JP3654290B2 - Capillary array electrophoresis device - Google Patents

Description

Δθはレーザビームの入射位置ｘによって異なり、キャピラリーの外半径Ｒ、内半径ｒ、キャピラリー外部の屈折率ｎ₁、キャピラリー材質の屈折率ｎ₂、キャピラリー内部の屈折率ｎ₃でコントロールできる。ｘ＝０のレーザビームに対しては常にΔθ＝０であり、ｘの増加にともなって｜Δθ｜も単調に増加する（但し、ここではｘ≦ｒを想定している）。つまりキャピラリーそのものが焦点の定まらないレンズの作用をもち、Δθ＞０ならば凹レンズ、、Δθ＜０ならば凸レンズとなる。ｎ₁＝ｎ₂＝ｎ₃のときは、もちろんｘによらずΔθ＝０となる。
【００３５】
複数本のキャピラリーを１平面状に配列した配列体に、複数本のキャピラリーを通過するように配列体の側方よりレーザビームを照射した場合、レーザパワーを多数のキャピラリーに到達させるためには、キャピラリーによる屈折角Δθが小さいほど、特に、各キャピラリーがΔθ＜０の凸レンズ作用をもつほど有利である。１本のキャピラリーに着目したとき、入射レーザビームよりも透過レーザビームが絞られていれば、効率良く隣のキャピラリーにレーザパワーを到達させることができるからである。通常の電気泳動条件では、常にｎ₂＞ｎ₃であるから、（数９）からｎ₁が小さいほどΔθは小さくなる。つまり、キャピラリー外部がｎ₁＝１．００である空気のような透明気体で満たされていることが好適な条件となる。
【００３６】
一方、レーザビームは各境界点で反射も受ける。つまり、入射光のレーザパワーの一部は反射光となり、残りが屈折光（透過光）となる。入射光のレーザパワーに対する反射光のレーザパワーの比率を反射率Ｒ_ef、入射光のレーザパワーに対する屈折光のレーザパワーの比率を透過率Ｔ_raとすると、これらは入射角θ_iと屈折角θ_t（即ち両媒質の屈折率）の関数で、（数１０）、（数１１）で与えられる。
【００３７】
【数１０】

【００３８】
【数１１】

ここでＲ_ef＋Ｔ_ra＝１である。但し、ここでは入射光に偏光がない場合を示した。レーザパワーは境界点を通過する毎に、（数１０）、（数１１）に従って順次減少する。そこで、電気泳動時に試料成分が通過するキャピラリー内部のレーザビームの光路長（図２において点Ｐ₂と点Ｐ₃を結ぶ長さ）にその位置でのレーザパワーを乗じることにより、レーザビームのこのキャピラリーにおける励起効率（このキャピラリー内を泳動する標識された試料成分のレーザビームによる標識の励起効率）を見積もった。また、無限小幅のレーザビームが入射する位置を、キャピラリー配列軸（ｘ＝０）からの距離ｘとし、この距離ｘを変化させることにより、有限幅のレーザビームのこのキャピラリーにおける励起効率を見積もった。更に、複数のキャピラリーが平面状に配列された場合に、以上の計算を連続的に行なうことにより、複数のキャピラリーの各々のキャピラリーにおける励起効率がどのように変化するかを調べた。
【００３９】
具体例として、キャピラリーの構成を、外径０．３７５ｍｍ（Ｒ＝０．１８７５ｍｍ）、内径０．１ｍｍ（ｒ＝０．０５ｍｍ）、長さ５０ｃｍの溶融石英円筒管（ｎ₂＝１．４６）を用いた。変性剤として７Ｍのウレアを含んだ４％Ｔ（Ｔｏｔａｌ ｍｏｎｏｍｅｒ ｃｏｎｃｅｎｔｒａｔｉｏｎ）、５％Ｃ（Ｃｒｏｓｓｌｉｎｋｉｎｇ ｍａｔｅｒｉａｌ ｃｏｎｃｅｎｔｒａｔｉｏｎ）の濃度のアクリルアミドを注入した後、ゲル化（ｎ₃＝１．３６）させた。試料注入側のキャピラリー末端より３０ｃｍの位置で、長さ１０ｍｍ、全周にわたり各キャピラリーのポリイミド被覆を除去して蛍光を計測するための窓を予め作製してレーザ照射部位とした。
【００４０】
これらキャピラリーゲル１０本の計測部分を図３に示すように、０．３７５ｍｍピッチに揃え、各キャピラリー１を密着させてガラス平板５上に固定して平面状に配列した。蛍光計測位置のキャピラリー外部は空気（ｎ₁＝１．００）とした。蛍光計測はキャピラリー配列平面に垂直な方向より、第１レンズ６、バンドパスフィルター７、第２レンズ８を介して２次元検出器９により行なった。本実験条件では、キャピラリーによるレーザビーム２の反射が大きいため、平板ゲル電気泳動等の蛍光計測時に用いるバンドパスフィルターよりもレーザ波長の透過率を下げる必要があった。そこで、レーザ波長の透過率が１０^-5以下であるバンドパスフィルターを用いた。
【００４１】
図４は蛍光計測部分のキャピラリー軸に対する断面図を示し、レーザをビーム径０．１ｍｍに集光し、キャピラリー配列体の側方よりキャピラリー配列軸に沿って入射させた。このとき、（数９）からｘ≦ｒのレーザビームに対して、常にΔθ＜０となり、各キャピラリーは凸レンズ作用をもった。０．１ｍｍのビーム幅を０．０１ｍｍ間隔の合計１１本の無限小幅のビームコンポーネントに分けて考えた。即ち、ビームコンポーネントはキャピラリー配列軸（レーザビーム中心軸、ｘ＝０）からの距離ｘが、０．００、±０．０１、±０．０２、±０．０３、±０．０４、±０．０５ｍｍの位置で１本目のキャピラリーに入射する。図５は、各ビームコンポーネントのキャピラリー透過光が、どのような光路を描いてキャピラリー配列中を進行するかをシュミレーションした結果を示す。図５は、蛍光計測部分のキャピラリー軸に対する断面図を示し、上段はレーザ光源側の５本のキャピラリー、下段はレーザ出射側の５本のキャピラリーを表わしている。即ち、レーザビームは、上段の左側のキャピラリーに入射して、下段の右側のキャピラリーから出射する。
【００４２】
全ての光路の計算は、１０本のキャピラリーが上記のように配列ているとして連続的に行なった。レーザビームの中心軸に位置するビームコンポーネン（ｘ＝０．００）は全ての境界点での入射角が０度であるため、屈折を全く受けず、Δθ＝０であり、キャピラリー配列軸上を直線的に進行する。ｘ＝０．００以外のビームコンポーネントは、キャピラリーの凸レンズ作用のために基本的にキャピラリー配列軸に向かう方向に屈折を受け、光路はキャピラリー配列軸を中心として上下に振動しながらキャピラリー配列中を進行した。振動の振幅や周期はビームコンポーネントによって異なったが、各ビームコンポーネントの光路は、キャピラリー配列軸を対称軸として上下対称であった。以上の結果、全てのビームコンポーネントは１０本のキャピラリー全てについて、キャピラリー外部に逸脱することなしに、キャピラリー内部を透過し、キャピラリー内を泳動する試料成分の励起に寄与していることが判明した。即ち、本発明のキャピラリー電気泳動装置は、各キャピラリーゲルにおいて照射されるレーザビームは、ほぼ収束するので、複数のキャピラリーゲルのそれぞれにおいて、近似的に収束するレーザビームの光路を形成するので、マルチフォーカスキャピラリー電気泳動装置と呼ぶにふさわしいと言える。
【００４３】
使用するキャピラリーの本数を変化させた場合、各キャピラリーにおける励起効率の減少の度合を評価するために、各キャピラリー毎に、キャピラリー内部を透過する光路の長さ（光路長）を求め、この光路長にその位置でのレーザパワーを乗じ、全てのビームコンポーネントについて積算し、この積算値を各キャピラリーにおける励起効率とした。図６はこのようにして求めた、各キャピラリーにおける励起効率を示す（各キャピラリーにおける励起効率は、レーザ光源側の１本目のキャピラリーにおける励起効率により規格化されている）。図６では、各キャピラリーにはレーザ光源側から順番に１から１０の番号を付けてある。
【００４４】
全てのビームコンポーネントのレーザパワーは、屈折率の変化する境界点を透過する毎に反射により減衰するため、キャピラリー毎の励起効率は、図６のようにレーザ光源から離れるに従い指数間数的に減少した。実際に計測される各キャピラリーからの蛍光強度はこの励起効率に比例する。図６に示すように、１０本目のキャピラリーの励起効率は１本目の５４．１％にも及んだ。この値からレーザビームのキャピラリー１本当たりの平均透過率を求めると、９３．４％の高効率であった（０．９３４⁹＝０．５４１）。検出器の感度とダイナミックレンジの関係から、同時計測可能な励起効率の減少を２０％までとすると、本計算実験条件では、２４本までのキャピラリーの同時計測が可能であった（０．９３４²³＝０．２１）。
【００４５】
他の具体例として、キャピラリー内径を０．１ｍｍ（ｒ＝０．０５ｍｍ）とし、キャピラリー外径を０．１１ｍｍ（Ｒ＝０．０５５ｍｍ）、０．２ｍｍ（Ｒ＝０．１ｍｍ）、０．３７５ｍｍ（Ｒ＝０．７５ｍｍ）、０．７５ｍｍ（Ｒ＝０．３７５ｍｍ）、１．０ｍｍ（Ｒ＝０．５ｍｍ）とする場合について、上記と同様の計算を行なった（他の条件は全て上記と同一とした）。但し、１０本のキャピラリーの配列ピッチは各キャピラリーの外径と一致させ、各キャピラリーを密着配列した。図７は、レーザ光源から最も遠い位置に配列された１０本目のキャピラリーにおける励起効率のキャピラリーの外径／内径の比率（Ｒ／ｒ）に関する変化を表す（励起効率は、図６と同様にレーザ光源に最も近い位置に配列された１本目キャピラリーにおける励起効率により規格化されている）。本計算実験では、キャピラリー内径を０．１ｍｍに統一して行なっているが、他の内径のキャピラリーを用いた場合でも、キャピラリーの外径／内径の比率（Ｒ／ｒ）が同じならば本質的に同じ結果が得られる（但し、入射するレーザビーム幅はキャピラリーの内径とほぼ同じとする）。
【００４６】
図８は、図７の結果からレーザビームのキャピラリー１本あたりの平均透過率を求め、励起効率の減少を２０％までとする同時計測可能な最大キャピラリーの本数を先と同様に求めた結果を示す。図７、図８の結果から、キャピラリーの外径／内径の比率Ｒ／ｒが４付近の値（正確には、３．７５）をとるとき、最も効率的に多数のキャピラリーを同時計測でき、この値から離れるに従い同時計測できるキャピラリーの数が減少することが判明した。キャピラリーの外径／内径の比率Ｒ／ｒが小さいとき、同時計測できるキャピラリーの数が減少する理由は、レーザビーム幅とキャピラリー外径の値が近いたために、キャピラリー外径から逸脱するビームコンポーネントが存在したためである。キャピラリーの外径／内径の比率Ｒ／ｒが大きいとき、同時計測できるキャピラリーの数が減少する理由は、キャピラリーのガラス部分のみを透過して、キャピラリー内部を透過しないビームコンポーネントが存在したためである。
【００４７】
分析又は解析する試料を大量、複数種類にわたり調製して処理する場合、一般にマイクロタイタープレートが広く用いられている。このマイクロタイタープレートは、１２×８の格子状に配列された９６個の穴のあいたプレートで、世界的な標準規格になっている。従って、電気泳動計測において同時に処理する試料数を１２又は８の倍数とすることは、試料調製と電気泳動計測の大量処理をリンクさせる場合、非常に重要である。即ち、同時に処理して計測する試料数は８、１２、１６、２４、…であること好ましい。図８の結果から、各キャピラリーにおける励起効率の減少を２０％までとする同時計測を可能とするための、キャピラリーの外径／内径の比率Ｒ／ｒに関する条件は、同時に処理して計測する試料数を、８、１２、１６、２４とする場合、それぞれ、１＜Ｒ／ｒ≦１０、１＜Ｒ／ｒ≦１０、１≦Ｒ／ｒ≦７、３≦Ｒ／ｒ≦５となる。
【００４８】
次に、本発明のもうひとつのキーポイントである、レーザビームの散乱による背景光を低減させる条件について説明する。レーザビームの散乱は主に、各キャピラリーの屈折率が変化する境界点でのレーザビームの反射が原因である。反射光は反射の法則に従い、入射角と反射角が等しい方向に進行する。その結果、様々な方向に反射光が進行するが、なかにはキャピラリーが配列する平面に垂直な方向に配置した光検出系に直接入射する反射光が存在する。これは蛍光計測時の背景光を著しく増大させ、影響大であり、計測感度の低下につながる。そこで、この光検出系に直接入射する反射光のレーザパワーを全てのキャピラリーについて積算することによりその影響を見積もった。
【００４９】
図９は、上記の図７、図８の結果を得たと同様の計算実験条件として、光検出系に直接入射する反射光のレーザパワーを１０本のキャピラリーについて積算して求め、積算結果のキャピラリーの外径／内径の比率（Ｒ／ｒ）に関する変化を示す。光検出系に直接入射する反射光のレーザパワーは、上述したように、蛍光計測時の背景光を著しく増大させ、計測感度の低下につながるので、値が小さいほど高感度に蛍光計測を行なうことができる。図９の結果から、キャピラリーの外径／内径の比率Ｒ／ｒが４付近で、最も背景光が小さくなり高感度に蛍光計測でき、この値から離れるに従い背景光が増大して計測感度が低下した。キャピラリーの外径／内径の比率Ｒ／ｒが小さいときに背景光が大きく増大した理由は、レーザビーム幅とキャピラリー外径の値が近いたために、大きな入射角でキャピラリーに照射されるビームコンポーネントの割合が増えたためである。以上の結果から、２≦Ｒ／ｒの条件が高感度計測に有効であることが判明した。
【００５０】
光検出系に直接入射する反射光のレーザパワーを下げるためには、屈折率の変化する各境界点での反射率を下げれば良い。そのための一つの手段として、レーザが照射される位置のキャピラリーの外部を水、水溶液等の透明液体で満たし、キャピラリーによる屈折角を大きくするすことがある。即ち、凹レンズ方向に作用させることになり、多くのキャピラリーにレーザパワーを到達させる点においては不利になる。従って、これらの兼ね合いで条件を評価すべきである。シュミレーションした結果、Ｒ／ｒ≦２のキャピラリーについては、キャピラリー外部を液体で満たすことによって、背景光が減少する効果の方が大きかった。その結果、多数のキャピラリーを並べて同時分析できるので、高速、高スループット、高感度な分析を実現できる。
【００５１】
本実施形態で用いたサイズ以外の円筒形状キャピラリーを用いても、もちろん同様の効果が得られる。
【００５２】
（実施形態２）
（実施形態１）では平面状に配列したキャピラリーは互いに密着させたが、ここでは各キャピラリーの間隔をキャピラリーの外径以上にした。具体例として、キャピラリーに外径０．２ｍｍ（Ｒ＝０．１ｍｍ）、内径０．１ｍｍ（ｒ＝０．０５ｍｍ）、長さ５０ｃｍの溶融石英円筒管（ｎ₂＝１．４６）を１０本用いた。配列間隔は、キャピラリー外径の１倍（実施形態１と同じ）、２倍、３倍、４倍、５倍、即ち、０．２、０．４、０．６、０．８、１．０ｍｍの各場合について調べた。その他の実験条件は（実施形態１）と同一とした。
【００５３】
図１０は、レーザ光源から最も遠い位置に配列された１０本目のキャピラリーにおける励起効率をキャピラリーの配列間隔を変数として示した（励起効率は、図６と同様にレーザ光源に最も近い位置に配列された１本目のキャピラリーにおける励起効率により規格化されている）。図１０の結果から、キャピラリーの配列間隔が増大するほど、１０本目のキャピラリーにおける励起効率は低下し、多数のキャピラリーの同時計測に不利な条件となった。つまり、キャピラリーの配列間隔をキャピラリー外径と一致させるとき、即ち、キャピラリーを密着配列するときに、最も効率的に多数のキャピラリーの同時計測ができることが判明した。この最適条件（キャピラリーを密着配列する）における１０本目のキャピラリーのＳ／Ｎが、１本目のキャピラリーのＳ／Ｎの半分になる条件を実用範囲とすると、励起効率は１／４になる条件まで許容範囲となる。この励起効率は１／４になる条件は、図１０からキャピラリーの配列間隔が０．８ｍｍ以下、即ち配列間隔がキャピラリー外径の４倍以下であれば満たされる。
【００５４】
（実施形態３）
（実施形態１）のシュミレーション結果に基づき、本実施形態ではＤＮＡ試料の電気泳動を実際に行なった。まず、平面状に配列された５本のキャピラリーゲルを使用し電気泳動を行ない、蛍光検出する部分のキャピラリーゲルを水中に配置して、蛍光計測することによりＤＮＡ塩基配列決定を実施した。図１１は、使用した装置の基本構成を示す。キャピラリーゲルは、長さ５０ｃｍ、外径０．２ｍｍ（Ｒ＝０．１ｍｍ）、内径０．１ｍｍ（ｒ＝０．０５ｍｍ）の溶融石英管（ｎ₂＝１．４６）に、変性剤として７Ｍのウレアを含んだ４％Ｔ（Ｔｏｔａｌ ｍｏｎｏｍｅｒ ｃｏｎｃｅｎｔｒａｔｉｏｎ）、５％Ｃ（Ｃｒｏｓｓｌｉｎｋｉｎｇ ｍａｔｅｒｉａｌ ｃｏｎｃｅｎｔｒａｔｉｏｎ）の濃度のアクリルアミドを注入した後ゲル化（ｎ₃＝１．３６）させて調製した。試料注入側のキャピラリー末端より３０ｃｍの位置で、長さ１０ｍｍ、全周にわたり、ポリイミド被覆を除去して、蛍光を計測する窓を予め作製してレーザ照射部位とした。図１１の示すように、このキャピラリーゲル５本を０．２ｍｍピッチに揃えて平面状に配列し、水（ｎ₁＝１．３３）を満たした蛍光計測セル１２内に固定した。但し、図１１では、キャピラリーゲル１はキャピラリーゲルの計測窓近傍のみを示している。
【００５５】
図１２は蛍光計測セル１２のキャピラリー軸に対する断面図を示し、ＹＡＧレーザ（５３２ｎｍ、２０ｍＷ）及びＨｅ−Ｎｅレーザ（５９４ｎｍ、１０ｍＷ）を同軸にした後ビーム径０．１ｍｍに集光しビーム２とし、キャピラリー配列体の側方よりキャピラリー配列軸に沿って入射させた。蛍光計測セル１２内部にはアクリルアミドゲル１０が充填されたキャピラリー１が平面上に配置され、キャピラリー１の外部には水１４が満たされている。
【００５６】
図１１に示すように、蛍光計測は、キャピラリーゲルが配列する平面に対して垂直方向より行なった。水平方向に幅０．８ｍｍで１列に並ぶ５個の蛍光発光点群を第一カメラレンズ６でほぼ平行光束にし、蛍光発光点群の像を垂直方向に４つに分割する像分割プリズム及び４つの像を結ぶ光束に対応させた４種の組み合わせフィルター１４を透過させ、第二カメラレンズ８で結像させた。なお、この蛍光選別法については特開平２−２６９９３６号公報に詳しく記されている。５×４＝２０個に２次元状に展開された蛍光発光点群は、冷却型の２次元ＣＣＤカメラ９で一度に露光計測した。露光時間１．０秒、データ取得間隔１．５秒で連続計測を行った。
【００５７】
５種のＤＮＡ試料の塩基配列決定はサンガー法に従って行なった。調製されたＤＮＡ試料成分には、末端塩基種Ｃ、Ｇ、Ａ、Ｔに対応して４種の蛍光体Ｃｙ３（極大発光波長５６５ｎｍ）、ＴＲＩＴＣ（極大発光波長５８０ｎｍ）、Ｔｅｘａｓ Ｒｅｄ（極大発光波長６１５ｎｍ）、Ｃｙ５（極大発光波長６６５ｎｍ）を標識した。ここで、Ｃｙ３、Ｃｙ５はバイオロジカル・ディテクション・システム社（米国）より販売されており、ＴＲＩＴＣ、Ｔｅｘａｓ Ｒｅｄはモレキュラー・プローブ社より販売されている。これら４種の蛍光が波長選別されて２次元ＣＣＤカメラに結像されるように、４種の組み合わせフィルターを用いた。末端塩基種に対応した４種の反応物を試料種毎に混合した後、エタノール沈殿によって１０倍に濃縮して試料溶媒をホルムアミドに置換した。５本のキャピラリーゲルの試料注入端をそれぞれ５種のＤＮＡ試料液中に浸し、キャピラリーゲルの両端に１００Ｖ／ｃｍ（５ｋＶ）の一定電界強度を２秒間印加して試料注入を行なった。電気泳動は一定電界強度１００Ｖ／ｃｍ（５ｋＶ）で約３時間行なった。２次元ＣＣＤカメラで得られた４種の蛍光に対応する４種の信号の時間変化をコンピュータで解析し、５種の試料のＤＮＡ塩基配列を決定した。
【００５８】
図１３は、本実施形態で得られたＤＮＡシーケンス結果のうち、Ｔｅｘａｓ Ｒｅｄで標識されたＡ成分の電気泳動パターンであり、泳動時間３０分〜６０分の電気泳動パターンで、プライマーから約１３０塩基長までのＡ成分のパターンを示している。但し、分析した試料は５種ともに標準試料のＭ１３ｍｐ１８である。図１３の最上段のキャピラリー−１が最もレーザ光源側のキャピラリーであり、以下数字の順番にレーザ光源から遠ざかる位置のキャピラリーの電気泳動パターンを示している。ピーク強度はレーザ光源から遠ざかるに従い減少した。キャピラリー−５のピーク強度は、キャピラリー−１の１／３以下になっているが、それでも十分に高いＳ／Ｎ（塩基長９１に相当する位置で、Ｓ／Ｎ＝７３４である）でピーク検出が可能であった。なお、キャピラリー−１ののピークにおけるＳ／Ｎは、塩基長９１に相当する位置で、Ｓ／Ｎ＝３３００である。
【００５９】
本実験条件ではレーザが照射される位置のキャピラリー外部を水としているため、外部を空気とした場合と比較して、キャピラリー界面でのレーザ反射が減少し、背景光信号が１桁近く減少した。このことは（実施形態１）で説明したことが実験的に確認されたことを意味する。他の蛍光体で標識された成分の電気泳動パターンも同様に得られており、５本のキャピラリーゲル電気泳動によるＤＮＡ塩基配列決定を高感度に実現できた。
【００６０】
次に、平面状に配列された１０本のキャピラリーゲルを使用して電気泳動を行ない、泳動する試料成分からの発する蛍光を空気中で検出し計測することにより、ＤＮＡ塩基配列決定を実施した。使用するキャピラリーは外径０．３７５ｍｍ（Ｒ＝０．１８７５ｍｍ）、内径０．１ｍｍ（ｒ＝０．０５ｍｍ）の溶融石英管（ｎ₂＝１．４６）とし、１０本のキャピラリー配列は、図４に示すように、ガラス平面上に０．３７５ｍｍピッチで固定し、図１１、図１２に示す蛍光計測セルは使用しない。即ち、キャピラリー外部の媒質は空気（ｎ₁＝１．００）である。これら以外の実験条件は先の実験と同一とした。
【００６１】
図１４、１５は、本実施形態で得られたＤＮＡシーケンス結果のうち、Ｔｅｘａｓ Ｒｅｄで標識されたＡ成分の電気泳動パターンであり、泳動時間３０分〜６０分の電気泳動パターンで、プライマーから約１００塩基長までのＡ成分のパターンを示している。但し、分析した試料は１０種ともに標準試料のＭ１３ｍｐ１８である。図１４、図１５において、図１４に示す最上段キャピラリー−１が最もレーザ光源側のキャピラリーであり、以下数字の順番にレーザ光源から遠ざかるキャピラリーの電気泳動パターンを示しており、図１５に示す最下段のキャピラリー−１０のいずれの１０本のキャピラリーとも十分なＳ／Ｎで計測できた。塩基長９を与えるピークおいて、Ｓ／Ｎは、キャピラリー−１において２３７、キャピラリー−４において２３７、キャピラリー−７において１１０、キャピラリー−１０において１４０であった（キャピラリー−１０でのＳ／Ｎがキャピラリー−７のＳ／Ｎより大きいのは測定のバラツキと考えられる）。他の蛍光体で標識された成分の電気泳動パターンも図１４、１５同様に得られており、１０本のキャピラリーゲル電気泳動によるＤＮＡ塩基配列決定を高感度に実現できた。
【００６２】
（実施形態４）
本実施形態では、断面が楕円形である楕円筒形状キャピラリーを用いた。このキャピラリーを用いることにより、その配列方法によって２つの異なる効果が生まれる。第１の配列方法は、キャピラリー断面の楕円の短径とキャピラリーの配列平面を平行にすることである。この場合、レーザビームのキャピラリーへの入射角が円筒形キャピラリーを用いた場合と比較して小さくなるので、蛍光を検出する光検出系に直接入射する反射光のレーザパワーが減り、背景光信号が減少する。これにより蛍光検出感度が増大する効果が得られる。第２の配列方法は、キャピラリー断面の楕円の長径とキャピラリーの配列平面を平行にすることである。この場合、キャピラリーを密着配列しながら隣り合うキャピラリーの泳動路（キャピラリー内部）どうしの間隔を大きくできるので、お互いのクロストークがなくなるので複数のキャピラリーの独立計測に有利となる。キャピラリーの密着配列は（実施形態３）に示したように、効率的な多数のキャピラリーの同時計測に有効である。次に、第１の配列方法を用いた具体例を示す。
【００６３】
具体例として、断面が外長径０．７５ｍｍ（外長半径Ｒ₁＝０．３７５ｍｍ）、外短径（外短半径Ｒ₂＝０．１８７５ｍｍ）、内長径０．２ｍｍ（内長半径ｒ₁＝０．１ｍｍ）、内短径０．０５ｍｍ（内短半径ｒ₂＝０．０２５ｍｍ）の楕円形のキャピラリーを用いた。このキャピラリー断面の内部の面積は、（実施形態１）で使用した外径０．３７５ｍｍ（Ｒ＝０．１８７５ｍｍ）、内径０．１ｍｍ（ｒ＝０．０５ｍｍ）の円筒形のキャピラリーの断面の内部の面積と等しい（７．８５×１０^-3ｍｍ²）。この形状のキャピラリーはポリマイクロ社（米国）によって製造可能である。その他の実験条件は（実施形態１）と同様で、全長５０ｃｍのキャピラリーの材質は溶融石英（ｎ₂＝１．４６）を用い、キャピラリー内部は７Ｍのウレアを含んだ４％Ｔ、５％Ｃの濃度のアクリルアミドゲル（ｎ₃＝１．３６）を充填した。試料注入側のキャピラリー末端より３０ｃｍの位置で、長さ１０ｍｍ、全周にわたり、ポリイミド被覆を除去して蛍光を計測する窓を予め作製してレーザ照射部位とした。
【００６４】
この１０本のキャピラリーゲルの蛍光を計測する部位を、図３、図４と同様に平面状に配列したが、この際に全てのキャピラリーの楕円の短径をキャピラリーの配列平面と平行とし、長径をキャピラリーの配列平面と垂直になるように配列した。この配置方法では、上記したように光検出系に直接入射する反射光の低減に効果がある。キャピラリーの配列ピッチは、楕円の短径と一致させ、密着配列とした。蛍光計測部位のキャピラリー外部は空気（ｎ₁＝１．００）とした。図３と同様にして、蛍光計測はキャピラリーの配列平面に垂直な方向より、第１レンズ、バンドパスフィルター、第２レンズを介して２次元検出器により行なった。レーザをビーム径０．１ｍｍに集光し、キャピラリー配列体の側方よりキャピラリー配列軸に沿って入射させた。
【００６５】
この実験条件に対して、（実施形態１）と同様のシュミレーション実験を行なった。図１６は、１１本のビームコンポーネントの光路を計算した結果である。図１６は、蛍光計測部位のキャピラリー軸に対する断面図であり、１番左のキャピラリーが最もレーザ光源側のキャピラリーであり、以下右に行くキャピラリーほどレーザ光源から遠ざかるキャピラリーである。全ての光路の計算は１０本のキャピラリーが上記の配列がなされているとして連続的に行った。本計算実験条件では、レーザビームのキャピラリーによる屈折角がΔθ〜０であるため、即ち、キャピラリーのレンズ作用が小さいため、各ビームコンポーネントはあまり屈折を受けることなしに、キャピラリー配列中を進行した。全てのビームコンポーネントは１０本のキャピラリー全てについて、キャピラリー外部に逸脱することなしに、キャピラリー内部を透過し、キャピラリー内を泳動する試料成分の励起に寄与することが判明した。
【００６６】
図６と同様に、励起効率の減少の度合のキャピラリーの本数による変化を評価するために、各キャピラリー毎に、キャピラリー内部を透過する光路の長（光路長）さを求め、この光路長にその位置でのレーザパワーを乗じ、全てのビームコンポーネントについて積算し励起効率とした。
【００６７】
図１７は、このようにして求めた各キャピラリーにおける励起効率を示した（レーザ光源側の１本目のキャピラリーの値により規格化されている）。図１７で、各キャピラリーにはレーザ光源側から順番に１から１０の番号を付けた。図６と同様に、キャピラリー毎の励起効率はレーザ光源から離れるに従い指数間数的に減少した。１０本目のキャピラリーの励起効率は、１本目の５３．４％にも及び、この減衰の程度は図６の場合とほとんど等しかった。つまり、キャピラリー１本当たりの平均透過率は９３．３％であり（０．９３３⁹＝０．５３６）、本計算実験条件では、２４本までのキャピラリーの同時計測が可能であった（０．９３３²³＝０．２０）。
【００６８】
一方、光検出系に直接入射する反射光のレーザパワーを１０本のキャピラリーについて積算した結果は（実施形態１）の場合と異なり、ゼロであった。これは屈折率が変化する各境界点でのレーザビームの入射角が十分に小さくなったためである。従って、円筒形のキャピラリーから楕円筒形のキャピラリーにすることにより、背景光を大幅に削減でき、より高感度に蛍光計測できることが判明した。
【００６９】
本実施形態で用いた形状寸法以外の楕円筒形キャピラリーを用いても、もちろん同様の効果が得られる。また、（実施形態１、３）で円筒形キャピラリーについて示したように、レーザ照射位置のキャピラリー外部を水等の透明液体で満たすことの効果は、楕円筒形状キャピラリーについても同様に得られる。即ち、キャピラリーに入射するレーザビームの反射率が低減することにより、多数のキャピラリーを効率的に同時計測できるようになる。
【００７０】
（実施形態５）
本実施形態では、（実施形態４）の計算実験条件で、各キャピラリーのレーザ照射部分に減反射のコーティングを施して計算実験を行なった。アクリルアミドゲル充填前に、楕円筒形キャピラリーの蛍光を計測するための窓（被覆が除去された部分）にフッ化マグネシウム（ＭｇＦ₂）（屈折率ｎ₀＝１．３８）を１８４ｎｍの厚さで単層蒸着した。この処理により、ゲル充填後のキャピラリー１本あたりの平均透過率は、９３．３％から９７．０％に増大した。このとき、レーザ光源より１本目のキャピラリーにおける励起効率に対する１０本目のキャピラリーにおける励起効率は、５３．４％から７６．０％に増大した（０．９７０⁹＝０．７６）。これに伴い、励起効率の減少を２０％までとする同時計測可能な最大キャピラリー本数は２４本から５３本まで増大した（０．９７０⁵²＝０．２１）。
【００７１】
本実施形態で用いたキャピラリーのレーザ照射部分に減反射のコーティングを（実施形態１）で説明した円筒形状キャピラリーや、後述する（実施形態６）の角筒形状キャピラリーに応用しても、もちろん同様の効果が得られる。また、コーティングの方法、種類は本実施形態で用いたものに限るものではない。
【００７２】
（実施形態６）
本実施形態では、図１８に示すような角筒形状キャピラリー１を用いる。角筒形状キャピラリーの断面は、外周が一辺０．２ｍｍの正方形、内周が一辺０．１ｍｍの正方形である。この形状のキャピラリーは、ポリマイクロ社（米国）によって製造可能である。その他の実験条件は（実施形態１）と同様で、全長５０ｃｍのキャピラリーの材質は溶融石英（ｎ₂＝１．４６）を用い、キャピラリー内部に７Ｍのウレアを含んだ４％Ｔ、５％Ｃの濃度のアクリルアミドゲル１０（ｎ₃＝１．３６）を充填した。試料注入側のキャピラリー末端より３０ｃｍの位置で、長さ１０ｍｍ、全周にわたり、ポリイミド被覆を除去して蛍光を計測する窓を予め作製してレーザ照射部位とした。このキャピラリーゲル１０本の蛍光を計測する窓の部位を０．４ｍｍピッチに揃えてガラス平板５上に固定して平面状に配列させた。蛍光を計測する位置のキャピラリー外部は空気（ｎ₁＝１．００）１１とした。図３と同様に、蛍光計測はキャピラリーの配列平面に垂直な方向より、第１レンズ、バンドパスフィルター、第２レンズを介して２次元検出器により行なった。図１８は、蛍光を計測する部分のキャピラリー軸に対する断面図であり、レーザをビーム径０．１ｍｍに集光しビーム２として、キャピラリー配列体の側方よりキャピラリー配列軸に沿って入射させた。
【００７３】
本計算実験条件では、レーザビームの各キャピラリーへの入射角は常にゼロであるため、レーザビームの屈折の影響は無視できる。屈折率が変化する各境界点での反射の影響を考慮すると、キャピラリー１本あたりの平均透過率は９２．９％と高効率であり、レーザ光源から１０本目のキャピラリーにおける励起効率は、１本目のキャピラリーにおける励起効率５１．５％であった（０．９２９⁹＝０．５１５）。検出器の感度とダイナミックレンジの関係から同時計測可能な励起効率の減少を２０％までとすると、本計算実験条件では、２２本までのキャピラリーの同時計測が可能であることが判明した（０．９２９²¹＝０．２１）。
【００７４】
一方、光検出系に直接入射する反射光のレーザパワーを１０本のキャピラリーについて積算した結果は、（実施形態４）の場合と同様に、ゼロであった。これは屈折率が変化する各境界点でのレーザビームの入射角が常にゼロであるためである。従って、円筒形状のキャピラリーから角筒形状のキャピラリーにすることにより、背景光を大幅に削減でき、より高感度に蛍光計測できる。
【００７５】
（実施形態７）
本実施形態では、（実施形態６）の計算実験条件において、蛍光を計測する位置のキャピラリー外部を水（ｎ₁＝１．３３）とした。その他の計算実験条件は（実施形態６）と同一とした。本計算実験条件では、キャピラリー外部の屈折率とキャピラリーの屈折率との差が、（実施形態６）の場合よりも小さくなったために、屈折率が変化する各境界点でのレーザビームの反射率が大幅に低減した。その結果、キャピラリー１本あたりの平均透過率は、９２．９％から９９．３％に増大した。このとき、レーザ光源より１本目のキャピラリーにおける励起効率に対する１０本目のキャピラリーにおける励起効率は、５１．５％から９３．９％に増大した（０．９９３⁹＝０．９３９）。これに伴い、励起効率の減少を２０％までとする同時計測可能な最大キャピラリー本数は、２２本から２３０本まで増大した（０．９９３²²⁹＝０．２０）。
【００７６】
次に、（実施形態６）の計算実験条件において、蛍光を計測する位置のキャピラリー外部を石英ガラス（ｎ₁＝１．４６）とした。本計算実験条件は、複数の角筒形状キャピラリーの計測部分を溶着して実現してもよいし、石英ガラスのブロックにキャピラリーの内部に相当する穴を複数あけて実現してもよい。その他の計算実験条件は（実施形態６）と同一とした。本計算実験条件では、キャピラリー外部の屈折率とキャピラリーの屈折率との差が、（実施形態６）の場合よりも小さくなったために、屈折率が変化する各境界点でのレーザビームの反射率が大幅に低減した。その結果、キャピラリー１本あたりの平均透過率は９２．９％から９９．８％に増大した。このとき、レーザ光源より１本目のキャピラリーにおける励起効率に対する１０本目のキャピラリーにおける励起効率は、５１．５％から９８．２％に増大した（０．９９８⁹＝０．９８２）。これに伴い、励起効率の減少を２０％までとする同時計測可能な最大キャピラリー本数は、２６本から８０４本まで増大した（０．９９８⁸⁰³＝０．２０）。
【００７７】
本実施形態では、蛍光を計測する位置のキャピラリー外部を水あるいは石英ガラスで満たしたが、これ以外の液体あるいは固体であっても、それが透明材質であれば同様の効果を発揮する。
【００７８】
（実施形態８）
本実施形態では、（実施形態６）の計算実験条件において、図１９に示すように、レーザビーム２が１０本のキャピラリーを透過した位置に全反射ミラー１５を配置し、レーザビーム２を折り返して逆経路で再び１０本のキャピラリーを透過させた。その他の計算実験条件は（実施形態６）と同一とした。
【００７９】
Ｎ本のキャピラリーを配列したとき、レーザ光源からｎ番目（ｎ≦Ｎ）のキャピラリーにおける励起効率Ｔ_effは、レーザビームのキャピラリー１本あたりの平均透過率をＴ_ramとすると、（数１２）で表わされる（なお、以下の説明では、Ｘ↑（Ｙ）は、ＸのＹ乗を表すものとする）。
【００８０】
Ｔ_eff＝Ｔ_ram↑（ｎ−１）＋Ｔ_ram↑（２Ｎ−ｎ） …（数１２）
（数１２）の第１項は、レーザ光源から離れる方向に進行するレーザビームによる励起を表わし、第２項は、全反射ミラーで折り返してレーザ光源に近づく方向に進むレーザビームによる励起を表わす。（数１２）は（実施形態６）の場合の１本目のキャピラリーにおける励起効率により規格化されており、Ｔ_eff＞１であることは、（実施形態６）の場合の１本目のキャピラリーにおける励起効率以上の励起効率となることを意味している。（数１２）によれば、励起効率Ｔ_effは（実施形態４）と同様にｎに関して単調減少であり、ｎ＝Ｎ、即ち、レーザ光源から最も離れたキャピラリーで最小となる。このときの励起効率Ｔ_effminは、（数１３）となる。
【００８１】
Ｔ_effmin＝Ｔ_ram↑（Ｎ−１）＋Ｔ_ram↑（Ｎ） …（数１３）
（数１３）は、（実施形態６）の場合、即ち、全反射ミラーを用いない場合の約２倍であることを意味する。本計算実験条件では、Ｎ＝１０、Ｔ_ra＝０．９２９であるから、１０本目のキャピラリーにおける励起効率は、５１．５％から９９．４％に増大した（０．９２９⁹＋０．９２９⁹＝０．９９４）。これに伴い、励起効率の減少を２０％までとする同時計測可能な最大キャピラリー本数は２２本から３２本まで増大した（０．９２９³¹＋０．９２９³²＝０．２０）。本実施形態で示した手法を円筒形状キャピラリーや楕円筒形状キャピラリーに適用しても、もちろん同様の効果が得られる。
【００８２】
（実施形態９）
本実施形態では、（実施形態６）の計算実験条件において、図２０に示すように、レーザビーム２をハーフミラー１６等のビームスプリッターで２本に分割した後、反射ミラー１７−１、１７−２、１７−３を使用して、１０本のキャピラリー配列したキャピラリー配列体の平面方向の２つの側面の両側から対向させて照射した。両側から照射するレーザビームはともにビーム径を０．１ｍｍにレンズ（図示せず）で絞り、互いに同軸になるように調整した。その他の計算実験条件は（実施形態６）と同一とした。Ｎ本のキャピラリーを平面上に配列したとき、キャピラリーの配列の片側からｎ番目（ｎ≦Ｎ）のキャピラリーにおける励起効率Ｔ_effは、レーザビームのキャピラリー１本あたりの平均透過率をＴ_ramとすると、（数１４）で表わされる。
【００８３】
２Ｔ_eff＝Ｔ_ram↑（ｎ−１）＋Ｔ_ram↑（Ｎ−ｎ） …（数１４）
（数１４）の第１項、第２項は、それぞれ分割された２本のレーザビームによる励起を示す。（数１４）は（実施形態６）の場合の１本目のキャピラリーにおける励起効率により規格化されている。（数１４）によれば、励起効率Ｔ_effはｎ＝（Ｎ＋１）／２、即ち、複数のキャピラリーが配列する配列中心置のキャピラリーで最小となり、他のキャピラリーにおける励起効率は上記の配列中心位置に関して対称である。励起効率の最小値Ｔ_effminは、（数１５）となる。
【００８４】
Ｔ_effmin＝Ｔ_ram↑（（Ｎ＋１）／２） …（数１５）
（数１５）は、（実施形態６）の場合、即ち、レーザビームを分割しない場合の励起効率の最小値Ｔ_ram↑（ｎ−１）（ｎ＝Ｎのとき）（Ｔ_ram≦１）より明らかに大きい。本計算実験条件では、Ｎ＝１０、Ｔ_ram＝０．９２９であるから、励起効率は５本目のキャピラリーで最小となり、７１．８％である。これに対して（実施形態６）では、励起効率は１０本目のキャピラリーで最小となり、５１．５％であった。従って、本手法によって多数のキャピラリーの同時計測をより効率的に行なうことができることが判明した。これに伴い、励起効率の減少を２０％までとする同時計測可能な最大キャピラリー本数は、２２本から４３本まで増大した（０．９２９²¹＝０．２１）。本実施形態で示した手法を円筒形状キャピラリーや楕円筒形状キャピラリーに適用しても、もちろん同様の効果が得られる。
【００８５】
（実施形態１０）
本実施形態では、（実施形態６）の計算実験条件において、図２１に示す用に、２台のレーザ光源１８−１、１８−２を用い、レーザビーム２−１、２−２を、１０本のキャピラリーが配列したキャピラリー配列体の２つの側面の両側から対向させて照射した。両レーザビーム２−１、２−２は、ともにビーム幅を０．１ｍｍにレンズ（図示せず）で絞り、互いに同軸になるように調整した。その他の計算実験条件は（実施形態６）と同一とした。Ｎ本のキャピラリーを配列したとき、キャピラリー配列の片側からｎ番目（ｎ≦Ｎ）のキャピラリーにおける励起効率Ｔ_effは、レーザビームのキャピラリー１本あたりの平均透過率をＴ_ramとすると、（数１６）で表わされ、
Ｔ_eff＝Ｔ_ram↑（ｎ−１）＋Ｔ_ram↑（Ｎ−ｎ） …（数１６）
（実施形態９）の場合において得られる（数１４）で示される励起効率の２倍の励起効率で複数のキャピラリー内を泳動する試料成分を励起できる。（数１６）の第１項、第２項は、それぞれ２台のレーザ光源からのレーザビームによる励起を示す。（数１６）は（実施形態６）の場合の１本目のキャピラリーにおける励起効率により規格化されており、Ｔ_eff＞１であることは、（数１４）で示される励起効率以上の励起効率になることを意味している。（数１６）によれば、励起効率Ｔ_effはｎ＝（Ｎ＋１）／２、即ち、複数のキャピラリーの配列中心位置に位置するキャピラリーで最小となり、他のキャピラリーにおける励起効率は中心位置に関して対称である。励起効率の最小値Ｔ_effminは、（数１７）となる。
Ｔ_effmin＝２・Ｔ_ram↑（（Ｎ＋１）／２） …（数１７）
これは（実施形態６）の場合、即ち、１台のレーザ光源を用いた場合の励起効率の最小値Ｔ_ram↑（ｎ−１）（ｎ＝Ｎのとき）（Ｔ_ram≦１）より明らかに大きい。本計算実験条件では、Ｎ＝１０、Ｔ_ram＝０．９２９であるから、励起効率は５本目のキャピラリーで最小となり、１４３．７％である。これに対して（実施形態４）では、励起効率は１０本目のキャピラリーで最小となり、５１．５％であった。従って、本手法によって多数のキャピラリーの同時計測をより効率的に行うことができることが判明した。これに伴い、励起効率の減少を２０％までとする同時計測可能な最大キャピラリー本数は、２２本から６３本まで増大した（２×０．９２９³¹＝０．２０）。本実施形態で示した手法を円筒形状キャピラリーや楕円筒形状キャピラリーに適用しても、もちろん同様の効果が得られる。
【００８６】
（実施形態１１）
本実施形態では、図２２に示すように、２種類の形状の異なるキャピラリーを接続して１つの電気泳動路を形成し、これを複数本平面上に配列して同時に各キャピイラリーからの蛍光を計測する手法の具体例を示す。１０本のキャピラリーのそれぞれの電気泳動分離部分は、断面が外径０．２ｍｍ（Ｒ＝０．１ｍｍ）、内径０．１ｍｍ（ｒ＝０．０５ｍｍ）の円形である分離用キャピラリーの円筒形状キャピラリー１９を用い、蛍光を計測する部分のキャピライーは、断面が外周が一辺０．２ｍｍの正方形、内周が一辺０．１ｍｍの正方形である計測用キャピラリーの角筒形状キャピラリー２０を用いた。各電気泳動路を構成する分離用キャピラリーと計測用キャピラリーとを接続し、ガラス平面上に０．２ｍｍピッチで配列させた。１０組の接続された分離キャピラリーと計測キャピラリーの中心軸は、それぞれ一致するようにした。両キャピラリーの材質はいずれも石英（ｎ₂＝１．４６）で、接続面はあらかじめ研磨してフラットにし、変性剤として７Ｍのウレアを含んだ４％Ｔ（Ｔｏｔａｌ ｍｏｎｏｍｅｒ ｃｏｎｃｅｎｔｒａｔｉｏｎ）、５％Ｃ（Ｃｒｏｓｓｌｉｎｋｉｎｇ ｍａｔｅｒｉａｌ ｃｏｎｃｅｎｔｒａｔｉｏｎ）の濃度のアクリルアミドを注入した後ゲル化させた。キャピラリーの接続部分は電気的に断線しないように緩衝液（図示せず）に浸し、分離キャピラリーを泳動してきた試料分離成分が、そのまま対向する計測キャピラリーを泳動するようにした。分離キャピラリーは長さ２５ｃｍ、計測キャピラリーは長さ１５ｃｍとした。計測キャピラリーが接続された位置から５ｃｍの位置で、長さ１０ｍｍ、全周にわたり、計測キャピラリーのポリイミド被覆は予め除去して、蛍光を計測する窓とし、この位置にレーザを照射した。つまり泳動距離は２５＋５＝３０ｃｍとした。これら以外の計算実験条件は（実施形態６）と同一とした。つまり、キャピラリー１本あたりの平均透過率は９２．９％の高効率であり、レーザ光源から１０本目のキャピラリーにおける励起効率は１本目のキャピラリーの５１．５％であった（０．９２９⁹＝０．５１５）。検出器の感度とダイナミックレンジの関係から同時計測可能な励起効率の減少を２０％までとすると、本計算実験条件で２２本までのキャピラリーの同時計測が可能であること判明した（０．９２９²¹＝０．２１）。
【００８７】
一般に、キャピラリーは、外径が大きく内径が小さいほど、即ち肉厚になるほどガラスの体積にほぼ比例して高価になる。また、楕円筒形状や角筒形状キャピラリーは円筒形状キャピラリーと比較して高価になる。つまり、本実施形態で示したように、多数のキャピラリーの同時計測に有利であるが、高価なキャピラリーを計測キャピラリーに用い、安価なキャピラリーを分離キャピラリーに用いればランニングコストを下げることができる。また、分離キャピラリーと計測キャピラリーとの脱着が可能となるので、計測終了後に分離キャピラリーのみを交換して次の計測を行えば、更にコストを低減できる。このことは本実施形態のもうひとつの効果である。もちろん、上記以外の形状のキャピラリーを組み合わせても同様の効果が得られる。
【００８８】
以上の実施形態では、電気泳動分離媒体に全てアクリルアミドゲルを用いたが、これ以外の分離媒体として溶液状のものを使用してもよいし、ポリマーを使用してもよい。ポリマーには、アクリルアミドおよびその誘導体のポリマーや、セルロース及びその誘導体等の多糖類のポリマーがある。キャピラリー中のポリマーを交換可能な状態にすれば、キャピラリー本体を繰り返し利用できるのでコストや労力を低減できる。
【００８９】
以上の実施形態では、キャピラリーの材質に全て溶融石英を用いたが、もちろんこれ以外の材質を用いてもよい。特にガラスは様々な屈折率の材質が利用でき、キャピラリーの形状と同様に、キャピラリーのレンズ作用をコントロールできる。つまり、より効率的な多数のキャピラリーの同時計測が可能である。また、キャピラリーの形状は円筒、楕円筒、角筒に限って示したが、もちろんこれ以外の形状であってもかまわない。さらに、本発明は、以上の実施形態に限定されるものではなく、以上の実施形態で説明した特徴事項を組み合わせてもよいことは、言うまでもない。
【００９０】
キャピラリーゲル電気泳動は平板ゲル電気泳動と比較して高電圧を印加できるので高速分析が可能である。このキャピラリーを複数本並べて一度に分析するマルチフォーカスキャピラリーアレイ電気泳動は多数の試料を同時に高速分析できる高スループットな分析手法である。試料のキャピラリーへの導入は、キャピラリーアレイ電気泳動では電界注入により行えるので、平板ゲル電気泳動と比較して非常に簡単になる。また、キャピラリーに直接レーザを照射して蛍光計測するオンカラム計測は、電気泳動分解能を低下させる要因が少なく、また励起効率が良いため高感度な蛍光計測手法である。
【００９１】
【発明の効果】
本発明では、オンカラム蛍光計測を行いながら、レーザビームのスキャンを行わずに、複数のキャピラリーを実質的に同時にレーザ照射し、一括して蛍光計測できるので、高感度、高速、高スループットな分析を達成できる。しかも、装置構成やキャピラリー配列は非常に簡単であり、実用的である。電気泳動媒体にポリマー等の詰め替え可能なものを使用すれば、キャピラリーアレイそのものは動かさずに繰り返し利用できるので、コストや労力を大幅に削減できる実用面での効果が甚大である。
【図面の簡単な説明】
【図１】本発明のキャピラリーアレイのオンカラム計測を行なうキャピラリーアレイ電気泳動装置の主要部の構成を示す、（ａ）平面図、（ｂ）断面図。
【図２】本発明のキャピラリー中のレーザビームの光路を示す断面図。
【図３】本発明の実施形態１の装置構成を示す図。
【図４】本発明の実施形態１での蛍光を計測する部分の断面図。
【図５】本発明の実施形態１でのキャピラリーアレイ中の光路の計算結果を示す図。
【図６】本発明の実施形態１でのキャピラリーの位置と励起効率の関係を示す図。
【図７】本発明の実施形態１でのキャピラリー外径／内径の比率と励起効率の関係を示す図。
【図８】本発明の実施形態１でのキャピラリー外径／内径の比率と同時計測可能なキャピラリー本数の関係を示す図。
【図９】本発明の実施形態１でのキャピラリー外径／内径の比率と光検出系に直接入射する反射光強度の計算結果を示す図。
【図１０】本発明の実施形態２でのキャピラリー配列間隔と励起効率の関係を示す図。
【図１１】本発明の実施形態３の装置の主要な構成を示す図。
【図１２】本発明の実施形態３での蛍光を計測する部分の断面図。
【図１３】本発明の実施形態３で得られた電気泳動パターンを例を示す図。
【図１４】本発明の実施形態３で得られた電気泳動パターンを例を示す図。
【図１５】本発明の実施形態３で得られた電気泳動パターンを例を示す図。
【図１６】本発明の実施形態４のキャピラリーアレイ中の光路の計算結果を示す図。
【図１７】本発明の実施形態４でのキャピラリー位置と励起効率の関係を示す図。
【図１８】本発明の実施形態６の蛍光を計測する部分の断面図。
【図１９】本発明の実施形態８の蛍光を計測する部分の断面図。
【図２０】本発明の実施形態９の蛍光を計測する部分の断面図。
【図２１】本発明の実施形態１０の蛍光を計測する部分の断面図。
【図２２】本発明の実施形態１１の蛍光を計測する部分の断面図。
【符号の説明】
１…キャピラリー、２…レーザ、３…入射レーザのキャピラリー配列軸からのずれの距離、４…キャピラリー配列軸、５…ガラス平面、６…第一レンズ、７…フィルター、８…第二レンズ、９…二次元ＣＣＤカメラ、１０…アクリルアミドゲル、１１…空気、１２…蛍光計測セル、１３…像分割プリズムおよび組み合わせフィルター、１４…水、１５…全反射ミラー、１６…ハーフミラー、１７−１、１７−２、１７−３…反射ミラー、１８−１、１８−２…レーザ光源、２−１、２−２…レーザビーム、１９…分離用キャピラリーの円筒形状キャピラリー、２０…計測用キャピラリーの角筒形状キャピラリー。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for separating and analyzing DNA, RNA, protein, etc., and in particular, a capillary array effective for determining the base sequence of DNA or RNA or measuring the polymorphism of restriction enzyme fragments based on the diversity of individual base sequences. The present invention relates to an electrophoresis apparatus.
[0002]
[Prior art]
Analytical techniques such as DNA and RNA are becoming increasingly important in the fields of medicine and biology including genetic analysis and genetic diagnosis. Particularly recently, development of high-speed, high-throughput DNA analyzers has been progressing in connection with genome analysis projects. In DNA analysis, a fluorescently labeled sample is separated by molecular weight by gel electrophoresis, a sample is irradiated with a laser to detect fluorescence emitted from the fluorescent label, and a series of signals obtained are analyzed. In gel electrophoresis, a slab gel in which acrylamide is polymerized between two glass plates with an interval of about 0.3 mm is widely used (slab gel electrophoresis). The sample injected into the upper end of the slab gel is migrated in the lower end direction while being separated in molecular weight by the electric field applied to both ends of the slab gel. The position of the slab gel at a certain distance from the starting point of the electrophoresis is irradiated with the laser on all sides of the slab gel at once to excite the separation component of the fluorescently labeled sample passing through the laser irradiation part. Detection of fluorescence by laser irradiation is performed periodically at regular time intervals. By analyzing this detection result, the DNA base sequence is determined.
[0003]
Recently, a capillary gel in which a gel is polymerized in a fused silica capillary tube has been used in place of a slab gel. Capillary gel electrophoresis is attracting attention as a method capable of applying a large electric field as compared with the above-described slab gel electrophoresis and capable of high speed and high separation (Analytical Chemistry 62, 900 (1990)). Usually, in a capillary gel electrophoresis apparatus, a single capillary tube is used, the inside of the capillary near its lower end is irradiated with laser, and on-column measurement is performed to detect fluorescence emitted from a fluorescently labeled sample. Since the entire outer surface of the capillary is coated with polyimide, the glass is exposed by removing the coating at the position where fluorescence is detected (US Pat. No. 5,312,535 (May 17, 1994)). By the laser irradiated to the exposed position of the glass, the separated components of the fluorescently labeled sample that migrates inside the capillary by electrophoresis are excited to emit fluorescence. The DNA base sequence is determined by periodically measuring and analyzing this fluorescence at regular time intervals.
[0004]
However, the above-mentioned on-column measurement apparatus has a drawback that since only one capillary can be used at a time because the refraction of the laser beam on the capillary surface is large, the throughput is not increased. Recently, several examples of high-throughput capillary array gel electrophoresis apparatuses that array a plurality of capillaries and simultaneously analyze many samples at high speed have been reported.
[0005]
The first example is a capillary array scanning method (Nature, 359, 167 (1992)), which is a method of performing on-column fluorescence measurement by irradiating a plurality of capillaries one by one in order. This device employs a confocal structure in which the position where the laser beam is most narrowed in the capillary and the position of the light source incident on the fluorescence measuring instrument (fluorescence light receiving optical system) are used, and each capillary is measured independently. it can. The laser irradiation and fluorescence light receiving optical systems are fixed, the stage holding the capillary array is moved in a one-dimensional direction, and each capillary is irradiated with laser in order.
[0006]
A second example is a capillary array sheath flow method (Nature, 361, 565-566 (1993), Japanese Patent Laid-Open No. 6-138037). In this device, the lower end of the capillary array is immersed in a buffer solution, the sample components separated by gel electrophoresis are eluted as they are in the buffer solution, and the fluorescence emitted from the sample components is detected in the space where there is no capillary. , Off-column measurement is performed (in contrast to on-column measurement in which fluorescence emitted from a fluorescence-labeled sample component is detected by laser irradiation of the capillary, in the present specification, fluorescence is labeled in a space portion where the capillary does not exist. The method of detecting fluorescence emitted from sample components is called off-column measurement for simplicity.
[0007]
Also, by slowly flowing the buffer solution in the direction of migration of the sample components, the separated components eluted from different capillary gels are mixed together in the buffer solution, or the two components separated in one capillary gel are mixed. Mixing in buffer is prevented. In the vicinity of the capillary array exit, laser irradiation is performed on the space where there is no capillary but buffer is present to avoid the problem of laser beam scattering on the capillary surface, and the components eluted from multiple capillaries It is possible to detect fluorescence at substantially the same time.
[0008]
In the third example, in order to simultaneously perform on-column measurement of a plurality of capillaries without mechanically scanning the plurality of capillaries, the laser light from a single laser light source is divided by a beam splitter or the like, so that the plurality of capillaries Are simultaneously irradiated (Analytical Chemistry 65, 956 (1993)).
[0009]
In the fourth example, a laser beam is expanded in a capillary array direction by a cylindrical lens and irradiated to a plurality of capillaries at once (Analytical Chemistry 66, 1424 (1994)).
[0010]
[Problems to be solved by the invention]
In the capillary array scan method of the first example, since the fluorescence measurement is performed one by one in order, the time allocated to the fluorescence detection per one capillary is compared with the normal on-column measurement using one capillary. Will decrease. When n capillary arrays are used, the time allotted to fluorescence detection per line is 1 / n at the maximum compared to the normal on-column measurement, and the glass part of the capillary where the sample separation component does not actually pass is also included. Since it is scanned, it becomes 1 / n or less. As a result, there arises a problem that the fluorescence detection sensitivity is lowered. That is, in the first example, in order to obtain the same fluorescence detection sensitivity as that of a normal on-column measurement using a single capillary, a time of n times or more of the fluorescence detection time in the normal on-column measurement is required. Will do.
[0011]
In addition, the time interval between adjacent peaks in the electrophoresis pattern of components separated in one capillary becomes smaller as the analysis becomes faster, but with respect to the time interval between adjacent peaks, n capillary arrays If the time required for one scan becomes too large to be ignored, there arises a problem that the separation ability of the electrophoretic pattern of the separated components is lowered. Further, the apparatus of the first example has a structure that has a stage movable part for mechanically scanning, and frequently causes failures.
[0012]
In the capillary array sheath flow method of the second example, there is a problem that the fluorescence intensity obtained from the components separated by molecular weight becomes smaller as the molecular weight becomes larger than in the case of on-column fluorescence measurement. This problem arises for the following reason. In order to prevent the separated components eluted in the buffer solution from the lower end of the capillary gel from mixing in the buffer solution by diffusion or the like, it is necessary to constantly flow the buffer solution in the migration direction of the sample component at a certain speed or higher. is there. On the other hand, the migration speed of the sample components that migrate while the molecular weight is separated in the capillary gel decreases as the molecular weight increases. The smaller the migration speed of the sample component in the capillary gel of the component relative to the flow rate of the buffer solution, the larger the sample component is stretched in the migration direction when it is eluted from the capillary gel into the buffer solution. . As a result, the fluorescence intensity is reduced, which causes a decrease in the measurement sensitivity of fluorescence emitted from the sample component controlled by the fluorescence table.
[0013]
In the third and fourth examples, the intensity of the laser beam irradiated per capillary is lowered, and thus there is a problem that the sample components separated by electrophoresis cannot be detected with high sensitivity.
[0014]
An object of the present invention is to solve the above-described problems of the prior art by performing on-column fluorescence measurement while not scanning a plurality of capillaries mechanically or without optically scanning a laser beam. An object of the present invention is to provide a capillary array electrophoresis apparatus capable of simultaneously and simultaneously measuring fluorescence emitted from fluorescently labeled sample components that are irradiated with laser at substantially the same time and migrate through a plurality of capillaries.
[0015]
[Means for Solving the Problems]
The capillary array electrophoresis apparatus of the present invention is a multi-focus capillary array in which a plurality of capillaries are arranged on the same plane, and laser light is irradiated from a direction parallel to the plane to simultaneously excite the plurality of capillaries and perform on-column measurement. Characterized by electrophoresis apparatus. More specifically, the capillary array electrophoresis apparatus of the present invention has the following features.
[0016]
(1) In an electrophoresis apparatus that measures fluorescence by irradiating a plurality of capillaries with laser, on-column fluorescence measurement is performed by irradiating a position where the inside of the capillary is filled with an electrophoretic separation medium, and at least the laser irradiation position (laser irradiation) of the capillary is performed. The transparent section of the capillary is circular in cross-section, and the outside of the capillary transparent section is transparent gas) and is made of a transparent material. At least the laser irradiation position of the capillary is arranged in a plane, and the laser is arranged in the capillary Irradiate parallel to the array plane from the side of the body and simultaneously irradiate multiple capillaries. The ratio of the outer diameter to the inner diameter of the capillary at the laser irradiation position is set to 1 to 7, or 3 to 5. The arrangement interval of the capillaries in the transparent portion should be 4 times or less the outer diameter of the capillaries. The transparent gas can be replaced by a transparent liquid outside the transparent part of the capillary. In this case, the ratio of the outer diameter to the inner diameter of the capillary in the transparent part is set to 2 or less, and the arrangement interval of the capillaries in the transparent part is outside the capillary. The diameter is 4 times or less.
[0017]
(2) In an electrophoresis apparatus that measures fluorescence by irradiating a plurality of capillaries with laser, on-column fluorescence measurement is performed by irradiating a position where the inside of the capillary is filled with an electrophoretic separation medium, and at least the laser irradiation position (laser irradiation) of the capillary is measured. The transparent section of the capillaries is elliptical in cross section), and at least the laser irradiation positions of the capillaries are arranged in a plane, and the laser is irradiated in parallel to the arrangement plane from the side of the array in which the capillaries are arranged. And irradiating a plurality of capillaries simultaneously. Multiple capillaries are arranged in a plane with the ellipse minor radius parallel to the capillary array plane and the major radius perpendicular to the capillary array plane, or the ellipse major radius is parallel to the capillary array plane and the minor radius is capillary aligned A plurality of capillaries are arranged in a plane perpendicular to the plane. The outside of the transparent portion of the capillary is a transparent gas or a transparent liquid.
[0018]
(3) In an electrophoresis apparatus for measuring fluorescence by irradiating a plurality of capillaries with laser, on-column fluorescence measurement is performed by irradiating a position where the inside of the capillary is filled with an electrophoretic separation medium, and at least the laser irradiation position (laser irradiation) of the capillary is measured. The cross section of the capillary of the transparent portion to be formed is a square or rectangular shape or a shape formed of at least two sets of substantially parallel lines), and is made of a transparent material, and at least the laser irradiation positions of the capillaries are arranged in a plane (cross-sectional shape) Are arranged so that one side is parallel to the capillary array plane), a laser is irradiated in parallel to the array plane from the side of the array in which the capillaries are arrayed, and a plurality of capillaries are irradiated simultaneously. The outside of the capillary of the transparent part is a transparent gas, a transparent liquid, or a transparent solid. In the case of a transparent solid, the outside is preferably made of glass, and particularly preferably made of fused quartz.
[0019]
Furthermore, in the electrophoresis apparatus of (1) to (3), an acrylamide gel or a derivative thereof, or a polymer is filled as an electrophoresis medium in the capillary. In the electrophoresis apparatus, at least two types of capillaries having different cross-sectional shapes may be connected between the sample injection end and the position where fluorescence is detected. In the electrophoretic apparatus, at least the surface of the measurement part for detecting fluorescence of the capillary is coated with a low-reflection single layer deposition.
[0020]
In the apparatus described above, laser irradiation may be performed by irradiating all of the plurality of capillaries and returning the laser beam transmitted by the total reflection mirror, and irradiating all of the plurality of capillaries again through the reverse path, or by irradiating the laser beam with a beam splitter. It is possible to divide into two by using and to irradiate the two sides of the capillary array so as to oppose each other, or by using two laser beams from two laser light sources, Irradiation may be performed opposite to the two directions, and the transmittance of the excitation laser wavelength of the filter for fluorescence measurement is 10 ^-Five The following.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
In FIG. 1, a plurality of capillaries 1 (six examples are shown in FIG. 1) are arranged on the same plane, parallel to this plane, and from two directions that are substantially perpendicular to the migration direction of the sample components. The main part of a multi-focus capillary array electrophoresis apparatus that irradiates and excites fluorescently labeled sample components that migrate in a plurality of capillaries substantially simultaneously and detects sample components by on-column measurement is shown. FIG. 1A is a diagram viewed from the direction of the plane of arrangement of a plurality of capillaries, and FIG. 1B is a sectional view thereof. The plurality of capillaries irradiated with the laser 2 are either separation capillaries for separating sample components or measuring capillaries connected to each of the plurality of separation capillaries to detect sample components separated by electrophoresis It is. The laser 2 irradiates a portion at a predetermined distance from the starting point of sample migration in a capillary (separation capillary) filled with a gel or a predetermined portion of a measurement capillary filled with a gel.
[0022]
In the multifocus capillary array electrophoresis apparatus of the present invention, the electrophoresis separation analysis or the sample to be analyzed includes a plurality of fluorescently labeled components. Further, when the component itself contained in the sample emits fluorescence by laser irradiation, no fluorescent label is required. In each drawing referred to below including FIG. 1, the power source for applying an electric field for migrating the sample components, each means such as an electrode and an electrode tank for storing an electrolytic solution, and the inside of the capillary 1 are also shown. After detecting fluorescence from the sample component to be migrated, an arithmetic processing device that processes these detection signals, a display that displays the results of the arithmetic processing, a control device that controls each part of the multifocus capillary array electrophoresis device, a laser light source, etc. Is omitted. In the following description, a laser 2 narrowed in a direction connecting the central axes of a plurality of capillaries for measurement or a plurality of separation capillaries arranged on a plane is incident, and the laser 2 is refracted. The axis on which the laser 2 travels when it is assumed that the laser beam travels straight without being scattered is called the capillary array axis.
[0023]
(Embodiment 1)
In this embodiment, a cylindrical capillary having a circular cross section is used. The features of the multifocus capillary array electrophoresis apparatus of the present invention are:
(1) let the laser power reach many capillaries;
(2) Reduction of background light due to scattering of the laser beam.
[0024]
First, the conditions for realizing (1) were clarified by a simulation experiment described below. FIG. 2 is a cross-sectional view of one cylindrical capillary having an outer radius R and an inner radius r. A state in which the optical path is changed by refraction by the capillary 1 of the laser beam 2 having an infinitesimal width incident at a position x away from the capillary array axis 4 is shown. The laser beam passes through the boundary point where the refractive index changes four times (or twice) per capillary. That is, the incident position P from the outside of the capillary 1 to the capillary 1 ₁ The incident position P from the capillary 1 to the inside of the capillary 1 ₂ The emission position P from the inside of the capillary 1 to the capillary 1 _Three The exit position P from the capillary 1 to the outside of the capillary 1 _Four The laser beam 2 is refracted at the four points. However, depending on the optical path of the laser beam 2, the inside of the capillary does not pass. ₂ , P _Three May not exist. P ₁ , P ₂ , P _Three , P _Four The incident angle and refraction angle of the laser beam at ₁ , Θ ₂ , Θ _Three , Θ _Four , Θ _Five , Θ ₆ , Θ ₇ , Θ ₈ It is. Let n be the refractive index of the medium outside the capillary. ₁ , The refractive index of the capillary material is n ₂ Let n be the refractive index of the medium inside the capillary. _Three And
[0025]
Using the geometric relationship shown in FIG. 2 and Snell's law at each interface, (Equation 1) to (Equation 8) are obtained.
[0026]
[Expression 1]
θ ₁ = Sin ^-1 {X / R} (Equation 1)
[0027]
[Expression 2]
θ ₂ = Sin ^-1 {(N ₁ / N ₂ ) Sinθ ₁ } (Formula 2)
[0028]
[Equation 3]
θ _Three = Sin ^-1 {(R / r) sinθ ₂ } (Formula 3)
[0029]
[Expression 4]
θ _Four = Sin ^-1 {(N ₂ / N _Three ) Sinθ _Three } (Formula 4)
[0030]
[Equation 5]
θ _Five = Θ _Four ... (Formula 5)
[0031]
[Formula 6]
θ ₆ = Θ _Three ... (Equation 6)
[0032]
[Expression 7]
θ ₇ = Θ ₂ ... (Equation 7)
[0033]
[Equation 8]
θ ₈ = Θ ₁ ... (Equation 8)
Assuming that the angle formed by the laser beam before entering the capillary and the laser beam after passing through is the refraction angle Δθ, Δθ is given by (Equation 9) according to FIG.
[0034]
[Equation 9]

Δθ varies depending on the incident position x of the laser beam, and the outer radius R of the capillary, the inner radius r, and the refractive index n outside the capillary. ₁ , Refractive index n of capillary material ₂ , Refractive index n inside the capillary _Three Can be controlled with. For a laser beam with x = 0, Δθ = 0 is always set, and | Δθ | also increases monotonously as x increases (here, x ≦ r is assumed). That is, the capillary itself acts as a lens with an unfocused lens. If Δθ> 0, it becomes a concave lens, and if Δθ <0, it becomes a convex lens. n ₁ = N ₂ = N _Three In this case, of course, Δθ = 0 regardless of x.
[0035]
To irradiate a laser beam from the side of the array so that the plurality of capillaries are arranged in a single plane so as to pass through the plurality of capillaries, the laser power can reach a number of capillaries. The smaller the refraction angle Δθ by the capillaries, the more advantageous, in particular, that each capillary has a convex lens action of Δθ <0. This is because, when focusing on one capillary, if the transmitted laser beam is narrower than the incident laser beam, the laser power can efficiently reach the adjacent capillary. Under normal electrophoresis conditions, always n ₂ > N _Three Therefore, from (Equation 9) to n ₁ Is smaller, the smaller Δθ is. In other words, the outside of the capillary is n ₁ It is preferable that the gas is filled with a transparent gas such as air, which is = 1.00.
[0036]
On the other hand, the laser beam is also reflected at each boundary point. That is, a part of the laser power of the incident light becomes reflected light, and the rest becomes refracted light (transmitted light). The ratio of the laser power of the reflected light to the laser power of the incident light is expressed by the reflectance R _ef , The ratio of the laser power of the refracted light to the laser power of the incident light, the transmittance T _ra Then these are incident angles θ _i And refraction angle θ _t It is given by (Equation 10) and (Equation 11) as a function of (ie, the refractive index of both media).
[0037]
[Expression 10]

[0038]
[Expression 11]

Where R _ef + T _ra = 1. However, the case where incident light has no polarization is shown here. The laser power decreases sequentially according to (Equation 10) and (Equation 11) every time it passes through the boundary point. Therefore, the optical path length of the laser beam inside the capillary through which the sample component passes during electrophoresis (point P in FIG. 2). ₂ And point P _Three Is multiplied by the laser power at that position to estimate the excitation efficiency of the laser beam in this capillary (excitation efficiency of the label by the laser beam of the labeled sample component that migrates in this capillary). Further, the position where the infinitesimal laser beam is incident is set as the distance x from the capillary array axis (x = 0), and the excitation efficiency of the laser beam with a finite width is estimated by changing the distance x. . Furthermore, when a plurality of capillaries are arranged in a plane, the above calculation is continuously performed to examine how the excitation efficiency of each of the plurality of capillaries changes.
[0039]
As a specific example, the structure of the capillary is a fused quartz cylindrical tube (n) having an outer diameter of 0.375 mm (R = 0.1875 mm), an inner diameter of 0.1 mm (r = 0.05 mm) and a length of 50 cm. ₂ = 1.46) was used. After injecting acrylamide at a concentration of 4% T (Total monomer concentration) and 5% C (Crosslinking material concentration) containing 7 M urea as a denaturing agent, gelation (n _Three = 1.36). At a position 30 cm from the capillary end on the sample injection side, 10 mm in length, the polyimide coating of each capillary was removed over the entire circumference, and a window for measuring fluorescence was prepared in advance to serve as a laser irradiation site.
[0040]
As shown in FIG. 3, the measurement portions of these 10 capillary gels were arranged at a pitch of 0.375 mm, and the capillaries 1 were brought into close contact with each other and fixed on the glass flat plate 5 and arranged in a plane. The outside of the capillary at the fluorescence measurement position is air (n ₁ = 1.00). The fluorescence measurement was performed by the two-dimensional detector 9 through the first lens 6, the band pass filter 7 and the second lens 8 from the direction perpendicular to the capillary array plane. Under the present experimental conditions, since the laser beam 2 is largely reflected by the capillary, it is necessary to lower the transmittance of the laser wavelength than the bandpass filter used in fluorescence measurement such as slab gel electrophoresis. Therefore, the transmittance of the laser wavelength is 10 ^-Five The following bandpass filter was used.
[0041]
FIG. 4 is a cross-sectional view of the fluorescence measurement portion with respect to the capillary axis. The laser was focused to a beam diameter of 0.1 mm and incident along the capillary array axis from the side of the capillary array. At this time, from (Equation 9), Δθ <0 is always satisfied for the laser beam of x ≦ r, and each capillary has a convex lens action. The beam width of 0.1 mm was divided into a total of 11 infinitely small beam components with an interval of 0.01 mm. That is, the beam component has a distance x from the capillary array axis (laser beam central axis, x = 0) of 0.00, ± 0.01, ± 0.02, ± 0.03, ± 0.04, ± 0. .It enters the first capillary at a position of 05 mm. FIG. 5 shows the result of simulating what optical path the capillary light transmitted through each beam component travels through the capillary array. FIG. 5 is a cross-sectional view of the fluorescence measurement portion with respect to the capillary axis. The upper part shows five capillaries on the laser light source side, and the lower part shows five capillaries on the laser emission side. That is, the laser beam enters the upper left capillary and exits from the lower right capillary.
[0042]
All optical paths were calculated continuously assuming that 10 capillaries were arranged as described above. The beam component (x = 0.00) located at the central axis of the laser beam has no incident angle at all the boundary points, so it is not refracted at all, and Δθ = 0, and is on the capillary array axis. Proceed linearly. Beam components other than x = 0.00 are refracted basically in the direction toward the capillary array axis due to the convex lens action of the capillary, and the optical path travels through the capillary array while oscillating up and down around the capillary array axis. did. The amplitude and period of vibration differed depending on the beam component, but the optical path of each beam component was vertically symmetric with respect to the capillary array axis. As a result, it was found that all the beam components contributed to the excitation of the sample components that permeate the inside of the capillary and migrate inside the capillary without deviating outside the capillary for all ten capillaries. That is, in the capillary electrophoresis apparatus of the present invention, since the laser beam irradiated on each capillary gel is substantially converged, an optical path of the laser beam that is approximately converged is formed in each of the plurality of capillary gels. It can be said that it is suitable to be called a focus capillary electrophoresis device.
[0043]
When the number of capillaries used is changed, in order to evaluate the degree of decrease in excitation efficiency in each capillary, the length of the optical path (optical path length) that passes through the capillary is obtained for each capillary. Was multiplied by the laser power at that position and integrated for all beam components, and this integrated value was used as the excitation efficiency in each capillary. FIG. 6 shows the excitation efficiency in each capillary determined in this way (the excitation efficiency in each capillary is normalized by the excitation efficiency in the first capillary on the laser light source side). In FIG. 6, the capillaries are numbered 1 to 10 in order from the laser light source side.
[0044]
Since the laser power of all beam components is attenuated by reflection every time it passes through the boundary where the refractive index changes, the excitation efficiency for each capillary decreases exponentially with increasing distance from the laser light source as shown in FIG. did. The fluorescence intensity actually measured from each capillary is proportional to the excitation efficiency. As shown in FIG. 6, the excitation efficiency of the tenth capillary reached 54.1% for the first capillary. When the average transmittance per capillary of the laser beam was determined from this value, it was a high efficiency of 93.4% (0.934). ⁹ = 0.541). From the relationship between the sensitivity of the detector and the dynamic range, assuming that the decrease in excitation efficiency that can be measured simultaneously is 20%, up to 24 capillaries can be measured simultaneously under the present experimental conditions (0.934). ^{twenty three} = 0.21).
[0045]
As other specific examples, the inner diameter of the capillary is 0.1 mm (r = 0.05 mm), and the outer diameter of the capillary is 0.11 mm (R = 0.055 mm), 0.2 mm (R = 0.1 mm), 0.375 mm. (R = 0.75 mm), 0.75 mm (R = 0.375 mm), and 1.0 mm (R = 0.5 mm) were calculated in the same manner as above (all other conditions were as above) The same). However, the arrangement pitch of the 10 capillaries was matched with the outer diameter of each capillary, and the capillaries were closely arranged. FIG. 7 shows the change of the excitation efficiency of the tenth capillary arranged at the farthest position from the laser light source with respect to the outer diameter / inner diameter ratio (R / r) of the capillary (excitation efficiency is the same as in FIG. 6). It is standardized by the excitation efficiency in the first capillary arranged at the position closest to the light source). In this calculation experiment, the inner diameter of the capillary is unified to 0.1 mm. However, even when a capillary having another inner diameter is used, it is essential if the ratio of the outer diameter / inner diameter (R / r) of the capillary is the same. (The incident laser beam width is approximately the same as the inner diameter of the capillary).
[0046]
FIG. 8 shows the result of obtaining the average transmittance per one capillary of the laser beam from the result of FIG. 7 and obtaining the maximum number of capillaries that can be measured simultaneously with a reduction in excitation efficiency of up to 20%. Show. From the results of FIGS. 7 and 8, when the ratio R / r of the outer diameter / inner diameter of the capillary takes a value around 4 (exactly 3.75), it is possible to measure a large number of capillaries most efficiently simultaneously. It was found that the number of capillaries that can be measured simultaneously decreases as the distance from this value increases. When the capillary outer diameter / inner diameter ratio R / r is small, the number of capillaries that can be measured simultaneously decreases because the laser beam width and the capillary outer diameter are close to each other. Because it existed. The reason why the number of capillaries that can be simultaneously measured decreases when the ratio R / r of the outer diameter / inner diameter of the capillary is large is that there is a beam component that transmits only the glass portion of the capillary but does not transmit the inside of the capillary.
[0047]
In the case of preparing and processing a large number of samples to be analyzed or analyzed, a microtiter plate is generally widely used. This microtiter plate is a plate with 96 holes arranged in a 12 × 8 grid and has become a global standard. Therefore, it is very important to set the number of samples processed simultaneously in electrophoresis measurement to a multiple of 12 or 8 when linking sample preparation and mass processing of electrophoresis measurement. That is, it is preferable that the number of samples processed and measured simultaneously is 8, 12, 16, 24,. From the results shown in FIG. 8, the conditions relating to the ratio R / r of the outer diameter / inner diameter of the capillaries for enabling simultaneous measurement with a reduction in excitation efficiency of each capillary up to 20% are the samples to be processed and measured simultaneously. When the numbers are 8, 12, 16, and 24, 1 <R / r ≦ 10, 1 <R / r ≦ 10, 1 ≦ R / r ≦ 7, and 3 ≦ R / r ≦ 5, respectively.
[0048]
Next, conditions for reducing background light due to laser beam scattering, which is another key point of the present invention, will be described. The scattering of the laser beam is mainly caused by the reflection of the laser beam at the boundary point where the refractive index of each capillary changes. The reflected light follows the law of reflection and travels in the direction where the incident angle and the reflection angle are equal. As a result, reflected light travels in various directions, and there is reflected light that directly enters a light detection system arranged in a direction perpendicular to the plane in which the capillaries are arranged. This significantly increases the background light at the time of fluorescence measurement, has a large influence, and leads to a decrease in measurement sensitivity. Therefore, the influence was estimated by integrating the laser power of the reflected light directly incident on the light detection system for all capillaries.
[0049]
FIG. 9 shows a calculation experimental condition similar to that obtained for the results of FIGS. 7 and 8 described above, where the laser power of the reflected light directly incident on the light detection system is obtained by integrating the 10 capillaries, and the result of the integration of the capillaries The change regarding the outer diameter / inner diameter ratio (R / r) is shown. As described above, the laser power of the reflected light that is directly incident on the light detection system significantly increases the background light at the time of fluorescence measurement, leading to a decrease in measurement sensitivity. Therefore, the smaller the value, the more sensitive the fluorescence measurement. Can do. From the results shown in FIG. 9, when the ratio R / r of the outer diameter / inner diameter of the capillary is around 4, the background light becomes the smallest and fluorescence measurement can be performed with high sensitivity. As the distance from this value increases, the background light increases and the measurement sensitivity decreases. did. The reason why the background light greatly increases when the ratio R / r of the outer diameter / inner diameter of the capillary is small is that the value of the laser beam width and the outer diameter of the capillary are close, so that the beam component irradiated to the capillary at a large incident angle. This is because the ratio has increased. From the above results, it was found that the condition of 2 ≦ R / r is effective for high sensitivity measurement.
[0050]
In order to reduce the laser power of the reflected light directly incident on the light detection system, the reflectance at each boundary point where the refractive index changes may be lowered. One means for this is to fill the outside of the capillary at the position where the laser is irradiated with a transparent liquid such as water or an aqueous solution to increase the refraction angle by the capillary. In other words, it acts in the direction of the concave lens, which is disadvantageous in reaching the laser power to many capillaries. Therefore, the conditions should be evaluated based on these considerations. As a result of the simulation, the effect of reducing the background light by filling the outside of the capillary with the liquid was larger for the capillary with R / r ≦ 2. As a result, since a large number of capillaries can be arranged and analyzed simultaneously, a high-speed, high-throughput, high-sensitivity analysis can be realized.
[0051]
Of course, the same effect can be obtained even when a cylindrical capillary other than the size used in the present embodiment is used.
[0052]
(Embodiment 2)
In (Embodiment 1), the capillaries arranged in a plane are brought into close contact with each other, but here the interval between the capillaries is set to be equal to or larger than the outer diameter of the capillaries. As a specific example, a fused quartz cylindrical tube having an outer diameter of 0.2 mm (R = 0.1 mm), an inner diameter of 0.1 mm (r = 0.05 mm), and a length of 50 cm (n ₂ = 1.46) was used. The arrangement interval is 1 times the capillary outer diameter (same as in Embodiment 1), 2 times, 3 times, 4 times, 5 times, that is, 0.2, 0.4, 0.6, 0.8, 1. Each case of 0 mm was examined. Other experimental conditions were the same as in (Embodiment 1).
[0053]
FIG. 10 shows the excitation efficiency of the tenth capillary arranged at the farthest position from the laser light source as a variable of the capillary arrangement interval (the excitation efficiency is arranged at the position closest to the laser light source as in FIG. 6). In addition, it is standardized by the excitation efficiency in the first capillary). From the results in FIG. 10, the excitation efficiency in the tenth capillary decreased as the capillary array interval increased, which was a disadvantageous condition for simultaneous measurement of a large number of capillaries. That is, it has been found that simultaneous measurement of a large number of capillaries can be performed most efficiently when the arrangement interval of the capillaries is matched with the outer diameter of the capillaries, that is, when the capillaries are closely arranged. If the S / N of the tenth capillary under this optimum condition (the capillaries are closely arranged) is half the S / N of the first capillary, the excitation efficiency will be 1/4. This is an acceptable range. The condition for the excitation efficiency to be ¼ is satisfied when the arrangement interval of the capillaries is 0.8 mm or less, that is, the arrangement interval is 4 times or less of the outer diameter of the capillary as shown in FIG.
[0054]
(Embodiment 3)
Based on the simulation result of (Embodiment 1), electrophoresis of a DNA sample was actually performed in this embodiment. First, electrophoresis was performed using five capillary gels arranged in a plane, and the DNA base sequence was determined by placing the capillary gel for fluorescence detection in water and measuring the fluorescence. FIG. 11 shows the basic configuration of the apparatus used. The capillary gel is a fused quartz tube (n) having a length of 50 cm, an outer diameter of 0.2 mm (R = 0.1 mm), and an inner diameter of 0.1 mm (r = 0.05 mm). ₂ = 1.46) was injected with acrylamide at a concentration of 4% T (Total monomer concentration) and 5% C (Crosslinking material concentration) containing 7 M urea as a denaturing agent, and then gelled (n _Three = 1.36). At a position 30 cm from the capillary end on the sample injection side, the polyimide coating was removed over the entire circumference of 10 mm, and a window for measuring fluorescence was prepared in advance to serve as a laser irradiation site. As shown in FIG. 11, the five capillary gels are aligned in a plane with a pitch of 0.2 mm, and water (n ₁ = 1.33) and fixed in the fluorescence measurement cell 12. However, in FIG. 11, the capillary gel 1 shows only the vicinity of the measurement window of the capillary gel.
[0055]
FIG. 12 shows a cross-sectional view of the fluorescence measuring cell 12 with respect to the capillary axis. A YAG laser (532 nm, 20 mW) and a He-Ne laser (594 nm, 10 mW) are made coaxial and then condensed to a beam diameter of 0.1 mm to form a beam 2. The incident light was incident along the capillary array axis from the side of the capillary array. A capillary 1 filled with acrylamide gel 10 is disposed on a plane inside the fluorescence measurement cell 12, and water 14 is filled outside the capillary 1.
[0056]
As shown in FIG. 11, the fluorescence measurement was performed from the direction perpendicular to the plane on which the capillary gels were arranged. An image dividing prism for dividing five fluorescent light emitting point groups arranged in a row with a width of 0.8 mm in a horizontal direction into substantially parallel light beams by the first camera lens 6 and dividing an image of the fluorescent light emitting point group into four in the vertical direction; Four kinds of combination filters 14 corresponding to the light beams connecting the four images were transmitted and imaged by the second camera lens 8. This fluorescence selection method is described in detail in JP-A-2-269936. The fluorescence emission point group that was two-dimensionally expanded to 5 × 4 = 20 was subjected to exposure measurement at once with a cooling type two-dimensional CCD camera 9. Continuous measurement was performed with an exposure time of 1.0 second and a data acquisition interval of 1.5 seconds.
[0057]
The nucleotide sequences of the five DNA samples were determined according to the Sanger method. The prepared DNA sample components include four phosphors Cy3 (maximum emission wavelength 565 nm), TRITC (maximum emission wavelength 580 nm), Texas Red (maximum emission wavelength) corresponding to terminal base species C, G, A, T. 615 nm), Cy5 (maximum emission wavelength 665 nm). Here, Cy3 and Cy5 are sold by Biological Detection Systems (USA), and TRITC and Texas Red are sold by Molecular Probes. Four kinds of combination filters were used so that these four kinds of fluorescence were wavelength-selected and imaged on a two-dimensional CCD camera. Four reactants corresponding to the terminal base species were mixed for each sample species, and then concentrated 10-fold by ethanol precipitation to replace the sample solvent with formamide. The sample injection ends of five capillary gels were immersed in five types of DNA sample solutions, respectively, and a sample was injected by applying a constant electric field strength of 100 V / cm (5 kV) to both ends of the capillary gel for 2 seconds. Electrophoresis was performed at a constant field strength of 100 V / cm (5 kV) for about 3 hours. The time changes of the four types of signals corresponding to the four types of fluorescence obtained by the two-dimensional CCD camera were analyzed by a computer, and the DNA base sequences of the five types of samples were determined.
[0058]
FIG. 13 shows the electrophoresis pattern of the component A labeled with Texas Red among the DNA sequencing results obtained in the present embodiment. The electrophoresis pattern has an electrophoresis pattern of 30 to 60 minutes, and about 130 bases from the primer. The pattern of the A component up to the length is shown. However, the five samples analyzed were M13mp18 as a standard sample. The uppermost capillary 1 in FIG. 13 is the most capillary on the laser light source side, and shows the electrophoresis pattern of the capillary at a position away from the laser light source in the following numerical order. The peak intensity decreased with increasing distance from the laser light source. The peak intensity of Capillary-5 is 1/3 or less that of Capillary-1, but it is still detected with a sufficiently high S / N (position corresponding to base length 91, S / N = 734). Was possible. In addition, S / N in the peak of capillary-1 is a position corresponding to the base length 91, and S / N = 3300.
[0059]
In this experimental condition, the outside of the capillary at the position where the laser is irradiated is water, so that the laser reflection at the capillary interface is reduced and the background light signal is reduced by almost an order of magnitude compared to the case where the outside is air. This means that what has been described in (Embodiment 1) has been experimentally confirmed. Electrophoretic patterns of components labeled with other phosphors were obtained in the same manner, and DNA base sequencing by five capillary gel electrophoresis could be realized with high sensitivity.
[0060]
Next, electrophoresis was performed using 10 capillary gels arranged in a plane, and the fluorescence generated from the sample components to be migrated was detected and measured in air to determine the DNA base sequence. The capillary used is a fused silica tube (n = 0.375 mm (R = 0.1875 mm) and 0.1 mm (r = 0.05 mm) inner diameter (n ₂ = 1.46), the 10 capillary arrays are fixed on a glass plane at a pitch of 0.375 mm as shown in FIG. 4, and the fluorescence measurement cells shown in FIGS. 11 and 12 are not used. That is, the medium outside the capillary is air (n ₁ = 1.00). The other experimental conditions were the same as the previous experiment.
[0061]
FIGS. 14 and 15 are electrophoresis patterns of the component A labeled with Texas Red among the DNA sequence results obtained in the present embodiment. The pattern of A component up to 100 base length is shown. However, all 10 types of analyzed samples are M13mp18 as a standard sample. 14 and 15, the uppermost capillary 1 shown in FIG. 14 is the capillary closest to the laser light source, and shows the electrophoresis pattern of the capillaries moving away from the laser light source in the order of the numbers. Measurement was possible with sufficient S / N for all ten capillaries of the lower capillary-10. In the peak giving the base length 9, S / N was 237 in capillary-1, 237 in capillary-4, 110 in capillary-7, and 140 in capillary-10 (S / N in capillary-10 was It is considered that the S / N of Capillary-7 is larger than that of measurement). Electrophoretic patterns of components labeled with other phosphors were also obtained in the same manner as in FIGS. 14 and 15, and DNA base sequencing by 10 capillary gel electrophoresis could be realized with high sensitivity.
[0062]
(Embodiment 4)
In this embodiment, an elliptical cylindrical capillary having an elliptical cross section is used. By using this capillary, two different effects are produced depending on the arrangement method. The first arrangement method is to make the minor axis of the ellipse of the capillary cross section parallel to the arrangement plane of the capillaries. In this case, since the incident angle of the laser beam on the capillary is smaller than that when a cylindrical capillary is used, the laser power of the reflected light directly incident on the light detection system for detecting fluorescence is reduced, and the background light signal is reduced. Decrease. As a result, an effect of increasing the fluorescence detection sensitivity can be obtained. The second arrangement method is to make the major axis of the ellipse of the capillary cross section parallel to the arrangement plane of the capillaries. In this case, it is possible to increase the interval between the migration paths (adjacent capillaries) of adjacent capillaries while closely arranging the capillaries, which eliminates crosstalk between the capillaries, which is advantageous for independent measurement of a plurality of capillaries. As shown in (Embodiment 3), the close-contact arrangement of capillaries is effective for simultaneous simultaneous measurement of a large number of capillaries. Next, a specific example using the first arrangement method is shown.
[0063]
As a specific example, the cross section has an outer major diameter of 0.75 mm (outer major radius R ₁ = 0.375 mm), outer short radius (outer short radius R) ₂ = 0.1875 mm), inner long diameter 0.2 mm (inner long radius r) ₁ = 0.1 mm), inner minor diameter 0.05 mm (inner minor radius r) ₂ = 0.025 mm) elliptical capillaries were used. The inside area of this capillary cross section is the inside of the cross section of a cylindrical capillary having an outer diameter of 0.375 mm (R = 0.1875 mm) and an inner diameter of 0.1 mm (r = 0.05 mm) used in (Embodiment 1). Is equal to the area (7.85 × 10 ^-3 mm ² ). Capillaries of this shape can be manufactured by Polymicro (USA). The other experimental conditions were the same as in (Embodiment 1), and the material of the capillary with a total length of 50 cm was fused quartz (n ₂ = 1.46), and the inside of the capillary is an acrylamide gel containing 7M urea and having a concentration of 4% T and 5% C (n _Three = 1.36). At a position 30 cm from the capillary end on the sample injection side, a window for measuring the fluorescence by removing the polyimide coating over the entire circumference of 10 mm was prepared in advance and used as a laser irradiation site.
[0064]
The sites for measuring the fluorescence of these 10 capillary gels were arranged in a planar shape as in FIGS. 3 and 4, but at this time, the short axis of all the capillaries was parallel to the capillary array plane, Were arranged so as to be perpendicular to the arrangement plane of the capillaries. This arrangement method is effective in reducing the reflected light that directly enters the light detection system as described above. The arrangement pitch of the capillaries coincided with the minor axis of the ellipse, and a close arrangement was made. The outside of the capillary at the fluorescence measurement site is air (n ₁ = 1.00). Similarly to FIG. 3, the fluorescence measurement was performed by a two-dimensional detector from the direction perpendicular to the capillary array plane through the first lens, the band pass filter, and the second lens. The laser was focused to a beam diameter of 0.1 mm and incident along the capillary array axis from the side of the capillary array.
[0065]
Under these experimental conditions, a simulation experiment similar to (Embodiment 1) was performed. FIG. 16 shows the result of calculating the optical path of 11 beam components. FIG. 16 is a cross-sectional view of the fluorescence measurement site with respect to the capillary axis. The leftmost capillary is the most capillary on the laser light source side, and the capillary that goes to the right is further away from the laser light source. All optical paths were calculated continuously assuming that 10 capillaries were arranged as described above. In this calculation experimental condition, since the refraction angle of the laser beam by the capillary is Δθ˜0, that is, the lens action of the capillary is small, each beam component traveled through the capillary array without receiving much refraction. It was found that all the beam components contributed to the excitation of the sample components that passed through the capillary and migrated in the capillary without deviating outside the capillary for all ten capillaries.
[0066]
As in FIG. 6, in order to evaluate the change in the degree of decrease in excitation efficiency due to the number of capillaries, the length of the optical path (optical path length) passing through the inside of the capillary is obtained for each capillary, The laser power at the position was multiplied and integrated for all beam components to obtain the excitation efficiency.
[0067]
FIG. 17 shows the excitation efficiency in each capillary thus obtained (normalized by the value of the first capillary on the laser light source side). In FIG. 17, the capillaries are numbered 1 to 10 in order from the laser light source side. As in FIG. 6, the excitation efficiency for each capillary decreased exponentially with increasing distance from the laser light source. The excitation efficiency of the tenth capillary reached 53.4% of the first capillary, and the degree of attenuation was almost equal to that in FIG. That is, the average transmittance per capillary is 93.3% (0.933). ⁹ = 0.536), up to 24 capillaries can be measured simultaneously under the calculation experimental conditions (0.933). ^{twenty three} = 0.20).
[0068]
On the other hand, unlike the case of (Embodiment 1), the result of integrating the laser power of the reflected light directly incident on the light detection system with respect to the ten capillaries was zero. This is because the incident angle of the laser beam at each boundary point where the refractive index changes is sufficiently small. Therefore, it has been found that by changing from a cylindrical capillary to an elliptical capillary, the background light can be greatly reduced and fluorescence can be measured with higher sensitivity.
[0069]
Of course, the same effect can be obtained even if an elliptical cylindrical capillary other than the shape and dimensions used in the present embodiment is used. In addition, as described for the cylindrical capillaries in (Embodiments 1 and 3), the effect of filling the outside of the capillary at the laser irradiation position with a transparent liquid such as water can be similarly obtained for an elliptical cylindrical capillary. That is, by reducing the reflectance of the laser beam incident on the capillaries, a large number of capillaries can be efficiently and simultaneously measured.
[0070]
(Embodiment 5)
In the present embodiment, the calculation experiment was performed under the calculation experiment condition of (Embodiment 4) by applying a coating for reducing reflection to the laser irradiation portion of each capillary. Before filling the acrylamide gel, magnesium fluoride (MgF) is placed on the window (portion from which the coating has been removed) for measuring the fluorescence of the elliptical capillary. ₂ ) (Refractive index n ₀ = 1.38) was deposited in a single layer with a thickness of 184 nm. This treatment increased the average transmittance per capillary after gel filling from 93.3% to 97.0%. At this time, the excitation efficiency in the tenth capillary with respect to the excitation efficiency in the first capillary from the laser light source increased from 53.4% to 76.0% (0.970). ⁹ = 0.76). Along with this, the maximum number of capillaries that can be measured simultaneously with a decrease in excitation efficiency of up to 20% increased from 24 to 53 (0.970). ⁵² = 0.21).
[0071]
Of course, the same applies when the anti-reflection coating is applied to the laser irradiation portion of the capillary used in this embodiment to the cylindrical capillary described in (Embodiment 1) or the rectangular tube-shaped capillary described in (Embodiment 6) described later. The effect is obtained. Further, the coating method and type are not limited to those used in the present embodiment.
[0072]
(Embodiment 6)
In this embodiment, a rectangular tube-shaped capillary 1 as shown in FIG. 18 is used. The cross section of the rectangular tube-shaped capillary is a square with an outer circumference of 0.2 mm on one side and a square with an inner circumference of 0.1 mm on one side. Capillaries of this shape can be manufactured by Polymicro (USA). The other experimental conditions were the same as in (Embodiment 1), and the material of the capillary with a total length of 50 cm was fused quartz (n ₂ = 1.46), 4% T, 5% C acrylamide gel 10 (n) containing 7 M urea inside the capillary _Three = 1.36). At a position 30 cm from the capillary end on the sample injection side, a window for measuring the fluorescence by removing the polyimide coating over the entire circumference of 10 mm was prepared in advance and used as a laser irradiation site. The portions of the windows for measuring the fluorescence of the 10 capillary gels were aligned on a flat glass plate 5 at a pitch of 0.4 mm and arranged in a plane. The outside of the capillary where the fluorescence is measured is air (n ₁ = 1.00) 11. Similarly to FIG. 3, the fluorescence measurement was performed by a two-dimensional detector through a first lens, a band pass filter, and a second lens from a direction perpendicular to the capillary array plane. FIG. 18 is a cross-sectional view with respect to the capillary axis of the portion where fluorescence is measured. The laser is focused to a beam diameter of 0.1 mm and incident as a beam 2 along the capillary array axis from the side of the capillary array.
[0073]
In this calculation experimental condition, the incident angle of the laser beam to each capillary is always zero, so that the influence of refraction of the laser beam can be ignored. Considering the influence of reflection at each boundary point where the refractive index changes, the average transmittance per capillary is as high as 92.9%, and the excitation efficiency in the 10th capillary from the laser light source is 1st. The excitation efficiency in the capillary was 51.5% (0.929 ⁹ = 0.515). From the relationship between the sensitivity of the detector and the dynamic range, assuming that the reduction in excitation efficiency that can be measured simultaneously is 20%, up to 22 capillaries can be measured simultaneously under the present experimental conditions (0. 929 ^{twenty one} = 0.21).
[0074]
On the other hand, the result of integrating the laser power of the reflected light directly incident on the light detection system for the ten capillaries was zero, as in the case of (Embodiment 4). This is because the incident angle of the laser beam at each boundary point where the refractive index changes is always zero. Therefore, by changing from a cylindrical capillary to a square cylindrical capillary, background light can be greatly reduced, and fluorescence measurement can be performed with higher sensitivity.
[0075]
(Embodiment 7)
In the present embodiment, under the calculation experiment conditions of (Embodiment 6), the outside of the capillary at the position where fluorescence is measured is placed in water (n ₁ = 1.33). The other calculation experiment conditions were the same as in (Embodiment 6). In this calculation experimental condition, the difference between the refractive index outside the capillary and the refractive index of the capillary is smaller than in the case of (Embodiment 6), so the reflectance of the laser beam at each boundary point where the refractive index changes. Was greatly reduced. As a result, the average transmittance per capillary increased from 92.9% to 99.3%. At this time, the excitation efficiency in the tenth capillary with respect to the excitation efficiency in the first capillary from the laser light source increased from 51.5% to 93.9% (0.993). ⁹ = 0.939). Along with this, the maximum number of capillaries that can be measured simultaneously with a decrease in excitation efficiency of up to 20% increased from 22 to 230 (0.993). ²²⁹ = 0.20).
[0076]
Next, under the calculation experiment conditions of (Embodiment 6), the outside of the capillary at the position where fluorescence is measured is made of quartz glass (n ₁ = 1.46). This calculation experimental condition may be realized by welding measurement portions of a plurality of rectangular tube-shaped capillaries, or may be realized by making a plurality of holes corresponding to the inside of the capillary in a quartz glass block. The other calculation experiment conditions were the same as in (Embodiment 6). In this calculation experimental condition, the difference between the refractive index outside the capillary and the refractive index of the capillary is smaller than in the case of (Embodiment 6), so the reflectance of the laser beam at each boundary point where the refractive index changes. Was greatly reduced. As a result, the average transmittance per capillary increased from 92.9% to 99.8%. At this time, the excitation efficiency in the tenth capillary with respect to the excitation efficiency in the first capillary from the laser light source increased from 51.5% to 98.2% (0.998). ⁹ = 0.982). Along with this, the maximum number of capillaries that can be measured simultaneously with a decrease in excitation efficiency of up to 20% increased from 26 to 804 (0.998). ⁸⁰³ = 0.20).
[0077]
In this embodiment, the outside of the capillary at the position where fluorescence is measured is filled with water or quartz glass. However, even if it is a liquid or a solid other than this, the same effect is exhibited as long as it is a transparent material.
[0078]
(Embodiment 8)
In the present embodiment, under the calculation experiment conditions of (Embodiment 6), as shown in FIG. 19, the total reflection mirror 15 is disposed at a position where the laser beam 2 has passed through 10 capillaries, and the laser beam 2 is folded back. Ten capillaries were again passed through the reverse path. The other calculation experiment conditions were the same as in (Embodiment 6).
[0079]
When N capillaries are arranged, the excitation efficiency T in the nth (n ≦ N) capillary from the laser light source _eff Is the average transmittance of the laser beam per capillary T _RAM (Expression 12) (In the following description, X ↑ (Y) represents X to the Y power).
[0080]
T _eff = T _RAM ↑ (n-1) + T _RAM ↑ (2N−n) (Equation 12)
The first term of (Equation 12) represents excitation by a laser beam traveling in a direction away from the laser light source, and the second term represents excitation by a laser beam that is folded back by a total reflection mirror and proceeds in a direction approaching the laser light source. (Equation 12) is normalized by the excitation efficiency in the first capillary in the case of (Embodiment 6), and T _eff > 1 means that the excitation efficiency is equal to or higher than the excitation efficiency of the first capillary in the case of (Embodiment 6). According to (Equation 12), the excitation efficiency T _eff Is a monotonic decrease with respect to n, as in (Embodiment 4), and n = N, that is, the smallest in the capillary farthest from the laser source. Excitation efficiency T at this time _effmin Becomes (Equation 13).
[0081]
T _effmin = T _RAM ↑ (N-1) + T _RAM ↑ (N) (Equation 13)
(Equation 13) means that in the case of (Embodiment 6), that is, about twice that in the case where the total reflection mirror is not used. In this calculation experimental condition, N = 10, T _ra = 0.929, the excitation efficiency in the 10th capillary increased from 51.5% to 99.4% (0.929 ⁹ +0.929 ⁹ = 0.994). Along with this, the maximum number of capillaries that can be measured simultaneously with a decrease in excitation efficiency of up to 20% increased from 22 to 32 (0.929). ³¹ +0.929 ³² = 0.20). Of course, the same effect can be obtained even if the method shown in the present embodiment is applied to a cylindrical capillary or an elliptical capillary.
[0082]
(Embodiment 9)
In this embodiment, under the calculation experiment conditions of (Embodiment 6), as shown in FIG. 20, after the laser beam 2 is split into two by a beam splitter such as a half mirror 16, reflection mirrors 17-1, 17- 2 and 17-3, irradiation was performed by facing the two side surfaces of two side surfaces in the planar direction of the capillary array in which 10 capillaries were arrayed. The laser beams irradiated from both sides were adjusted to be coaxial with each other by reducing the beam diameter to 0.1 mm with a lens (not shown). The other calculation experiment conditions were the same as in (Embodiment 6). When N capillaries are arranged on a plane, the excitation efficiency T in the nth (n ≦ N) capillaries from one side of the capillary array _eff Is the average transmittance of the laser beam per capillary T _RAM Then, it is expressed by (Equation 14).
[0083]
2T _eff = T _RAM ↑ (n-1) + T _RAM ↑ (N−n) (Equation 14)
The first and second terms in (Expression 14) indicate excitation by two divided laser beams. (Equation 14) is normalized by the excitation efficiency of the first capillary in the case of (Embodiment 6). According to (Equation 14), the excitation efficiency T _eff N = (N + 1) / 2, that is, the minimum is the capillary at the center of the array in which a plurality of capillaries are arranged, and the excitation efficiencies in the other capillaries are symmetric with respect to the above-mentioned array center position. Minimum excitation efficiency T _effmin Becomes (Equation 15).
[0084]
T _effmin = T _RAM ↑ ((N + 1) / 2) (Equation 15)
(Equation 15) is the minimum value T of the excitation efficiency in the case of (Embodiment 6), that is, when the laser beam is not split. _RAM ↑ (n-1) (when n = N) (T _RAM Obviously greater than ≦ 1). In this calculation experimental condition, N = 10, T _RAM = 0.929, the excitation efficiency is minimum with the fifth capillary, 71.8%. On the other hand, in (Embodiment 6), the excitation efficiency was the minimum with the tenth capillary and was 51.5%. Therefore, it has been found that this method can perform simultaneous measurement of a large number of capillaries more efficiently. Along with this, the maximum number of capillaries that can be measured simultaneously with a decrease in excitation efficiency of up to 20% increased from 22 to 43 (0.929). ^{twenty one} = 0.21). Of course, the same effect can be obtained even if the method shown in the present embodiment is applied to a cylindrical capillary or an elliptical capillary.
[0085]
(Embodiment 10)
In the present embodiment, under the calculation experiment conditions of (Embodiment 6), the two laser light sources 18-1 and 18-2 are used as shown in FIG. Irradiation was performed so as to oppose both sides of the two side surfaces of the capillary array in which the capillaries were arrayed. Both laser beams 2-1 and 2-2 were adjusted to be coaxial with each other by narrowing the beam width to 0.1 mm with a lens (not shown). The other calculation experiment conditions were the same as in (Embodiment 6). When N capillaries are arranged, the excitation efficiency T in the nth (n ≦ N) capillary from one side of the capillary array _eff Is the average transmittance of the laser beam per capillary T _RAM Then, it is expressed by (Equation 16),
T _eff = T _RAM ↑ (n-1) + T _RAM ↑ (N−n) (Equation 16)
Sample components that migrate in a plurality of capillaries can be excited with an excitation efficiency that is twice the excitation efficiency shown in (Equation 14) obtained in the case of (Embodiment 9). The first term and the second term in (Equation 16) indicate excitation by laser beams from two laser light sources, respectively. (Equation 16) is normalized by the excitation efficiency in the first capillary in the case of (Embodiment 6), and T _eff > 1 means that the excitation efficiency is equal to or higher than the excitation efficiency represented by (Equation 14). According to (Equation 16), the excitation efficiency T _eff N = (N + 1) / 2, that is, the minimum in the capillary located at the center position of the plurality of capillaries, and the excitation efficiency in the other capillaries is symmetric with respect to the center position. Minimum excitation efficiency T _effmin Becomes (Equation 17).
T _effmin = 2 ・ T _RAM ↑ ((N + 1) / 2) (Equation 17)
This is the minimum value T of the excitation efficiency in the case of (Embodiment 6), that is, when one laser light source is used. _RAM ↑ (n-1) (when n = N) (T _RAM Obviously greater than ≦ 1). In this calculation experimental condition, N = 10, T _RAM = 0.929, the excitation efficiency is minimum with the fifth capillary, 143.7%. On the other hand, in (Embodiment 4), the excitation efficiency was the minimum with the tenth capillary and was 51.5%. Therefore, it has been found that this method can perform simultaneous measurement of a large number of capillaries more efficiently. Along with this, the maximum number of capillaries that can be measured simultaneously with a decrease in excitation efficiency of up to 20% increased from 22 to 63 (2 × 0.929). ³¹ = 0.20). Of course, the same effect can be obtained even if the method shown in the present embodiment is applied to a cylindrical capillary or an elliptical capillary.
[0086]
(Embodiment 11)
In this embodiment, as shown in FIG. 22, two types of capillaries having different shapes are connected to form one electrophoresis path, which is arranged on a plurality of planes, and fluorescence from each capillary is simultaneously measured. A specific example of the technique is shown. Each electrophoresis separation portion of the 10 capillaries is a cylindrical capillary of a separation capillary having a circular cross-section of an outer diameter of 0.2 mm (R = 0.1 mm) and an inner diameter of 0.1 mm (r = 0.05 mm). No. 19 was used, and the capillary of the portion for measuring fluorescence was a rectangular tube-shaped capillary 20 of a measuring capillary having a cross section of a square having an outer periphery of 0.2 mm on a side and an inner periphery of a square having a side of 0.1 mm. Separation capillaries and measurement capillaries constituting each electrophoresis path were connected and arranged on a glass plane at a pitch of 0.2 mm. The central axes of 10 sets of connected separation capillaries and measurement capillaries were made to coincide with each other. Both capillaries are made of quartz (n ₂ = 1.46), the connecting surface was previously polished and flattened, and acrylamide at a concentration of 4% T (Total monomer concentration) and 5% C (Crosslinking material concentration) containing 7 M urea as a denaturant was injected. After gelation. The connecting portion of the capillary was immersed in a buffer solution (not shown) so as not to be electrically disconnected, and the sample separation component that had migrated through the separation capillary migrated through the opposing measuring capillary as it was. The separation capillary was 25 cm long, and the measurement capillary was 15 cm long. At a position 5 cm from the position where the measuring capillary was connected, the polyimide coating on the measuring capillary was removed in advance for 10 mm in length and over the entire circumference to form a window for measuring fluorescence, and this position was irradiated with laser. That is, the migration distance was 25 + 5 = 30 cm. The calculation experiment conditions other than these were the same as in (Embodiment 6). That is, the average transmittance per capillary was 92.9%, and the excitation efficiency of the 10th capillary from the laser light source was 51.5% of the first capillary (0.929). ⁹ = 0.515). From the relationship between the sensitivity of the detector and the dynamic range, it was found that simultaneous measurement of up to 22 capillaries is possible under the present calculation experimental conditions, assuming that the decrease in excitation efficiency that can be measured simultaneously is 20% (0.929). ^{twenty one} = 0.21).
[0087]
Generally, the capillary becomes more expensive in proportion to the volume of the glass as the outer diameter is larger and the inner diameter is smaller, that is, the wall thickness is increased. In addition, an elliptical cylindrical shape or a rectangular cylindrical capillary is more expensive than a cylindrical capillary. In other words, as shown in the present embodiment, it is advantageous for simultaneous measurement of a large number of capillaries, but if an expensive capillary is used as a measurement capillary and an inexpensive capillary is used as a separation capillary, the running cost can be reduced. In addition, since the separation capillary and the measurement capillary can be detached, the cost can be further reduced if only the separation capillary is replaced after the measurement is completed and the next measurement is performed. This is another effect of this embodiment. Of course, the same effect can be obtained by combining capillaries with shapes other than those described above.
[0088]
In the above embodiment, acrylamide gel is used as the electrophoretic separation medium, but a solution or a polymer may be used as the other separation medium. Examples of the polymer include polymers of acrylamide and derivatives thereof, and polymers of polysaccharides such as cellulose and derivatives thereof. If the polymer in the capillary is made exchangeable, the capillary body can be used repeatedly, thereby reducing costs and labor.
[0089]
In the above embodiment, fused silica is used as the material for the capillaries, but other materials may be used as a matter of course. In particular, glass can use materials with various refractive indexes, and can control the lens action of the capillary as well as the shape of the capillary. That is, more efficient simultaneous measurement of a large number of capillaries is possible. In addition, although the capillaries are shown in a cylindrical shape, an elliptical tube, and a rectangular tube, other shapes may be used as a matter of course. Furthermore, it goes without saying that the present invention is not limited to the above embodiment, and the features described in the above embodiment may be combined.
[0090]
Capillary gel electrophoresis can be applied with a higher voltage than plate gel electrophoresis, so that high-speed analysis is possible. Multifocus capillary array electrophoresis, in which a plurality of capillaries are arranged side by side, is a high-throughput analysis technique that can simultaneously analyze a large number of samples at high speed. The introduction of the sample into the capillary can be performed by electric field injection in capillary array electrophoresis, so that it is very simple compared to slab gel electrophoresis. In addition, on-column measurement, in which fluorescence is measured by directly irradiating a capillary with a laser, is a highly sensitive fluorescence measurement method because there are few factors that lower electrophoresis resolution and good excitation efficiency.
[0091]
【The invention's effect】
In the present invention, while performing on-column fluorescence measurement, a plurality of capillaries can be irradiated with laser at substantially the same time without scanning a laser beam, and fluorescence measurement can be performed collectively, so that high sensitivity, high speed, and high throughput analysis can be performed. Can be achieved. Moreover, the apparatus configuration and capillary arrangement are very simple and practical. If a refillable material such as a polymer is used as the electrophoretic medium, the capillary array itself can be used repeatedly without moving, so the practical effect that can greatly reduce the cost and labor is enormous.
[Brief description of the drawings]
FIG. 1A is a plan view and FIG. 1B is a cross-sectional view showing a configuration of a main part of a capillary array electrophoresis apparatus that performs on-column measurement of a capillary array of the present invention.
FIG. 2 is a cross-sectional view showing an optical path of a laser beam in the capillary of the present invention.
FIG. 3 is a diagram showing an apparatus configuration according to the first embodiment of the present invention.
FIG. 4 is a cross-sectional view of a portion for measuring fluorescence in Embodiment 1 of the present invention.
FIG. 5 is a diagram showing a calculation result of an optical path in a capillary array in Embodiment 1 of the present invention.
FIG. 6 is a diagram showing the relationship between the capillary position and the excitation efficiency in the first embodiment of the present invention.
FIG. 7 is a diagram showing a relationship between a capillary outer diameter / inner diameter ratio and excitation efficiency in Embodiment 1 of the present invention.
FIG. 8 is a diagram showing a relationship between a capillary outer diameter / inner diameter ratio and the number of capillaries capable of simultaneous measurement in the first embodiment of the present invention.
FIG. 9 is a diagram showing a calculation result of a capillary outer diameter / inner diameter ratio and reflected light intensity directly incident on the light detection system in the first embodiment of the present invention.
FIG. 10 is a diagram showing a relationship between capillary arrangement intervals and excitation efficiency in Embodiment 2 of the present invention.
FIG. 11 is a diagram showing a main configuration of an apparatus according to a third embodiment of the present invention.
FIG. 12 is a cross-sectional view of a portion for measuring fluorescence in Embodiment 3 of the present invention.
FIG. 13 is a diagram showing an example of an electrophoresis pattern obtained in Embodiment 3 of the present invention.
FIG. 14 is a diagram showing an example of an electrophoresis pattern obtained in Embodiment 3 of the present invention.
FIG. 15 is a diagram showing an example of an electrophoresis pattern obtained in Embodiment 3 of the present invention.
FIG. 16 is a diagram showing a calculation result of an optical path in the capillary array according to the fourth embodiment of the present invention.
FIG. 17 is a diagram showing a relationship between capillary position and excitation efficiency in Embodiment 4 of the present invention.
FIG. 18 is a cross-sectional view of a portion for measuring fluorescence according to Embodiment 6 of the present invention.
FIG. 19 is a cross-sectional view of a portion for measuring fluorescence according to an eighth embodiment of the present invention.
FIG. 20 is a cross-sectional view of a portion for measuring fluorescence according to the ninth embodiment of the present invention.
FIG. 21 is a sectional view of a portion for measuring fluorescence according to the tenth embodiment of the present invention.
FIG. 22 is a sectional view of a portion for measuring fluorescence according to the eleventh embodiment of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Capillary, 2 ... Laser, 3 ... Distance of deviation of incident laser from capillary array axis, 4 ... Capillary array axis, 5 ... Glass plane, 6 ... First lens, 7 ... Filter, 8 ... Second lens, 9 ... 2D CCD camera, 10 ... Acrylamide gel, 11 ... Air, 12 ... Fluorescence measurement cell, 13 ... Image division prism and combination filter, 14 ... Water, 15 ... Total reflection mirror, 16 ... Half mirror, 17-1, 17 -2, 17-3: reflection mirror, 18-1, 18-2 ... laser light source, 2-1, 2-2 ... laser beam, 19 ... cylindrical capillary of separation capillary, 20 ... square tube of measurement capillary Shape capillary.

Claims (5)

A plurality of capillaries at least partially arranged in a plane;
Means for irradiating a laser at a measurement position of the capillary from a direction parallel to a plane formed by the axes of the plurality of capillaries;
Means for detecting fluorescence emitted inside each capillary,
Each of the capillaries is circular with respect to the axis of the measurement position, the outer radius of the capillary at the position where the laser passes is R, the inner radius is r, the refractive index is n ₂ , and the laser passes. The refractive index of the medium outside the capillary at the position is n ₁ , the refractive index of the medium inside the capillary at the position where the laser passes is n _3, and each of the incident positions of the laser to the capillary is When the distance from the plane formed by each axis of the capillary is x and x ≦ r, {−sin ⁻¹ (x / R) + sin ⁻¹ (xn ₁ / (Rn ₂ )) − sin ⁻¹ (xn ₁ / (rn ₂ )) + sin ⁻¹ (xn ₁ / (rn ₃ )) <0. The capillary array electrophoresis apparatus according to claim 1, wherein the n ₂ is larger than the n ₃ . The capillary array electrophoresis apparatus according to claim 2, wherein the n ₁ is 1.00. The capillary array electrophoresis apparatus according to claim 1, wherein the external medium is any one of a transparent gas, a transparent liquid, and a transparent solid. 2. The capillary array electrophoresis apparatus according to claim 1, wherein the capillary contains an electrophoretic separation medium, and the electrophoretic separation medium is replaceable.

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