JP2004144479A - Multi-capillary array electrophoresis system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enhance analytical precision in an electrophorestic system wherein irradiation light is emitted to pass successively through a plurality of capillaries to a detecting part of a multi-capillary array comprising the plurality of capillaries. <P>SOLUTION: A liquid or solid having a refractive index higher than that of air and not higher than that of water is interposed in a space between the capillaries to reduce a scattering level of detected light caused by the capillaries. Refraction and reflection are regulated when the irradiation light passes through the capillaries, so as to reduce optical loss caused by the transmission of the light through the plurality of capillaries, and light intensity emitted to a sample is thereby prevented from being reduced. <P>COPYRIGHT: (C)2004,JPO

Description

【００７１】
キャピラリアレイ１の光照射部８はアレイ台１５上に形成されている。アレイ台上の９６本のキャピラリ１６すべてがアレイ台１５に接触するように、かつ、隣接キャピラリと接触するようにアレイ台１５上に配列され、押さえ板１７とともに、アレイ台１５に対して接着固定されている。これにより、実施例２と同様に、各キャピラリと取付基準面の位置関係が一義的に定まる。
【００７２】
キャピラリ１６は、実施例１と同様にポリマ薄膜で覆われているが、光照射部６０−２においては、ポリマ被膜が除去され、石英管１８がむき出しの状態になっている。実施例３にかかる発明では、この石英が露出している光照射部６０−２が、屈折率１．２９　のフッ素化ポリマであるＦポリマで覆われている。または、屈折率１．３２　のフッ素化ポリマであるデュポン社製Ｔｅｆｌｏｎ　ＡＦ　１６００（登録商標）（以下「Ｆ′ポリマ」という）で覆われていてもよい。
【００７３】
キャピラリ１６をアレイ台１５に対して固定した後にＦポリマ６０をキャピラリ間に充填した。両端のキャピラリの外側に位置する２つのポリマブロック６１，６２の間の空間において、レーザ光２４，２５の通過領域を完全に覆うようにＦポリマ６０を充填した。
【００７４】
ここで、Ｆポリマの化学構造式Ｅを示す。なおｎは自然数である。
【化２】

【００７５】
また、Ｆポリマ、及びＦ′ポリマには次の特徴がある。
（１）温度安定性が高い（Ｈｉｇｈ　ｔｅｍｐｅｒａｔｕｒｅ　ｓｔａｂｉｌｉｔｙ）
（２）科学的耐性に秀でている（Ｅｘｃｅｌｌｅｎｔ　ｃｈｅｍｉｃａｌ　ｒｅｓｉｓｔａｎｃｅ）
（３）表面エネルギーが低い（Ｌｏｗ　ｓｕｒｆａｃｅ　ｅｎｅｒｇｙ）
（４）水吸収が低い（Ｌｏｗ　ｗａｔｅｒ　ａｂｓｏｒｐｔｉｏｎ）
（５）透明で光伝達率が高い（ｔｒａｎｓｐａｒｅｎｔ　ｈｉｇｈ　ｔｒａｎｓｍｉｓｓｉｏｎ）
（６）屈折率が低い（ｖｅｒｙ　ｌｏｗ　ｒｅｆｒａｃｔｉｖｅ　ｉｎｄｅｘ）
（７）ガス浸透性が高い（Ｈｉｇｈ　ｇａｓ　ｐｅｒｍｅａｂｉｌｉｔｙ）
【００７６】
図２０にＦポリマの透過スペクトラムを示す。図よりＦポリマは紫外線から赤外線の範囲で高い透過性を有することがわかる。従って、Ｆポリマ６０は、蛍光であるアルゴンレーザの波長４８８．０ｎｍ，５１４．５ｎｍの光を吸収せず、また、この光を照射しても蛍光を発しないため、充填媒体として適している。
【００７７】
図２２にＦポリマ及びＦ′ポリマの屈折率を示す。Ｆポリマの温度２０℃におけるナトリウムＤ線による屈折率は１．２９　であり、Ｆ′ポリマの温度２０℃におけるナトリウムＤ線による屈折率は１．３２　である。屈折率は、接触液体としてα−ｂｒｏｍｏ−ｎａｐｔｈａｌｅｎｅ　を利用したＡｂｂｅ反射測定器を用いて測定した。これら屈折率は、固体有機重合体の中で最も低い値として知られており、ＡＦ２４００の屈折率は、ＧｒｏｈとＺｉｍｍｅｒｍ　が提唱した固体有機重合体屈折率の理論的最低限界値に近い値である。
【００７８】
このように、屈折率が１．２５から１．３２、好ましくは１．２９　のフッ素含有ポリマを、キャピラリの隙間に充填することにより、レーザ光２４，２５が石英管１８表面を透過する際の屈折と反射による光強度の損失を防ぐことができ、実施例１とほぼ同様の信号を得ることができる。
【００７９】
実施例４
図９ａに、実施例４のレーザ照射部付近の側面図を示す。
本実施例では、図９ａに示すように、充填したＦポリマ６０の表面形状を、その断面形状がキャピラリ１６と同心の円状になるように成型した。その他の構造は実施例３と同様である。ここで、蛍光集光レンズ３１のＦ値が１．８　であるとする。Ｆポリマ６０の表面が平面である場合（図９ｂ）には、空気とＦポリマ６０の界面６５において信号光が屈折するため、図示するとおりキャピラリ断面において±１２°の範囲の信号光しか蛍光集光レンズ３１に到達しない。一方、Ｆポリマ６０の表面形状がキャピラリ１６と同心円状である場合には、キャピラリ断面において±１５．５°　の範囲の信号光が蛍光集光レンズに到達する。また、キャピラリ中の検査試料から生じた蛍光が石英管とＦポリマ６０の境界面、及びＦポリマ６０と外気（空気）との境界面を通過する際、蛍光はこれら境界面に対して垂直に入射する。従って、蛍光は屈折しないため、収差による問題が生じない。このように、充填したＦポリマ６０の表面形状を、その断面形状がキャピラリの同心円の一部となるように成型することにより、測定部３４−３（ＣＣＤ３４）が検出する蛍光の強度を増加することができ、分離能が向上する。
【００８０】
また、充填したＦポリマ６０の表面形状を、所定の曲面となるように形成することでも分離能を向上することができる。たとえば、その１断面が所定の曲線（楕円，双曲線，中心軸がキャピラリ中心軸と異なる円）となるように曲面を形成する。これにより、Ｆポリマ６０をレンズとして作用させ、検査試料から生じる蛍光をより多く検出機構３４−２に収束させることができる。尚、収差による問題は、表面形状を非球面レンズとして形成したりすること等で回避できる。
【００８１】
以上のように、Ｆポリマ６０の表面形状を所定の曲面に形成し、蛍光の進行方向を制御して、検出機構に入射する蛍光強度を上げることにより、分離能を向上させることができる。
【００８２】
実施例５
図１０に、実施例５の検出部付近の側面図を示す。本実施例では、キャピラリ間であってレーザ光が通過する空間を満たす液体は、キャピラリアレイのセル構造に包含されておらず、キャピラリアレイ電気泳動装置本体側に具備された液体を包含するセル構造に保持されている。この場合、装置のセル内にキャピラリをディップし、キャピラリアレイを固定する。その他、特に記載のない事項については、実施例１と同様である。
【００８３】
キャピラリアレイがセル構造を有さない点を除いて、アレイ台１５上に形成される検出部の基本的な構造は、実施例１と同様であり、９６本のキャピラリ１６すべてがアレイ台１５に接触するように、かつ、隣接キャピラリと接触するようにアレイ台１５上に配列され、押さえ板１７とともに、アレイ台１５に対して接着固定されている。
【００８４】
電気泳動装置のキャピラリアレイ取付部は、Ｆ溶液を満たすことができる容器部であるセルと、レーザ光を透過することができるレーザ光透過部であるセルの石英窓７２，７３と、蛍光を透過することができる蛍光透過部であるセル下部の石英窓７１と、取付基準面Ｐ′４１を備えている。
【００８５】
電気泳動装置は、キャピラリアレイを下向きに取り付けるようになっている。アレイ台１５の面Ｐ４０を装置の取付基準面Ｐ′４１に接触させることによりアレイ台１５と電気泳動装置の相対位置合わせを行うことができる。
【００８６】
電気泳動装置のセルは、Ｆ溶液１９で満たされ、レーザ光２４，２５はセルの石英窓７２，７３を通してＦ溶液１９中を水平方向に進行している。キャピラリからの発光は下向きであり、セル下部の石英窓７１を通して、実施例１と同様な検出機構により検出される。
【００８７】
気泡がレーザ光路上に侵入することを防ぐために、アレイ台には穴加工７０が施してある。穴加工７０とは、アレイ台１５を貫通する穴を形成することである。また、光照射部の上方に位置するアレイ台１５に、溝を形成することでも同様の効果を達成することができる。これにより、キャピラリの光照射部の近傍の気泡が上方に移動できるため、該気泡はレーザ光路上に残留しない。
【００８８】
本実施例により、マルチキャピラリアレイの光照射部近傍をＦ媒体に浸すことができるため、実施例１とほぼ同様の測定結果を得ることができ、電気泳動装置の分離能を向上させることができる。
【００８９】
実施例６
実施例６は、複数キャピラリの両側からレーザ光を照射する電気泳動装置において、透過したレーザ光を直線偏光とする偏光素子，透過したレーザ光の偏光方向を回転させる偏光回転素子を適切に配置することにより、レーザ光がレーザ発振機に戻りレーザ発振を不安定にするという問題を回避・低減するものである。
【００９０】
図１１に、実施例６の概略を示す。キャピラリアレイの構成は実施例１と同様である。キャピラリアレイの検出部付近とレーザ光の導入経路のみ表示し、レーザ用のシャッタ，フィルタ等は表示していない。
【００９１】
本実施例にかかるマルチキャピラリアレイ電気泳動装置は、実施例１と同様の構成であるキャピラリアレイと、複数のキャピラリを貫く２つのレーザ光を照射し、この２つのレーザ光の進行方向を対向するように調整する光照射機構と、キャピラリアレイを通過したレーザ光がレーザ本体に戻ることを防ぐための光遮断機構をそなえる。
【００９２】
光照射機構は、コヒーレント光であるレーザ光８０を照射する光源であるレーザ本体８７と、一つのレーザ光８０を２つのレーザ光２４，２５に２等分できるハーフミラー８１と、レーザの進行方向を変更するミラー９１と、集光レンズ８３，８４よりなる。レーザ光の経路の途中に、ミラー，ハーフミラー，レンズなどがあってもかまわない。
【００９３】
光遮断機構は、透過する光の偏光方向を変更できる偏光回転素子であるハーフ波長板（水晶λ／２板，雲母波長板など）８８，８９と、所定の偏光のみ透過する偏光フィルタ９０よりなる。
【００９４】
レーザ本体８７から照射されたレーザ光８０は、ハーフミラー８１によって２等分される。これら２つのレーザ光は、キャピラリアレイ８２に対して両側の側面から照射する。ここで、ハーフミラー８１による反射光をレーザ光２４，透過光をレーザ光２５とする。レーザ光２４の集光レンズを集光レンズ８３，レーザ光２５の集光レンズを集光レンズ８４とする。アレイの端に位置し、レーザ光２４が導入されるキャピラリを以下、第１キャピラリ８５，レーザ光２５が導入されるキャピラリを以下、第９６キャピラリ８６とする。レーザ光２４，２５は、９６本のそれぞれのキャピラリの中心軸を含む平面（以下、「アレイ面」という）内にあり、キャピラリに対し垂直に入射する。レーザ光２４と２５は同軸になっており、一方のレーザ光のうちキャピラリを通過した光は、もう一方の入射レーザ光の同軸上を通り、レーザ本体８７に戻るように光軸を調整した。レーザ光２４，２５はともに直線偏光であり、その偏光方向はキャピラリ軸に対し垂直方向である。以上の構成（偏光素子と偏光回転素子を設置しない場合）において、キャピラリアレイからの反射光、または、キャピラリアレイを通過した透過光がレーザ本体に戻り、レーザ発振が不安定化し、分離能が悪化するという問題が発生する。尚、透過戻り光強度，反射戻り光強度ともに、入射光強度の６％程度であった。
【００９５】
この戻り光を抑制するために、上下両方からのそれぞれのレーザ光２４，２５に対し、レーザ集光レンズのキャピラリ側に偏光回転素子としてハーフ波長板である水晶λ／２板，雲母波長板８８，８９などを設置した。偏光フィルタおよび偏光回転素子の光軸周りの回転角度調整を次のように行うことが望ましい。偏光フィルタは、レーザからの光の透過光強度を最大にするように角度調整する。偏光回転素子については、偏光回転角度が４５°になるようにこの素子の回転角度を調整する。偏光を４５°回転させる光学素子を２つ通過することにより、入射光の偏光方向は９０°回転する。２つ目の偏光フィルタは偏光方向が９０°回転した光の透過光強度が最小になるように角度調整する。尚、偏光回転素子の回転角度，偏光フィルタの偏光方向は上記数値に厳密に限定されるものではなく、戻り光強度の問題を解決するために十分であれば良い。
【００９６】
本実施例では、分岐点よりもキャピラリアレイ側に偏光回転素子を設置し、分岐点よりもレーザ側に偏光フィルタを設置する。レーザ本体８７の近くに設置された偏光フィルタは、レーザからの光の強度を最大にするように角度調整してある。キャピラリアレイを透過してループ状の光路を戻ってきた光は偏光方向が９０°回転しているので、偏光フィルタは戻り光に対しては透過光強度が最小になる。
【００９７】
尚、偏光フィルタ、及び偏光回転素子の設置は、これに限定されるものではない。たとえば、分岐点よりもキャピラリ側に、偏光フィルタ，偏光回転素子の両方を設置してもよい。この場合、それぞれの光学素子は２個ずつ必要になる。また、分岐点よりもレーザ側に偏光フィルタを設置してもよい。この場合には１つの偏光フィルタが必要になる。また、集光レンズと偏光回転素子の配置を交換し、集光レンズを偏光回転素子よりキャピラリ側に配置してもよい。この場合、レーザは、偏光回転素子、集光レンズの順に透過する。
【００９８】
これにより、レーザ本体８７より照射されたレーザ光は、偏光フィルタ（透過光が最大になるように角度調整する）を通過し、ハーフミラーで２つに分岐し、偏光回転素子を通過した後に、２方向からキャピラリアレイを照射する。キャピラリ軸に対して垂直方向のレーザ光の偏光がハーフ波長板８８により４５°回転され、キャピラリ軸に対して４５°の角度でキャピラリアレイに入射する。上記光経路では、キャピラリアレイの地点において上下２つのレーザ光の偏光方向は互いに垂直になる。キャピラリ内径内からの信号光強度は、入射レーザ光の偏光方向に依存するものの、上下２つのレーザ光はキャピラリ軸に対してともに４５°になるため、９６本のキャピラリの信号光強度分布は上下対称になる。
【００９９】
キャピラリアレイを透過したレーザ光は、偏光回転素子（最初の偏光回転素子と同一方向に偏光を回転する）を通過する。キャピラリアレイの両側に配置された偏光回転素子は、レーザ光を同じ方向に偏光させる性質を有するため、キャピラリアレイの一端から入射するレーザ光は、キャピラリを通過後に再度偏光素子により偏光方向が回転し、他方のレーザ光の偏光方向とほぼ直角の関係になる。
【０１００】
次に、ハーフミラーを経由して偏光フィルタに進入する。ここで、２つ目の偏光フィルタは１つ目の偏光フィルタと同一のものであり、戻り光は、反対側から入射することになる。但し、戻り光の偏光は９０°回転している。言い換えると、レーザ光２４はハーフ波長板８８により、キャピラリ軸に対して垂直方向のレーザ光の偏光が４５°回転され、キャピラリ軸に対して４５°の角度でキャピラリアレイに入射するようになっている。レーザ光２４は、９６本のキャピラリを透過した後、ハーフ波長板８９でさらに４５°偏光が回転する。２つのハーフ波長板８８，８９を通過することによりレーザ光２４の偏光方向は９０°回転する。同様にレーザ光２５の透過光も偏光方向が９０°回転する。そして、透過光は偏光フィルタに進入する。
【０１０１】
しかし、この偏光フィルタは、レーザ本体８７からのレーザ光の強度を最大にするように角度調整してあり、キャピラリアレイを透過してループ状の光路を戻ってきた光は偏光方向が９０°回転しているので、偏光フィルタは戻り光に対しては透過光強度が最小になるようになっている。
【０１０２】
従って、この偏光フィルタを通過することができず、レーザヘッドに到達しないため、戻り光がレーザに戻ることを抑制することができる。９６本のキャピラリを通過する過程において直線偏光が乱れ、キャピラリ通過前の偏光成分は２５％程度減少する。しかしながら、本発明により透過戻り光を７５％抑制することができた。
【０１０３】
本実施例では、キャピラリアレイを通過した光がレーザ発振器に戻り、レーザ発振を不安定にするという問題が解決でき、比較的安定したレーザ発振を得ることができた。
【０１０４】
実施例７
実施例６においては、キャピラリにより反射した戻り光を抑制できないという問題があった。実施例７は、複数キャピラリの両側からレーザ光を照射する電気泳動装置において、キャピラリへのレーザ入射角度をキャピラリと垂直にしないことで、キャピラリにより反射したレーザ光がレーザ発振機に戻りレーザ発振を不安定にするという問題を回避・低減するものである。
【０１０５】
図１２に、実施例７の模式図を示す。本実施例は、光照射部の光軸構成以外は実施例６と基本的に同様である。しかし、前述した光照射機構がキャピラリに対し垂直から２°ずれた角度でレーザ光を照射し、また、光選択部材であるピンホール板９７を供えている。この光選択部材は、光源からキャピラリに進行するレーザ光を透過できる開口部と、キャピラリから光源に進行する戻りレーザ光は遮断できる障壁部を備えている。
【０１０６】
レーザ光２４，２５は、アレイ面内にあり、キャピラリに対し垂直から２°ずれた角度で入射する。レーザ光２４と２５はキャピラリアレイに照射される際に同軸になっており、一方のレーザ光のうちキャピラリを通過した光は、もう一方の入射レーザ光と同軸上を通り、レーザ本体８７に戻るように光軸を調整している。これにより、レーザ光２４，２５によるキャピラリやセルからの反射光９５，９６は、図１２に示すように入射レーザ光軸とは別の光軸上を伝搬するため、レーザ光２４，レーザ光２５のいずれとも同軸とはならない。
【０１０７】
レーザ本体８７のレーザ光出射口付近に、キャピラリ入射前のレーザ光は透過し、キャピラリで反射したレーザ光は透過しない光選択手段である直径１．４ｍｍのピンホールを有するピンホール板９７を取付ける。レーザ本体８７からのレーザ光はピンホールを通過するが、反射光はピンホールを通過できないため、反射光９５，９６がレーザ本体８７に戻ることを防ぐことができる。従って、キャピラリアレイやセルからの反射光がレーザ発振器に戻り、レーザ発振を不安定にするという問題を解決できる。
【０１０８】
尚、本実施例と実施例６とは組み合わせることにより、透過戻り光は実施例６の方法で抑制され、反射戻り光は本実施例の方法で抑制することができるため、安定したレーザ発振を得ることができる。
【０１０９】
実施例８
実施例８にかかる発明は、キャピラリ周囲の充填媒質の形状を所定の曲面に形成することで、キャピラリから生じる蛍光が充填媒質境界面を通過する際に発散することを回避するものである。
【０１１０】
図１６に、実施例８のレーザ照射部付近の上面図を示す。その他の構造は実施例３と同様である。図１６ａに示すように、充填した光伝達媒体であるＦポリマ６０の表面形状が、キャピラリ内のレーザ照射部分１００から見て凸形状となるように成型されている。
【０１１１】
ここで、蛍光集光レンズ３１のＦ値が１．８　であるとする。Ｆポリマ６０の表面が平面である場合（図１６ｂ）には、空気とＦポリマ６０の界面６５において信号光が屈折するため、図示するとおりキャピラリ断面において約±１１°の範囲の信号光しか蛍光集光レンズ３１に到達しない。
【０１１２】
一方、Ｆポリマ６０の表面形状が図１６ａのように凸形状である場合には、その表面形状の曲率半径に依存してキャピラリ断面において約±１１〜１５°の範囲の信号光が蛍光集光レンズに到達する。このように、充填したＦポリマ６０の表面形状を凸形状等の曲面にすることにより、検出機構により検出されるキャピラリ１６からの照射光強度を増加させることができる。なお、照射光強度を効果的に増大させるためには、Ｆポリマ６０の表面凸形状の円弧の中心とレーザ光軸が一致することが望ましい。検査試料が発した蛍光が、Ｆポリマ６０の凸形状表面に対して垂直方向に進行するため、その凸形状表面（境界面）を通過する際に進行方向が変化しないためである。これにより、収差の問題も回避することができる。
【０１１３】
また、充填したＦポリマ６０の表面形状を、所定の曲面となるように形成することでも、分離能を向上することができる。たとえば、その１断面が所定の曲線（楕円，双曲線，中心がレーザ光軸と異なる円）となるように曲面を形成する。このようにすると、蛍光が、検出部であるＣＣＤカメラにより多く収束するため、ＣＣＤが検出する蛍光強度が増大する。つまり、キャピラリ周囲のＦポリマ６０の表面形状を所定の曲面に形成することで、Ｆポリマ６０がレンズとして作用し、蛍光の進行方向を調整することができる。尚、収差による問題は、Ｆポリマ６０の表面形状にかかるレンズが、複数の焦点を持つように形成することで回避できる。
【０１１４】
尚、Ｆポリマの表面とは空気に接するものに限らず、Ｆポリマとは異なる屈折率を有する媒質との接触面をいう。また、Ｆポリマとキャピラリとの間に、屈折率の異なる別媒質が存在してもよい。
本実施例は実施例４と同時に実施することもできる。
【０１１５】
実施例９
実施例９は、キャピラリ周囲の充填媒質を被覆する容器の外型を所定の曲面に形成することで、キャピラリから生じる蛍光が容器表面を通過する際に発散することを回避するものである。
【０１１６】
図１７に、実施例９の光照射部付近の上面図を示す。その他の構造は実施例１と同様である。図１７ａに示すように、セル蓋２０の表面形状を、キャピラリ内のレーザ照射部分１００から見て凸形状になるようにする。
【０１１７】
ここで、蛍光集光レンズ３１のＦ値が１．８　であるとする。セル蓋２０の表面形状が平面である場合（図１７ｂ）には、空気とセル蓋２０の界面において信号光が屈折するため、図示するとおりキャピラリ断面において約±１１°の範囲の信号光しか蛍光集光レンズ３１に到達しない。
【０１１８】
一方、セル蓋２０の表面形状が図１７ａのように凸形状である場合には、その表面形状の曲率半径に依存してキャピラリ断面において約±１１〜１５°の範囲の信号光が蛍光集光レンズに到達する。このように、セル蓋２０の表面形状を、凸形状にすることで検出されるキャピラリ１６からの照射光強度を増加させることができる。なお、照射光強度を効果的に増大させるためには、セル蓋の表面凸形状の円弧の中心とレーザ光軸が一致することが望ましい。
【０１１９】
また、セル蓋の表面形状をより大きな曲率の曲面とすることで、蛍光が検出機構内のＣＣＤカメラにより収束し、照射光強度を増大させることができる。つまり、キャピラリ周囲の充填媒質を被覆するセル蓋等容器の形状を所定の曲面に形成することで、セル蓋等の容器がレンズとして作用し、蛍光の進行方向を調整することができる。
【０１２０】
尚、セル蓋の表面とは空気に接するものに限らず、セル蓋等の容器とは異なる屈折率を有する媒質との接触面をいう。また、セル蓋と境界面を形成する充填媒質の間に、屈折率の異なる別媒質が存在してもよい。
【０１２１】
実施例１０
実施例１０にかかる発明は、一方が平面で他方が曲面の部材であるシリンドリカルレンズを、表面形状が平面であるマルチキャピラリアレイに備え付けることで、実施例９におけるセル蓋の表面凸形状の機能を、装置に装着されたシリンドリカルレンズ２０２が担うものである。
【０１２２】
図１８に、実施例１０のレーザ照射部付近の上面図を示す。本実施例では、実施例１にかかるマルチキャピラリアレイのセル蓋が、電気泳動装置本体に装着されたシリンドリカルレンズ２０２に覆われている。その他の構造は実施例１と同様である。
【０１２３】
実施例９におけるセル蓋の表面凸形状の機能を装置に装着されたシリンドリカルレンズ２０２が担うことができるため、消耗品であるキャピラリアレイのセル蓋は平面ガラス板でよいことになる。また、シリンドリカルレンズ２０２が装置に装着されているため、シリンドリカルレンズ２０２の表面凸形状の中心とレーザ光軸が一致するため、照射光強度がキャピラリアレイの取付精度に依存しないというメリットがある。
【０１２４】
実施例１１
図１９に、実施例１１のレーザ照射部付近の正面図（１９ａ）と正面図におけるＡ−Ａ′断面（１９ｂ）を示す。本実施例では、１本のキャピラリ中からの試料から生じる蛍光を透過する検出窓を有する背景光遮蔽部材が、光照射部は石英製のアレイ台１５上に並列配置されたキャピラリの上方に配置されている。その他の構造は実施例１と同様である。
【０１２５】
９６本のキャピラリ１６すべてがアレイ台１５に接触するようにアレイ台１５に配列され、シリコン板１０１とともに、アレイ台１５に対して接着固定されている。シリコン板１０１には、シリコン異方性エッチング技術により、柵１０２により仕切られた検出窓１０３と、キャピラリ１６の配列位置決めのために用いられるＶ溝１０４が形成されている。Ｖ溝１０４にキャピラリ１６が嵌るため、キャピラリ１６を所定の間隔で高精度に配列することができ、また、柵１０２により仕切られた検出窓１０３とキャピラリ１６との位置合わせを容易に行うことができる。
【０１２６】
また、アレイ台１５にはシリコン板の位置決めガイド１０５が形成されているが、これは測定部の検出許容範囲に対応している。すなわち、全てのキャピラリ１６がシリコン板の位置決めガイド１０５の内側にあれば、全てのキャピラリ１６が検出機構の検出許容範囲内に入るということである。アレイ台１５にシリコン板の位置決めガイド１０５を形成することにより、アレイ台１５に対するキャピラリ１６の位置決めを容易に行うことが可能となる。
【０１２７】
キャピラリ１６は、内径５０μｍ，外径１２６μｍの石英管１８が厚さ１２μｍのポリマ被膜で覆われた構造をとり、全外径は１５０μｍである。キャピラリ内部にはＤＮＡの分離媒体であるポリマ水溶液（屈折率１．４１）が充填されている。光照射部においては、ポリマ被膜が除去され、石英管１８がむき出しの状態になっている。レーザ光を照射すると、レーザ光の乱反射光の一部がキャピラリ１６のポリマ被膜にあたることにより蛍光を発したり、ＤＮＡサンプルが発した蛍光の一部が乱反射したりする。このポリマ被膜からの蛍光やＤＮＡサンプルからの蛍光の乱反射光が検出機構で受光されると、背景光の増大によりＳＮ比が低下し、分離能が低下するしかし、所望の照射光は検出窓１０３を透過するが、背景光は柵１０２により遮られるため、背景光の増大を防止することが可能となる。さらには、アレイ台１５からの反射光が検出窓１０３を通過し、検出機構で受光されることを防ぐために、アレイ台１５には反射防止膜１０６が形成されている。これにより、さらに背景光を低減させることができるため、分離能をさらに向上させることが可能となる。
【０１２８】
光照射部のキャピラリ周辺は、Ｆ溶液１９で満たされている。Ｆ溶液１９は、石英製のセル蓋２０，アレイ台１５、および、それらを固定する接着剤で完全に密封されている。
【０１２９】
温度変化に伴うＦ溶液１９の体積膨張によりセルの密封構造が破壊されることを防ぐために、密封構造の中には発泡倍率が３０倍（体積の約３０分の２９が気泡）の高圧縮が可能な発泡体１０７を混入させた。Ｆ溶液１９の体膨張率は０．００１２ｍｍ^３／ｍｍ^３・℃　であるから、キャピラリアレイの保存温度（室温２５℃）と使用温度（６０℃）との差を３５℃とすれば、Ｆ溶液１９の保存温度から使用温度への温度上昇（３５℃）による体積増加は約０．０４ｍｍ^３／ｍｍ^３（約４％）となる。発泡体１０７の大きさは、セル容積の約１０％の大きさに設定した。
【０１３０】
発泡体１０７は、アレイ台１５に設けられた溝に、アレイ台１５とキャピラリ１６に挟まれるようにして配置されている。また、発泡体１０７は、検出機構の検出範囲内であるが、シリコン板１０１により検出機構から隠された位置に配置されている。これにより、発泡体１０７の蛍光による背景光の増大を防止することができる。また、発泡体１０７は、セルの密封構造内にＦ溶液を注入するための穴１０９に、それを塞ぐ蓋として配置することもできる。これにより、アレイ台１５への発泡体１０７の為のみの特別加工が不要になる。
【０１３１】
気泡がレーザ光路上に侵入することを防ぐために、セル内部には気泡が存在しないことが望ましいが、セル内部から気泡を完全に除去することは容易ではない。そこで、仮にＦ溶液１９で満たされたセル内部に気泡が混入したとしても、この気泡がレーザ光路上に侵入することを防ぐために、セルに石英製の気泡排除ブロック２３を形成している。
【０１３２】
入射レーザ光とセルのレーザ入射面の角度を一定に保つ必要がある。アレイ台１５にはセル蓋の位置決め用の溝であるガイド１０８を形成されており、アレイ台１５に対するセルの位置決めを容易かつ確実に行うことができる。
【０１３３】
以上より、背景光を低減したＳＮ比の高いキャピラリアレイを実現することができた。
【０１３４】
【発明の効果】
本発明によれば、複数のキャピラリ内の試料を高精度に分析することができる。
【図面の簡単な説明】
【図１】実施例１のキャピラリアレイにおける光照射部付近の側面図（１ａ）と正面図（１ｂ）である。
【図２】本発明が適用されるキャピラリアレイ電気泳動装置の概観図である。
【図３】キャピラリアレイからの発光の検出系の概観図である。
【図４】実施例１により得られるＣＣＤに結像されたイメージと、９３本のキャピラリからの発光強度分布である。
【図５】実施例１により得られた電気泳動結果である。
【図６】実施例２のキャピラリアレイ取付部の正面図（６ａ）と正面図におけるＡ−Ａ′断面（６ｂ）である。
【図７】Ｆ溶液における直線ＶＷ上の温度勾配の計算結果である。
【図８】実施例３の検出部付近の側面図（８ａ）と正面図（８ｂ）である。
【図９】実施例４のレーザ照射部付近の側面図（９ａ）と、実施例３のレーザ照射部付近の側面図（９ｂ）である。
【図１０】実施例５の検出部付近の側面図である。
【図１１】実施例６の模式図である。
【図１２】実施例７の模式図である。
【図１３】充填媒体の屈折率と発光強度比のシミュレート結果である。
【図１４】実施例１により得られるＣＣＤに結像されたイメージと、９６本のキャピラリからの発光強度分布である。
【図１５】９６本のキャピラリ中の一本についての電気泳動結果である。
【図１６】実施例８の模式図である。
【図１７】実施例９の模式図である。
【図１８】実施例１０の模式図である。
【図１９】実施例１１のレーザ照射部付近の正面図（図１ａ）と正面図におけるＡ−Ａ′断面（図１ｂ）である。
【図２０】Ｆポリマの透過波長と透明度の関係を表したグラフである。
【図２１】Ｆ溶液の化学的特長を表した表である。
【図２２】Ｆポリマの屈折率を表した表である。
【図２３】実施例２のキャピラリアレイ取付部変形例の正面図（２３ａ）と正面図におけるＡ−Ａ′断面（２３ｂ）である。
【符号の説明】
１，８２…キャピラリアレイ、２…負電極、３…負電極側のバッファー液、４…ゲルブロック、５…ゲルブロックへの接続部、６…バルブ、７…アース電極、
８…光照射部、９，２４，２５，８０…レーザ光、１０…シリンジ、１１…オーブン、１１−１…光源、１１−２…高圧電源、１１−３…試料導入部、１１−４…第１バッファー容器、１１−５…流動媒体注入機構、１１−６…検出部、１１−７…第２バッファー容器、１１−８…測定部、１２…アース電極側のバッファー、１５…アレイ台、１６…キャピラリ、１７…キャピラリ押さえ板、１８…石英管、１９…キャピラリ周辺を満たす３Ｍ社製の含フッ素溶液フロリナートＦＣ４３、２０…セル蓋、２０−２…気泡収容空間、２１…接着剤、２２…気泡、
２３…気泡がレーザ光路上に侵入することを防ぐための石英製の気泡排除ブロック、３１…蛍光集光レンズ、３２…グレーティング、３３…フォーカスレンズ、
３４…ＣＣＤ、３４−２…検出機構、３５…蛍光、３６…キャピラリからの発光が蛍光集光レンズによって平行光となった光束、４０…アレイ台１５上の基準面Ｐ、４０−２…キャピラリアレイを配列する基準平面、４１…取付基準面Ｐ′、
４４…押し当て棒、４５，５１−１０…バネ、４６…アレイ台において面Ｐに対して垂直でＸ軸に対し平行の基準面Ｑ、４７，４８…取付基準線ＱＱ′、４７−２，４８−２…取付基準線Ｑ″Ｑ′′′、４９…面Ｑに最も近いキャピラリ、
５０…セル蓋の石英表面のうちレーザ光が通過する面Ｒ、５１…セルの石英表面のうちレーザ光が通過する面Ｓ、５１−６…蛍光透過部、５１−７…レーザ光透過部、５１−８…押圧部Ａ、５１−９…押圧部Ｂ、５１−１１…取付部蓋、５２…セルの石英表面のうちレーザ光が通過する面Ｔ、５３…セルの石英表面のうちレーザ光が通過する面Ｕ、５４…アレイ台においてＦ溶液に接する部分の一点、
５５…セル蓋においてＦ溶液に接する部分の一点、６０…キャピラリアレイの光照射部を覆うフッ素化ポリマ、６０−２…光照射部、６１，６２…両端のキャピラリの外側に位置し、フッ素化ポリマをせき止めるポリマブロック、６５…空気とＦポリマ６０の界面、７０…気泡がレーザ光路上に侵入することを防ぐためにアレイ台に施した穴加工、７１…セル下部の蛍光検出用石英窓、７２，７３…レーザ光透過用石英窓、８１…ハーフミラー、８３，８４…レーザ集光レンズ、
８５，８６…レーザが入射する端キャピラリ、８７…レーザ本体、８８，８９…ハーフ波長板、９０…偏光フィルタ、９１…ミラー、９５，９６…レーザ光によるキャピラリやセルからの反射光、９７…ピンホール板、１０１…シリコン板、
１０２…柵、１０３…検出窓、１０４…Ｖ溝、１０５…シリコン板の位置決めガイド、１０６…反射防止膜、１０７…発泡体、１０８…セル蓋の位置決めガイド、１０９…Ｆ溶液注入穴、２０２…シリンドリカルレンズ。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an electrophoresis apparatus that separates a sample such as a fluorescently labeled DNA by electrophoresis and analyzes the sample.
[0002]
[Prior art]
For the purpose of determining the base sequence and base length of DNA, etc., an electrophoresis method using a capillary made of a quartz tube and a polymer coating covering the tube is used. A sample containing DNA to be measured is injected into a separation medium such as polyacrylamide in a quartz capillary, and a voltage is applied to both ends of the capillary. The DNA synthesized product in the sample moves in the capillary and is separated according to the molecular weight and the like, and a DNA band is generated in the capillary. A fluorescent dye is added to each DNA band, and the DNA band develops a color by irradiation with a laser beam, and this is read by a fluorescence measuring means to determine a DNA sequence. Separation and analysis of proteins can be performed in the same manner to examine the structure of the proteins.
[0003]
One of the laser beam irradiation methods is as follows. A method of irradiating one or both ends of a capillary array composed of a plurality of capillaries arranged on a plane substrate with laser light, and the laser light propagates successively to adjacent capillaries and traverses the capillary array. There is. Here, the coating of the surface of the capillary such as polyimide is removed at and near the location where the laser light is irradiated in the capillary.
[0004]
However, when a laser beam passes through a boundary surface having a different refractive index, such as a contact surface between a capillary and air, the laser beam is attenuated due to the effect of dispersion and reflection of the laser beam due to a difference in the refractive index of the material constituting the boundary surface. . Therefore, in the process of transmitting the laser beam through several capillaries, the laser beam decreases exponentially, and the analysis accuracy decreases.
[0005]
Therefore, there is a concept of suppressing the loss of laser light due to refraction and reflection by filling a light transmission medium having a predetermined refractive index between the capillaries and adjusting the refractive index and the reflectance.
[0006]
Here, in a known example of the laser light irradiation method, there is a known example in which a space between the capillaries and at least a part of a space through which the laser light passes is filled with a medium other than air (see the following patent). Reference 1, Patent Document 2 below, Patent Document 3 below). In these specifications, the conditions of the refractive index of the medium to be filled between the capillaries are described according to parameters such as the inner diameter and outer diameter of the quartz of the capillaries, the outer diameter of the quartz, and the refractive index of the separation medium filled in the capillaries. In this specification, the contents of these specifications are incorporated to the extent that the interpretation of rights is not limited.
[0007]
However, in the above specification, the conditions of the refractive index of the medium to be filled between the capillaries are described in accordance with parameters such as the inner diameter and outer diameter of the capillary of the capillary, the refractive index of the separation medium to be filled in the capillary, and the like. . In Patent Literature 1 below, a liquid, for example, water, an aqueous solution, air, or vacuum is described as a medium to be filled between capillaries. In Patent Literature 2 below, only water is described as a substance having a refractive index of 1.3 to 1.5 as a filler between capillaries. Further, in Patent Literature 3 below, as a filling material between capillaries, water and a buffer solution having a refractive index of 1.33-1.37 as a transparent liquid, air as a transparent gas, and quartz glass as a vitreous body are exemplified. .
[0008]
[Patent Document 1]
U.S. Pat. No. 5,790,727
[Patent Document 2]
U.S. Pat. No. 5,582,705
[Patent Document 3]
U.S. Pat. No. 5,833,827 (JP-A-9-152418, JP-A-9-96623)
[0009]
[Problems to be solved by the invention]
The present invention relates to improving the analysis accuracy of an electrophoresis apparatus that irradiates a detection unit of a multi-capillary array including a plurality of capillaries so that irradiation light passes through a plurality of capillaries simultaneously.
[0010]
[Means for Solving the Problems]
The present invention is directed to an electrophoresis device that irradiates a detection unit of a capillary array having a plurality of capillaries for separating a sample by electrophoresis so that the irradiation light passes through the plurality of capillaries simultaneously, and detects sample information generated thereby. In the apparatus, a liquid or a solid having a refractive index higher than that of air and not higher than that of water is interposed in a space between the capillaries to reduce the amount of scattered irradiation light by the capillaries. This adjusts the refraction and reflection of the irradiation light when passing through the capillary, reduces the light loss caused by passing through the plurality of capillaries, and prevents the light intensity irradiated on the sample from decreasing. In the present invention, the substance filled between the capillaries is a liquid, semi-solid or solid at normal temperature and normal pressure, and a substance having a refractive index smaller than that of water is preferable.
[0011]
In the above-mentioned known example, quartz glass (refractive index: 1.46) is described, but it is not possible to sufficiently reduce the reflection and scattering of irradiation light applied to the capillary. Although the above-mentioned Patent Document 2 describes an aqueous solution, there is no specific description. Further, the above-mentioned known examples describe that air, vacuum and the like can be used, but they have too small a refractive index and cannot be used. Therefore, the substance to be filled between the capillaries has at most the same refractive index as water, and preferably a substance having a refractive index smaller than that of water. Further, a substance having a refractive index larger than that of air or vacuum, and preferably having a refractive index sufficiently larger than 1 is desirable.
[0012]
More specifically, a fluorine-containing compound (which may be a liquid, a semi-solid, or a polymer) which is liquid at normal temperature and normal pressure and has a refractive index of 1.25 to 1.32 is preferable. This fluorine-containing compound will be described later in detail.
[0013]
Further, the present invention provides an electrophoresis apparatus in which the plurality of capillaries are immersed in a liquid, wherein a mechanism for supplying the expansion of the liquid is added to the container of the liquid so that damage to the container due to temperature rise of the liquid or liquid Prevent leaks. Further, by controlling the temperature of the liquid, the refractive index gradient of the liquid in a portion through which the irradiation light passes is adjusted, the irradiation light is suppressed from being curved, and the intensity of the light irradiated to the sample is reduced. To prevent.
[0014]
Further, the present invention provides an electrophoresis apparatus in which a photoconductive medium having a predetermined refractive index is provided around a capillary, by forming the surface shape of the photoconductive medium into a predetermined curved surface, so that the traveling direction of light generated from the sample can be changed. Adjustment can increase the light intensity detected by the detection mechanism.
[0015]
The above and other objects and novel features of the present invention will be described in detail with reference to the drawings.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Example 1
FIG. 2 shows the overall configuration of the multi-capillary electrophoresis apparatus according to the present embodiment.
The multi-capillary electrophoresis apparatus according to the present embodiment includes a multi-capillary array 1 composed of a capillary containing a separation medium for separating a test sample, a buffer for immersing the negative electrode 2 of the multi-capillary array and the sample introduction unit 11-3. A first buffer container 11-4 for holding the liquid 3, a gel block 4 having a valve 6, a second buffer container 11-7 for holding a buffer solution 12 for immersing the gel block 4 and the ground electrode 7, and a capillary array A syringe 10 for injecting a gel as an electrophoresis medium into the inside, a detection unit 11-8 for acquiring information depending on a sample, and a light source 11 for irradiating a laser irradiation 9 which is coherent light to the light irradiation unit 8 -1, a measuring unit (not shown) for acquiring fluorescence generated by the sample, a thermostatic chamber 11 for adjusting the temperature of the capillary array, and a separation medium Composed of high-voltage power supply 11-2 for applying a voltage.
[0017]
The multi-capillary array 1 has a quartz capillary, which is a tubular member, filled with a test sample containing a sample such as a DNA molecule and an aqueous polymer solution as a separation medium for separating DNA molecules in the test sample. Have a book. At one end of the multi-capillary array 1, a sample introduction section 11-3 for introducing a sample into the capillary is formed, and an electrode 2 to which a negative voltage can be applied is arranged. At the other end, there is a connecting portion 5 connected to the gel block 4 and capable of moving the separation medium between the gel block 4 and the multi-capillary array 1. A detection unit 11-8 including a light irradiation unit 8 to be irradiated with laser light is provided between the sample introduction unit 11-3 and the connection unit 5.
[0018]
The gel block 4 and the syringe 10 are a fluid medium injection mechanism 11-5 for injecting an aqueous polymer solution as an electrophoretic medium into a capillary. When the capillary is filled with the aqueous polymer solution as the electrophoresis medium, the valve 6 is closed and the syringe 10 is pushed in so that the aqueous polymer solution in the syringe 10 is injected into the capillary.
[0019]
The capillary array 1, the gel block 4, the buffer solution 3, the electrode 2, the buffer 12 on the side of the ground electrode, the ground electrode 7, and the high-voltage power supply 11-2 constitute a voltage application mechanism for electrophoresis of the test sample. When performing electrophoresis, the negative electrode 2 is immersed in the buffer solution 3 and the valve 6 is opened. Thus, the negative electrode 2, the buffer solution 3, the multi-capillary array 1 (more precisely, the aqueous polymer solution in the capillary), the gel block (more precisely, the aqueous polymer solution in the gel block), and the buffer 12 on the ground electrode side , And a grounding electrode 7 are formed. A voltage is applied to this current path by the high voltage power supply 11-2. When a voltage is applied to the current path, the test sample in the aqueous polymer solution undergoes electrophoresis and is separated according to its molecular weight and other properties.
[0020]
The optical system of the electrophoresis apparatus includes a light source 11-1, a measurement unit 11-8 including the light irradiation unit 8, and a detection mechanism for detecting fluorescence generated from the detection unit. The light source 11-1 oscillates laser light 9 (488.0 nm and 514.5 nm light from an argon ion laser) that is coherent light. In the detection units 11 to 88, the light irradiation units 8 where laser light 9 passes through the capillary are arranged in parallel. Then, the laser beam 9 is emitted from both upper and lower directions to the detection unit 11-8 so as to penetrate the light irradiation units 8 of the plurality of capillaries simultaneously. The laser light 9 excites the test sample, and the test sample emits fluorescence. This fluorescence is detected by a detection mechanism including the CCD 34, so that information depending on the test sample such as the DNA molecule sequence can be obtained.
[0021]
FIG. 3 shows a mechanism 34-2 for detecting fluorescence from the test sample in the capillary array, and the light irradiation unit 8. The detection mechanism 34-2 includes a fluorescence condenser lens 31, a grating 32, a focus lens 33, and a CCD. Fluorescent light 35 from the test sample in the capillary 16 generated by irradiating the light irradiating section 8 with the laser light 9 is converted into parallel light 36 by the fluorescent light condensing lens 31, is separated by the grating 32, and is separated by the focus lens 33 into the CCD 34. Imaged on top. The state of the elements (detection unit 11-8, grating 32, CCD 34) relating to the image formation is shown in the right diagram of FIG. 96 capillary images are arranged in the Y-axis direction, and light emission from each capillary is split in the X-axis direction.
[0022]
The thermostat (oven) 11 is a temperature control mechanism for controlling the temperature of the capillary array 1. Thereby, most of the capillary array 1 is kept at a constant temperature, for example, 60 ° C.
[0023]
The detection unit 11-8 will be described. FIG. 1 shows a side view (1a) and a front view (1b) of the detection unit 11-8. The detection unit 11-8 includes 96 capillaries 16, an array base 15, a cell lid 20, a holding plate 17, a bubble elimination block 23, a filling medium (F solution 19), and bubbles 22. .
[0024]
The arrangement of the capillaries will be described. On the array base 15 made of quartz, a reference plane for arranging the capillaries is formed. The 96 capillaries are arranged on the array table 15 such that all of the capillaries are in contact with the reference plane and adjacent capillaries are in contact with each other. The capillary 16 is bonded and fixed to the array base 15 by being sandwiched between a holding plate 17 for fixing the capillary 16 and the array base 15. As a result, the capillaries are arranged in parallel in a plane, and the deviation of the center axis of each capillary becomes 6 μm or less. Therefore, when the laser light 9 is irradiated so as to simultaneously penetrate as many as 96 capillaries, the influence of the loss of the laser light 9 due to refraction or reflection can be reduced.
[0025]
The structure of the capillary will be described. The capillary 16 has a structure in which a quartz tube 18 having an inner diameter of 50 μm and an outer diameter of 126 μm is covered with a polymer coating having a thickness of 12 μm, and has a total outer diameter of 150 μm. The inside of the capillary is filled with a polymer aqueous solution (refractive index: 1.41) as a DNA separation medium. In the light irradiation section 8, the polymer coating is removed, and the quartz tube 18 is exposed. When the laser beam 9 is irradiated, a part of the irregularly reflected light hits the polymer film of the capillary, and this polymer film may emit fluorescence. However, the fluorescent light from the polymer coating is blocked by the holding plate 17 so as not to reach the measuring section. Therefore, highly accurate detection with a high SN ratio can be performed.
[0026]
Here, in the present embodiment, the array base 15, the quartz cell lid 20, and the adhesive 21 for fixing them form a sealed structure, and a sealed container capable of holding a specific liquid or solid photoconductive medium. (Cell). By filling the cell with the light transmitting medium, the space between the capillaries through which the laser light passes can be filled with the light transmitting medium. In other words, the light irradiation section 8 of the capillary 16 is immersed in the light transmission medium. During electrophoresis, a high voltage is applied to the capillary, so that the capillary adsorbs dust in the charged air. However, since the vicinity of the light irradiating portion of the capillary 16 is isolated from the outside air by a sealed container, light irradiation is performed. Dust does not adhere to the part 8.
[0027]
The choice of this light transmission medium has a very large effect on the analytical performance of the electrophoretic analysis. In the description, problems caused by the propagation of laser light and the loss of laser light will be described. In the present embodiment, the capillary array is irradiated with laser beams 24 and 25 from both upper and lower ends of the detection unit. The two laser beams 24 and 25 are irradiated so as to overlap each other. Each of the irradiated laser beams propagates one after another to the adjacent capillaries, passes through the 96 capillaries, and traverses the light irradiation section 8 of the capillaries. Here, when the laser beams 24 and 25 travel along the interface between the capillary 16 and the light transmission medium, the laser beams 24 and 25 pass through the interface between the media having different refractive indexes, and the laser beams due to refraction and reflection. Light intensity is lost due to scattering of light. When the light intensity of the laser beams 24 and 25 becomes weak due to this loss, the fluorescence generated from the sample in the capillary 16 becomes small, so that the DNA molecule arrangement or the like cannot be analyzed with high sensitivity. Hereinafter, in principle, this is expressed as "the separation ability is reduced".
[0028]
As a solution to the laser light loss, a method of increasing the fluorescence by increasing the intensity of the laser light can be considered. However, if the laser beam intensity is too high, the test sample such as DNA molecules will be denatured, so this solution has a limit.
[0029]
Further, in an electrophoresis apparatus in which laser light penetrates a plurality of capillaries, a difference in the intensity of laser light applied to each capillary becomes a problem. As the laser beam passes through the capillary, its intensity decreases exponentially due to refraction and reflection. Therefore, the fluorescence generated from the capillary with the highest laser beam intensity and the weakest capillary (from both sides of the capillary array). Irradiates a difference in the magnitude of the fluorescence generated from the center capillary). At this time, since the detection range of the CCD 34 needs to be set so as to contain all the fluorescence, if the difference between the fluorescence intensities is large, the analysis performance of the CCD 34 cannot be effectively used, and the analysis performance deteriorates. Therefore, if the laser beam intensity difference between the capillaries is large, effective analysis of the test sample cannot be performed. In particular, since the loss due to refraction and reflection increases exponentially as the number of capillaries through which laser light passes increases, the loss of light intensity due to refraction and reflection is a major problem in an electrophoresis apparatus having 24 or more capillaries. Become.
[0030]
The loss due to refraction and the loss due to reflection depend on the difference in refractive index at the interface. If the difference in refractive index is large (the reflectance is large), attenuation due to reflection occurs when the laser beam passes through the boundary surface, and if the difference in refractive index is small, attenuation occurs due to dispersion of the laser beam when passing through the boundary surface. That's why. Therefore, the inventors have considered that a method of filling a medium having a predetermined refractive index between the capillaries and suppressing the sum of losses due to refraction and reflection is effective.
[0031]
Here, the loss due to reflection will be described. In general, the reflectance (R) when light travels from the medium 1 (refractive index: n1) to the medium 2 (refractive index: n2) at an incident angle of 0 can be obtained by the following equation.
Reflectance: R = [(n1-n2) / (n1 + n2)]²
[0032]
For example, when the filling medium around the capillary is air, the reflectance at the interface between air (n = 1.00) and quartz (n = 1.46) is 3.49%, so that the laser beam Each time one light passes through the boundary surface, 3.49% of light is reflected, resulting in a loss of light intensity. This loss increases exponentially as the number of capillaries through which the laser beam passes increases. In order to reduce the loss due to reflection, the refractive index of the filling medium should be close to the refractive index of the capillary.
[0033]
Further, the loss due to refraction is attenuation due to dispersion of laser light (a state where light spreads) when passing through the boundary surface. That is, when the cross section of the capillary is elliptical or circular, the boundary surface between the capillary and the filling medium acts as a lens, and the laser light concentrates on the hollow portion of the capillary filled with the separation medium (light density increases). However, when the refractive index of the capillary and the surrounding medium is small, the lens effect due to the boundary surface is reduced, and the loss occurs because the laser light diverges. Generally, medium 1 (refractive index: n₁) To medium 2 (refractive index: n₂Angle of incidence θ when light travels₁, Emission angle θ₂Is obtained by the following equation.
n₁sin θ₁= N₂sin θ₂
[0034]
Therefore, generally, in order to reduce the loss due to refraction by strengthening the lens effect of the capillary, it is sufficient to increase the difference in the refractive index between the filling medium and the capillary.
[0035]
Based on the above consideration, the inventor simulated the relationship between the refractive index N of the light transmission medium around the capillary and the emission intensity ratio. The simulation conditions are as follows. For a multi-capillary array having 96 capillaries having the same detection unit structure as in the present embodiment, the inside of the capillary is filled with a polymer aqueous solution having a refractive index of 1.41, and the laser light is emitted only from the capillary. Is irradiated. It was assumed that the capillary alignment error was ± 6 μm, the laser diameter was 72 μm, and the optical axis deviation was 10 μm.
[0036]
FIG. 13 shows the simulation result. The horizontal axis is the refractive index of the light transmission medium, and the vertical axis is the emission intensity ratio. The emission intensity ratio is obtained by averaging the relative values of the amounts of light passing through the respective capillaries with respect to the incident laser light when the laser light propagates through the capillaries one after another for 96 capillaries. In the figure, ■ indicates the expected value of the luminescence intensity ratio (the average value of the luminescence intensity of a plurality of capillaries), and the vertical line including ■ indicates the distribution of the luminescence intensity.
[0037]
It can be understood that the emission intensity ratio has a mountain shape having a peak near a refractive index of 1.29 ° with respect to the refractive index of the filling medium. When the refractive index is smaller than 1.29 °, the contribution of loss due to reflection is large. When the refractive index is larger than 1.29 °, the contribution of the loss due to refraction is large. If the emission intensity ratio allowed in the electrophoretic analysis is 0.35 ° or more, it can be understood that a material having a refractive index in the range of 1.25 to 1.32 is suitable as the light transmission medium around the capillary. . In particular, a light transmission medium having a refractive index of 1.29 ° is optimal.
[0038]
It is desirable that the light transmission medium does not absorb laser light. This is to suppress the attenuation of the light intensity even when the laser light is transmitted through the light transmission medium. This attenuation also increases as the number of capillaries increases and the laser light path in the optical transmission medium becomes longer.
[0039]
Further, it is desirable that the light transmission medium does not emit fluorescence. This is because, when the light transmission medium itself emits fluorescence by the laser light, the emitted light becomes noise, which hinders detection of the fluorescence emitted from the test sample.
[0040]
The inventor has searched for an optimal substance as a light transmission medium satisfying the above conditions. Then, it reached Fluorinert Electronic Liquid FC-43 (hereinafter referred to as “F solution 19”) manufactured by 3M. “Fluorinert” is a registered trademark of 3M Company. The F solution 19 is a fully fluorinated liquid having a refractive index of about 1.29 °, low viscosity, colorless and transparent, and very stable chemically. In addition, it does not absorb the irradiation light, and emits only fluorescence equal to or less than the Raman scattering of water when irradiated with the irradiation light. Therefore, it is a substance very suitable as a light transmission medium of a multi-capillary electrophoresis apparatus having a plurality of capillaries. Further, it has the following characteristics, and these sometimes work favorably. 1) Excellent electrical insulation and thermal conductivity. 2) Very low surface tension and excellent permeability. 3) Does not dissolve in various solvents regardless of high or low temperature. 4) Nonflammable, non-toxic, odorless and safe. 5) It is inert and hardly attacks electronic components, metals, plastics, rubber, etc. FIG. 21 shows the chemical characteristics of the F solution at 25 [° C.].
[0041]
The inside of the sealed container of the capillary array according to the present embodiment is filled with the F solution 19, and the light irradiation unit 8 is immersed in the F solution 19. As a result, a loss due to refraction and reflection when the laser beam passes through the surface of the capillary 16 can be reduced, and a decrease in the fluorescence intensity generated by the test sample can be avoided. Therefore, a multi-capillary having a plurality of capillaries, particularly, a capillary having 24 or more capillaries In the electrophoresis apparatus, the analysis ability can be improved.
[0042]
In the sealed structure, bubbles 22 are filled together with the F solution 19. This is to prevent the hermetic structure from being broken by the volume expansion of the F solution 19 due to the temperature change. However, since the bubbles 22 are movable, they may enter the laser beam path and affect the analysis. In order to prevent the bubbles 22 from entering the laser beam path, a bubble elimination block 23 made of quartz, which is a bubble elimination means, is formed in a sealed structure. The bubble elimination block 23 is disposed at a position where the laser beams 24 and 25 are transmitted when the detection unit 11-8 is mounted on the electrophoresis apparatus, at a position above the bubble removal block 23. Thereby, a space 20-2 for accommodating the bubbles 22 is formed above the sealed container and at a position where the laser beams 24 and 25 do not transmit. At the time of measurement, the bubbles move above the cell and stay in this space 20-2, so that the bubbles 22 can be prevented from crossing the laser beam path. When the light irradiating unit 8 is mounted on the electrophoresis apparatus so that the reference surface of the array table 15 is horizontal and the reference surface is down, a groove-shaped reference surface of the array table 15 near the light irradiating unit 20-3. Form a space. Bubbles are likely to accumulate in the gap between the capillaries near the light irradiation part, but if the volume of the groove-shaped space is equal to or greater than the volume of the bubbles 22, the bubbles will remain in this space. Can be avoided. The same effect can be obtained by equipping the electrophoresis apparatus with the light irradiation unit 8 obliquely, that is, by equipping the electrophoresis apparatus so that the bubble 22 moves to one corner in the sealed container and to a position where the laser beams 24 and 25 do not transmit. Is obtained. That is, when the detection unit is provided in the electrophoresis apparatus, a closed structure is formed so that the bubbles remain at positions where the laser beams 24 and 25 do not transmit, thereby avoiding contact between the bubbles 22 and the laser beams 24 and 25. be able to. Accordingly, it is possible to avoid a loss due to refraction and reflection due to the passage of the laser beam through the boundary surface between the bubble 22 and the F solution 19. FIG. 14 shows an image formed on the CCD of this embodiment and the distribution of light emission intensity from the 96 capillaries. FIG. 4 shows an image formed on a CCD in the case of 93 capillaries and a light emission intensity distribution from the 93 capillaries. The emission shown in FIG. 14 is an emission spectrum (mainly Raman scattering of water) of the separation medium. As shown in the figure, light emission from 96 capillaries can be detected simultaneously. FIG. 15 shows the results of electrophoresis of one of the 96 capillaries. FIG. 5 shows the results of electrophoresis of one of the 93 capillaries. Here, a DNA sample having a known base length, which is a so-called size marker, was used as a measurement target. The temperature in the thermostat was 60 ° C., the length between the light irradiation part and the sample introduction end was 36 cm, and the voltage applied to the capillary was 319 V per cm.
[0043]
One of the labels indicating the DNA separation ability of the electrophoresis apparatus is a crossover point. This means the base length at which the separation length corresponding to one base in the light irradiation part is equal to the full width at half maximum of the DNA band of one base, and the larger the value, the higher the electrophoretic separation ability. The crossover point in FIG. 15 was 410 bases. Similar results were obtained for the other 95 capillaries. The crossover point in FIG. 5 was 410 bases, and similar results could be obtained for the other 92 capillaries.
[0044]
As described above, in the present embodiment, it is possible to provide a multi-capillary electrophoresis apparatus having a high analytical capability in which the crossover point for each capillary is 410 bases.
[0045]
Example 2
The angle between the incident laser light and the laser incident surface of the cell must be kept constant. If the angle varies for each capillary array, the incident angle of the laser beam to the cell varies, and as a result, the laser light path in the cell varies. For example, when the laser incident surface of the cell is quartz and the medium filling the capillary is a liquid with a refractive index of 1.29 °, if the laser incident surface of the cell varies by 4 mrad, the laser propagates 20 mm from the incident point on the cell. The displacement of the laser beam path at the point where it was set is 20 μm. When the laser is displaced in the direction of the capillary axis, the two laser beams irradiated in the upper and lower directions are not coaxial, and the laser beam diameter is effectively increased. As a result, performances such as a decrease in DNA detection separation ability (discrimination ability of adjacent DNA bands) are caused. Further, when the laser light is displaced in a direction perpendicular to the capillary axis (hereinafter, referred to as “Z-axis direction”), the amount of laser light irradiation into the inner diameter of the capillary decreases, and the signal intensity decreases. In order to avoid such performance degradation, it is necessary to keep the angle between the incident laser beam and the laser incident surface of the cell constant.
[0046]
In particular, in the multi-capillary electrophoresis apparatus according to Example 1, the inner diameter of the capillary filled with the separation medium and the test sample is 50 μm, and the tolerance of the central axis of the capillary (the variation in the distance between the central axis of the capillary and the reference plane) is ± 6 μm. The laser beam diameter is 72 μm. Therefore, unless the positional relationship between the capillary and the laser beam is precisely adjusted on the order of μ, the separation ability cannot be ensured. Further, it is preferable that the multi-capillary array be detachable from the electrophoresis apparatus main body. However, it is necessary to easily adjust the position of the capillary and the laser beam at the time of mounting. For this reason, in the electrophoresis apparatus according to the second embodiment, the mounting reference plane to the electrophoresis apparatus in the multi-capillary array and the laser incident surface of the cell are made to have a fixed angle, and the mounting reference plane and the mounting reference plane on the apparatus side are always set. It has a structure that can be attached at a fixed angle. More specifically, by bringing a plane in which a plurality of capillaries are arranged into contact with a mounting reference plane in a multi-capillary array mounting portion of the electrophoresis apparatus, relative positioning of the flat substrate and the electrophoresis apparatus in a direction perpendicular to the mounting reference plane is performed. And, by contacting one plane perpendicular to the plane in which the capillaries are arranged in the planar substrate with another mounting reference plane in the multi-capillary array mounting section, the capillary in the direction perpendicular to the perpendicular direction. By performing relative positioning between the array and the electrophoresis apparatus, the angle between the incident laser light and the laser incident surface of the cell can be kept constant.
[0047]
FIG. 6 shows a front view (6a) of the capillary array mounting portion of the second embodiment and a cross section AA '(6b) in the front view. The other points are the same as in the first embodiment. The XYZ axes are defined as shown. The X axis is a direction parallel to the capillary axis, the Z axis is a direction perpendicular to a reference plane on which a plurality of capillaries are arranged, and the Y axis is a direction perpendicular to both the X axis and the Z axis.
[0048]
The array base 15 made of quartz has a reference plane 40-2 on which the capillaries can be arranged by contacting the polymer coating of the capillaries, an attachment reference plane P40 that contacts the electrophoresis apparatus main body, and an attachment reference plane Q46. The mounting reference plane Q46 of the array base 15 is perpendicular to the mounting reference plane P40 and parallel to the X axis. It is desirable that the reference plane 40-2 and the attachment reference plane P40 are substantially parallel and the distance between them is within 6 μm. If the reference plane 40-2 and the mounting reference plane P40 are formed on the same plane as in this embodiment, it is preferable because the capillary array can be manufactured with high accuracy.
[0049]
The capillary array mounting portion in the main body of the electrophoresis apparatus has a mounting reference plane P'41 and mounting reference lines QQ'47 and 48 in contact with the mounting reference plane P40 and the mounting reference plane Q46 of the multi-capillary array, respectively. A laser beam transmitting portion 51-7 through which the laser beams 24 and 25 incident on the sample 8 pass, a fluorescence transmitting portion 51-6 through which the fluorescence emitted from the sample passes, and a pressing portion A51 comprising a mounting portion cover 51-11 and a spring 51-10. -8, a pressing portion B51-9 including a pressing rod 44 and a spring 45 is provided.
[0050]
The pressing portion A51-8 can move when the multi-capillary array is attached to or detached from the capillary array mounting portion. At the time of mounting, the mounting reference plane P40 of the array base 15 is brought into contact with the mounting reference plane P'41 in the multi-capillary array mounting section of the electrophoresis apparatus, and the array base 15 is moved in the -Z-axis direction (mounting) by the pressing portion A51-8. By pressing against the reference plane P40 (from the reference plane P'41 to the mounting reference plane P'41), the relative positioning between the array table 15 and the electrophoresis apparatus in the Z-axis direction can be performed with an accuracy on the order of μm. By this alignment, reproducibility of the laser irradiation position of the capillary when the capillary array is detached can be ensured. In particular, when the reference plane 40-2 and the attachment reference plane P40 are on the same plane, the positional relationship between the center axis of each capillary and the transmission paths of the laser beams 24 and 25 can be adjusted with extremely high precision.
[0051]
The mounting reference plane Q46 of the array base 15 is brought into contact with the mounting reference lines QQ'47, 48 by contacting two capillary mounting reference members that are semicircular when viewed from above the Z axis. Then, through a pressing rod 44 having two capillary pressing members having a semicircular shape when viewed from above the Z axis, a spring 45 causes a Y-axis direction (from the mounting reference plane Q46 to the mounting reference lines QQ'47, 48). Direction) is pressed against the array table 15. As described above, since one straight line touches at two points when viewed from the Z direction, the relative positioning between the array table 15 and the electrophoresis apparatus in the Y axis direction can be performed with high accuracy.
[0052]
Further, in the capillary array, the capillaries are arranged so that the mounting reference plane Q46 and each capillary are parallel, and the distance between the capillary 49 closest to the mounting reference plane Q46 and the mounting reference plane Q46 is set to any capillary array. It was fixed. By doing so, the positional relationship between each capillary and the mounting reference lines QQ'47, 48 is uniquely determined for all remaining 95 capillaries starting from the closest capillary 49. Therefore, the imaging position on the CCD 34 does not depend on the capillary array, and the size of the light receiving surface of the CCD 34 can be minimized.
Alternatively, a range in which all capillaries are arranged on the array table is set, and the capillaries are arranged so that the attachment reference plane Q46 and each capillary are parallel within the range. The distance in the range closest to the mounting reference plane Q46 and the distance in the range farthest from the reference plane Q46 were constant in any of the capillary arrays. Such a structure can be realized, for example, by forming two parallel blocks on an array base as shown in FIG. 8 and setting an arrangement range between the blocks, and arranging 96 capillaries in this range. it can. In this case, the distance between the inner wall of the block on the capillary side and the mounting reference plane Q46 is kept constant. By doing so, the imaging position on the CCD 34 does not depend on the capillary array, and the light receiving surface size of the CCD 34 can be minimized.
[0053]
The parallelism between the R50, S51, T52, and U53 surfaces through which the laser beams 24 and 25 pass and the surface Q46 on the surface of the quartz cell lid 20 is 2 × 10.^-3rad or less. If the parallelism varies from one capillary array to another, the incident angles of the laser beams 24 and 25 to the cell lid 20 vary, and as a result, the laser light path in the cell varies. When the laser beam path varies in the X-axis direction, the laser beams 24 and 25 in the two upper and lower directions are not coaxial, and the laser beam diameter is effectively increased. As a result, performances such as a decrease in DNA detection separation ability (discrimination ability of adjacent DNA bands) are caused. Further, when the laser beams 24 and 25 are displaced in the Z-axis direction, the amount of laser beam irradiation into the inner diameter of the capillary decreases, and the signal intensity decreases. In order to avoid such performance deterioration, the R50, S51, T52, and U53 planes are each substantially parallel to the plane Q46 as described above.
[0054]
In order to suppress variations in the laser beam path, it is not sufficient that the R50, S51, T52, and U53 surfaces are parallel to the surface Q, and the mounting angle of the array table 15 to the electrophoresis apparatus is fixed with good reproducibility. Need to be This condition is realized by the above-described method in which the surface Q46 is pressed against the lines Q47 and Q'48.
[0055]
As a method of attaching a capillary array to a capillary array attachment portion, there is also the following method.
[0056]
FIG. 23 shows a front view (23a) of a capillary array mounting portion according to a modified example of the second embodiment and an AA ′ cross section (23b) in the front view. The XYZ axes are defined as shown. The reference surface P40 on the array table 15 (the surface with which the capillary polymer film is in contact) is brought into contact with the mounting reference surface P'41 in the capillary array mounting portion of the electrophoresis apparatus main body, so that the array table in the Z-axis direction is provided. Relative positioning of 15 and the electrophoresis apparatus was performed. By this alignment, reproducibility of the capillary irradiation position of the laser beam when attaching and detaching the capillary array can be ensured.
[0057]
Further, the array table 15 is pressed in the Y-axis direction by a spring 45 via a pressing rod 44, and the cell surface U53 (perpendicular to the surface P40 and parallel to the X-axis) is attached to the capillary array of the electrophoresis apparatus. The relative position of the array table 15 and the electrophoresis apparatus in the Y-axis direction was adjusted by contacting the mounting reference lines Q "Q" "47-2 and 48-2 in the section. The capillaries are arranged so that the surface U53 and each capillary are parallel to each other, and the distance between the capillary 49 closest to the surface U53 and the surface U53 is constant in any of the capillary arrays. The image position does not depend on the capillary array, and the size of the light receiving surface of the CCD 34 can be minimized.
Alternatively, a range in which all capillaries are arranged on the array table is set, and the capillaries are arranged so that the attachment reference plane Q46 and each capillary are parallel within the range. The distance in the range closest to the mounting reference plane Q46 and the distance in the range farthest from the reference plane Q46 were constant in any of the capillary arrays. Such a structure can be realized, for example, by forming two parallel blocks on an array base as shown in FIG. 8 and setting an arrangement range between the blocks, and arranging 96 capillaries in this range. it can. In this case, the distance between the inner wall of the block on the capillary side and the mounting reference plane Q46 is kept constant. By doing so, the imaging position on the CCD 34 does not depend on the capillary array, and the light receiving surface size of the CCD 34 can be minimized.
[0058]
On the quartz surface of the cell lid 20, the parallelism between the R50, S51, and T52 surfaces through which the laser beams 24 and 25 pass and the surface U53 is 2 × 10.^-3rad or less. When the parallelism varies for each capillary array, the incident angles of the laser beams 24 and 25 to the cell lid 20 vary, and as a result, the laser light path in the cell varies. When the laser beam path varies in the X-axis direction, the laser beams 24 and 25 in the two upper and lower directions are not coaxial, and the laser beam diameter is effectively increased. As a result, performances such as a decrease in DNA detection separation ability (discrimination ability of adjacent DNA bands) are caused. Further, when the laser beams 24 and 25 are displaced in the Z-axis direction, the amount of laser beam irradiation into the inner diameter of the capillary decreases, and the signal intensity decreases. In order to avoid such performance deterioration, R50, S51, and T52 are each substantially parallel to the plane U53 as described above.
[0059]
In order to suppress variations in the laser light path, it is not sufficient that the planes R50, S51, and T52 are parallel to the plane U53, and the mounting angle of the array table 15 to the electrophoresis apparatus is made constant with good reproducibility. There is a need. This condition is realized by the above-described method in which the surface U53 is pressed against the lines Q'47 and Q "48.
[0060]
In the method shown in FIG. 6, it is necessary to attach and bond the cell lid 20 to the array base 15 so that the surface R50 of the cell lid 20 and the surface Q46 of the array base 15 are parallel. In this method, since the surface U53 of the cell lid 20 directly contacts the mounting reference lines Q'47, Q "48 of the capillary array mounting portion, the degree of parallelism between the array table 15 and the cell lid 20 is increased. .
[0061]
The inventor has also found that when a temperature gradient occurs in the filling medium between the capillaries, the resolution of the electrophoresis apparatus deteriorates. Then, it was found that the reason was the curvature of the laser beam due to the refractive index gradient in the filling medium. Hereinafter, this problem and the solution will be described.
[0062]
If the temperature distribution of the light transmission medium (F solution 19) in the Z-axis direction exists, the refractive index gradient of the F solution 19 occurs in the Z-axis direction. Due to the refractive index gradient, the traveling directions of the laser beams 24 and 25 having components only in the Y-axis direction have Z-components, though they are minute. As a result of the laser light 24, 25 traveling in the Y direction being displaced in the Z-axis direction due to the refractive index gradient, the laser light is displaced from the capillary array in the process of propagating through the capillary. For this reason, the intensity of the laser beam applied to the capillary decreases, the signal intensity (the intensity of the fluorescence from the sample) decreases, and the problem arises that the resolution of the electrophoresis apparatus deteriorates. In particular, this problem becomes more prominent as the distance of the laser beams 24 and 25 passing through the optical transmission medium becomes longer (as the number of capillaries increases).
[0063]
The inventor thought that by properly adjusting the internal surface temperature of the sealed container, this problem could be solved without using a complicated configuration. Therefore, the relationship between the internal surface temperature difference and the temperature gradient at a predetermined position in the filling medium was investigated. FIG. 7 shows the result. FIG. 7 shows a temperature difference between the point V54 and the point W55 in FIG. 6 and a temperature gradient on the straight line VW. The horizontal axis is the Z coordinate on the straight line VW, and Z = 0 corresponds to the center position of the capillary. The straight line VW is perpendicular to the array table and passes through the center of the space between the capillaries. The distance between V and W, that is, the F solution layer thickness is 0.8 mm. The vertical axis is the temperature gradient. ΔT is a temperature difference between the point V54 and the point W55 (ΔT = [temperature at point V] − [temperature at point W]). Each curve represents a temperature gradient on the straight line VW in the F solution when ΔT is −20, −5K, −2K, 2K, 5K, and 20K. The thermal conductivity of the F solution is 0.066 W / mK, the specific heat is 1050 J / kgK, and the density is 1880 kg / m³. Further, the heat generation of one capillary during the electrophoresis was calculated assuming 1 mW per 10 mm length.
[0064]
As can be seen from FIG. 7, the absolute value of the temperature gradient is smaller when ΔT is positive (the temperature at point V54 is higher than the temperature at point W55) than when it is negative. That is, it was found that the displacement of the temperature gradient was smaller when the temperature of the flat substrate of the array table 15 was higher than that of the cell lid 20 as compared with the opposite case.
[0065]
Based on this result, a multi-capillary array was designed as shown in FIG. In the multi-capillary array according to the present embodiment, the thickness of the quartz array base 15 which is the first flat plate portion of the closed vessel is equal to the thickness of the quartz cell lid 20 which is the second flat plate portion (the capillary to be detected). (The thickness of a transparent plate which is a part of the cell through which the signal light from the array passes). By making the cell lid 20 made of quartz and the array base 15 made of sapphire, ΔT can be kept positive even if the array base 15 is thinner than the cell lid. That is, the capillary array is designed so that the heat transfer efficiency of the first flat plate portion is higher than the heat transfer efficiency of the second flat plate portion. Accordingly, when the multi-capillary array mounting portion is maintained at a constant temperature such as 60 ° C., the surface P ′ of the multi-capillary array mounting portion maintained at 60 ° C. (the mounting reference surface P ′ 41 and the mounting reference surface P ′ 41). The heat transmitted from the portion in contact with the lines QQ'47, 48) to the surface P (the reference mounting surface P40 and the reference mounting surface Q46) is more efficiently transferred to the array table than the cell lid, and ΔT becomes positive. To keep it. Thus, the temperature gradient at the point where the laser light passes through the light transmission medium between the capillaries is reduced, and the refractive index gradient is reduced. Therefore, the laser light path is hardly bent, and the linearity can be maintained.
[0066]
Preferably, the multi-capillary array is designed such that the temperature gradient of the filling medium at the place where the laser light is transmitted becomes zero during electrophoresis. Here, the term “zero temperature gradient” refers to a state in which the laser beam can travel straight because there is almost no temperature gradient, and refers to a state in which the curvature of the laser beam hardly deteriorates the separation ability.
[0067]
As described above, in the electrophoresis apparatus in which the filling medium is filled around the capillary, the separation ability can be improved.
[0068]
Example 3
In Example 3, as a light transmission medium, Teflon {AF # 2400 (hereinafter, “F polymer”) manufactured by DuPont, which is a fluorinated polymer having a refractive index of 1.29 °, or DuPont, which is a fluorinated polymer having a refractive index of 1.32 ° One feature is that Teflon AF 1600 (hereinafter referred to as “F ′ polymer”) is used. “Teflon” is a registered trademark of DuPont. FIG. 8 shows a side view (8a) and a front view (8b) near the detection unit of the third embodiment. The configuration other than the light irradiation unit 8 is the same as that of the first embodiment.
[0069]
Further, a medium having a structure having at least one repeating unit among the following chemical structures A, B, C, and D can also be used. These have all hydrogen atoms replaced by fluorine atoms, and are excellent as a light transmission medium like the F solution.
[0070]
Embedded image

[0071]
The light irradiation section 8 of the capillary array 1 is formed on an array table 15. The 96 capillaries 16 on the array table are arranged on the array table 15 so as to be in contact with the array table 15 and in contact with adjacent capillaries, and are adhesively fixed to the array table 15 together with the holding plate 17. Have been. Thereby, similarly to the second embodiment, the positional relationship between each capillary and the mounting reference plane is uniquely determined.
[0072]
The capillary 16 is covered with a polymer thin film as in the first embodiment, but the polymer coating is removed from the light irradiation unit 60-2, and the quartz tube 18 is exposed. In the invention according to the third embodiment, the light irradiating section 60-2 where the quartz is exposed is covered with the F polymer which is a fluorinated polymer having a refractive index of 1.29 °. Alternatively, it may be covered with Teflon AF # 1600 (registered trademark) (hereinafter referred to as "F 'polymer") manufactured by DuPont, which is a fluorinated polymer having a refractive index of 1.32%.
[0073]
After fixing the capillary 16 to the array table 15, the F polymer 60 was filled between the capillaries. In the space between the two polymer blocks 61 and 62 located outside the capillaries at both ends, the F polymer 60 was filled so as to completely cover the passage area of the laser beams 24 and 25.
[0074]
Here, the chemical structural formula E of the F polymer is shown. Note that n is a natural number.
Embedded image

[0075]
The F polymer and the F 'polymer have the following characteristics.
(1) High temperature stability (High temperature stability)
(2) Excels in scientific resistance (Excellent chemical resistance)
(3) Low surface energy (Low @ surface @ energy)
(4) Low water absorption (Low water absorption)
(5) Transparent and high light transmission rate (transparent @ high @ transmission)
(6) Low refractive index (very low refractive index)
(7) High gas permeability (High @ gas @ permeability)
[0076]
FIG. 20 shows the transmission spectrum of the F polymer. From the figure, it can be seen that the F polymer has high transmittance in the range from ultraviolet rays to infrared rays. Therefore, the F polymer 60 is suitable as a filling medium because it does not absorb light having a wavelength of 488.0 nm or 514.5 nm of an argon laser, which is fluorescent, and does not emit fluorescent light even when irradiated with this light.
[0077]
FIG. 22 shows the refractive index of the F polymer and the F ′ polymer. The refractive index of the F polymer at a temperature of 20 ° C. by the sodium D line is 1.29 °, and the refractive index of the F ′ polymer at a temperature of 20 ° C. by the sodium D line is 1.32 °. The refractive index was measured using an Abbe reflectometer using α-bromo-napthalene as the contact liquid. These refractive indices are known as the lowest values among solid organic polymers, and the refractive index of AF2400 is a value close to the theoretical minimum limit of the solid organic polymer refractive index proposed by Groh and Zimmerm. .
[0078]
As described above, the gap between the capillaries is filled with the fluorine-containing polymer having a refractive index of 1.25 to 1.32, preferably 1.29 °, so that the laser beams 24 and 25 are transmitted through the surface of the quartz tube 18. Loss of light intensity due to refraction and reflection can be prevented, and almost the same signal as in the first embodiment can be obtained.
[0079]
Example 4
FIG. 9A is a side view of the vicinity of the laser irradiation unit according to the fourth embodiment.
In this example, as shown in FIG. 9A, the surface shape of the filled F polymer 60 was molded such that the cross-sectional shape became a circle concentric with the capillary 16. Other structures are the same as in the third embodiment. Here, it is assumed that the F value of the fluorescent light condensing lens 31 is 1.8 °. When the surface of the F polymer 60 is flat (FIG. 9B), the signal light is refracted at the interface 65 between the air and the F polymer 60, so that only the signal light within the range of ± 12 ° in the cross section of the capillary as shown in FIG. It does not reach the optical lens 31. On the other hand, when the surface shape of the F polymer 60 is concentric with the capillary 16, the signal light within the range of ± 15.5 ° reaches the fluorescent condenser lens in the cross section of the capillary. In addition, when the fluorescence generated from the test sample in the capillary passes through the boundary between the quartz tube and the F polymer 60 and the boundary between the F polymer 60 and the outside air (air), the fluorescence is perpendicular to the boundary. Incident. Therefore, since the fluorescence is not refracted, there is no problem due to aberration. As described above, by molding the surface shape of the filled F polymer 60 so that its cross-sectional shape becomes a part of the concentric circle of the capillary, the intensity of the fluorescence detected by the measuring unit 34-3 (CCD 34) is increased. And the resolution is improved.
[0080]
The separation ability can also be improved by forming the surface shape of the filled F polymer 60 to have a predetermined curved surface. For example, a curved surface is formed such that one section thereof has a predetermined curve (ellipse, hyperbola, circle whose central axis is different from the central axis of the capillary). This allows the F polymer 60 to act as a lens, thereby converging more fluorescence generated from the test sample to the detection mechanism 34-2. The problem due to aberration can be avoided by forming the surface shape as an aspherical lens.
[0081]
As described above, the resolution can be improved by forming the surface shape of the F polymer 60 into a predetermined curved surface, controlling the traveling direction of the fluorescent light, and increasing the intensity of the fluorescent light incident on the detection mechanism.
[0082]
Example 5
FIG. 10 is a side view illustrating the vicinity of the detection unit according to the fifth embodiment. In the present embodiment, the liquid between the capillaries and filling the space through which the laser light passes is not included in the cell structure of the capillary array, but includes the cell structure including the liquid provided on the capillary array electrophoresis apparatus main body side. Is held in. In this case, the capillary is dipped in the cell of the device, and the capillary array is fixed. Other items not particularly described are the same as in the first embodiment.
[0083]
Except that the capillary array does not have a cell structure, the basic structure of the detection unit formed on the array base 15 is the same as that of the first embodiment, and all the 96 capillaries 16 are attached to the array base 15. It is arranged on the array table 15 so as to be in contact with and adjacent to the capillary, and is adhered and fixed to the array table 15 together with the pressing plate 17.
[0084]
The capillary array mounting portion of the electrophoresis apparatus has a cell which is a container portion which can fill the F solution, a quartz window 72, 73 of a cell which is a laser light transmitting portion which can transmit laser light, and a fluorescent light transmitting portion. It has a quartz window 71 at the lower part of the cell, which is a fluorescence transmitting portion that can be used, and an attachment reference plane P'41.
[0085]
The electrophoresis apparatus is configured to mount the capillary array downward. By bringing the surface P40 of the array table 15 into contact with the mounting reference plane P'41 of the apparatus, the relative positioning of the array table 15 and the electrophoresis apparatus can be performed.
[0086]
The cell of the electrophoresis apparatus is filled with the F solution 19, and the laser beams 24 and 25 travel in the F solution 19 in the horizontal direction through the quartz windows 72 and 73 of the cell. Light emitted from the capillary is directed downward, and is detected by a detection mechanism similar to that of the first embodiment through the quartz window 71 below the cell.
[0087]
In order to prevent bubbles from entering the laser beam path, the array base is provided with a hole processing 70. The hole processing 70 is to form a hole penetrating the array table 15. A similar effect can be achieved by forming a groove in the array table 15 located above the light irradiation unit. Thereby, the bubbles near the light irradiation part of the capillary can move upward, so that the bubbles do not remain on the laser beam path.
[0088]
According to this embodiment, the vicinity of the light irradiation part of the multi-capillary array can be immersed in the F medium, so that substantially the same measurement results as those of the first embodiment can be obtained, and the separation ability of the electrophoresis apparatus can be improved. .
[0089]
Example 6
In the sixth embodiment, in an electrophoresis apparatus that irradiates laser light from both sides of a plurality of capillaries, a polarization element that converts transmitted laser light into linearly polarized light and a polarization rotation element that rotates the polarization direction of transmitted laser light are appropriately arranged. This avoids or reduces the problem that laser light returns to the laser oscillator and makes laser oscillation unstable.
[0090]
FIG. 11 shows an outline of the sixth embodiment. The configuration of the capillary array is the same as in the first embodiment. Only the vicinity of the detection section of the capillary array and the laser light introduction path are displayed, and the laser shutter, filter, and the like are not displayed.
[0091]
The multi-capillary array electrophoresis apparatus according to the present embodiment irradiates a capillary array having the same configuration as that of the first embodiment with two laser beams penetrating a plurality of capillaries, and opposes the traveling directions of the two laser beams. And a light blocking mechanism for preventing the laser light passing through the capillary array from returning to the laser main body.
[0092]
The light irradiation mechanism includes a laser main body 87 that is a light source that irradiates a laser beam 80 that is a coherent light, a half mirror 81 that can divide one laser beam 80 into two laser beams 24 and 25, and a laser traveling direction. , And condensing lenses 83 and 84. A mirror, a half mirror, a lens, and the like may be provided in the path of the laser beam.
[0093]
The light blocking mechanism includes half-wave plates (quartz λ / 2 plate, mica wave plate, etc.) 88 and 89, which are polarization rotating elements capable of changing the polarization direction of transmitted light, and a polarization filter 90 that transmits only predetermined polarized light. .
[0094]
The laser light 80 emitted from the laser main body 87 is split into two by a half mirror 81. These two laser beams irradiate the capillary array 82 from both sides. Here, the light reflected by the half mirror 81 is referred to as a laser beam 24, and the transmitted light is referred to as a laser beam 25. The condenser lens for the laser light 24 is a condenser lens 83, and the condenser lens for the laser light 25 is a condenser lens 84. The capillary positioned at the end of the array and into which the laser light 24 is introduced is hereinafter referred to as a first capillary 85, and the capillary into which the laser light 25 is introduced is hereinafter referred to as a 96th capillary 86. The laser beams 24 and 25 are in a plane including the central axis of each of the 96 capillaries (hereinafter, referred to as an “array surface”), and are perpendicularly incident on the capillaries. The laser beams 24 and 25 are coaxial, and the optical axis of one of the laser beams is adjusted so that the light that has passed through the capillary passes on the same axis as the other incident laser beam and returns to the laser main body 87. The laser beams 24 and 25 are both linearly polarized light, and the polarization direction is perpendicular to the capillary axis. In the above configuration (when the polarization element and the polarization rotation element are not installed), the reflected light from the capillary array or the transmitted light that has passed through the capillary array returns to the laser body, destabilizes laser oscillation and degrades the separation ability. Problem arises. In addition, both the transmitted return light intensity and the reflected return light intensity were about 6% of the incident light intensity.
[0095]
In order to suppress this return light, a quartz λ / 2 plate which is a half-wave plate as a polarization rotating element and a mica wave plate 88 are provided on the capillary side of the laser condenser lens with respect to the laser beams 24 and 25 from both the upper and lower sides. , 89 and so on. It is desirable to adjust the rotation angle of the polarization filter and the polarization rotation element about the optical axis as follows. The polarizing filter adjusts the angle so as to maximize the transmitted light intensity of the light from the laser. For the polarization rotation element, the rotation angle of this element is adjusted so that the polarization rotation angle becomes 45 °. By passing through two optical elements that rotate the polarization by 45 °, the polarization direction of the incident light is rotated by 90 °. The second polarization filter adjusts the angle so that the transmitted light intensity of the light whose polarization direction is rotated by 90 ° is minimized. The rotation angle of the polarization rotation element and the polarization direction of the polarization filter are not strictly limited to the above values, but may be any values as long as they are sufficient to solve the problem of the return light intensity.
[0096]
In this embodiment, a polarization rotator is installed on the capillary array side of the branch point, and a polarization filter is installed on the laser side of the branch point. The angle of the polarizing filter installed near the laser body 87 is adjusted so as to maximize the intensity of light from the laser. Since the polarization direction of the light that has passed through the capillary array and returned through the loop-shaped optical path is rotated by 90 °, the intensity of the transmitted light of the polarization filter becomes minimum with respect to the returned light.
[0097]
The installation of the polarization filter and the polarization rotation element is not limited to this. For example, both the polarization filter and the polarization rotation element may be provided closer to the capillary than the branch point. In this case, two optical elements are required. Further, a polarizing filter may be provided on the laser side of the branch point. In this case, one polarizing filter is required. Alternatively, the arrangement of the condenser lens and the polarization rotation element may be exchanged, and the condenser lens may be arranged closer to the capillary than the polarization rotation element. In this case, the laser beam passes through the polarization rotator and the condenser lens in this order.
[0098]
As a result, the laser light emitted from the laser main body 87 passes through a polarizing filter (adjusts the angle so that transmitted light is maximized), is split into two by a half mirror, and passes through a polarization rotating element. The capillary array is irradiated from two directions. The polarization of the laser beam in the direction perpendicular to the capillary axis is rotated by 45 ° by the half-wave plate 88, and is incident on the capillary array at an angle of 45 ° with respect to the capillary axis. In the optical path, the polarization directions of the upper and lower laser beams at the point of the capillary array are perpendicular to each other. Although the signal light intensity from inside the capillary inner diameter depends on the polarization direction of the incident laser light, since the upper and lower two laser lights are both at 45 ° to the capillary axis, the signal light intensity distribution of the 96 capillaries is higher and lower. Become symmetric.
[0099]
The laser beam transmitted through the capillary array passes through a polarization rotation element (rotates polarization in the same direction as the first polarization rotation element). Since the polarization rotation elements arranged on both sides of the capillary array have the property of polarizing the laser light in the same direction, the laser light incident from one end of the capillary array is rotated again by the polarization element after passing through the capillary. , And a substantially right angle with the polarization direction of the other laser beam.
[0100]
Next, the light enters the polarizing filter via the half mirror. Here, the second polarization filter is the same as the first polarization filter, and the return light enters from the opposite side. However, the polarization of the return light is rotated by 90 °. In other words, the polarization of the laser light in the direction perpendicular to the capillary axis is rotated by 45 ° by the half-wave plate 88, so that the laser light 24 enters the capillary array at an angle of 45 ° with respect to the capillary axis. I have. After transmitting the laser light 24 through the 96 capillaries, the half-wave plate 89 rotates the polarized light further by 45 °. By passing through the two half-wave plates 88 and 89, the polarization direction of the laser light 24 is rotated by 90 °. Similarly, the polarization direction of the transmitted light of the laser beam 25 is rotated by 90 °. Then, the transmitted light enters the polarizing filter.
[0101]
However, this polarizing filter is angle-adjusted so as to maximize the intensity of the laser light from the laser main body 87, and the light that has passed through the capillary array and returned through the loop-shaped optical path has its polarization direction rotated by 90 °. Therefore, the polarizing filter is designed to minimize the transmitted light intensity with respect to the return light.
[0102]
Therefore, since the light cannot pass through the polarizing filter and does not reach the laser head, it is possible to suppress return light from returning to the laser. In the process of passing through the 96 capillaries, the linearly polarized light is disturbed, and the polarization component before passing through the capillaries is reduced by about 25%. However, the present invention was able to suppress the transmitted light by 75%.
[0103]
In the present embodiment, the problem that the light that has passed through the capillary array returns to the laser oscillator and the laser oscillation becomes unstable can be solved, and a relatively stable laser oscillation can be obtained.
[0104]
Example 7
In the sixth embodiment, there is a problem that the return light reflected by the capillary cannot be suppressed. Example 7 is an electrophoresis apparatus that irradiates laser light from both sides of a plurality of capillaries, by setting the laser incident angle on the capillary not perpendicular to the capillary, so that the laser light reflected by the capillary returns to the laser oscillator and causes laser oscillation. The problem of instability is avoided or reduced.
[0105]
FIG. 12 is a schematic diagram of the seventh embodiment. This embodiment is basically the same as the sixth embodiment except for the optical axis configuration of the light irradiation unit. However, the above-described light irradiation mechanism irradiates the capillary with laser light at an angle shifted by 2 ° from the vertical, and has a pinhole plate 97 as a light selection member. The light selection member includes an opening through which laser light traveling from the light source to the capillary can pass, and a barrier portion capable of blocking return laser light traveling from the capillary to the light source.
[0106]
The laser beams 24 and 25 are in the array plane and enter the capillary at an angle shifted by 2 ° from the perpendicular. The laser beams 24 and 25 are coaxial when irradiating the capillary array, and of the one laser beam, the light that has passed through the capillary passes coaxially with the other incident laser beam and returns to the laser main body 87. The optical axis is adjusted as follows. Accordingly, the reflected lights 95 and 96 from the capillaries and cells by the laser lights 24 and 25 propagate on an optical axis different from the incident laser optical axis as shown in FIG. Are not coaxial.
[0107]
A pinhole plate 97 having a pinhole with a diameter of 1.4 mm, which is a light selecting means that transmits laser light before entering the capillary and does not transmit laser light reflected by the capillary, is mounted near the laser light emission port of the laser main body 87. . Although the laser beam from the laser main body 87 passes through the pinhole, the reflected light cannot pass through the pinhole, so that the reflected lights 95 and 96 can be prevented from returning to the laser main body 87. Therefore, the problem that the reflected light from the capillary array or the cell returns to the laser oscillator and makes the laser oscillation unstable can be solved.
[0108]
By combining the present embodiment and the sixth embodiment, transmitted return light can be suppressed by the method of the sixth embodiment, and reflected return light can be suppressed by the method of the sixth embodiment. Obtainable.
[0109]
Example 8
The invention according to the eighth embodiment is to prevent the fluorescence generated from the capillary from diverging when passing through the boundary of the filling medium by forming the shape of the filling medium around the capillary into a predetermined curved surface.
[0110]
FIG. 16 is a top view of the vicinity of the laser irradiation unit according to the eighth embodiment. Other structures are the same as in the third embodiment. As shown in FIG. 16A, the F polymer 60, which is the filled light transmitting medium, is molded so that the surface shape is convex when viewed from the laser irradiation portion 100 in the capillary.
[0111]
Here, it is assumed that the F value of the fluorescent light condensing lens 31 is 1.8 °. When the surface of the F polymer 60 is flat (FIG. 16B), the signal light is refracted at the interface 65 between the air and the F polymer 60, so that only the signal light within a range of about ± 11 ° in the cross section of the capillary as shown in FIG. It does not reach the condenser lens 31.
[0112]
On the other hand, when the surface shape of the F polymer 60 is convex as shown in FIG. 16A, the signal light in the range of about ± 11 to 15 ° in the cross section of the capillary is collected by fluorescence depending on the radius of curvature of the surface shape. Reach the lens. As described above, by making the surface shape of the filled F polymer 60 a curved surface such as a convex shape, the intensity of irradiation light from the capillary 16 detected by the detection mechanism can be increased. In order to effectively increase the irradiation light intensity, it is desirable that the center of the arc of the surface convex shape of the F polymer 60 coincides with the laser optical axis. This is because the fluorescence emitted by the test sample travels in a direction perpendicular to the convex surface of the F polymer 60, and the traveling direction does not change when passing through the convex surface (boundary surface). Thereby, the problem of aberration can be avoided.
[0113]
In addition, by forming the surface shape of the filled F polymer 60 so as to have a predetermined curved surface, the separation ability can be improved. For example, a curved surface is formed such that one section thereof has a predetermined curve (ellipse, hyperbola, circle whose center is different from the laser optical axis). In this case, the fluorescence converges more to the CCD camera serving as the detection unit, and the fluorescence intensity detected by the CCD increases. In other words, by forming the surface shape of the F polymer 60 around the capillary into a predetermined curved surface, the F polymer 60 acts as a lens and can adjust the traveling direction of the fluorescence. The problem due to the aberration can be avoided by forming the lens relating to the surface shape of the F polymer 60 to have a plurality of focal points.
[0114]
The surface of the F polymer is not limited to a surface in contact with air, but refers to a contact surface with a medium having a refractive index different from that of the F polymer. Further, another medium having a different refractive index may exist between the F polymer and the capillary.
This embodiment can be performed simultaneously with the fourth embodiment.
[0115]
Example 9
In the ninth embodiment, the outer mold of the container that covers the filling medium around the capillary is formed with a predetermined curved surface, so that the fluorescence generated from the capillary is prevented from diverging when passing through the surface of the container.
[0116]
FIG. 17 shows a top view of the vicinity of the light irradiation unit of the ninth embodiment. Other structures are the same as in the first embodiment. As shown in FIG. 17A, the surface shape of the cell lid 20 is made to be convex when viewed from the laser irradiation portion 100 in the capillary.
[0117]
Here, it is assumed that the F value of the fluorescent light condensing lens 31 is 1.8 °. When the surface shape of the cell lid 20 is flat (FIG. 17b), the signal light is refracted at the interface between the air and the cell lid 20, so that only the signal light within a range of about ± 11 ° in the cross section of the capillary as shown in FIG. It does not reach the condenser lens 31.
[0118]
On the other hand, when the surface shape of the cell lid 20 is convex as shown in FIG. 17A, depending on the radius of curvature of the surface shape, the signal light within the range of about ± 11 to 15 ° in the capillary cross section is collected by fluorescence. Reach the lens. As described above, the intensity of the irradiation light from the capillary 16 detected by making the surface shape of the cell lid 20 convex can be increased. In order to effectively increase the irradiation light intensity, it is desirable that the center of the arc of the surface convex shape of the cell lid coincides with the laser optical axis.
[0119]
Further, by making the surface shape of the cell lid a curved surface having a larger curvature, the fluorescence is converged by the CCD camera in the detection mechanism, and the intensity of the irradiation light can be increased. In other words, by forming the shape of the container such as the cell lid that covers the filling medium around the capillary into a predetermined curved surface, the container such as the cell lid acts as a lens, and the traveling direction of the fluorescence can be adjusted.
[0120]
The surface of the cell lid is not limited to a surface in contact with air, but refers to a contact surface with a medium having a refractive index different from that of a container such as a cell lid. Further, another medium having a different refractive index may exist between the cell lid and the filling medium forming the boundary surface.
[0121]
Example 10
In the invention according to the tenth embodiment, the function of the surface convex shape of the cell lid in the ninth embodiment is provided by mounting a cylindrical lens, one of which is a flat surface and the other a curved surface member, on a multi-capillary array having a flat surface shape. And the cylindrical lens 202 mounted on the apparatus.
[0122]
FIG. 18 is a top view of the vicinity of the laser irradiation unit of the tenth embodiment. In the present embodiment, the cell cap of the multi-capillary array according to the first embodiment is covered by a cylindrical lens 202 mounted on the electrophoresis apparatus main body. Other structures are the same as in the first embodiment.
[0123]
The function of the surface convex shape of the cell lid in the ninth embodiment can be performed by the cylindrical lens 202 mounted on the apparatus, so that the cell lid of the consumable capillary array may be a flat glass plate. Further, since the cylindrical lens 202 is mounted on the apparatus, the center of the convex surface of the cylindrical lens 202 coincides with the laser optical axis, so that there is an advantage that the irradiation light intensity does not depend on the mounting accuracy of the capillary array.
[0124]
Example 11
FIG. 19 shows a front view (19a) of the vicinity of the laser irradiation part of Example 11 and an AA ′ section (19b) in the front view. In this embodiment, a background light shielding member having a detection window through which fluorescence generated from a sample from one capillary is transmitted is disposed above the capillaries arranged in parallel on a quartz array base 15. Have been. Other structures are the same as in the first embodiment.
[0125]
All of the 96 capillaries 16 are arranged on the array table 15 so as to contact the array table 15, and are adhered and fixed to the array table 15 together with the silicon plate 101. The silicon plate 101 is formed with a detection window 103 partitioned by a fence 102 and a V-shaped groove 104 used for alignment positioning of the capillary 16 by a silicon anisotropic etching technique. Since the capillaries 16 are fitted in the V-shaped grooves 104, the capillaries 16 can be arranged with high precision at predetermined intervals, and the alignment between the detection windows 103 partitioned by the fences 102 and the capillaries 16 can be easily performed. it can.
[0126]
Further, a positioning guide 105 of a silicon plate is formed on the array table 15, which corresponds to a detection allowable range of the measuring unit. That is, if all the capillaries 16 are inside the positioning guide 105 of the silicon plate, all the capillaries 16 are within the detection allowable range of the detection mechanism. By forming the positioning guide 105 of the silicon plate on the array table 15, it is possible to easily position the capillary 16 with respect to the array table 15.
[0127]
The capillary 16 has a structure in which a quartz tube 18 having an inner diameter of 50 μm and an outer diameter of 126 μm is covered with a polymer film having a thickness of 12 μm, and has a total outer diameter of 150 μm. The inside of the capillary is filled with a polymer aqueous solution (refractive index: 1.41) as a DNA separation medium. In the light irradiation part, the polymer coating is removed, and the quartz tube 18 is exposed. When the laser light is irradiated, part of the irregularly reflected laser light hits the polymer coating of the capillary 16 to emit fluorescence, and part of the fluorescence emitted from the DNA sample is irregularly reflected. If the fluorescence from the polymer coating or the irregularly reflected light from the DNA sample is received by the detection mechanism, the S / N ratio decreases due to the increase in background light, and the resolution decreases. However, since the background light is blocked by the fence 102, it is possible to prevent the background light from increasing. Further, an anti-reflection film 106 is formed on the array base 15 to prevent the reflected light from the array base 15 from passing through the detection window 103 and being received by the detection mechanism. As a result, the background light can be further reduced, so that the resolution can be further improved.
[0128]
The periphery of the capillary of the light irradiation part is filled with the F solution 19. The F solution 19 is completely sealed with a quartz cell lid 20, the array base 15, and an adhesive for fixing them.
[0129]
In order to prevent the sealed structure of the cell from being destroyed by the volume expansion of the F solution 19 due to the temperature change, a high compression with an expansion ratio of 30 times (approximately 29/30 of the volume is a bubble) is provided in the sealed structure. A possible foam 107 was incorporated. The body expansion coefficient of the F solution 19 is 0.0012 mm³/ Mm³Since the temperature is ° C., if the difference between the storage temperature of the capillary array (room temperature 25 ° C.) and the use temperature (60 ° C.) is 35 ° C., the temperature rise from the storage temperature of the F solution 19 to the use temperature (35 ° C.) Volume increase by about 0.04mm³/ Mm³(About 4%). The size of the foam 107 was set to about 10% of the cell volume.
[0130]
The foam 107 is arranged in a groove provided in the array base 15 so as to be sandwiched between the array base 15 and the capillary 16. The foam 107 is located at a position within the detection range of the detection mechanism but hidden from the detection mechanism by the silicon plate 101. Thereby, it is possible to prevent an increase in background light due to the fluorescence of the foam 107. In addition, the foam 107 can be arranged as a lid for closing the hole 109 for injecting the F solution into the sealed structure of the cell. This eliminates the need for special processing on the array base 15 only for the foam 107.
[0131]
In order to prevent bubbles from entering the laser beam path, it is desirable that no bubbles exist inside the cell, but it is not easy to completely remove the bubbles from inside the cell. Therefore, even if bubbles are mixed into the cell filled with the F solution 19, a bubble elimination block 23 made of quartz is formed in the cell in order to prevent the bubbles from entering the laser beam path.
[0132]
It is necessary to keep the angle between the incident laser light and the laser incident surface of the cell constant. A guide 108 serving as a groove for positioning the cell lid is formed on the array table 15, and the cells can be positioned with respect to the array table 15 easily and reliably.
[0133]
As described above, it was possible to realize a capillary array having a low SN ratio and a high SN ratio.
[0134]
【The invention's effect】
According to the present invention, samples in a plurality of capillaries can be analyzed with high accuracy.
[Brief description of the drawings]
FIG. 1 is a side view (1a) and a front view (1b) of a vicinity of a light irradiation unit in a capillary array according to a first embodiment.
FIG. 2 is a schematic view of a capillary array electrophoresis apparatus to which the present invention is applied.
FIG. 3 is a schematic view of a detection system for light emission from a capillary array.
FIG. 4 shows an image formed on a CCD obtained in Example 1 and a light emission intensity distribution from 93 capillaries.
FIG. 5 shows the results of electrophoresis obtained in Example 1.
6A and 6B are a front view (6a) of a capillary array mounting portion according to a second embodiment and an AA 'section (6b) in the front view.
FIG. 7 is a calculation result of a temperature gradient on a straight line VW in the F solution.
8A and 8B are a side view (8a) and a front view (8b) of the vicinity of a detection unit according to the third embodiment.
FIG. 9 is a side view (9a) of the vicinity of the laser irradiation unit of the fourth embodiment and a side view (9b) of the vicinity of the laser irradiation unit of the third embodiment.
FIG. 10 is a side view of the vicinity of a detection unit according to a fifth embodiment.
FIG. 11 is a schematic diagram of a sixth embodiment.
FIG. 12 is a schematic diagram of a seventh embodiment.
FIG. 13 is a simulation result of the ratio of the refractive index of the filling medium to the emission intensity.
FIG. 14 shows an image formed on a CCD obtained in Example 1 and a light emission intensity distribution from 96 capillaries.
FIG. 15 shows the results of electrophoresis of one of 96 capillaries.
FIG. 16 is a schematic diagram of an eighth embodiment.
FIG. 17 is a schematic view of a ninth embodiment.
FIG. 18 is a schematic diagram of Example 10.
19A and 19B are a front view (FIG. 1A) of the vicinity of a laser irradiation unit and a cross section taken along the line AA 'in FIG.
FIG. 20 is a graph showing the relationship between the transmission wavelength and the transparency of F polymer.
FIG. 21 is a table showing the chemical characteristics of the F solution.
FIG. 22 is a table showing the refractive index of F polymer.
23A and 23B are a front view (23a) and a cross-sectional view (23b) taken along the line AA 'in a modified example of the capillary array mounting portion according to the second embodiment.
[Explanation of symbols]
1, 82: Capillary array, 2: Negative electrode, 3: Buffer solution on negative electrode side, 4: Gel block, 5: Connection to gel block, 6: Valve, 7: Earth electrode,
8: Light irradiation unit, 9, 24, 25, 80: Laser light, 10: Syringe, 11: Oven, 11-1: Light source, 11-2: High voltage power supply, 11-3: Sample introduction unit, 11-4: 1st buffer container, 11-5: fluid medium injection mechanism, 11-6: detecting unit, 11-7: second buffer container, 11-8: measuring unit, 12: buffer on the ground electrode side, 15: array base, 16: Capillary, 17: Capillary presser plate, 18: Quartz tube, 19: Fluorine-containing solution Fluorinert FC43 made by 3M to fill around the capillary, 20: Cell lid, 20-2: Bubble accommodating space, 21: Adhesive, 22 ... bubbles,
23: A bubble elimination block made of quartz for preventing bubbles from entering the laser beam path, 31: Fluorescent condensing lens, 32: Grating, 33: Focus lens,
Reference numeral 34 denotes a CCD, 34-2 denotes a detection mechanism, 35 denotes fluorescent light, 36 denotes a light flux in which light emitted from the capillary is converted into parallel light by a fluorescent light condensing lens, 40 denotes a reference plane P on the array table 15, 40-2 denotes a capillary. Reference plane for arranging the array, 41 ... Mounting reference plane P ',
44: pressing rod, 45, 51-10: spring, 46: reference plane Q perpendicular to plane P and parallel to the X axis on the array base, 47, 48: mounting reference line QQ ', 47-2, 48-2: mounting reference line Q "Q" ", 49: capillary closest to surface Q,
50: surface R of the quartz surface of the cell lid through which the laser beam passes; 51: surface S of the quartz surface of the cell through which the laser beam passes; 51-6: a fluorescence transmitting portion; 51-7: a laser beam transmitting portion; 51-8: Pressing portion A, 51-9: Pressing portion B, 51-11: Mounting portion cover, 52: Surface T of the quartz surface of the cell through which laser light passes, 53: Laser light of the quartz surface of the cell Surface U, 54... At a point in contact with the F solution on the array base,
55: one point of the cell lid in contact with the F solution; 60: fluorinated polymer covering the light irradiation part of the capillary array; 60-2: light irradiation part; 61, 62 ... fluorination located outside the capillaries at both ends A polymer block for damping the polymer; 65, an interface between air and the F polymer 60; 70, processing of holes formed in the array table to prevent air bubbles from entering the laser beam path; 71, a quartz window for fluorescence detection below the cell; 73, quartz window for transmitting laser light, 81, half mirror, 83, 84, laser condenser lens,
85, 86: End capillary into which laser enters, 87: Laser main body, 88, 89: Half wavelength plate, 90: Polarizing filter, 91: Mirror, 95, 96 ... Light reflected from the capillary or cell by laser light, 97 ... Pinhole plate, 101 ... silicon plate,
102: fence, 103: detection window, 104: V-groove, 105: positioning guide of silicon plate, 106: antireflection film, 107: foam, 108: positioning guide of cell lid, 109: F solution injection hole, 202 ... Cylindrical lens.

Claims (28)

A separation medium for separating a sample is filled, and a plurality of capillaries having an introduction unit for introducing the sample and a light irradiation unit irradiated with irradiation light are provided, and the plurality of light irradiation units are aligned. Multi-capillary array,
A voltage application mechanism that applies a voltage to a current path including the introduction section and the light irradiation section,
A light irradiation mechanism that irradiates irradiation light continuously penetrating the plurality of light irradiation units arranged in parallel,
An electrophoresis apparatus having a detection mechanism for detecting light emitted by the sample,
An electrophoresis apparatus, wherein the periphery of the light irradiation unit is surrounded by a substance having a refractive index higher than the refractive index of air and lower than the refractive index of water. The electrophoretic device according to claim 1,
An electrophoresis apparatus, wherein the substance is water, an aqueous solution, or a fluorine-containing compound. The electrophoretic device according to claim 1,
An electrophoresis apparatus, wherein the substance is a fluorine-containing compound which is liquid at normal temperature and normal pressure. The electrophoretic device according to claim 1,
An electrophoresis apparatus, wherein the substance is a fluorine-containing polymer. The electrophoretic device according to claim 1,
An electrophoresis apparatus, wherein the substance is a fluorine-containing compound having a refractive index of 1.29 to 1.32. The electrophoretic device according to claim 1,
An electrophoresis apparatus comprising 24 or more capillaries. The electrophoretic device according to claim 1,
An electrophoresis apparatus comprising a background light shielding member having a detection window through which the light emitted from the sample passes, between the measurement mechanism and the capillary. A separation medium for separating the sample is filled, an introduction unit for introducing the sample, a plurality of capillaries having a light irradiation unit irradiated with irradiation light, the light irradiation unit arranged and arranged. A multi-capillary array irradiated with continuously penetrating irradiation light,
A multi-capillary array in which the periphery of the capillary of the light irradiation section is surrounded by a substance having a refractive index higher than that of air and lower than that of water. The multi-capillary array according to claim 8,
The multi-capillary array, wherein the substance is water, an aqueous solution, or a fluorine-containing compound. The multi-capillary array according to claim 8,
A multi-capillary array, wherein the substance is a fluorinated compound which is liquid at normal temperature and normal pressure. The electrophoretic device according to claim 8,
The multi-capillary array, wherein the substance is a fluorine-containing polymer. The multi-capillary array according to claim 8,
A multi-capillary array, wherein the substance is a fluorine-containing compound having a refractive index of 1.29 to 1.32. The multi-capillary array according to claim 8,
A multi-capillary array comprising 24 or more capillaries. A separation medium for separating a sample is filled, and a plurality of capillaries having an introduction unit for introducing the sample and a light irradiation unit irradiated with irradiation light are provided, and the plurality of light irradiation units are aligned. Multi-capillary array,
A voltage application mechanism that applies a voltage to a current path including the introduction section and the light irradiation section,
A light irradiation mechanism that irradiates irradiation light continuously penetrating the plurality of light irradiation units arranged in parallel,
An electrophoresis apparatus having a measurement mechanism for detecting light emitted by the sample and a container around the light irradiation unit, which holds a liquid having a refractive index larger than air and not larger than that of water. ,
The temperature of the liquid sandwiched between the plurality of capillaries and present in the space through which the irradiation light passes, in the direction perpendicular to both the major axis direction of the capillary in the light detection unit and the traveling direction of the irradiation light. An electrophoresis device whose gradient is almost zero. A separation medium for separating the sample is filled, an introduction unit for introducing the sample, and a plurality of capillaries having a light irradiation unit irradiated with irradiation light,
A first flat plate portion in which a plurality of the light irradiating portions are arranged in parallel, and a second flat plate portion; A container for holding a liquid having a refractive index greater than air and not greater than the refractive index of water;
A voltage application mechanism that applies a voltage to a current path including the introduction section and the light irradiation section,
A light irradiation mechanism for irradiating irradiation light continuously penetrating the plurality of irradiation units,
An electrophoresis apparatus having a detection mechanism for detecting the light emitted from the sample,
An electrophoresis apparatus in which the temperature of the first flat plate is higher than the temperature of the second flat plate. A multi-capillary array including a plurality of capillaries having an introduction unit for introducing the sample and a light irradiation unit to which irradiation light is applied, which is filled with a separation medium for separating the sample,
A voltage application mechanism that applies a voltage to a current path including the introduction section and the light irradiation section,
A light irradiation mechanism that irradiates irradiation light continuously penetrating the plurality of light irradiation units arranged in parallel,
An electrophoresis apparatus comprising: a detection mechanism for detecting light emitted from the sample; and a sealed container around the light irradiation unit for holding a liquid having a refractive index larger than air and not larger than that of water. hand,
The liquid and gas are held inside the sealed container,
An electrophoresis apparatus in which a bubble accommodating space for accommodating the gas during electrophoresis is located at a position where the irradiation light does not pass during electrophoresis. A multi-capillary array including a plurality of capillaries having an introduction unit for introducing the sample and a light irradiation unit to which irradiation light is applied, which is filled with a separation medium for separating the sample,
A voltage application mechanism that applies a voltage to a current path including the introduction section and the light irradiation section,
A light irradiation mechanism that irradiates irradiation light continuously penetrating the plurality of light irradiation units arranged in parallel,
An electrophoresis apparatus comprising: a detection mechanism for detecting light emitted from the sample; and a sealed container around the light irradiation unit for holding a liquid having a refractive index larger than air and not larger than that of water. hand,
An electrophoresis apparatus in which a foam whose volume shrinks exists inside the sealed container. The electrophoretic device according to claim 17,
An electrophoresis apparatus in which the foam is present at a liquid inlet for injecting the liquid into the sealed container. A multi-capillary array including a plurality of capillaries having an introduction unit for introducing the sample and a light irradiation unit to which irradiation light is applied, which is filled with a separation medium for separating the sample,
A voltage application mechanism that applies a voltage to a current path including the introduction section and the light irradiation section,
A light irradiation mechanism that irradiates irradiation light continuously penetrating the plurality of light irradiation units arranged in parallel,
An electrophoresis apparatus having a detection mechanism for detecting light emitted by the sample,
A light transmission medium having a refractive index different from the refractive index of the capillary, larger than air, and not larger than water in a space between the light irradiation units and in which the irradiation light passes; Exists,
An electrophoresis apparatus, wherein a surface shape of a portion of the surface of the light transmission medium through which the light detected by the detection mechanism passes is a curved surface. The electrophoretic device according to claim 19,
An electrophoretic layer device, wherein the cross-sectional shape of the curved surface is a circle centered on the intersection of the capillary and the irradiation light. The electrophoretic device according to claim 19,
An electrophoresis apparatus, wherein the cross-sectional shape of the curved surface is a circle, an ellipse, or a hyperbola. A multi-capillary array including a plurality of capillaries having an introduction unit for introducing the sample and a light irradiation unit to which irradiation light is applied, which is filled with a separation medium for separating the sample,
A voltage application mechanism that applies a voltage to a current path including the introduction section and the light irradiation section,
A light irradiation mechanism that irradiates irradiation light continuously penetrating the plurality of light irradiation units arranged in parallel,
A detection mechanism for detecting fluorescence emitted by the sample,
An electrophoresis apparatus, comprising: a container that holds a liquid having a refractive index larger than air and not higher than the refractive index of water around the light irradiation unit,
An electrophoresis apparatus, wherein a surface shape of a portion of the surface of the container through which the light detected by the detection mechanism passes is a curved surface. A multi-capillary array including a plurality of capillaries having an introduction unit for introducing the sample and a light irradiation unit to which irradiation light is applied, which is filled with a separation medium for separating the sample,
A voltage application mechanism that applies a voltage to a current path including the introduction section and the light irradiation section,
A light irradiation mechanism for irradiating two or more irradiation lights continuously penetrating the plurality of light irradiation units arranged in parallel,
Having a detection mechanism for detecting light emitted by the sample,
A liquid or a solid having a refractive index different from that of the capillary, larger than air, and not larger than water is present in a space between the plurality of light irradiation units and through which the irradiation light passes. Electrophoresis device,
A polarizing plate that passes the irradiation light before being incident on the light irradiation unit and blocks the irradiation light after being incident on the light irradiation unit,
An electrophoresis apparatus comprising: a rotating plate that changes a polarization direction of the irradiation light. The electrophoretic device according to claim 23,
An electrophoresis apparatus in which the polarization direction of the irradiation light immediately before entering the light irradiation unit is inclined by about 45 ° with respect to a plane including the plurality of light irradiation units arranged in parallel. The electrophoretic device according to claim 23,
The irradiation light is incident at an angle other than a right angle with respect to a long axis direction of the capillary in the light irradiation unit,
An electrophoretic device including a light selection member having an opening through which the irradiation light passes before entering the light irradiation unit, and a barrier unit that blocks the irradiation light reflected by the light irradiation unit. A separation medium for separating the sample is filled, and has a plurality of capillaries having an introduction portion for introducing the sample, and a light irradiation portion to which irradiation light is applied, and water having a refractive index larger than the refractive index of air. A multi-capillary array including a sealed container capable of holding a substance having a refractive index smaller than the refractive index around the light irradiation unit,
A multi-capillary array mounting portion capable of attaching and detaching the sealed container,
A voltage application mechanism that applies a voltage to a current path including the introduction section and the light irradiation section,
A light irradiation mechanism that irradiates irradiation light continuously penetrating the plurality of light irradiation units arranged in parallel,
An electrophoresis apparatus having a detection mechanism for detecting light emitted by the sample,
The sealed container includes a first plane substantially parallel to a reference plane in which a plurality of the light irradiation units are arranged and a second plane substantially perpendicular to the first plane,
The electrophoresis apparatus, wherein the multi-capillary array mounting portion includes a first mounting reference surface that contacts the first plane and a second mounting reference surface that contacts the second plane. The electrophoretic device according to claim 26,
An electrophoresis apparatus in which the reference plane and the first plane are on the same plane. The electrophoretic device according to claim 26,
An electrophoretic device through which the irradiation light passes through the second plane.

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