JP4320908B2 - DNA chip reader - Google Patents

Description

【００３２】
【数２】
r(x,y)={Ipp(x,y)-G*Ips(x,y)}/{Ipp(x,y)+2G*Ips(x,y)} … (3)
【００３３】
ここで、Ips、Ippは、それぞれ励起光の偏光がｐ偏光のときの第１と第２のＣＣＤのカメラ３６、３８の画素の受光量であり、Iss、Ispは、それぞれ励起光の偏光がｓ偏光の時の第１と第２のＣＣＤカメラ３６、３８の画素の受光量である。
【００３４】
ＤＮＡチップ１上でハイブリダイゼーションが生じていると、この異方性比が増大する。 よって、この異方性比の値を調べることによって、ハイブリダイゼーションが生じたＤＮＡチップ上のスポットを知ることができる。
【００３５】
【発明が解決しようとする課題】
しかしながら、図４に示すようなＤＮＡチップ読み取り装置では、ダイクロイックミラー３２や偏光ビームスプリッタ３４等を使用した光学系を使用しているため、装置が大型化してしまうという欠点があった。また、同じ測定点を測定するに際し、光源をｐ偏光とｓ偏光に切り替えて測定を行う必要があり、この切替は機械的に行われるので時間を要し、全体としてスループットが低下してしまうという問題点もあった。
【００３６】
本発明はこのような事情に鑑みてなされたもので、以上説明したようなＤＮＡチップの読み取り装置等に使用される、物体の偏光異方性を測定する装置を小型化するとともに、迅速な測定を行えるようにし、もって、小型でスループットの良いＤＮＡチップの読み取り装置を提供することを課題とする。
【００３７】
【課題を解決するための手段】
前記課題を解決するための第１の手段は、光源からの偏光をＤＮＡチップに照射し、ＤＮＡチップから発生する蛍光の異方性を計測することにより、当該ＤＮＡチップ上のハイブリダイゼーションを測定するＤＮＡチップ読み取り装置であって、光源と、基板に設けられた導波路と、当該導波路中に設けられたオンオフ可能なＴＥ／ＴＭモード変換器と、対物レンズと、前記導波路中に設けられたＴＥ／ＴＭモードスプリッタと、２つの受光器を有してなり、光源からの光は、前記導波路、ＴＥ／ＴＭモード変換器及び対物レンズを介して被測定物体に照射され、ＤＮＡが発する蛍光は、前記対物レンズを介して前記導波路に入射し、前記導波路中で分岐されて、ＴＥ／ＴＭモードスプリッタによりＴＥモード光とＴＭモード光に分離され、それぞれ２つの受光器に受光されるようにされていることを特徴とするＤＮＡチップ読み取り装置（請求項１）である。
【００３８】
本発明においては、基本的な光学装置として、基板上に形成された導波路、ＴＥ／ＴＭモード変換器、ＴＥ／ＴＭモードスプリッタを使用している。光源から導波路に導入されたＴＥモード、又はＴＭモードの偏光は、導波路を伝播してＴＥ／ＴＭモード変換器に入る。このＴＥ／ＴＭモード変換器は、外部からの電圧操作により、モード変換を行ったり行わなかったりすることができ、これにより、ＴＥ／ＴＭモード変換器を出る偏光を、電気信号による操作で、ＴＥモードとしたりＴＭモードとしたりすることができる。
【００３９】
ＴＥ／ＴＭモード変換器を出た偏光は、再び導波路を通ってその出射端より放出され、対物レンズを介してＤＮＡチップアレイを照明する。すると、前述したように、ＤＮＡチップにおけるハイブリダイゼーションに応じた偏光異方性を有する蛍光が、ＤＮＡチップから発生する。
【００４０】
この蛍光は、対物レンズより、前記導波路の出射端に集められる。このとき、ＤＮＡチップ面の像が、前記導波路の出射端に結像するように、対物レンズを調整しておくことが好ましい。前記導波路の出射端に集められた蛍光は、この導波路を逆方向に進行するが、この導波路には途中に分岐点が設けられており、この蛍光は２つに分岐され、その一方が受光器側に導かれる。
【００４１】
受光器側の導波路の途中には、ＴＥ／ＴＭモードスプリッタが設けられているので、これにより、蛍光はＴＥモードの光とＴＭモードの光に分離され、導波路を介してそれぞれの受光器によって受光される。
【００４２】
このような装置を用いて蛍光の異方性比ｒ（x,y）を測定するには、まず、ＴＥ／ＴＭモード変換器を動作させない状態で、２つの測定器によりＴＥモードの蛍光とＴＭモードの蛍光の強さを測定し、次にＴＥ／ＴＭモード変換器を動作させて、２つの測定器によりＴＥモードの蛍光とＴＭモードの蛍光の強さを測定する。これにより、４つの測定量を得て、前記(2)、(3)式により蛍光の異方性比ｒ（x,y）を算出する。そして、測定位置を変えることにより、ＤＮＡチップの各点での異方性比ｒ（x,y）の測定を行う。
【００４３】
このような光学装置においては、基板上に微細な導波路、ＴＥ／ＴＭモード変換器、ＴＥ／ＴＭモードスプリッタがフォトリソグラフィ等を使用して形成されているので、全体の構成を微小なものにすることができる。また、照射光源として利用する偏光のモードの切替を電気的に行うことができるので、測定のスループットが向上する。
【００４４】
前記課題を解決するための第２の手段は、光源からの偏光をＤＮＡチップに照射し、ＤＮＡチップから発生する蛍光の異方性を計測することにより、当該ＤＮＡチップ上のハイブリダイゼーションを測定するＤＮＡチップ読み取り装置であって、ＴＥモード光、ＴＭモード光をそれぞれ発する光源と、基板に設けられた導波路と、対物レンズと、前記導波路中に設けられたＴＥ／ＴＭモードスプリッタと、２つの受光器を有してなり、２つの光源からの光路は、前記導波路中で１つの導波路にまとめられ、各光源からの光は、まとめられた前記１つの導波路から対物レンズを介して被測定物体に照射され、ＤＮＡが発する蛍光は、前記対物レンズを介して前記導波路に入射し、前記導波路中で分岐されてＴＥ／ＴＭモードスプリッタによりＴＥモード光とＴＭモード光に分離され、それぞれ２つの受光器に受光されるようにされていることを特徴とするＤＮＡチップ読み取り装置（請求項２）である。
【００４５】
前記第１の手段においては、光源を１つとし、ＴＥ／ＴＭモード変換器によって照射光の偏光モードを変えていたが、本手段においては、ＴＥ／ＴＭモード変換器を用いず、異なるモードの偏光を発生する光源を２つ用い、それぞれの光源からの光路を導波路中で合流させている。そして、２つの光源を切り替えて使用することにより、照射される偏光のモードを変える。その他の作用効果については、前記第１の手段と同じであるので、説明を省略する。
【００４６】
前記課題を解決するための第３の手段は、前記第１の手段又は第２の手段であって、光源及び受光素子の少なくとも１つが前記基板の端面に配設されていることを特徴とするもの（請求項３）である。
【００４７】
基板上に形成された導波路に光源からの光を導入したり、導波路からの光を受光器に導くのには、従来は光ファイバー等を使用していた。本発明においては、光源及び受光素子を前記基板の端面に配設しているので、光ファイバー等の光伝達手段が不必要となり、全体の構成がコンパクトとなる。
【００４８】
前記課題を解決するための第４の手段は、前記第１の手段又は第２の手段であって、光源及び受光素子の少なくとも１つが、前記基板中に形成されていることを特徴とするもの（請求項４）である。
【００４９】
本手段においては、リソグラフィー等により、光源及び受光素子の少なくとも一つが、導波路やＴＥ／ＴＭモード変換器、ＴＥ／ＴＭモードスプリッタが形成されている基板中に形成されている。よって、全体の構成をさらにコンパクトなものとすることができる。
【００５０】
前記課題を解決するための第５の手段は、前記第１の手段から第４の手段のいずれかであって、前記導波路と前記受光素子の間に光学フィルタが配設されていることを特徴とするもの（請求項５）である。
【００５１】
本手段においては、導波路と前記受光素子の間に光学フィルタが配設されているので、ＤＮＡチップから反射されて受光器に達しようとする照射光（励起光）と、ＤＮＡチップが発する蛍光を分離することができ、蛍光の検出性能を向上させることができる。
【００５２】
前記課題を解決するための第６の手段は、前記第１の手段から第５の手段のいずれかであって、前記導波路の分岐点からＴＥ／ＴＭモードスプリッタに至る導波路中に、前記導波路に入射した光源からの光の、ＤＮＡチップからの反射光を反射するコリニアグレーティング波長フィルタを有することを特徴とするＤＮＡチップ読み取り装置（請求項６）である。
【００５３】
本手段においては、コリニアグレーティング波長フィルタにより、ＤＮＡチップから反射された照射光（励起光）を反射して通過させないようにすることができるので、ＤＮＡチップが発する蛍光の検出性能を向上させることができる。
【００５４】
【発明の実施の形態】
以下、本発明の実施の形態の例を図を用いて説明する。図１は、本発明の実施の形態の１例であるＤＮＡチップ読み取り装置の全体構成を示す概要図である。図１において、１はＤＮＡチップ、２は試料台、３はＸ軸ガイドレール、４はモータ、５はＸ軸ウォームギア、６はＹ軸ウォームギア、７はモータ、８は読み取りヘッド、９は制御装置、１０は表示装置である。
【００５５】
ＤＮＡチップ１は、試料台２の上に固定されている。ＤＮＡチップ１は、蛍光マーキングを施されたＤＮＡプローブを有しており、更に、検体によるハイブリダイゼーション処理が終了している。試料台２の両側には、Ｘ軸ガイドレール３およびモーター４に固定されたＸ軸ウオームギヤ５がそれぞれ配設されている。Ｘ軸ガイドレール３とX軸ウオームギヤ５をまたぐようにＹ軸ウオームギヤ６が配置され、このＹ軸ウオームギヤ６はモーター７で駆動される。Ｙ軸ウオームギヤ６は、Ｘ軸ウオームギヤ５により、Ｘ軸に沿って自由に動かすことができる。このＹ軸ウオームギヤ６には、読取光学系を内包した読み取りヘッド８が取り付けられている。読み取りヘッド８は、Ｙ軸ウオームギヤ６によってＹ軸に沿って自由に動かすことができる。
【００５６】
制御装置９は、読み取りヘッド８がＤＮＡチップ１全体をＸ−Ｙ方向に走査するようにモーター４、７ を制御する。具体的には、特定のＸ座標で読み取りヘッド８をＹ軸全体に１方向走査し、特定のＸ座標を有する測定点のデータを採取する。その後、１ピッチ分だけＸ座標を進め、今度は、先程と反対のＹ軸方向に読み取りヘッド８を走査する。そして、これにより、新しいＸ座標を有する測定点のデータを採取する。これをＤＮＡチップ１の測定対象領域全体を走査するまで繰返す。制御装置９は、前記走査の間、読み取りヘッド８から出力される蛍光強度を読み取りヘッド８のＸ−Ｙ座標とともに記録し、表示装置１０に表示する。
【００５７】
図２に、図１に示された読み取りヘッド８の構成の概要を示す。図２において、１はＤＮＡチップ、１１は基板、１２は励起用光源、１３は光導波路、１４、１５、１６は電極、１７は導波路分岐部、１８は光導波路、１９は対物レンズ、２０は光導波路、２１は導波路型ＴＥ／ＴＭモードスプリッタ、２２はアルミクラッド、２３、２４は光導波路、２５、２６は光検出器、２７、２８は波長フィルタである。
【００５８】
読み取りヘッド内には、光導波路１３、１８、２０、２３、２４が表面に形成された基板１１が配設されている。基板１１は、Ｘ-cutＺ軸伝搬のニオブ酸リチウム（LiNbＯ₃）基板であり、幅10mm、高さ20mm、厚さ１mmである。光導波路は、チタン拡散法にて作製されている。
【００５９】
基板１１の端面に結合された励起用光源１２からの光は、光導波路１３を伝搬する。励起用光源１２には、その出射光の偏光方向がＴＥモードで光導波路１３を伝搬するように配置されたレーザーを使用している。
【００６０】
３つの電極１４、１５、１６はＴＥ／ＴＭモードコンバータとして働き、光導波路１３を伝搬するＴＥモード光をＴＭモード光に変換する働きをする。すなわち、これらの電極間に電圧が印加されない場合は、ＴＥモード光がそのまま通過し、電圧を印加するとＴＥモード光がＴＭモード光に変換される。電極１４は接地電極であり、電極１５にはＴＥ／ＴＭモード変換用電圧が印加される。また、電極１６には、ＴＥ／ＴＭモード間複屈折補償のための電圧が印加される。（詳細については、例えば、社団法人電子情報通信学会、信学技報、OQE93-146(1993-12)、P67-72に報告されている。）
【００６１】
導波路１３を通った光は、導波路分岐部１７を経て、光導波路１８の端面から出射する。導波路１８の端面から出射した光は、対物レンズ１９を介してＤＮＡチップ１上を照明する。ＤＮＡチップ１上のスポットで発生した蛍光は、対物レンズ１９により、光導波路１８の端面に集光され、光導波路１８中を伝搬する。
【００６２】
光導波路１８を伝搬した蛍光の一部は、導波路分岐部１７で分岐され、その一部が光導波路２０に入射する。光導波路２０を伝搬した蛍光は、周知の導波路型ＴＥ／ＴＭモードスプリッタ２１に入射する。ここで用いられている導波路型ＴＥ／ＴＭモードスプリッタ２１は、アルミ装荷の方向性結合器型のものであり、アルミクラッド２２が方向性結合器の片側に配置されている。この導波路型ＴＥ／ＴＭモードスプリッタ２１によって、ＤＮＡチップ１からの蛍光のうちＴＥモード成分は光導波路２３を伝搬し、ＴＭモード成分は、光導波路２４を伝搬する。
【００６３】
光導波路２３、２４のそれぞれの出射端には、光検出器２５、２６が配置されている。光検出器２５、２６と光導波路２３、２４の間には、励起光の波長の光を遮断し、ＤＮＡチップ１からの蛍光を透過する波長フィルター２７、２８が配置されている。光検出器２５、２６からの出力は、図１の制御装置９に入力される。
【００６４】
図２におけるＴＥ／ＴＭモードコンバータ部分の断面図を図３に示す。図３において、図２と同じ構成要素には同じ符号を示し、その説明を省略する。１１’はSiＯ₂薄膜である。
【００６５】
ニオブ酸リチウム（LiNbＯ₃）基板１１の表面にTiがドープされ、Ti:LiNbＯ₃からなる光導波路１３が形成されている。基板１１の表面には、SiＯ₂薄膜１１’からなる絶縁層が形成され、その上に電極１４、１５、１６が形成されている。光導波路１３の幅は数μｍ、深さは約１μｍである。よって、その端面から放出される照射光は、点光源からの光とみなすことができる。
【００６６】
図１における制御装置９は、電極１５、１６に印加する電圧をオンオフすることにより、励起光の偏光をＴＥモード又はＴＭモードに切り替える。制御装置９は、あるＸ，Ｙ座標において、まず、励起光をＴＥモードにしてその時の２つの光検出器２５、２６の出力を記録し、次に励起光をＴＭモードにして、２つの光検出器２５、２６の出力を記録する。
【００６７】
続いて、前述のように、モータ４、７を駆動して次の走査点に読み取りヘッド８を移動して、その点での出力を同様に記録する。これをＤＮＡチップ１の全走査領域に対して行い、各Ｘ，Ｙ座標について、光検出器２５、２６の出力を合計４つずつ記録する。これらの４つの出力は、前述のIpp、Ips、Iss、Ispに対応しており、前述の式(2)、(3)を用いて各Ｘ，Ｙ座標における蛍光の異方性比を求めることができる。制御装置９は、このようにして求めた異方性比を測定点のＸ，Ｙ座標に対応させて２次元の画像として表示装置１０に表示する。
【００６８】
本実施の形態においては、読み取りヘッド８が小さな基板１１上に集積化しているため、装置全体を小型にすることができる。また、電気光学効果を用いて偏光素子を物理的に回転させることなく励起光の偏光方向を切り替えることができるため、高速な読み取りも期待できる。
【００６９】
なお、以上の実施の形態においては、基板１１の材料として、ニオブ酸リチウムを使用していたが、タンタル酸リチウム、ガリウム砒素、インジウムリン等の材料を使用することができる。特に、ガリウム砒素、インジウムリンを用いた場合には、励起光発生用の光源及び光検出器を、リソグラフィ技術を用いて基板１１中に組み込むことが可能になるので、読み取りヘッド８の構成をさらに小型化にすることができる。
【００７０】
また、以上の実施の形態においては、光検出器２５、２６と光導波路２３、２４の間には、励起光の波長の光を遮断し、ＤＮＡチップ１からの蛍光を透過する波長フィルター２７、２８を設けていたが、この代わりに、又はこれに加えて、励起光の波長の光を反射するコリニアグレーティング波長フィルタを、図２の光導波路２０、又は光導波路２３、２４中に設けてもよい。
【００７１】
さらに、以上の実施の形態においては、１つの励起光用光源を使用し、ＴＥ／ＴＭモードコンバータによって、ＴＥモードの光とＴＭモードの光を切り替えるようにしていたが、ＴＥモードの励起光を発する光源と、ＴＭモードの励起光を発する光源の２つを使用し、これらからの光をそれぞれ光導波路に入射させ、２つの光導波路を結合させて１つの導波路とし、その端面から放出される光を対物レンズに導くようにしてもよい。この場合は、２つの光源を切り替えて使用する。
【００７２】
【発明の効果】
以上説明したように、本発明のうち請求項１に係る発明及び請求項２に係る発明においては、全体の構成を微小なものにすることができ、かつ測定のスループットを向上させることができる。そのため、臨床や税関現場でのＤＮＡ判定を促進することができる。
【００７３】
請求項３に係る発明及び請求項４に係る発明においては、光ファイバー等の光伝達手段が不必要となり、全体の構成がコンパクトとなる。
請求項５に係る発明及び請求項６に係る発明においては、蛍光の検出性能を向上させることができる。
【図面の簡単な説明】
【図１】本発明の実施の形態の１例であるＤＮＡチップ読み取り装置の全体構成を示す概要図である。
【図２】図１に示された読み取りヘッド８の構成の概要を示す図である。
【図３】図２におけるＴＥ／ＴＭモードコンバータ部分の断面を示す図である。
【図４】従来のＤＮＡチップ読み取り装置の構成の概要を示す図である。
【符号の説明】
１…ＤＮＡチップ、２…試料台、３…Ｘ軸ガイドレール、４…モータ、５…Ｘ軸ウォームギア、６…Ｙ軸ウォームギア、７…モータ、８…読み取りヘッド、９…制御装置、１０…表示装置、１１…基板、１１’…SiＯ₂薄膜、１２…励起用光源、１３…光導波路、１４、１５、１６…電極、１７…導波路分岐部、１８…光導波路、１９…対物レンズ、２０…光導波路、２１…導波路型ＴＥ／ＴＭモードスプリッタ、２２…アルミクラッド、２３、２４…光導波路、２５、２６…光検出器、２７、２８…波長フィルタ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus for optically reading a DNA chip that specifies the structure of a gene component using complementary interaction between the gene components, and more specifically, the polarization from a light source is converted into a DNA chip. The present invention relates to a DNA chip reader that measures hybridization on the DNA chip by measuring the anisotropy of fluorescence generated from the DNA chip.
[0002]
[Prior art]
In recent years, genomes of organisms have been used in a very wide range of fields, such as identification of pathogens in the medical field, treatment of diseases, and improvement of species by genetic recombination in the agricultural field. When using such a genome, it is essential to elucidate the structure of the target organism at the gene level.
[0003]
As a device for specifying the structure of the specimen at the gene level, that is, the nucleotide sequence of the site of interest in the gene, what is called a DNA chip or a DNA chip (in this specification, these are called DNA chips), And its reading device has been developed and put into practical use. For example, General Scanning Inc. of the United States sells a DNA chip named ScanArray series and its reading device. In such an apparatus, the nucleotide sequence of the specimen is identified as follows.
[0004]
A DNA chip is obtained by immobilizing oligonucleotides (DNA probes) having a predetermined base sequence on a plate-like substrate in spots at appropriate intervals. Usually, the size of one spot is about 20 to 200 μm, and several millions of the same oligonucleotide is immobilized in one spot. The number of bases of the oligonucleotide to be immobilized is generally about 20.
[0005]
As the substrate, glass, Si substrate, or plastic is used. Of these, the most widely used is a microscope slide glass as a substrate. A typical glass slide substrate has a size of 75 mm × 25 mm and a thickness of 1 mm. In one entire DNA chip, the spots as described above are arranged in a matrix at intervals of 50 μm to 500 μm. The oligonucleotides contained in each spot have different nucleotide sequences.
[0006]
As a method of immobilizing oligonucleotides on a substrate, there are a method of immobilizing oligonucleotides while synthesizing them one by one on a substrate, and a method of immobilizing oligonucleotides synthesized separately in advance on a substrate. .
[0007]
A typical technique in the former method is a photolithography technique. The latter method includes mechanical micro spotting technology. Inkjet technology is used for both methods.
[0008]
The photolithographic technique is a technique developed by Fordor et al. (See Science, No. 251, page 767 (1991)) and utilizes a photoreactive protecting group. This protecting group has a function of inhibiting the binding of each base monomer to the same type or another type of base monomer, and no new base binding reaction occurs at the base end to which this protecting group is bonded. This protecting group can be easily removed by light irradiation.
[0009]
First, an amino group having this protecting group is immobilized on the entire surface of the substrate. Next, only a spot to which a desired base is to be bonded is selectively irradiated with light using a method similar to the photolithography technique used in a normal semiconductor process. As a result, only the portion of the base irradiated with light can introduce the next base by subsequent binding. Here, a desired base having the same protecting group at its end is bound.
[0010]
Next, the shape of the photomask is changed, and another spot is selectively irradiated with light. Thereafter, a base having a protecting group is bonded in the same manner. A DNA chip is produced by repeating this until a desired base sequence is obtained for each spot.
[0011]
Next, a method for manufacturing a DNA chip using ink jet technology will be described. The ink jet technique is a technique for ejecting very small droplets to a predetermined position on a two-dimensional plane using heat and piezoelectric effects, and is widely used mainly in printer apparatuses.
[0012]
For manufacturing a DNA chip, an ink jet apparatus having a structure in which a piezoelectric element is combined with a glass capillary is used. By applying a voltage to the piezoelectric element connected to the liquid chamber, the liquid in the chamber is ejected as droplets from the capillary connected to the chamber by the change in volume of the piezoelectric element. The size of the ejected droplet is determined by the diameter of the capillary, the volume change of the piezoelectric element, and the physical properties of the liquid, but generally the diameter is about 30 μm. In an inkjet apparatus using a piezoelectric element, such droplets can be ejected at a cycle of about 10 KHz.
[0013]
A DNA chip manufacturing apparatus using such an ink jet apparatus can drop a desired droplet at a desired spot on the DNA chip substrate by relatively moving the ink jet apparatus and the DNA chip substrate. There are roughly two types of DNA chip manufacturing apparatuses using an ink jet apparatus. One uses only one inkjet device.
[0014]
This ink jet apparatus is configured to drop a reagent for removing a protecting group at the end of an oligomer onto a desired spot. The protecting group of the spot into which the desired base is to be introduced is removed using this ink jet apparatus to make it active, and then the desired base binding reaction operation is performed on the entire DNA chip substrate. At this time, a desired base binds only to a spot having an oligomer whose terminal is activated by dropping of the reagent from the ink jet apparatus. Thereafter, an operation of protecting the terminal of the newly added base is performed. The procedure is then repeated until the desired nucleotide sequence is obtained, changing the spot from which the protecting group is removed.
[0015]
On the other hand, a DNA chip manufacturing apparatus using a multi-head inkjet apparatus has a configuration in which a desired base can be directly bonded to each spot by preparing an inkjet apparatus for each reagent containing each base. A higher throughput than the above-described DNA chip manufacturing apparatus can be obtained.
[0016]
Among the methods of immobilizing oligonucleotides synthesized in advance on a substrate, mechanical microspotting technology is a technology that mechanically presses and immobilizes a liquid containing oligonucleotides attached to the tips of stainless steel pins onto the substrate. . The spot obtained by this method is about 50 to 300 μm. After micro spotting, post-processing such as immobilization with UV light is performed.
[0017]
Using the DNA chip produced as described above, the nucleotide sequence of the site of interest to be examined is specified as follows. As described above, DNA or RNA extracted from the test object is added to the DNA chip on which the DNA probe is immobilized.
[0018]
DNA and RNA as specimens are subjected to a proliferation operation in advance, and are further marked with a fluorescent substance. Addition of the specimen to the DNA chip is usually performed in a liquid phase. If the sample in the additive solution has a nucleotide sequence that is complementary to the sequence of the oligonucleotide immobilized on the DNA chip, the partial duplex by hydrogen bonding between the base in the sample and the base on the chip Occurs. This double-strand formation by hydrogen bonding is called hybridization. Nucleotides contained in DNA are complementary to adenine and thymine, and cytosine and guanine, respectively.
[0019]
Thereafter, the specimen that has not undergone hybridization is washed and removed. As a result, the fluorescent marker added to the specimen remains only in the spot where the oligonucleotide having a sequence complementary to the nucleotide sequence contained in the specimen is immobilized. For the marking, a radioisotope may be used in addition to the fluorescent substance.
[0020]
This DNA chip is analyzed by an apparatus generally called an array reader. The array reader measures and displays the fluorescence intensity of each spot. There are two types of array reader methods: a method that measures the fluorescence intensity of multiple spots simultaneously using a CCD camera, and a two-dimensional scan on a DNA chip using a confocal laser microscope. It is a method to do. A photomultiplier tube and an avalanche photodiode are used for the photodetector of the confocal laser microscope.
[0021]
In general, the intensity of fluorescence is several orders of magnitude smaller than the irradiated excitation light. For this reason, it is necessary to remove only the excitation light and measure only the fluorescence during measurement. For this purpose, a method of separating the two by a dichroic mirror using the difference in wavelength between the excitation light and the fluorescence, or excitation by a beam splitter. A method of spatially separating light and fluorescence is used.
[0022]
A method of measuring the intensity at two wavelengths and estimating the intensity from the relative value is generally used for measuring the fluorescence intensity. In the system using a CCD camera, a lamp is used as excitation light, a wide area is excited at once, and fluorescence generated therefrom is combined as a two-dimensional image on the CCD by an optical system and measured. In the method using a confocal laser microscope, a laser is used as excitation light, only the inside of a focused laser spot is excited, and fluorescence generated from that portion is measured.
[0023]
Therefore, in the CCD camera system, it is about 1cm. ² Whereas a wide area can be measured at a time, a method using a confocal laser microscope can measure only a region having a diameter of about 5 to 30 μm at a time. Therefore, two-dimensional scanning of a DNA chip or a confocal laser microscope head is required, and throughput is reduced. This two-dimensional scanning is realized by scanning the stage on which the substrate is placed in the xy2 axis direction. A typical scanning time is about 5 to 15 minutes when a 20 × 60 mm region is scanned at a resolution of 10 μm.
[0024]
On the other hand, in the method using a confocal laser microscope, noise light flare due to contamination in the depth direction of the DNA chip can be removed by so-called confocal sectioning, and a high S / N ratio can be obtained compared to the CCD method. It is characteristic that
[0025]
In any method, the measured fluorescence intensity distribution on the DNA chip is output as two-dimensional image data having a resolution of about 16 bits. At this time, a pseudo coloring process may be performed to facilitate image determination. A spot having a high fluorescence intensity in the output image indicates that the sample includes a nucleotide sequence complementary to the oligonucleotide sequence of the spot. In this way, the nucleotide sequence of the site of interest of the gene of the specimen can be known depending on which spot has a high fluorescence intensity.
[0026]
Among the detection methods using DNA probes as described above, a method using fluorescence polarization anisotropy for detection of hybridization has recently been proposed (S. Abe and S. Toyyonaga, “Detection of DNA hybridization with immobilized fluorescently labeled oligonucleotide probes using fluorescence polarization technique ", Proc. SPIE, Vol. 3602, page 430, 1999).
[0027]
The outline of this method will be described below according to the description in the above-mentioned literature. In this method, the DNA probe side is marked with a fluorescent marker, and hybridization is detected by utilizing the fact that the anisotropy ratio of the fluorescence polarization generated by the linearly polarized excitation light varies depending on the presence or absence of hybridization. The anisotropy ratio r is defined by the following equation.
r = (Ip-Ic) / (Ip + 2 Ic) (1)
Here, Ip and Ic represent the intensities of components parallel to and perpendicular to the linearly polarized light of the excitation light, among the fluorescence generated by the excitation light. In order to detect this anisotropy ratio r, for example, the one shown in FIG. 4 is used as a DNA chip reader and apparatus used in this method.
[0028]
The excitation light emitted from the light source 29 enters the polarizing filter 31 through the lens 30 and is linearly polarized light. The linearly polarized light is reflected by the dichroic mirror 32, passes through the objective lens 33, and illuminates the DNA chip 1 on which the hybridization operation has been performed in advance.
[0029]
The fluorescence excited by the excitation light is collected by the objective lens 33, passes through the dichroic mirror 32, and enters the polarization beam splitter 34. The polarized beam splitter 34 reflects the component (s-polarized light) of the excited fluorescence whose electric field oscillation direction is perpendicular to the paper surface and forms an image on the first CCD camera 36 by the lens 35. Further, of the excited fluorescence, a component (p-polarized light) whose electric field oscillation direction is parallel to the paper surface is transmitted through the polarization beam splitter 34 and imaged on the second CCD camera 38 by the lens 37. The images from the CCD cameras 36 and 38 are input to the image processing device 39.
[0030]
Such measurement is performed twice by switching the polarization direction of the excitation light in a direction parallel to the direction perpendicular to the paper surface to obtain a total of four images. Then, for these four images, an anisotropy ratio r (x, y) image is obtained by calculating a pixel value from the corresponding received light amount for each pixel according to the following equation, and is output to the output device 40.
[0031]
[Expression 1]

[0032]
[Expression 2]
r (x, y) = {Ipp (x, y) -G * Ips (x, y)} / {Ipp (x, y) + 2G * Ips (x, y)}… (3)
[0033]
Here, Ips and Ipp are the amounts of light received by the pixels of the first and second CCD cameras 36 and 38 when the polarization of the excitation light is p-polarization, and Iss and Isp are the polarizations of the excitation light, respectively. This is the amount of light received by the pixels of the first and second CCD cameras 36 and 38 in the case of s-polarized light.
[0034]
When hybridization occurs on the DNA chip 1, this anisotropy ratio increases. Therefore, by examining the value of this anisotropy ratio, the spot on the DNA chip where hybridization has occurred can be known.
[0035]
[Problems to be solved by the invention]
However, the DNA chip reading apparatus as shown in FIG. 4 has a drawback that the apparatus becomes large because an optical system using the dichroic mirror 32, the polarization beam splitter 34, and the like is used. Further, when measuring the same measurement point, it is necessary to perform measurement by switching the light source between p-polarized light and s-polarized light. Since this switching is performed mechanically, it takes time and overall throughput is reduced. There was also a problem.
[0036]
The present invention has been made in view of such circumstances, and a device for measuring the polarization anisotropy of an object used in a DNA chip reader or the like as described above is miniaturized and rapidly measured. Accordingly, it is an object of the present invention to provide a small-sized and high throughput DNA chip reader.
[0037]
[Means for Solving the Problems]
The first means for solving the above problem is to measure the hybridization on the DNA chip by irradiating the DNA chip with polarized light from the light source and measuring the anisotropy of the fluorescence generated from the DNA chip. A DNA chip reader, comprising: a light source; a waveguide provided on a substrate; an on / off TE / TM mode converter provided in the waveguide; an objective lens; and the waveguide. The TE / TM mode splitter and two light receivers are used, and the light from the light source is irradiated onto the object to be measured via the waveguide, the TE / TM mode converter and the objective lens, and DNA is emitted. Fluorescence is incident on the waveguide through the objective lens, branched in the waveguide, and separated into TE mode light and TM mode light by the TE / TM mode splitter, Be a DNA chip reader and wherein the (claim 1) which is to be received into respectively two light receivers.
[0038]
In the present invention, as a basic optical device, a waveguide formed on a substrate, a TE / TM mode converter, and a TE / TM mode splitter are used. The TE mode or TM mode polarized light introduced from the light source into the waveguide propagates through the waveguide and enters the TE / TM mode converter. The TE / TM mode converter can perform or not perform mode conversion by an external voltage operation, whereby the polarized light exiting the TE / TM mode converter can be converted into TE by operating with an electric signal. Mode or TM mode.
[0039]
The polarized light exiting the TE / TM mode converter is again emitted from the exit end through the waveguide and illuminates the DNA chip array through the objective lens. Then, as described above, fluorescence having polarization anisotropy corresponding to hybridization in the DNA chip is generated from the DNA chip.
[0040]
This fluorescence is collected from the objective lens at the exit end of the waveguide. At this time, it is preferable to adjust the objective lens so that an image of the DNA chip surface is formed at the exit end of the waveguide. The fluorescence collected at the exit end of the waveguide travels in the reverse direction in this waveguide, but this waveguide is provided with a branch point in the middle, and this fluorescence is branched into two. Is guided to the light receiver side.
[0041]
Since a TE / TM mode splitter is provided in the middle of the waveguide on the light receiver side, the fluorescence is separated into TE mode light and TM mode light. Is received by.
[0042]
In order to measure the fluorescence anisotropy ratio r (x, y) using such an apparatus, first, the TE mode fluorescence and the TM are measured by two measuring devices without operating the TE / TM mode converter. The mode fluorescence intensity is measured, and then the TE / TM mode converter is operated, and the TE mode fluorescence and the TM mode fluorescence intensity are measured by the two measuring instruments. As a result, four measurement amounts are obtained, and the fluorescence anisotropy ratio r (x, y) is calculated by the above equations (2) and (3). Then, the anisotropy ratio r (x, y) is measured at each point of the DNA chip by changing the measurement position.
[0043]
In such an optical device, a fine waveguide, a TE / TM mode converter, and a TE / TM mode splitter are formed on a substrate using photolithography, etc., so that the entire configuration is made minute. can do. In addition, since the polarization mode used as the irradiation light source can be electrically switched, the measurement throughput is improved.
[0044]
The second means for solving the above problem is to measure the hybridization on the DNA chip by irradiating the DNA chip with polarized light from the light source and measuring the anisotropy of the fluorescence generated from the DNA chip. A DNA chip reader, a light source that emits TE mode light and TM mode light, a waveguide provided on a substrate, an objective lens, a TE / TM mode splitter provided in the waveguide, 2 The optical paths from the two light sources are combined into one waveguide in the waveguide, and the light from each light source passes through the objective lens from the combined one waveguide. The fluorescence emitted from the DNA irradiated on the object to be measured is incident on the waveguide through the objective lens, branched in the waveguide, and T is transmitted by the TE / TM mode splitter. It is separated into mode light and TM mode light, respectively DNA chip reading apparatus characterized by being adapted to be received into two light receivers (claim 2).
[0045]
In the first means, a single light source is used, and the polarization mode of irradiation light is changed by the TE / TM mode converter. However, in this means, a TE / TM mode converter is not used, and different modes are used. Two light sources that generate polarized light are used, and optical paths from the respective light sources are merged in the waveguide. And the mode of the polarized light to be irradiated is changed by switching between the two light sources. Other operational effects are the same as those of the first means, and the description thereof is omitted.
[0046]
A third means for solving the problem is the first means or the second means, wherein at least one of a light source and a light receiving element is disposed on an end surface of the substrate. (Claim 3).
[0047]
Conventionally, an optical fiber or the like has been used to introduce light from a light source into a waveguide formed on a substrate or to guide light from a waveguide to a light receiver. In the present invention, since the light source and the light receiving element are disposed on the end face of the substrate, a light transmission means such as an optical fiber is unnecessary, and the overall configuration is compact.
[0048]
A fourth means for solving the above problem is the first means or the second means, wherein at least one of a light source and a light receiving element is formed in the substrate. (Claim 4).
[0049]
In this means, at least one of the light source and the light receiving element is formed in a substrate on which a waveguide, a TE / TM mode converter, and a TE / TM mode splitter are formed by lithography or the like. Thus, the overall configuration can be made more compact.
[0050]
A fifth means for solving the above problem is any one of the first to fourth means, wherein an optical filter is disposed between the waveguide and the light receiving element. This is a characteristic (claim 5).
[0051]
In this means, since an optical filter is disposed between the waveguide and the light receiving element, irradiation light (excitation light) reflected from the DNA chip to reach the light receiver and fluorescence emitted from the DNA chip. Can be separated, and the fluorescence detection performance can be improved.
[0052]
A sixth means for solving the above problem is any one of the first means to the fifth means, wherein the waveguide extends from the branch point of the waveguide to the TE / TM mode splitter. A DNA chip reader having a collinear grating wavelength filter for reflecting light from a light source incident on a waveguide and reflected light from a DNA chip (Claim 6).
[0053]
In this means, the collinear grating wavelength filter can prevent the irradiation light (excitation light) reflected from the DNA chip from being reflected and passed, so that the detection performance of the fluorescence emitted from the DNA chip can be improved. it can.
[0054]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a schematic diagram showing an overall configuration of a DNA chip reading apparatus which is an example of an embodiment of the present invention. In FIG. 1, 1 is a DNA chip, 2 is a sample stage, 3 is an X-axis guide rail, 4 is a motor, 5 is an X-axis worm gear, 6 is a Y-axis worm gear, 7 is a motor, 8 is a read head, and 9 is a control device. Reference numeral 10 denotes a display device.
[0055]
The DNA chip 1 is fixed on the sample stage 2. The DNA chip 1 has a DNA probe to which a fluorescent marking is applied, and the hybridization process using the sample is completed. On both sides of the sample stage 2, X-axis worm gears 5 fixed to the X-axis guide rail 3 and the motor 4 are respectively arranged. A Y-axis worm gear 6 is disposed so as to straddle the X-axis guide rail 3 and the X-axis worm gear 5, and the Y-axis worm gear 6 is driven by a motor 7. The Y-axis worm gear 6 can be freely moved along the X-axis by the X-axis worm gear 5. A reading head 8 including a reading optical system is attached to the Y-axis worm gear 6. The read head 8 can be freely moved along the Y axis by the Y axis worm gear 6.
[0056]
The control device 9 controls the motors 4 and 7 so that the read head 8 scans the entire DNA chip 1 in the XY direction. Specifically, the reading head 8 is scanned in one direction along the entire Y axis at a specific X coordinate, and data of a measurement point having the specific X coordinate is collected. Thereafter, the X coordinate is advanced by one pitch, and this time, the reading head 8 is scanned in the opposite Y-axis direction. And thereby, the data of the measurement point which has a new X coordinate are extract | collected. This is repeated until the entire measurement target area of the DNA chip 1 is scanned. The control device 9 records the fluorescence intensity output from the reading head 8 together with the XY coordinates of the reading head 8 during the scanning, and displays it on the display device 10.
[0057]
FIG. 2 shows an outline of the configuration of the read head 8 shown in FIG. In FIG. 2, 1 is a DNA chip, 11 is a substrate, 12 is an excitation light source, 13 is an optical waveguide, 14, 15 and 16 are electrodes, 17 is a waveguide branching part, 18 is an optical waveguide, 19 is an objective lens, 20 Is an optical waveguide, 21 is a waveguide type TE / TM mode splitter, 22 is an aluminum clad, 23 and 24 are optical waveguides, 25 and 26 are photodetectors, and 27 and 28 are wavelength filters.
[0058]
A substrate 11 having optical waveguides 13, 18, 20, 23, and 24 formed on the surface thereof is disposed in the read head. The substrate 11 is made of lithium niobate (LiNbO) with X-cut Z-axis propagation. _Three ) A substrate with a width of 10 mm, a height of 20 mm and a thickness of 1 mm. The optical waveguide is produced by a titanium diffusion method.
[0059]
Light from the excitation light source 12 coupled to the end face of the substrate 11 propagates through the optical waveguide 13. The excitation light source 12 uses a laser arranged such that the polarization direction of the emitted light propagates through the optical waveguide 13 in the TE mode.
[0060]
The three electrodes 14, 15 and 16 function as a TE / TM mode converter, and function to convert TE mode light propagating through the optical waveguide 13 into TM mode light. That is, when no voltage is applied between these electrodes, the TE mode light passes as it is, and when the voltage is applied, the TE mode light is converted into TM mode light. The electrode 14 is a ground electrode, and a TE / TM mode conversion voltage is applied to the electrode 15. Further, a voltage for TE / TM mode birefringence compensation is applied to the electrode 16. (Details are reported, for example, in the Institute of Electronics, Information and Communication Engineers, IEICE Technical Report, OQE93-146 (1993-12), P67-72.)
[0061]
The light passing through the waveguide 13 is emitted from the end face of the optical waveguide 18 through the waveguide branching portion 17. The light emitted from the end face of the waveguide 18 illuminates the DNA chip 1 through the objective lens 19. Fluorescence generated at the spot on the DNA chip 1 is condensed on the end face of the optical waveguide 18 by the objective lens 19 and propagates through the optical waveguide 18.
[0062]
A part of the fluorescence propagated through the optical waveguide 18 is branched by the waveguide branching portion 17, and a part thereof enters the optical waveguide 20. The fluorescence propagated through the optical waveguide 20 enters a well-known waveguide type TE / TM mode splitter 21. The waveguide type TE / TM mode splitter 21 used here is of an aluminum loaded directional coupler type, and an aluminum clad 22 is disposed on one side of the directional coupler. By this waveguide type TE / TM mode splitter 21, the TE mode component of the fluorescence from the DNA chip 1 propagates through the optical waveguide 23, and the TM mode component propagates through the optical waveguide 24.
[0063]
Photodetectors 25 and 26 are arranged at the exit ends of the optical waveguides 23 and 24, respectively. Between the photodetectors 25 and 26 and the optical waveguides 23 and 24, wavelength filters 27 and 28 that block the light having the wavelength of the excitation light and transmit the fluorescence from the DNA chip 1 are arranged. Outputs from the photodetectors 25 and 26 are input to the control device 9 of FIG.
[0064]
A cross-sectional view of the TE / TM mode converter portion in FIG. 2 is shown in FIG. 3, the same components as those in FIG. 2 are denoted by the same reference numerals, and the description thereof is omitted. 11 'is SiO ₂ It is a thin film.
[0065]
Lithium niobate (LiNbO _Three ) Ti is doped on the surface of the substrate 11, and Ti: LiNbO _Three The optical waveguide 13 which consists of is formed. On the surface of the substrate 11, SiO 2 ₂ An insulating layer made of a thin film 11 'is formed, and electrodes 14, 15, and 16 are formed thereon. The optical waveguide 13 has a width of several μm and a depth of about 1 μm. Therefore, the irradiation light emitted from the end face can be regarded as light from a point light source.
[0066]
The control device 9 in FIG. 1 switches the polarization of the excitation light to the TE mode or the TM mode by turning on and off the voltage applied to the electrodes 15 and 16. At a certain X, Y coordinate, the control device 9 first sets the excitation light to the TE mode, records the outputs of the two photodetectors 25 and 26 at that time, and then sets the excitation light to the TM mode, Record the output of the detectors 25,26.
[0067]
Subsequently, as described above, the motors 4 and 7 are driven to move the reading head 8 to the next scanning point, and the output at that point is similarly recorded. This is performed for all the scanning areas of the DNA chip 1, and the outputs of the photodetectors 25 and 26 are recorded for each X and Y coordinate, a total of four. These four outputs correspond to the above-mentioned Ipp, Ips, Iss, and Isp, and the above-described equations (2) and (3) are used to obtain the fluorescence anisotropy ratio at each X and Y coordinate. Can do. The control device 9 displays the anisotropy ratio thus obtained on the display device 10 as a two-dimensional image in correspondence with the X and Y coordinates of the measurement point.
[0068]
In the present embodiment, since the read head 8 is integrated on a small substrate 11, the entire apparatus can be reduced in size. Further, since the polarization direction of the excitation light can be switched without physically rotating the polarizing element using the electro-optic effect, high-speed reading can be expected.
[0069]
In the above embodiment, lithium niobate is used as the material of the substrate 11. However, materials such as lithium tantalate, gallium arsenide, and indium phosphide can be used. In particular, when gallium arsenide or indium phosphide is used, it becomes possible to incorporate a light source and a photodetector for generating excitation light into the substrate 11 using a lithography technique. The size can be reduced.
[0070]
In the above embodiment, between the photodetectors 25 and 26 and the optical waveguides 23 and 24, the wavelength filter 27 that blocks the light having the wavelength of the excitation light and transmits the fluorescence from the DNA chip 1, However, instead of this, or in addition to this, a collinear grating wavelength filter that reflects light having the wavelength of the excitation light may be provided in the optical waveguide 20 or the optical waveguides 23 and 24 in FIG. Good.
[0071]
Furthermore, in the above embodiment, one pump light source is used, and the TE / TM mode converter is used to switch between the TE mode light and the TM mode light. Two light sources, a light source emitting light and a light source emitting TM mode excitation light, are incident on the optical waveguide, and the two optical waveguides are combined to form one waveguide, which is emitted from the end face. The light may be guided to the objective lens. In this case, the two light sources are switched and used.
[0072]
【The invention's effect】
As described above, in the invention according to claim 1 and the invention according to claim 2 of the present invention, the overall configuration can be made minute, and the measurement throughput can be improved. Therefore, it is possible to promote DNA determination at clinical or customs sites.
[0073]
In the invention according to claim 3 and the invention according to claim 4, no optical transmission means such as an optical fiber is required, and the overall configuration becomes compact.
In the invention according to claim 5 and the invention according to claim 6, the fluorescence detection performance can be improved.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an overall configuration of a DNA chip reading apparatus as an example of an embodiment of the present invention.
FIG. 2 is a diagram showing an outline of the configuration of the read head 8 shown in FIG.
FIG. 3 is a diagram showing a cross section of a TE / TM mode converter portion in FIG. 2;
FIG. 4 is a diagram showing an outline of the configuration of a conventional DNA chip reader.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... DNA chip, 2 ... Sample stand, 3 ... X-axis guide rail, 4 ... Motor, 5 ... X-axis worm gear, 6 ... Y-axis worm gear, 7 ... Motor, 8 ... Reading head, 9 ... Control apparatus, 10 ... Display Device, 11 ... substrate, 11 '... SiO ₂ Thin film, 12 ... excitation light source, 13 ... optical waveguide, 14, 15, 16 ... electrode, 17 ... waveguide branch, 18 ... optical waveguide, 19 ... objective lens, 20 ... optical waveguide, 21 ... waveguide TE / TM mode splitter, 22 ... aluminum clad, 23, 24 ... optical waveguide, 25, 26 ... photo detector, 27, 28 ... wavelength filter

Claims (6)

A DNA chip reader for measuring hybridization on a DNA chip by irradiating the DNA chip with polarized light from a light source and measuring the anisotropy of fluorescence generated from the DNA chip. A waveguide provided, a TE / TM mode converter that can be turned on / off provided in the waveguide, an objective lens, a TE / TM mode splitter provided in the waveguide, and two light receivers. The light from the light source is irradiated onto the object to be measured through the waveguide, the TE / TM mode converter and the objective lens, and the fluorescence emitted from the DNA is applied to the waveguide through the objective lens. Incident, branched in the waveguide, separated into TE mode light and TM mode light by a TE / TM mode splitter, and received by two light receivers, respectively. DNA chip reading apparatus characterized by being. A DNA chip reader for measuring hybridization on a DNA chip by irradiating the DNA chip with polarized light from a light source and measuring the anisotropy of fluorescence generated from the DNA chip. A light source that emits mode light; a waveguide provided on the substrate; an objective lens; a TE / TM mode splitter provided in the waveguide; and two light receivers. Are integrated into one waveguide in the waveguide, and the light from each light source is irradiated to the object to be measured from the combined waveguide through the objective lens, and the fluorescence emitted from the DNA is The light enters the waveguide through the objective lens, is branched in the waveguide, and is separated into TE mode light and TM mode light by the TE / TM mode splitter. DNA chip reading apparatus characterized by being adapted to be received respectively in the two light receivers. 3. The DNA chip reading device according to claim 1, wherein at least one of a light source and a light receiving element is disposed on an end surface of the substrate. The DNA chip reader according to claim 1 or 2, wherein at least one of a light source and a light receiving element is formed in the substrate. 5. The DNA chip reading device according to claim 1, wherein an optical filter is disposed between the waveguide and the light receiving element. 6. apparatus. 6. The DNA chip reading device according to claim 1, wherein the light source is incident on the waveguide in the waveguide extending from the branch point of the waveguide to the TE / TM mode splitter. 7. And a collinear grating wavelength filter for reflecting light reflected from the DNA chip.

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