in which separation and fractionation are performed by electrophoresis while continuously injecting a sample and a support liquid into an electrophoresis tank, as described above. In the separation operation, a support liquid is flowed from the buffer solution inlet at a constant flow rate to create a liquid flow to a plurality of outlets, and a sample is injected from the sample inlet. Since a strong laminar flow is realized by a physical phenomenon peculiar to a narrow space inside the migration tank, when different liquids are continuously flowed, the liquids are difficult to diffuse and flow almost linearly toward the outlet. When a voltage is applied between a pair of electrodes formed at both ends of the electrophoresis tank, the substance contained in the sample flows while migrating in the migration direction (for example, either positive or negative electrode) determined by the substance according to the principle of electrophoresis. It flows out to the exit. Since the direction and speed of electrophoresis differ depending on the substance, the substance contained in the sample flows out to the outlet while being separated. Therefore, the sample can be sorted by taking out the liquid from the specific sorting port from the plurality of outlets.
[0008]
The “buffer solution” refers to an “electrophoretic solution” used for electrophoresis in the present invention.
When an acid or a base is added to the solution or diluted, it acts to relax the change in pH, that is, a buffering solution.
A mixture of a weak acid and a strong base, a weak base and a strong acid, or a weak acid and a weak base is a typical buffer solution, but its composition, concentration, and pH are not particularly limited, and are generally used for electrophoresis. pH can be used.
[0009]
Electric Close range The term “area in the liquid channel along the flow direction of the liquid channel and along the electrode” refers to the region where the sample flows and the sample is electrophoretically separated by the electrophoresis principle.
The electrophoretic element used in the present invention is not particularly limited as long as the object of the present invention can be achieved, but the electrophoretic element disclosed in Japanese Patent Application No. 2001-210560, which is a prior application, can be preferably used. This will be described later.
Embodiments relating to the present invention will be described later. Reference Example 1 and Examples 1-3 Will be described in detail.
[0010]
reference In Example 1, the “electrophoretic buffer solution flowing in the sample flow area” and “both electric currents” in claim 1 are used. Close range The “electrophoresis buffer solution” to be flowed into “corresponding to” corresponds to “buffer solution 1” and “buffer solution 2” respectively.
Includes reference examples According to the present invention, the following effects can be obtained.
The free flow electrophoresis method of the present invention is a Close range In addition, free flow electrophoresis is performed in a state in which a buffer solution having a high electrical conductivity flows and a buffer solution having a low electrical conductivity flows in the sample flow area. The voltage applied between the electrodes is applied in a large proportion to a buffer solution having a low electrical conductivity. For this reason, it becomes possible to apply a high voltage effectively in the vicinity where the sample flows, and the resolution is improved. Since the separation speed can be increased, the free flow electrophoresis element to be used can be miniaturized and manufactured at low cost, and in the future, a disposable free flow electrophoresis element will be possible. Moreover, since the ratio of voltage applied is low in the vicinity of both electrodes, heat generation due to Joule heat can be suppressed, and generation of bubbles in the electrodes can be suppressed.
[0011]
Therefore Includes reference examples The present invention Close range Running buffer and running buffer running through the sample flow area Electrical conductivity Use different electrophoresis buffers Close range It is possible to provide a free-flow electrophoresis method capable of improving the resolution by lowering the electric conductivity of the electrophoresis buffer flowing in the sample flow area with respect to the electrophoresis buffer flowing in the sample.
Hereinafter, preferred embodiments of the present invention will be described.
(1) First preferred embodiment ( Claim 1 Corresponding to)
The first preferred embodiment is: Includes reference examples In the free-flow electrophoresis method according to the present invention (claim 1), at least three kinds of electrophoresis buffers are used, the electrophoresis buffer flowing in the vicinity of the positive electrode, the electrophoresis buffer flowing in the vicinity of the negative electrode, and the sample flow area. Running buffer Electrical conductivity Sample component to be separated as a running buffer that flows in the vicinity of the negative electrode using a buffer solution that contains anions having a higher mobility than the sample component to be separated as a running buffer that flows in the vicinity of the positive electrode as a different running buffer It is characterized by using a buffer containing an anion having a smaller mobility.
A preferred first embodiment of the present invention is: Example 1 The embodiment of this corresponds.
[0012]
(Corresponding example)
Embodiments relating to the present invention are: First The embodiment corresponds. Book Example 1 Among them, “electrophoretic buffer solution flowing to the sample flow area” corresponds to “buffer solution 1”, “electrophoretic buffer solution flowing to the positive electrode region” corresponds to “buffer solution 2”, and “near electrode electrode” "Running buffer to be passed through the area" corresponds to "Buffer 3". The “sample to be separated” in this example corresponds to “an aqueous solution in which xylene cyanol FF and bromophenol blue are mixed”.
[0013]
(Function and effect)
According to a first preferred embodiment of the present invention, the free-flow electrophoresis method is negatively charged. Close range In addition, a buffer solution containing an anion having a high electrical conductivity and a mobility smaller than the sample component to be separated is positively charged. Close range A buffer solution containing an anion having a higher electric conductivity and a higher mobility than the sample component to be separated is subjected to free flow electrophoresis in a state where a buffer solution having a lower electric conductivity flows in the sample flow area. . Therefore, the voltage applied between the electrodes is applied in a large proportion to the buffer solution having low electrical conductivity. For this reason, it is possible to effectively apply a high voltage in the vicinity where the sample flows, and the improvement of the separation ability intended in the present invention can be achieved. Furthermore, since the separation speed can be increased, the free flow electrophoresis element to be used can be made smaller and inexpensively manufactured, and in the future, a disposable free flow electrophoresis element can be realized.
[0014]
Also, the anion contained in the electrophoresis buffer flowing in the vicinity of the negative electrode has a lower mobility than the sample component to be separated, and the anion contained in the electrophoresis buffer flowing in the vicinity of the positive electrode moves more than the sample component to be separated. The degree is great. For this reason, in the electrophoresis in the first preferred embodiment of the present invention, isotachophoretic separation is realized, and the sample converges while being separated by being sandwiched between the preceding ions and the terminal ions. For this reason, the separated components do not spread and can be recovered in a high concentration state. Here, isokinetic electrophoresis uses a buffer solution containing ions with high mobility and a buffer solution containing ions with low mobility to create a state in which all of the separated zones move at the same speed. In the electrophoresis method in which the sample is separated, the sample is sandwiched between two buffers and separated and converged.
[0015]
Therefore, the first preferred embodiment of the present invention comprises an electrophoresis buffer that flows in the vicinity of the positive electrode, an electrophoresis buffer that flows in the vicinity of the negative electrode, and an electrophoresis buffer that flows in the sample flow area. Electrical conductivity Use different electrophoresis buffers Close range The electroconductivity of the electrophoresis buffer that flows in the sample flow area is lower than that of the electrophoresis buffer that flows in the sample, and it contains anions that have higher mobility than the sample components that are separated as the electrophoresis buffer that flows in the vicinity of the positive electrode. Using a buffer solution containing an anion with a lower mobility than the sample component to be separated as a running buffer solution that flows in the vicinity of the negative electrode improves the resolution and provides a high-concentration fraction. Can be provided.
(2) Preferred second embodiment of the present invention ( Claim 2 Corresponding to)
The second preferred embodiment is the first preferred embodiment of the free flow electrophoresis method of the present invention ( Claim 1 ), A solution obtained by diluting the electrophoresis buffer solution flowing in the vicinity of the negative electrode is used as the electrophoresis buffer solution flowing in the sample circulation area.
[0016]
(Corresponding example)
The second preferred embodiment of the present invention is: Example 2 The embodiment of this corresponds. Book Example 2 Among them, the “electrophoresis buffer solution flowing to the sample flow area” corresponds to “buffer solution 1”, and the “electrophoresis buffer solution to flow to the negative electrode vicinity region” corresponds to “buffer solution 3”.
(Function and effect)
The free flow electrophoresis method of the present invention is a negative electrode. Close range In addition, a buffer solution containing an anion having a high electrical conductivity and a mobility smaller than the sample component to be separated is positively charged. Close range A buffer solution containing an anion having a higher electric conductivity and a higher mobility than the sample component to be separated is subjected to free flow electrophoresis in a state where a buffer solution having a lower electric conductivity flows in the sample flow area. . The voltage applied between the electrodes is applied in a large proportion to a buffer solution having a low electrical conductivity. For this reason, it becomes possible to apply a high voltage effectively in the vicinity where the sample flows, and the improvement of the resolution that is the object of the present invention can be achieved. Furthermore, since the separation speed can be increased, the free flow electrophoresis element to be used can be made smaller and inexpensively manufactured, and in the future, a disposable free flow electrophoresis element can be realized.
[0017]
Moreover, the anion contained in the electrophoresis buffer flowing in the vicinity of the negative electrode has a smaller mobility than the sample, and the anion contained in the electrophoresis buffer flowing in the vicinity of the positive electrode has a higher mobility than the sample. For this reason, in the second preferred embodiment of the present invention, isotachophoretic separation is realized in the electrophoresis, and the sample is sandwiched between the preceding ions and the terminal ions as in the first preferred embodiment. Converge while separating. For this reason, the separated components do not spread and can be recovered in a high concentration state.
[0018]
In addition, the running buffer solution that flows to the sample flow area has a composition obtained by diluting the running buffer solution that flows to the vicinity of the negative electrode, and since the contained anions are equal, the time required for convergence can be shortened. In addition, the electrophoresis buffer that flows in the sample flow area can be prepared by diluting the electrophoresis buffer that flows in the vicinity of the negative electrode, and the preparation of the buffer becomes simple.
[0019]
Therefore, the second preferred embodiment of the present invention comprises an electrophoresis buffer that flows in the vicinity of the positive electrode, an electrophoresis buffer that flows in the vicinity of the negative electrode, and an electrophoresis buffer that flows in the sample flow area. Electrical conductivity Use different electrophoresis buffers Close range Electrophoresis buffer that flows into the sample flow area has a lower electrical conductivity than the electrophoresis buffer that flows into the buffer, and the buffer contains anions that have greater mobility than the sample components that are separated as the electrophoresis buffer that flows near the positive electrode. Use a solution containing an anion whose mobility is lower than that of the sample component to be separated as an electrophoresis buffer that flows in the vicinity of the negative electrode. By using a solution obtained by diluting an electrophoresis buffer, it is possible to provide a free-flow electrophoresis method that improves separation performance and can efficiently obtain a high-concentration fraction.
[0020]
(3) Third preferred embodiment of the present invention ( Corresponding to claim 3 )
The third preferred embodiment of the present invention comprises a positive electrode in addition to the free flow electrophoresis method of the first preferred embodiment of the present invention. Close range The electroconductivity of the electrophoresis buffer flowing to the negative electrode and the electrophoresis buffer flowing to the negative electrode side are equal.
[0021]
(Corresponding example)
A third preferred embodiment of the present invention is: Example 3 The embodiment of this corresponds. Book Example 3 Among them, the “electrophoretic buffer solution flowing near the positive electrode” corresponds to “buffer solution 2”, and the “electrophoretic buffer solution flowing near the negative electrode” corresponds to “buffer solution 3”.
(Function and effect)
The free flow electrophoresis method of the third preferred embodiment of the present invention comprises negative electrode electrophoresis. Close range In addition, a buffer solution containing an anion having a high electrical conductivity and a mobility smaller than the sample component to be separated is positively charged. Close range A buffer solution containing an anion having a higher electric conductivity and a higher mobility than the sample component to be separated is subjected to free flow electrophoresis in a state where a buffer solution having a lower electric conductivity flows in the sample flow area. . The voltage applied between the electrodes is applied in a large proportion to a buffer solution having a low electrical conductivity. For this reason, it becomes possible to apply a high voltage effectively in the vicinity where the sample flows, and the improvement of the separation ability targeted by the present invention can be achieved. Since the separation speed can be increased, the free flow electrophoresis element to be used can be miniaturized and manufactured at low cost, and in the future, a disposable free flow electrophoresis element will be possible.
[0022]
Moreover, the anion contained in the electrophoresis buffer flowing in the vicinity of the negative electrode has a lower mobility than the sample, and the anion contained in the electrophoresis buffer flowing in the vicinity of the positive electrode has a higher mobility than the sample component to be separated. . Therefore, the electrophoresis in the third preferred embodiment of the present invention achieves isotachophoretic separation, and the sample component to be separated is separated from the preceding ions and the terminal as in the first preferred embodiment of the present invention. It converges while being separated by being sandwiched between ions. For this reason, the separated components do not spread and can be recovered in a high concentration state.
In addition, since the electrophoresis buffer that flows in the vicinity of the positive electrode and the electrophoresis buffer that flows in the vicinity of the negative electrode have the same electrical conductivity, heat generation due to Joule heat generated in both electrodes is equal, and bubbles generated in both electrodes are not unevenly emitted. Therefore, stable separation for a long time is possible.
[0023]
Therefore, in the third preferred embodiment of the present invention, an electrophoresis buffer that flows in the vicinity of the positive electrode, an electrophoresis buffer that flows in the vicinity of the negative electrode, and an electrophoresis buffer that flows in the sample flow area. Electrical conductivity Use different electrophoresis buffers Close range Electrophoresis buffer that flows into the sample flow area has a lower electrical conductivity than the electrophoresis buffer that flows into the buffer, and the buffer contains anions that have greater mobility than the sample components that are separated as the electrophoresis buffer that flows near the positive electrode. Using a buffer solution containing an anion with a lower mobility than the sample component to be separated as a running buffer that flows in the vicinity of the negative electrode, Close range Equalization of the electroconductivity of the electrophoresis buffer that flows to the negative electrode and the electrophoresis buffer that flows to the negative electrode side improves resolution, provides a high-concentration preparative solution, and enables stable use over a long period of time A free flow electrophoresis method can be provided.
[0024]
(Example)
A free flow electrophoresis element that can be suitably used in the free flow electrophoresis method of the present invention will be described. As this free flow electrophoretic element, substantially the same one as shown in FIGS. 1 to 4 of Japanese Patent Application No. 2000-210560 can be used. However, the free flow electrophoresis element that can be used in the free flow electrophoresis method of the present invention is not limited to this.
[0025]
FIG. 1 is a top view of a free flow electrophoresis element 1 used in the present invention. The free flow electrophoretic element 1 is constituted by a combination of an electrophoresis tank constituting substrate 2 and an injection port constituting substrate 3. FIG. 2 is a cross-sectional view of the line A in FIG. 1, FIG. 3 is a top view of the electrophoresis tank constituting substrate 2, and FIG. 4 is a back view of the inlet constituting substrate 3. This free-flow electrophoretic element 1 includes an electrophoresis tank 8 comprising a wide groove, two electrode chambers 9 comprising elongated grooves, and an electrophoresis tank constituting substrate 2 comprising a partition wall 10 separating the migration tank 8 and the electrode chamber 9. The plurality of buffer solution inlets 4a to 4u, the plurality of sample inlets 5a to 5e, the plurality of outlets 6a to 5u, and the inlet configuration substrate 3 including the pair of electrodes 7a and 7b are bonded to each other. . The electrode 7 is formed of a thin film on the surface of the inlet configuration substrate 3 facing the migration tank configuration substrate 2. The partition wall 10 is configured to have a gap between the opposing inlet configuration substrate 3. The plurality of buffer solution inlets 4a to 4u and the plurality of outlets 6a to 6u correspond to each other in the same number and are configured at the same interval when viewed in the direction perpendicular to the flow.
[0026]
The material for the electrophoresis tank constituting substrate 2 and the inlet constituting substrate 3 is made of borosilicate glass, and the electrode 7 is made of a platinum thin film. The migration tank 8 and the electrode chamber 9 were processed to a depth of 30 micrometers by a known wet etching method. The partition wall 10 was processed so that a space of 5 micrometers was formed between the opposing inlet composition substrate 3. The buffer solution inlet 4, the sample inlet 5, and the outlet 6 were processed by ultrasonic machining. The electrode 7 was formed by sputtering. The electrophoresis tank constituting substrate 2 and the injection port constituting substrate 3 were joined by an anodic bonding method through an amorphous silicon thin film (not shown).
[0027]
In this free flow electrophoresis element, the same number of buffer inlets and outlets correspond to each other and are configured at the same interval when viewed in the direction perpendicular to the flow. When the flow is continued, the flow becomes a strong laminar flow, so that it is difficult for the liquids to diffuse. When different buffer solutions are flowed from each buffer solution inlet, they will diffuse to the corresponding outlet. It will flow out. In isokinetic electrophoresis and isoelectric focusing, it is preferable to provide the buffer inlets closely with a small interval.
Next, a free flow electrophoresis method using the free flow electrophoresis element 1 will be described.
[0028]
First, the function of the free flow electrophoresis element 1 of the present invention will be described. Free flow electrophoresis is an electrophoresis method in which separation and fractionation are performed by electrophoresis while continuously injecting a sample and a support liquid into an electrophoresis tank. The electrophoresis tank corresponds to the electrophoresis tank 8 of the present invention. In the separation operation, a support liquid is flowed in advance from the plurality of buffer solution inlets 4 at a constant flow rate to create a liquid flow to the plurality of outlet ports 6, and a sample is injected from at least one of the plurality of sample inlet ports 5. Since a strong laminar flow is realized by a physical phenomenon peculiar to a narrow space inside the migration tank, when different liquids are continuously flowed, the liquids are difficult to diffuse and flow almost linearly toward the outlet. When a voltage is applied between the pair of electrodes 7, the substance contained in the sample flows out to the outlet 6 while migrating to either the positive or negative electrode side according to the principle of electrophoresis. Since the direction and speed of electrophoresis vary depending on the substance, the substance contained in the sample flows out to the outlet 6 while being separated. Therefore, the sample can be sorted by taking out the liquid from the specific sorting port out of the plurality of outlets 6. Bubbles generated from the electrode 7 due to the application of voltage flow through the electrode chamber to the outlet 6 without entering the migration tank 8 side by the partition wall 10.
[0029]
( reference Example 1) (corresponds to claim 1)
3 mM trishydroxymethylaminomethane-3 mM hydrochloric acid buffer as buffer 1, 10 mM trishydroxymethylaminomethane-10 mM hydrochloric acid buffer as buffer 2, and an aqueous solution in which xylene cyanol FF and bromophenol blue are mixed as samples The free flow electrophoresis was performed using the free flow electrophoresis element 1 described with reference to FIGS. As the free flow electrophoresis element, the one shown in FIGS. 1 and 2 was used. In the free flow electrophoretic element 1, the electrode 7a was connected to the cathode of the voltage application power source, and the electrode 7b was connected to the anode of the voltage application power source. Buffer solution is shade Close range Buffer inlets 4a-4e and anode Neighborhood The buffer solution 2 was supplied to the buffer solution inlets 4q to 4u, and the buffer solution 1 was supplied to the buffer solution inlets 4f to 4p in the sample flow area. The sample was injected with a pump from the sample inlet 5b. Free flow electrophoresis was performed by applying a voltage to the electrode 7 while injecting the buffer solutions 1 and 2 and the sample. The aliquot was obtained by collecting the effluent from the outlets 6a-5u. When free flow electrophoresis was performed by applying a voltage of several kV, xylene cyanol FF in the sample separated and flowed out to the outlet from the cathode and bromophenol blue to the outlet from the anode.
[0030]
When a flow of several kV was applied and free flow electrophoresis was performed, xylene cyanol FF in the sample separated into the outflow from the cathode, and promophenol blue separated into the outflow port from the anode.
When a single buffer solution is used, the interval between the outflow ports where xylene cyanol FF and bromophenol blue flow out is separated by one out of the number of outflows, whereas in this embodiment the outflow port is separated. In this way, a separation of 4 portions was obtained. Thus, the separation rate was superior to the case where a single buffer was used, and separation with high separation capability was possible.
[0031]
Book reference In the example Close range The free flow electrophoresis is performed in a state in which the buffer solution 2 and the buffer solution 1 flow into the sample flow area. Since the buffer solution 2 has a higher concentration than the buffer solution 1, the electric conductivity is high. The voltage applied to the electrode 7 is applied in a large proportion to the buffer solution 1 having low electrical conductivity. For this reason, it becomes possible to apply a high voltage effectively in the vicinity where the sample flows, and the resolution is improved. Moreover, since the ratio of voltage applied is low in the vicinity of both electrodes, heat generation due to Joule heat can be suppressed, and generation of bubbles in the electrodes can be suppressed.
[0032]
Therefore Includes Reference Example 1 The present invention Close range Running buffer and running buffer running through the sample flow area Electrical conductivity Use different electrophoresis buffers Close range It is possible to provide a free-flow electrophoresis method capable of improving the resolution by lowering the electric conductivity of the electrophoresis buffer flowing in the sample flow area with respect to the electrophoresis buffer flowing in the sample. Of course, various modifications can be made to the embodiment of the present invention.
For example, the type of the buffer solution is not limited and can be changed to a buffer solution usually used for electrophoresis. The concentration of the buffer solution is not limited as long as the conductivity of the buffer solution 2 is higher than that of the buffer solution 1. In addition, the buffer solution 1 and the buffer solution 2 do not have to have the same composition, and may have different compositions. The injection port of the buffer solution 2 is not limited, and can flow from both electrodes with an appropriate width. The location of the sample inlet is not limited as long as the buffer solution 1 is flowing.
[0033]
In addition, this invention reference The free flow electrophoresis element 1 used in the example has a plurality of buffer solution inlets, and can be a free flow electrophoresis element capable of performing free flow electrophoresis in a stable laminar flow state. Its configuration is not particularly limited
( Example 1 ) ( Claim 1 Fall under)
5 mM trishydroxymethylaminomethane-5 mM 6-amino-n-caproic acid buffer as buffer 1, 20 mM trishydroxymethylaminomethane-20 mM hydrochloric acid buffer as buffer 2 and 50 mM trishydroxymethylaminomethane as buffer 3 Using an aqueous solution in which xylene cyanol FF and bromophenol blue are mixed as a sample with -500 mM CAPS buffer, reference Free flow electrophoresis was performed using the free flow electrophoresis element 1 used in Example 1. In the free flow electrophoretic element 1, the electrode 7a was connected to the cathode of the voltage application power source, and the electrode 7b was connected to the anode of the voltage application power source. Buffer solution is shade Close range The buffer solution 3 is pumped to the buffer solution inlets 4a to 4e, the buffer solution 2 is flown to the buffer solution inlets 4q to 4u on the anode side, and the buffer solution 1 is pumped to the buffer solution inlets 4f to 4p in the sample flow area. did. The sample was injected with a pump from the sample inlet 5b. Free flow electrophoresis was performed by applying a voltage to the electrode 7 while injecting the buffer solutions 1 and 2 and the sample. The aliquot was obtained by collecting the effluent from the outlets 6a-6u. When free flow electrophoresis is performed with a voltage of several kV, xylene cyanol FF and bromophenol blue in the sample converge after separation, xylene cyanol FF is at the outlet from the cathode, and bromophenol blue is from the anode. It separated and flowed out to the outlet.
[0034]
When free flow electrophoresis is performed with a voltage of several kV, xylene cyanol FF and bromophenol blue in the sample converge after separation, xylene cyanol FF is at the outlet from the cathode, and bromophenol blue is from the anode. It separated and flowed out to the outlet.
When a single buffer solution is used, the interval between the outflow ports from which xylene cyanol FF and bromophenol blue flow out is one out of the number of the outflow ports, whereas in this embodiment, the outflow port is separated. Thus, a separation of 4 portions was obtained. Also, Reference example 1 Under these conditions, xylene cyanol FF and bromophenol blue each flowed out over two outlets, but under this condition, they converged after separation and flowed out to only one outlet. Thus, the separation rate was superior to the case where a single buffer was used, and separation with high separation capability was possible. Moreover, since it has converged after separation, separation in a high concentration state became possible.
[0035]
(Action / Effect)
Next, the operation and effect of the embodiment of the present invention will be described.
In this embodiment, free-flow electrophoresis is performed in a state where the buffer solution 3 flows in the vicinity of the negative electrode side, the buffer solution 2 flows in the vicinity of the positive electrode side, and the buffer solution 1 flows in the sample flow area. Since the buffer solutions 3 and 2 are higher in concentration than the buffer solution 1, the electric conductivity is high. The voltage applied to the electrode 7 is applied in a large proportion to the buffer solution 1 having low electrical conductivity. For this reason, it is possible to effectively apply a high voltage in the vicinity where the sample flows, and the separation performance intended in the present invention is improved.
In addition, CAPS contained in buffer 3 has a lower mobility than xylene cyanol FF and bromophenol blue of the sample, and chloro ion contained in buffer 2 has a mobility higher than xylene cyanol FF and bromophenol blue of the sample. Is big. For this reason, the electrophoresis in this embodiment achieves isokinetic electrophoresis separation, and the sample converges while being separated by being sandwiched between chloro ions as the preceding ions and CAPS as the terminal ions. For this reason, the separated components do not spread and can be recovered in a high concentration state. Here, isokinetic electrophoresis uses a buffer solution containing ions with high mobility and a buffer solution containing ions with low mobility to create a state in which all of the separated zones move at the same speed. In the electrophoresis method in which the sample is separated, the sample is sandwiched between two buffers and separated and converged.
[0036]
Therefore, in the first preferred embodiment of the present invention, an electrophoresis buffer that flows in the vicinity of the positive electrode, an electrophoresis buffer that flows in the vicinity of the negative electrode, and an electrophoresis buffer that flows in the sample flow area. Electrical conductivity Use different electrophoresis buffers Close range Electrophoresis buffer that flows into the sample flow area has a lower electrical conductivity than the electrophoresis buffer that flows into the buffer, and the buffer contains anions that have greater mobility than the sample components that are separated as the electrophoresis buffer that flows near the positive electrode. Using a buffer solution containing an anion having a lower mobility than the sample component to be separated as an electrophoresis buffer that flows in the vicinity of the negative electrode, the separation performance is improved and a high-concentration preparative solution can be obtained. A free-flow electrophoresis method that can be obtained can be provided.
Of course, various modifications can be made to the embodiment of the present invention.
[0037]
For example, the buffer 2 is not particularly limited as long as it contains an anion having a higher mobility than the sample component to be separated. As the anionic species, acetic acid or the like can be used in addition to chloro ions. The buffer 3 is not particularly limited as long as it contains an anion having a mobility smaller than that of the sample component to be separated. In addition to CAPS, caproic acid, glutamic acid, β-alanine, phenol and the like can be used as the anionic species. The buffer solution 1 has a slower anion mobility than the anion mobility in the buffer solution 2, and the buffer solution 1 has the same or faster mobility as the anion mobility contained in the buffer solution 3. The kind is not limited. In both the buffer solutions 1, 2 and 3, the type of anion counterion is not particularly limited. In addition to trishydroxymethylaminomethane, for example, histidine, β-alanine, amediol, 2-amino-2-methylpropanol, barium ion, potassium ion and the like can be used. The concentration of the buffer solution 1 is not particularly limited as long as the electric conductivity is lower than that of the buffer solutions 2 and 3. The inlets of the buffer solutions 2 and 3 are not limited, and can flow from the electrodes with an appropriate width. The location of the sample inlet is not limited as long as the buffer solution 1 is flowing.
[0038]
In addition, a present Example is the conditions in the case of using anion as a sample component to be separated. When cations are targeted for separation, use a buffer containing a cation with a lower mobility than the sample component to be separated in the running buffer flowing in the vicinity of the positive electrode, and separate it into the running buffer in the vicinity of the negative electrode. By using a buffer containing a cation having a mobility higher than that of the sample component, a free flow electrophoresis method having the same effect can be provided.
[0039]
( Example 2 ) ( Claim 2 Fall under)
10 mM trishydroxymethylaminomethane-100 mM CAPS buffer as buffer 1, 5 mM trishydroxymethylaminomethane-10 mM acetic acid buffer as buffer 2, 50 mM trishydroxymethylaminomethane-500 mM CAPS buffer as buffer 3 As an aqueous solution mixed with xylene cyanol FF and bromophenol blue, reference Free flow electrophoresis was performed using the free flow electrophoresis element 1 described in Example 1. In the free flow electrophoretic element 1, the electrode 7a was connected to the cathode of the voltage application power source, and the electrode 7b was connected to the anode of the voltage application power source. Buffer solution is shade Close range Buffer solution 3 to the buffer solution inlets 4a to 4e Neighborhood The buffer solution 2 was supplied to the buffer solution inlets 4q to 4u, and the buffer solution 1 was supplied to the buffer solution inlets 4f to 4p in the sample flow area. The sample was injected with a pump from the sample inlet 5b. Free flow electrophoresis was performed by applying a voltage to the electrode 7 while injecting the buffer solutions 1 and 2 and the sample. The aliquot was obtained by collecting the effluent from the outlets 6a-6u. When free flow electrophoresis is performed with a voltage of several kV, xylene cyanol FF and bromophenol blue in the sample converge after separation, xylene cyanol FF is at the outlet from the cathode, and bromophenol blue is from the anode. It separated and flowed out to the outlet.
[0040]
When free flow electrophoresis is performed with a voltage of several kV, xylene cyanol FF and bromophenol blue in the sample converge after separation, xylene cyanol FF is at the outlet from the cathode, and bromophenol blue is from the anode. It separated and flowed out to the outlet. Reference example 1 Similarly, the separation rate was superior to the case where a single buffer was used, and separation with high separation capability was possible. Also Example 1 Similarly, since it has converged after separation, separation in a high concentration state became possible. Also, Example 1 Under this condition, the time required for convergence was 3 seconds after the sample injection, whereas in this example, the convergence was achieved in 1 second. Thus, convergence after separation occurred for a short time, and efficient separation became possible.
[0041]
(Action / Effect)
Next, the operation and effect of the embodiment of the present invention will be described.
In this example, negative electric Close range Buffer solution 3 Close range The free flow electrophoresis is performed in a state in which the buffer solution 2 and the buffer solution 1 flow into the sample flow area. Since the buffer solutions 3 and 2 are higher in concentration than the buffer solution 1, the electric conductivity is high. The voltage applied to the electrode 7 is applied in a large proportion to the buffer solution 1 having low electrical conductivity. For this reason, it is possible to effectively apply a high voltage in the vicinity where the sample flows, and the improvement of the separation ability intended in the present invention can be achieved.
[0042]
In addition, CAPS contained in buffer 3 has a lower mobility than xylene cyanol FF and bromophenol blue of the sample, and chloro ion contained in buffer 2 has a mobility higher than xylene cyanol FF and bromophenol blue of the sample. Is big. For this reason, electrophoresis in this example is Example 1 In the same manner as described above, isotachophoretic separation is realized, and the sample converges while being separated by being sandwiched between chloro ions as the preceding ions and CAPS as the final ions. For this reason, the separated components do not spread and can be recovered in a high concentration state.
Further, the buffer solution 1 has a composition obtained by diluting the buffer solution 3 five times, and since the contained anions are equal, it is possible to shorten the time required for convergence. Further, the buffer solution 1 can be prepared by diluting the buffer solution 3, and the buffer solution can be easily prepared.
[0043]
Therefore, the present invention provides an electrophoresis buffer that flows in the vicinity of the positive electrode, an electrophoresis buffer that flows in the vicinity of the negative electrode, and an electrophoresis buffer that flows in the sample distribution area. Electrical conductivity Use different electrophoresis buffers Close range Electrophoresis buffer that flows in the sample flow area is lower in conductivity than the electrophoresis buffer that flows in the buffer, and the buffer contains anions that have higher mobility than the sample that is separated into the electrophoresis buffer that flows in the vicinity of the positive electrode Use a buffer containing anions with a lower mobility than the sample to be separated into the running buffer that flows in the vicinity of the negative electrode, and use the running buffer that flows in the vicinity of the negative electrode in the running buffer that flows in the sample flow area. By using a solution obtained by diluting the solution, it is possible to provide a free flow electrophoresis method capable of improving the resolution and efficiently obtaining a high-concentration preparative solution.
The embodiment of the present invention is Example 1 Of course, various changes are possible as well.
[0044]
For example, the buffer 2 is not particularly limited as long as it contains an anion having a higher mobility than the sample. As the anionic species, acetic acid or the like can be used in addition to chloro ions. The buffer solution 3 is not particularly limited as long as it contains an anion having a smaller mobility than the sample. In addition to CAPS, caproic acid, glutamic acid, β-alanine, phenol and the like can be used as the anionic species. In both the buffer solutions 1, 2 and 3, the type of anion counterion is not particularly limited. In addition to trishydroxymethylaminomethane, for example, histidine, β-alanine, amediol, 2-amino-2-methylpropanol, barium ion, potassium ion and the like can be used. The concentration of the buffer solution 1 is not particularly limited as long as the buffer solution 3 is diluted. The inlets of the buffer solutions 2 and 3 are not limited, and can flow from the electrodes with an appropriate width. The location of the sample inlet is not limited as long as the buffer solution 1 is flowing.
[0045]
( Example 3 ) ( Claim 3 Fall under)
5 mM trishydroxymethylaminomethane-5 mM 6-amino-n-caproic acid buffer as buffer 1, 5 mM trishydroxymethylaminomethane-5 mM hydrochloric acid buffer as buffer 2, 50 mM trishydroxymethylaminomethane as buffer 3 -Free flow electrophoresis using the free flow electrophoresis element 1 described with reference to FIGS. 1 and 2 using an aqueous solution in which xylene cyanol FF and bromophenol blue are mixed as a sample with -500 mM 6-amino-n-caproic acid buffer. Went. In the preparation of the buffer solution, the buffer solution 2 and the buffer solution 3 were prepared so as to have the same electric conductivity. The electrical conductivity was measured and adjusted at a conductivity measurement rate under the condition of 25 ° C. The electric conductivity of the buffer solutions 2 and 3 in this example was 0.25 mS / cm. In the free flow electrophoretic element 1, the electrode 7a was connected to the cathode of the voltage application power source, and the electrode 7b was connected to the anode of the voltage application power source. Buffer solution is shade Close range The buffer 3 through the buffer inlets 4a to 4e, the buffer 2 through the anode buffer inlets 4q to u, and the buffer 1 through the buffer inlets 4f to 4p in the sample flow area. did. The sample was injected with a pump from the sample inlet 5b. Free flow electrophoresis was performed by applying a voltage to the electrode 7 while injecting the buffer solutions 1 and 2 and the sample. The aliquot was obtained by collecting the effluent from the outlets 6a-u. When free flow electrophoresis is performed with a voltage of several kV, xylene cyanol FF and bromophenol blue in the sample converge after separation, xylene cyanol FF is at the outlet from the cathode, and bromophenol blue is from the anode. It separated and flowed out to the outlet. The separation rate was superior to the case where a single buffer was used, and separation with high separation capability was possible. Moreover, since it has converged after separation, separation in a high concentration state became possible. Convergence also occurred in a short time, enabling efficient separation. Further, the generation of bubbles in the electrode 7 was stabilized, and stable separation for a long time was possible.
[0046]
When free flow electrophoresis is performed with a voltage of several kV, xylene cyanol FF and bromophenol blue in the sample converge after separation, xylene cyanol FF is at the outlet from the cathode, and bromophenol blue is from the anode. It separated and flowed out to the outlet. Reference example 1 Similarly, the separation rate was superior to the case where a single buffer was used, and separation with high separation capability was possible. Also Example 1 Similarly, since it has converged after separation, separation in a high concentration state became possible. Also, Example 2 Similarly, convergence occurred in a short time and efficient separation became possible. Also Example 2 Then, since the electric conductivities of the buffer solutions 2 and 3 are not equal, Joule heat is generated on the buffer solution 3 side having a lower electric conductivity than the buffer solution 2 side, and the amount of bubbles generated from the buffer solution 3 side is large. In contrast, in this embodiment, since the electric conductivity of the buffer solutions 2 and 3 is equal, the amount of heat generated on both electrodes is uniform, and the amount of bubbles generated is reduced. Stable. For this reason, long-time stable separation became possible.
[0047]
(Action / Effect)
Next, the operation and effect of the embodiment of the present invention will be described.
In this example, negative electric Close range Buffer solution 3 Close range The free flow electrophoresis is performed in a state in which the buffer solution 2 and the buffer solution 1 flow into the sample flow area. Since the buffer solutions 3 and 2 are higher in concentration than the buffer solution 1, the electric conductivity is high. The voltage applied to the electrode 7 is applied in a large proportion to the buffer solution 1 having low electrical conductivity. For this reason, it is possible to effectively apply a high voltage in the vicinity where the sample flows, and the improvement in the resolution aimed at by the present invention is achieved.
In addition, CAPS contained in buffer 3 has a lower mobility than xylene cyanol FF and bromophenol blue of the sample, and chloro ion contained in buffer 2 has a mobility higher than xylene cyanol FF and bromophenol blue of the sample. Is big. For this reason, the electrophoresis in the present embodiment achieves isotachophoretic separation in the same manner as in the second embodiment, and the sample is sandwiched between the chloro ion as the preceding ion and the CAPS as the terminal ion while being separated. Converge. Therefore, the separated components can be recovered in a high concentration state without spreading. Moreover, since the anion contained in the buffer solution 1 and the buffer solution 3 is equal, the time required for convergence is shortened. Moreover, since the buffer solution 2 and the buffer solution 3 have the same electric conductivity, the heat generated by the Joule heat generated in both electrodes is equal, and the bubble generation generated in both electrodes is stable without being biased. It becomes possible.
[0048]
Therefore, the present invention provides an electrophoresis buffer that flows in the vicinity of the positive electrode, an electrophoresis buffer that flows in the vicinity of the negative electrode, and an electrophoresis buffer that flows in the sample distribution area. Electrical conductivity Use different electrophoresis buffers Close range Electrophoresis buffer that flows into the sample flow area has a lower electrical conductivity than the electrophoresis buffer that flows into the buffer, and the buffer contains anions that have greater mobility than the sample components that are separated as the electrophoresis buffer that flows near the positive electrode. Using a buffer solution containing an anion with a lower mobility than the sample to be separated as a running buffer that flows in the vicinity of the negative electrode, Close range Running buffer and negative electrode Neighborhood By providing the same electrical conductivity as the electrophoresis buffer that flows in the cell, the separation performance is improved, a high-concentration preparative solution is obtained, and a free-flow electrophoresis method that can be used stably for a long time is provided. be able to.
The embodiment of the present invention is Example 1 Of course, various changes are possible as well.
[0049]
For example, the buffer solution 2 contains an anion having a higher mobility than the sample, and the composition and concentration are not particularly limited as long as it has the same electric conductivity as the buffer solution 3. As the anionic species, acetic acid or the like can be used in addition to chloro ions. The buffer solution 3 contains an anion having a mobility smaller than that of the sample, and the composition and concentration are not particularly limited as long as it has the same electric conductivity as the buffer solution 2. In addition to CAPS, caproic acid, glutamic acid, β-alanine, phenol and the like can be used as the anionic species. If the mobility of the anion contained in the buffer solution 1 is the same as or slower than the mobility of the anion in the buffer solution 2, and the mobility of the anion contained in the buffer solution 3 is the same or faster. The type is not limited. In both the buffer solutions 1, 2 and 3, the type of anion counterion is not particularly limited. In addition to trishydroxymethylaminomethane, for example, histidine, β-alanine, amediol, 2-amino-2-methylpropanol, barium ion, potassium ion and the like can be used. The concentration of the buffer solution 1 is not particularly limited as long as the electric conductivity is lower than that of the buffer solutions 2 and 3. The inlets of the buffer solutions 2 and 3 are not limited, and can flow from the electrodes with an appropriate width. The location of the sample inlet is not limited as long as the buffer solution 1 is flowing.
[0050]
【The invention's effect】
Includes reference examples According to claim 1 of the present invention, in the free flow electrophoresis method in which the sample is separated and sorted while continuously injecting the buffer and the sample into the electrophoresis tank, at least two kinds of electrophoresis buffers are used, Both electric Close range Running buffer and running buffer running through the sample flow area Electrical conductivity Use different electrophoresis buffers Close range Since the electric conductivity of the electrophoresis buffer flowing in the sample flow area is lower than that of the electrophoresis buffer flowing in the electrode, the voltage applied between the electrodes is applied to the buffer having a low electric conductivity in a large proportion. For this reason, it becomes possible to apply a high voltage effectively in the vicinity where the sample flows, and the resolution is improved. Since the separation speed can be increased, the free-flow electrophoresis element to be used must be miniaturized and inexpensively manufactured. But In the future, a disposable free flow electrophoresis element will be possible. Moreover, since the ratio of voltage applied is low in the vicinity of both electrodes, heat generation due to Joule heat can be suppressed, and generation of bubbles in the electrodes can be suppressed.
[0051]
Further, claim 1 of the present application. According to the present invention, at least three kinds of running buffer solutions are used, and the running buffer solution that flows in the vicinity of the positive electrode, the running buffer solution that flows in the vicinity of the negative electrode, and the running buffer solution that flows in the sample flow area. Electrical conductivity Sample component to be separated as a running buffer that flows in the vicinity of the negative electrode using a buffer solution that contains anions having a higher mobility than the sample component to be separated as a running buffer that flows in the vicinity of the positive electrode as a different running buffer A buffer solution containing an anion having a lower mobility is used. For this reason, claim 1 Reference examples included in In addition to the effects obtained with Claim 1 In the free-flow electrophoresis method, isokinetic electrophoresis separation is realized, and the sample converges while being separated by being sandwiched between preceding ions and terminal ions. For this reason, the separated components do not spread and can be recovered in a high concentration state.
[0052]
This application Claim 2 According to the second aspect of the present invention, in the free flow electrophoresis method, a solution obtained by diluting the electrophoresis buffer that flows in the vicinity of the negative electrode is used as the electrophoresis buffer that flows in the sample flow area. For this reason, this application Claim 2 In free-flow electrophoresis, Reference Example 1 and claim 1 including the same In addition to the effects obtained in, the running buffer that flows in the sample flow area is a diluted composition of the running buffer that flows in the vicinity of the negative electrode, and the contained anions are equal, reducing the time required for convergence. Is possible. In addition, the electrophoresis buffer that flows in the sample flow area can be prepared by diluting the electrophoresis buffer that flows in the vicinity of the negative electrode, and the preparation of the buffer becomes simple.
[0053]
Claim 3 According to the present invention, in the free flow electrophoresis method according to Reference Example 1 and claim 1 comprising the same, Close range Running buffer and negative electrode Neighborhood The electroconductivity of the running buffer flowing through is equal. Includes Reference Example 1 In addition to the effects obtained in claim 1, Claim 3 In the free-flow electrophoresis method, the electrophoretic buffer flowing in the vicinity of the positive electrode and the electrophoretic buffer flowing in the vicinity of the negative electrode have the same electrical conductivity, so the heat generated by Joule heat generated in both electrodes is equal, and the bubbles generated in both electrodes Since the exit is stable without being biased, stable separation for a long time is possible.
[Brief description of the drawings]
FIG. 1 is a plan view of a free-flow electrophoresis element that can be used in the free-flow electrophoresis method of the present invention.
FIG. 2 is a cross-sectional view taken along line AA of the free flow electrophoresis element shown in FIG.
3 is a top view of an electrophoresis tank constituting substrate of the free flow electrophoresis element of FIG. 1. FIG.
4 is a back view of the inlet configuration substrate 3 of the free flow electrophoresis element of FIG. 1; FIG.
[Explanation of symbols]
1 Free-flow electrophoresis element
2 Electrophoresis tank configuration substrate
3 Inlet composition board
4 (4a-4u) Buffer inlet
5 (5a-5e) Sample inlet
6 (6a-6u) Outlet
7 (7a-7b) Electrode
8 Electrophoresis tank (electrophoresis tank)
9 Electrode chamber
10 Bulkhead

Claims (3)

電気泳動槽に緩衝液と試料とを連続的に注入しながら試料の分離・分取を行うフリーフロー電気泳動法において、少なくとも３種の泳動緩衝液を用い、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液と試料流通部域に流す泳動緩衝液とを電気伝導度が異なる泳動緩衝液とし、両電極近域に流す泳動緩衝液に対し試料流通部域に流す泳動緩衝液の電気伝導度を低くするとともに、陽電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の大きな陰イオンを含んだ緩衝液を用い、陰電極近域に流す泳動緩衝液として分離する試料成分よりも移動度の小さな陰イオンを含んだ緩衝液を用いることを特徴とするフリーフロー電気泳動法。In the free-flow electrophoresis method that separates and sorts the sample while continuously injecting the buffer solution and the sample into the electrophoresis tank, the electrophoresis buffer solution that flows in the vicinity of the positive electrode using at least three kinds of electrophoresis buffers and a running buffer to flow to the electrophoresis buffer and the sample circulation unit area flowing through the negative electrode near region and the electric conductivity is different running buffer flow to sample flow section area relative to the running buffer to flow to both electrodes near range Electrophoretic buffer that lowers the conductivity of the running buffer and uses an anion that has a higher mobility than the sample component to be separated as the running buffer that flows near the positive electrode, and that flows near the negative electrode A free-flow electrophoresis method using a buffer solution containing an anion having a smaller mobility than a sample component to be separated as a liquid . 請求項１記載のフリーフロー電気泳動法において、試料流通部域に流す泳動緩衝液に、陰電極近域に流す泳動緩衝液を希釈した液を用いることを特徴とするフリーフロー電気泳動法。 2. The free-flow electrophoresis method according to claim 1, wherein a solution obtained by diluting an electrophoresis buffer that flows in the vicinity of the negative electrode is used as the electrophoresis buffer that flows in the sample flow area. 請求項１または２に記載のフリーフロー電気泳動法において、陽電極近域に流す泳動緩衝液と陰電極近域に流す泳動緩衝液の電気伝導度が等しいことを特徴とするフリーフロー電気泳動法。In free-flow electrophoresis method according to claim 1 or 2, free-flow electrophoresis, wherein the electrical conductivity of the running buffer to flow to the electrophoresis buffer and the negative electrode near zone to flow to the positive electrodes near frequency is equal to .
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