JP2010523976A - プラスチック製マイクロ流体分離および検出プラットフォーム - Google Patents
プラスチック製マイクロ流体分離および検出プラットフォーム Download PDFInfo
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Abstract
Description
本願は、2007年4月4日付けで出願した米国仮出願シリアル番号60/921,802;2007年8月13日付けで出願した米国仮出願シリアル番号60/964,502;および2008年2月12日付けで出願した米国仮出願シリアル番号61/028,073(各々をここに出典明示してそのすべてを本明細書の一部とみなす)の米国特許法第119条(e)下の出願日の利益を主張する。また、本願は、「Methods For Rapid Multiplexed Amplification Of Target Nucleic Acids」を表題とするアトニー整理番号08−318−US;および「Integrated Nucleic Acid Analysis」を表題とするアトニー整理番号07−801−USの本願と同日出願のそれらの2つの米国出願をここに出典明示してそのすべてを本明細書の一部とみなす。
本発明の様々な態様の適用は、核酸同定および配列決定の双方につき広範囲に広がる。ヒト同定における使用の例は、犯罪法医学および国土安全保障、例えば、軍事検問所、国境および港、空港ならびに集団災害現場での同定を含む。また、競走馬の育種および追跡、家畜の育種を含めた獣医学的同定適用ならびにペット同定は、開示された電気泳動チップの用途の範囲内である。
単純な組織病理学系統の結果に基づいて、外科医がどれくらい積極的でなければならないかに関する決定を行っている。本発明による電気泳動分離チップの使用は、組織病理学を1時間未満での癌の確定的な核酸診断に置き換えることができ、良好に通知された外科的決定がなされるのを可能とする。
実施例1
チップデザインおよび電気泳動
実施例1A:チップデザイン
本発明のデバイスの特定の具体例の模式図を図6に示す。このマイクロ流体デバイスは、各々ダブルTクロスインジェクターを持つ16のマイクロチャネルよりなった。チャネルの横断面の寸法(幅90μmおよび深さ40μm)ならびに陽極とクロスインジェクターとの間のチャネルの長さ(25cm)は、すべてのチャネルにつき等しかった。各チャネルについての分離長(交差と励起/検出窓との間の距離)は、16〜20cmの範囲にある。陰極ウェルとインジェクターとの間の横断面の面積は、全ての抵抗および、従って、陰極と交差との間の電場がバイアス下で本質的に等しいように調節した。これにより、試料によって経験された電場が、試料が負荷された分離チャネルにかかわらず同一であることを保証した。すべてのチャネルについての交差電圧は本質的に同一であった。試料注入のための試料入口および試料廃棄アームは、双方とも長さ2.5mmであった。双方のチャネル間のオフセットは500μmであった。
チップは、加熱エンボス法によりパターン化し、穿孔してアクセスホールを形成し、次いで、拡散結合させてチャネルを密閉した。マスターを化学的湿式エッチングプロセスを用いて、写真平版によりガラスに作製した。次いで、このガラスマスターを用いて、電気鋳造することによりニッケル・コバルトエンボスツールを作製して、ガラスマスターのネガティブ複製を生成した。
Zenor(商標)−1420Rフィルムのシート(5"×2"のサイズおよび188μmの厚み)を基材材料として用いた。これらのシートにおいては、陰極、陽極、試料および廃液アクセスホールを穿孔により形成した。この後、基材へエンボスツール上のチップデザイン特徴を加熱エンボスした。エンボス法は、図7に示すスタックを135℃の15分間の加熱水圧プレスおよび1250psiの圧縮圧力に入れることにより達成した。スタックは、1250psiの圧縮力下に保持し、放出に先立ち38℃に冷却させた。ノルボルネンモノマーを含有する薄い熱可塑性ポリマーでのこのチップの製作は、励起および検出窓での低バックグラウンド蛍光を生じさせた。高結合強度の分散結合の達成は、高粘度ふるい用マトリックスの使用を可能とした。
表面修飾は、最初に脱イオン水、続いて1M NaOHでマイクロチャネル表面を予め処理することにより達成した。窒素フラッシュを適用して、チャネルから流体を除去した。表面処理後に、チャネルを介して0.1%(w/v)のヒドロキシプロピルメチルセルロース(HPMC)溶液を流し、それに続いて、室温にて一晩インキュベートした。高純度窒素を用いて、チャネルを通じてフラッシュして、チャネルの内部の流体を除去した。
核酸分析の電気泳動の分離および分析は、Genebench−FX(商標)シリーズ100(Network Biosystems, Inc., Woburn, MA) で行った。この機器は、プラスチック分離チップおよびチップ支持体を受入れ、チップおよび機器の間の良好な光学的、電気的および熱カップリングを可能にするように構成した。チャンバーの温度を操作の全体にわたって50℃に維持した。
動電学的注入プラスチックチップ
実施例2A:チップデザイン
本発明の電気泳動の分離チップのもう一つの配置は、分離のために単一チャネルを用いる。各試料を動電学的な試料注入により分離チャネルに導入する。この別法のアプローチは、小試料容積の使用および分離プロセスにおけるかなりの単純化を可能とする。動電学的な試料注入についてのチップデザインの模式図を支持体および分離チップ領域を示す図16の分離図で示す。このデバイスは、分離長が有効には20cmである16のマイクロチャネルよりなる。各チャネルは、各末端にアクセスホールを有する。チャネルは幅90μmおよび深さ40μmである。
図16のデバイスは上記の断面図に記載された手順に従って作製される。要約すると、アクセスホール(直径が1mm)は188μmの厚みを持つCOPフィルム(Zeonor(商標)1420R)に形成される。次いで、チャネルパターン(幅90μmおよび深さ40μm)を加熱エンボス法により形成する。COP(Zeonor(商標)1420R)のカバーを基材に拡散結合して、チャネルを密閉する。
デバイスを上記の断面図に記載したチャネルに表面修飾を適用することにより、分離のために調製する。これに続いて、チャネルをふるい用マトリックスで充填する。試料を試料/陰極リザーバに負荷する。注入場を電極を介して試料に適用して、負に帯電したDNAを分離チャネルに注入する。チャネルへのDNAの注入に続いて、緩衝液(1X TTE;Ameresco)を試料容積の10倍容積で試料/陰極リザーバに添加する。電場を陰極および陽極を横切って印加して、分離チャネルの下の注入プラグからDNAを分離する。この添加は、試料/陰極にある試料を希釈するように機能し、緩衝液を負荷するに先立ち試料を取り出す必要性はない。分離および検出をGenebench−FX(商標)シリーズ100機器で行い、データ分析を先の実施例に記載されたソフトウェアで行う。
DNA配列決定
DNA配列決定分析については、DNAテンプレートをPCR酵素SpeedSTAR HS(Takara, Madison, WI)(U/μL):0.025、Fast緩衝液1:1x、dNTP:0.25mM、プライマー(順方向):250nMおよびプライマー(逆方向):250nMよりなる反応ミックス中で増幅する。所望のレベルの鋳型DNAをミックスに添加する。DI水またはTE緩衝液(トリス10mMまたはEDTA0.1mM)を10μLの全容積まで反応ミックスに添加する。PCR反応ミックスの熱サイクルは、製造者の推奨したプロトコールに従い、95℃での60秒間のホットスタート活性化、30サイクルの変性、アニーリングおよび伸長(98℃で5秒間、55℃で10〜15秒間、次いで、72℃での5〜10秒/kbp)よりなった。
Claims (29)
- 陽極部分、陰極部分、ならびに陽極部分と陰極部分との間のセンター部分を含む電気泳動分離チップであって、
陰極部分は、少なくとも1つの第1のビアを含み:
陽極部分は、少なくとも1つの第2のビアを含み:および
センター部分は、1または複数のマイクロチャネルおよび1つの検出窓を含み、各マイクロ流体チャネルは、分離領域および検出領域を有し;ここに、
各マイクロ流体チャネルは、少なくとも1つの第1のビアおよび少なくとも1つの第2のビアと流体連通し;
複数のマイクロ流体チャネルは、実質的に同一面にあり;
複数のマイクロ流体チャネルは、センター部分内で相互に交差せず;
検出窓は薄いプラスチックを含み;および
検出窓は、各マイクロ流体チャネルの検出領域の一部を覆う該チップ。 - 薄いプラスチック領域が、ポリエチレン、ポリ(アクリレート)、ポリ(カーボネート)および不飽和、部分的不飽和もしくは飽和の環状オレフィンポリマー(COP)、不飽和、部分的不飽和もしくは飽和の環状オレフィン共重合体(COC)またはノルボルネン熱可塑性ポリマーを含む請求項1記載のチップ。
- 薄いプラスチックが、ZEONOR(商標)、ZEONEX(商標)またはTOPAS(商標)ポリマーを含む請求項1記載のチップ。
- 薄いプラスチックが約300μm未満の厚みを有する請求項1記載のチップ。
- 薄いプラスチックが約500μm未満の厚みを有する請求項1記載のチップ。
- 薄いプラスチックが、1ミリメートル以下の厚みを有する請求項1記載のチップ。
- 各マイクロ流体チャネルが、2cm〜50cmの分離長を有する請求項1記載のチップ。
- 複数のマイクロ流体チャネルの各々が、注入チャネルをさらに含む請求項1記載のチップ。
- 薄いプラスチックが、約450〜500nmの間の波長で励起される場合に、本質的に500〜800nmの間の波長を有する蛍光を発しない請求項1記載のチップ。
- 薄いプラスチックが約488nmの波長で励起される請求項9記載のチップ。
- 断片サイジング適用のために生成された核酸試料中の複数の核酸種が、PCR増幅用の核酸テンプレートの単一コピーで始めて3を超える信号対雑音で検出できる請求項1記載のチップ。
- DNA配列決定適用のために生成された核酸試料中の複数の核酸種が、PCR増幅用の核酸テンプレートの単一コピーで始めて3を超える信号対雑音で検出できる請求項1記載のチップ。
- 複数のマイクロ流体チャネルの各々が、表面コーティングをさらに含む請求項1記載のチップ。
- 表面コーティングが、ヒドロキシプロピルメチルセルロース(HPMC)、ポリ(エチレンオキシド)(PEO)、ポリ(ビニルアルコール)(PVA)、ポリ(ジメチルアクリルアミド)(PDMA)、ポリ(ビニルピロリジノン)、ジメチルアクリルアミド DEA、ジエチルアクリルアミド、ポリ(ジエチルアクリルアミド)およびそれらの混合物である請求項13記載のチップ。
- 複数のマイクロ流体チャネルの各々が、ふるい用マトリックスをさらに含む請求項13記載のチップ。
- ふるい用マトリックスが、線形または架橋ポリ(N,N−ジアルキルアクリルアミド)、線形ポリアクリルアミド、ポリジメチルアクリルアミド、ポリビニルピロリジノンまたはそれらの組合せを含む請求項15記載のチップ。
- ふるい用マトリックスが、1〜15重量%のポリアクリルアミドを含む請求項16記載のチップ。
- 各陰極ウェルおよび各マイクロ流体チャネル間の多孔層を含み、ここに、多孔層は、陰極ウェルから各マイクロ流体チャネルへのガス気泡の通過を実質的にブロックできることをさらに含む請求項1記載のチップ。
- 多孔質層が、ガラスフリット、ポリマーフリット、ポリマー膜またはポリマーフィルターを含む請求項18記載のチップ。
- 核酸試料中の複数の核酸種を該核酸試料の電気泳動分析後に、単一塩基分解能で検出できる請求項1記載のチップ。
- 少なくとも2つの試料を複数のマイクロ流体チャネルに同時に注入するためのインジェクターをさらに含む請求項1記載のチップ。
- 陽極部分、陰極部分、ならびに陽極部分と陰極部分との間のセンター部分を含む頂部および底部表面を有する支持体を含む装置であって、ここに、
センター部分は検出窓にて開口部を含み、
陽極部分は少なくとも1つの陽極ウェルを含み、および
陰極部分は少なくとも1つの陰極ウェルを含み;
装置は、さらに、
頂部および底部表面を有する請求項1記載のチップを含み、ここに、
チップの頂部表面は、支持体の底部表面と接触し、
マイクロ流体チャネルはビアを介して陰極および陽極ウェルと流体連通し;および
チップは、支持体に固定して取付けられる該装置。 - 分離チップが、各々頂部および底部表面を有する基材層およびカバー層を含み、ここに、
基材層は、その頂部表面における複数のマイクロ流体溝を含み;および
カバー層は、チップ支持体の各陽極および陰極ウェルについての1つのビアを含み、ここに、
基材層の頂部表面は、カバー層の底部表面と接触し、それにより、分離チップ中の複数の
マイクロ流体チャネルを一緒に形成する請求項22記載の装置。 - 基材層およびカバー層が加熱結合される請求項23の装置。
- 複数の試料を電気泳動的に分離および検出する方法であって、
複数の試料を請求項1に記載のマイクロチップ上の複数のマイクロ流体チャネルの各々に供し;
複数のマイクロ流体チャネルを横切る電位を印加して、複数の分析試料の各々を含む検出可能な種を分離し;
検出窓にて複数の分離された試料を含む検出可能な種の各々を検出することを同時に含むことを特徴とする該方法。 - 複数のマイクロ流体チャネルの各々を横切って実質的に同一の電場を維持する工程をさらに含むことを特徴とする請求項25記載の方法。
- 本質的に同一の電場が、複数のマイクロ流体チャネルの各々の各部分の抵抗を平衡させることにより、複数のマイクロ流体チャネルの各々を横切って維持されることを特徴とする請求項26記載の方法。
- 検出可能な種が核酸を含むことを特徴とする請求項25記載の方法。
- 検出可能な種が、核酸に機能的に付着した色素を含むことを特徴とする請求項28記載の方法。
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