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JP2003514221A - 生体材料とのハイブリダイゼーションのためのバイオチャネルアッセイ - Google Patents

生体材料とのハイブリダイゼーションのためのバイオチャネルアッセイ

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Publication number
JP2003514221A
JP2003514221A JP2001536293A JP2001536293A JP2003514221A JP 2003514221 A JP2003514221 A JP 2003514221A JP 2001536293 A JP2001536293 A JP 2001536293A JP 2001536293 A JP2001536293 A JP 2001536293A JP 2003514221 A JP2003514221 A JP 2003514221A
Authority
JP
Japan
Prior art keywords
microfluidic device
microchannel
dna
binding pair
specific binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP2001536293A
Other languages
English (en)
Other versions
JP2003514221A5 (ja
Inventor
チャン−ロン・シー
バーバラ・フォーリー
フイナン・ユ
ビ−エン・チョーン
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Motorola Solutions Inc
Original Assignee
Motorola Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Motorola Inc filed Critical Motorola Inc
Publication of JP2003514221A publication Critical patent/JP2003514221A/ja
Publication of JP2003514221A5 publication Critical patent/JP2003514221A5/ja
Pending legal-status Critical Current

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Abstract

(57)【要約】 本発明は、多孔質ポリマー、ビーズまたはマイクロチャネル中に加工された構造物に結合したＤＮＡまたはＲＮＡのような特異的結合対メンバーを有する分離された領域を有するマイクロチャネルを有するミクロ流体デバイスに関する。本発明のマイクロチャネルは、プラスチックから加工されており、流体推進コンポーネントおよび検出器と有効に結合している。

Description

【発明の詳細な説明】
【０００１】 （発明の背景） （発明の分野） 本発明は、ミクロ流体デバイスの構造物、加工、およびミクロ流体デバイスに
おいて分析を行う方法に関する。
【０００２】 （発明に関連する技術背景） 抗原／抗体；相補ＤＮＡ／ＤＮＡ；ＤＮＡ／ＲＮＡ；ＲＮＡ／ＲＮＡ；ビオチ
ン／アビジン含有対のような特異的結合対を利用する分析は、当該技術分野で広
く知られている。ミクロ流体デバイスを製造する技法および利用する技法もまた
よく知られている。当該技術分野において、また、ＤＮＡの相補結合に基づくＤ
ＮＡ配列決定のための種々の技法が開示されている。
【０００３】 標的一本鎖ＤＮＡの固定ＤＮＡプローブ上への結合を利用するＤＮＡプローブ
アレイ技術は、幅広い用途をもっている。研究開発活動の大部分は、種々の技術
に力点をおいて行われてきた。例えば、いくつかの技術は、機械的手段によるプ
ローブ配置に焦点を合わせている。他の技術は、大きいアレイを生成する際に有
利であるインサイチュ・プローブ合成法に焦点を合わせている。さらに、他の技
法は、光重合および圧電液体計量分配技術を用いるゲルパッドアレイに焦点を合
わせている。
【０００４】 全てのＤＮＡハイブリダイゼーション技法に対する共通の問題は、各個のプロ
ーブ部位に対するストリンジェンシーの制御の欠如である。ＤＮＡハイブリダイ
ゼーションプロセスは、特異的な温度および塩度条件で生じ、ＤＮＡ配列によっ
て異なる。ＤＮＡプローブアレイについては、ＤＮＡプローブ配列が異なるので
、ハイブリダイゼーション認識化は、プローブアレイ全体についての均一なスト
リンジェンシー条件下で決して完全ではない。この問題点は、しばしば、一塩基
ミスマッチを引き起こす短い二本鎖について最も明白である。大きいプローブ部
位重複度を用いることにより、ミスマッチハイブリダイゼーションの作用を最小
化することができる。ストリンジェンシー管理は、オリゴヌクレオチドの電気泳
動移動量を管理することにより各プローブ部位について与えられた。この後のス
キームを成功裏に行うためには、ＤＮＡ分子または標識剤が直接電解または電解
の生成物により損傷されるのを防止するために念入りに処理された浸透層が必要
とされる。
【０００５】 さらに、現行のＤＮＡアレイ技法によっては、ハイブリダイゼーション効率を
最大にするのに有効な解決法が得られなかった。診断アッセイにおいて、標的Ｄ
ＮＡ分子は、しばしば、微量のものである。該アッセイの検出限界は、検出デバ
イスの感度により決定され、また、ハイブリダイゼーションの間にプローブに結
合した標的オリゴ体の量によっても決定される。能動的な混合が存在しない静止
のハイブリダイゼーションチャンバー中で、所定の標的分子が表面上でその相補
鎖とハイブリダイズする確率は、拡散速度および統計量により決定される。低い
標的濃度レベルでハイブリダイゼーションを完全にするために１０時間までの時
間を必要とする。標的分子を良好に用い、該ハイブリダイゼーションを増強する
ために、プローブアレイを流体流方向に対して垂直に置くフロースルー技法が提
案された。フロースルー技法の場合でさえ、標的分子の一部しか特異的ＤＮＡプ
ローブ部位と接触することができない。
【０００６】 本発明は、ミクロ流体チャネル中にＤＮＡプローブ部位を連続的に置いてＤＮ
Ａプローブがその結合相手と効率的に接触することができるようにすることによ
り上記技術的問題点を克服する。
【０００７】 米国特許第5,147,607号には、抗体またはＤＮＡなどの試薬をチャネルの種々
の位置に固定したプラスチック材料製のマイクロチャネルを有する種々の微量検
定デバイスが開示されている。マイクロチャネル壁に抗体を結合させる技法は開
示されているが、ＤＮＡを結合させる技法は開示されていない。プローブのマイ
クロチャネル壁への結合は、プローブと試験試料とを最適に接触させない。米国
特許第5,843,767号には、ＤＮＡ／ＤＮＡのような結合反応の離散検出用の微細
加工されたフロースルー多孔質装置が開示されている。WO 98/43739には、チャ
ンバー中に固定された試薬を有する多孔質フローチャネルが開示されている。
【０００８】 （図面の簡単な記載） 図１は、多孔質ゲルおよびスポットしたＤＮＡプローブを充填した流体チャネ
ルの略平面図を示す。 図２は、ミクロ流体チャネルの内側にある平版的に型押しされたゲルパッドを
示す。 図３は、ＤＮＡ結合用の成形プラスチックミクロ構造物を有するミクロ流体チ
ャネルを示す。 図４は、ビーズの別個セクションがＤＮＡのような特異的結合剤を有するビー
ズを充填したミクロ流体チャネルを示す。 図５は、単一の初期の流れが複数のチャネルに指向されている例を示す。 図６は、循環ミクロ流体チャネルデバイスを例示する。
【０００９】 （発明の概要） 本発明は、チャネルを通して流体を流すための流入口および流出口を有するマ
イクロチャネルを有するチップのような固体物質のセクションを含むミクロ流体
デバイスを含む。該マイクロチャネルは、多孔質ポリマー、マイクロチャネル中
に成形されたミクロ構造物または充填されたビーズ上に固定された特異的結合対
メンバーの分離された画定領域を有する。これらの構造物は、固定された結合対
メンバーとマイクロチャネルを通って流れる流体中の結合対メンバーとの最適な
接触をもたらす。多孔質ポリマー、ビーズまたはミクロ構造物は、流れをもたら
さなければならず、該チャネルをふさいではならない。マイクロチャネルは、検
出器およびチャネル中に液体を流すための流体推進コンポーネントと有効に結合
されており、流出口および流入口に電極を有していてもよい。
【００１０】 ＤＮＡ／ＤＮＡ；ＤＮＡ／ＲＮＡ、およびＲＮＡ／ＲＮＡ相補結合対が好まし
い。マイクロチャネルは、フルオロフォアで標識された標的ＤＮＡ、励起源、お
よび結合対から発光された蛍光を検出するための検出器と有効に結合されている
。本発明の目的は、蛍光標識された標的ＤＮＡを結合するための分離された画定
領域中に固定されたＤＮＡまたはＲＮＡプローブを有する上記で定義されたチッ
プを準備することによるＤＮＡまたはＲＮＡ配列決定方法を提供することである
。
【００１１】 本発明の目的は、また、遺伝的欠陥を判定する手段を提供することである。本
発明は、また、ＤＮＡ分析により病原体を同定する手段を提供する。
【００１２】 マイクロチャネルは、種々の形状、フィードバックアーム、バルブ、および流
体流を制御するためのベントを有していてもよい。チャネルは、一本であっても
、または複数であってもよい。本発明は、固定された結合物質と該チャネルを通
って流れる流体中の結合相手との間での有効な接触を提供する。本発明は、流量
調節による改良されたハイブリダイゼーションストリンジェンシー制御；流れに
より誘発された撹拌でハイブリダイゼーション速度を増大させることおよびミク
ロ流体チャネル内で標的とプローブとを接近させることによるアッセイ時間の短
縮；ならびに感度を改善するハイブリダイゼーション効率の増大を提供する。ま
た、加水分解による障害はない。
【００１３】 （発明の詳細な記載） 本発明のミクロ流体チャネルは、成形技法またはエンボス技法を用いたプラス
チック製の一般的に２００ミクロン未満のチャネルである。該チャネルは、ミク
ロ流体系のポンピングを支持するための寸法のものであることが必要である。ミ
クロ流体チャネルは、いずれの形状を有していてもよく、例えば、線形、Ｓ字曲
線、円弧形状などであってもよい。該チャネルの断面寸法は、正方形、長方形、
半円などであってもよい。再循環をもたらすためのバルブを有する複数の連結し
たマイクロチャネルであってもよい。
【００１４】 固体物質のセクションは、ガラス、セラミック、金属、珪素またはプラスチッ
ク製のチップであってもよい。チップは、好ましくは、エポキシ樹脂、ポリアク
リル酸樹脂、ポリエステル樹脂、ポリスチレン、ポリカーボネート、ポリ塩化ビ
ニルなどのプラスチックから加工されている。特異的結合対は、ＤＮＡ／ＤＮＡ
またはＤＮＡ／ＲＮＡ相補結合対である。
【００１５】 加圧ガス、減圧、電界、磁界および遠心力デバイスなどの流体推進コンポーネ
ントは、マイクロチャネルと有効に結合されており、該マイクロチャネルを通し
て流体を移動させる。さらに、充填された試験試料は、流れの方向もしくはその
対向方向にまたは流れに対して垂直方向に電界を調節することにより変更され得
る。かくして、マイクロチャネル中の流体の流速は、結合対の結合、例えば、Ｄ
ＮＡ／ＤＮＡまたはＤＮＡ／ＲＮＡ対のハイブリダイゼーションを促進するよう
に変更され得る。光学的、電気的または電気化学的検出器などの検出器もまた、
マイクロチャネルと有効に結合される。
【００１６】 図１は、ＤＮＡのような特異的結合対のメンバーを結合した離散分離領域３を
有する多孔質ゲル２を充填したＳ字曲線形状のミクロ流体チャネル１を例示する
。試料は、４でミクロ流体チャネル中に流入し、５で該チャネルから流出する。
この方法において、該チャネルには、アガロースまたはポリアミドのような多孔
質ゲル物質が充填されている。該ゲルの細孔は、希ゲル化溶液を用いることによ
り、有意な流体流が該ゲルを通ることができるように十分な大きさに調整される
。特異的結合対のメンバーは、該ゲル上にスポットされ、その結果、該プローブ
は、化学的に結合される。
【００１７】 図２は、チャネル内に型押しされたゲルパッド１１を有するミクロ流体チャネ
ル１０を例示する。該ゲルパッドは、平版技法を用いてアクリルアミドの光重合
によって形成される。
【００１８】 図３は、高表面積のミクロ構造物がチャネル中に成形されているミクロ流体チ
ャネル１５を例示する。図３ａは、別個領域における一連の柱状物１６を示し、
図３ｂは、チャネル１５中に成形されたドームの別個領域１７を示す。これらの
ミクロ構造物が化学的に修飾され、特異的結合物質が結合される。
【００１９】 図４は、プレーンビーズ２１、およびＤＮＡのような特異的結合物質を有する
ビーズ２２の領域を交互に充填したミクロ流体チャネル２０を例示する。
【００２０】 図５は、各々が上記した結合物質の別個領域２７を有する複数のミクロ流体チ
ャネル２６ａ、ｂ、ｃなどに枝分れしているミクロ流体チャネル２５を例示する
。この実施態様により、同一または異なる特異的結合物質に対するその反応性を
試験するために試料を並行して試験することができる。
【００２１】 図６は、再循環ミクロ流体チャネル３４を有するチップ３０を例示する。該ミ
クロ流体チャネルは、上記した特異的結合物質を有する離散領域３２、再循環ア
ーム３３および再循環後の排出用のバルブ３４を有する。この実施態様において
、試験試料は、結合相手の位置を通過して再循環される。かくして、希試料また
は遅反応性試料は、各々、特異的結合物質によって通過され得る。
【００２２】 微細加工プラスチックキャピラリー電気泳動（ＣＥ）デバイスは、当該技術分
野において示されている。熱可塑性成形ポリメチルメタクリレートＣＥデバイス
は、R. M. McCormick, et al,“Microchannel electrophoretic separations of
DNA in injection-molded plastic substrates,”Anal. Chem., vol. 69, pp.
2626, 1997 により開示されている。Eckstrom らは、ＰＤＭＳのような弾性ポリ
マーを研究した（PCT出願公開 WO 91/16966, 1991）。さらに最近、他の研究者
が、ＰＤＭＳデバイスにおけるＤＮＡラダーの電気泳動的分離を公表した。例え
ば、C. S. Effenhauser, et al,“Integrated Capillary Electrophoresis on F
lexible Silicone Microdevices,”Anal. Chem., vol. 69, pp. 3451, 1997 。M
astrangelo らは、表面マイクロマシン法を用いるパリレン−ポリカーボネート
支持体をベースとしたミクロＣＥデバイスの構築を開示している（“An Inexpen
sive Plastic Technology for Microfabricated Capillary Electrophoresis Ch
ip”presented at Micro-TAS'98, Banff, 1998）。かくして、これらの技法は、
マイクロチャネルを加工するのに利用可能である。本発明は、結合をより効率的
に促進するために、多孔質ポリマー、ビーズまたはマイクロチャネル中の構造物
により特異的結合物質を固定することを含む。
【００２３】 これらの実施例は、本発明を例示するものであり、趣旨または範囲においてそ
れを限定するものではない。
【図面の簡単な説明】
【図１】 多孔質ゲルおよびスポットしたＤＮＡプローブを充填した流体チ
ャネルの略平面図である。
【図２】 ミクロ流体チャネルの内側にある平版的に型押しされたゲルパッ
ドを示す図である。
【図３】 ＤＮＡ結合用の成形プラスチックミクロ構造物を有するミクロ流
体チャネルを示す図である。
【図４】 ビーズの別個セクションがＤＮＡのような特異的結合剤を有する
ビーズを充填したミクロ流体チャネルを示す図である。
【図５】 単一な初期の流れが複数のチャネルに指向されている例を示す図
である。
【図６】 循環ミクロ流体チャネルデバイスを例示する図である。
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｃ１２Ｑ 1/68 Ｇ０１Ｎ 33/566 Ｇ０１Ｎ 33/566 37/00 １０１ 37/00 １０１ Ｃ１２Ｎ 15/00 Ａ (81)指定国 ＥＰ(ＡＴ，ＢＥ，ＣＨ，ＣＹ， ＤＥ，ＤＫ，ＥＳ，ＦＩ，ＦＲ，ＧＢ，ＧＲ，ＩＥ，Ｉ Ｔ，ＬＵ，ＭＣ，ＮＬ，ＰＴ，ＳＥ，ＴＲ)，ＯＡ(ＢＦ ，ＢＪ，ＣＦ，ＣＧ，ＣＩ，ＣＭ，ＧＡ，ＧＮ，ＧＷ， ＭＬ，ＭＲ，ＮＥ，ＳＮ，ＴＤ，ＴＧ)，ＡＰ(ＧＨ，Ｇ Ｍ，ＫＥ，ＬＳ，ＭＷ，ＭＺ，ＳＤ，ＳＬ，ＳＺ，ＴＺ ，ＵＧ，ＺＷ)，ＥＡ(ＡＭ，ＡＺ，ＢＹ，ＫＧ，ＫＺ， ＭＤ，ＲＵ，ＴＪ，ＴＭ)，ＡＥ，ＡＧ，ＡＬ，ＡＭ， ＡＴ，ＡＵ，ＡＺ，ＢＡ，ＢＢ，ＢＧ，ＢＲ，ＢＹ，Ｂ Ｚ，ＣＡ，ＣＨ，ＣＮ，ＣＲ，ＣＵ，ＣＺ，ＤＥ，ＤＫ ，ＤＭ，ＤＺ，ＥＥ，ＥＳ，ＦＩ，ＧＢ，ＧＤ，ＧＥ， ＧＨ，ＧＭ，ＨＲ，ＨＵ，ＩＤ，ＩＬ，ＩＮ，ＩＳ，Ｊ Ｐ，ＫＥ，ＫＧ，ＫＰ，ＫＲ，ＫＺ，ＬＣ，ＬＫ，ＬＲ ，ＬＳ，ＬＴ，ＬＵ，ＬＶ，ＭＡ，ＭＤ，ＭＧ，ＭＫ， ＭＮ，ＭＷ，ＭＸ，ＭＺ，ＮＯ，ＮＺ，ＰＬ，ＰＴ，Ｒ Ｏ，ＲＵ，ＳＤ，ＳＥ，ＳＧ，ＳＩ，ＳＫ，ＳＬ，ＴＪ ，ＴＭ，ＴＲ，ＴＴ，ＴＺ，ＵＡ，ＵＧ，ＵＳ，ＵＺ， ＶＮ，ＹＵ，ＺＡ，ＺＷ (72)発明者 バーバラ・フォーリー アメリカ合衆国85048アリゾナ州フェニッ クス、サウス・フォックステイル・レイン 14842番 (72)発明者 フイナン・ユ アメリカ合衆国85226アリゾナ州チャンド ラー、ウエスト・パーク・アベニュー5760 番 (72)発明者 ビ−エン・チョーン アメリカ合衆国85226アリゾナ州チャンド ラー、ウエスト・ジェノア・ウェイ3380番 Ｆターム(参考） 4B024 AA11 AA19 CA01 CA09 CA11 HA13 HA14 4B029 AA07 AA23 BB15 BB20 CC01 CC02 CC03 CC08 FA12 FA15 4B063 QA01 QQ41 QR32 QR35 QR38 QR56 QR82 QS34 QS39 QX02 QX04

Claims (20)

    【特許請求の範囲】
  1. 【請求項１】 流体をマイクロチャネルの中へ輸送するための流入口および
    流体をマイクロチャネルの外へ輸送するための流出口を有するマイクロチャネル
    を有する固体物質のセクションを含むミクロ流体デバイスであって、該マイクロ
    チャネルが、多孔質ポリマー上、ビーズ上、または該マイクロチャネル中に加工
    されたミクロ構造物上に固定された特異的結合対メンバーの空間的に分離された
    画定領域を含有する、ミクロ流体デバイス。
  2. 【請求項２】 デバイスが珪素、ガラス、二酸化珪素、プラスチックまたは
    セラミックから加工される請求項１記載のミクロ流体デバイス。
  3. 【請求項３】 分離された画定領域が、特異的結合対メンバーを結合した多
    孔質ポリマーである請求項１記載のミクロ流体デバイス。
  4. 【請求項４】 分離された画定領域が特異的結合対メンバーを結合したビー
    ズを有する請求項１記載のミクロ流体デバイス。
  5. 【請求項５】 固定化された結合対メンバーを有する画定領域がマイクロチ
    ャネル中にヒドロゲルを導入することにより形成される請求項１記載のミクロ流
    体デバイス。
  6. 【請求項６】 結合対メンバーがヒドロゲルの空間的に分離された部分上に
    選択的に分配される請求項５の画定領域。
  7. 【請求項７】 マイクロチャネル中のヒドロゲルが写真平版を含む手段によ
    り型押しされる請求項５の画定領域。
  8. 【請求項８】 分離された画定領域がマイクロチャネル中に加工されたミク
    ロ構造物を有し、該ミクロ構造物が特異的結合対メンバーを結合している請求項
    １記載のミクロ流体デバイス。
  9. 【請求項９】 結合対メンバーがＤＮＡ、ＲＮＡ、ポリペプチド、核酸およ
    び抗体／抗原からなる群から選択される請求項１記載のミクロ流体デバイス。
  10. 【請求項１０】 特異的結合メンバーがＤＮＡまたはＲＮＡプローブである
    請求項１記載のミクロ流体デバイス。
  11. 【請求項１１】 特異的結合メンバーがＤＮＡである請求項１記載のミクロ
    流体デバイス。
  12. 【請求項１２】 さらに、マイクロチャネルと有効に結合された流体推進コ
    ンポーネントを含む請求項１記載のミクロ流体デバイス。
  13. 【請求項１３】 加圧ガス、減圧、電界、磁界または遠心力である請求項１
    ２における流体推進コンポーネント。
  14. 【請求項１４】 マイクロチャネルと有効に結合された検出器コンポーネン
    トを含む請求項１記載のミクロ流体デバイス。
  15. 【請求項１５】 検出器が光学的、電気的、または電気化学的検出器である
    請求項１４記載のミクロ流体デバイス。
  16. 【請求項１６】 試験試料中の特異的結合メンバーの検出方法であって、 ａ．請求項１記載のミクロ流体デバイスを準備し； ｂ．マイクロチャネルを通して試験試料を流して結合対を形成させ； ｃ．該結合対を検出すること を含む方法。
  17. 【請求項１７】 試験試料の流れがマイクロチャネル中において再循環され
    る請求項１６記載の方法。
  18. 【請求項１８】 試験試料の流速が、マイクロチャネルと有効に結合された
    流体推進コンポーネントにより調節される請求項１６記載の方法。
  19. 【請求項１９】 充填された試料の移動速度が、さらに、流れの方向または
    その対向方向にモジュール電界を印加することにより変更される請求項１６記載
    の方法。
  20. 【請求項２０】 充填された試験試料が、流れの方向に対して垂直方向に電
    界を印加することにより空間的画定領域で引き寄せられるかまたは反発される請
    求項１６記載の方法。
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US6960467B2 (en) 2005-11-01
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EP1350568B1 (en) 2005-01-26
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US20020094584A1 (en) 2002-07-18
WO2001034302A2 (en) 2001-05-17
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