JP2020515815A - デジタルマイクロ流体デバイスおよび方法 - Google Patents
デジタルマイクロ流体デバイスおよび方法 Download PDFInfo
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Abstract
Description
本特許出願は、12/28/2016に出願された「DIGITAL MICROFLUIDIC DEVICES AND METHODS」と題する米国特許仮出願第62/439,641号への優先権を主張する。本特許出願はまた、9/1/2017に出願された「DIGITAL MICROFLUIDICS DEVICES AND METHODS OF USING THEM」と題する米国特許仮出願第62/553,743号への優先権を主張する。本特許出願はまた、9/12/2017に出願された「DIGITAL MICROFLUIDICS DEVICES AND METHODS OF USING THEM」と題する米国特許仮出願第62/557,714号への優先権を主張する。これらの特許出願それぞれが全体として参照により本明細書に組み入れられる。
本明細書において挙げられるすべての刊行物および特許出願は、各個の刊行物または特許出願が参照により組み入れられることが明確かつ個別に示される場合と同じ程度に、全体として参照により本明細書に組み入れられる。
本開示は、デジタルマイクロ流体デバイスおよび対応する流体操作・抽出装置ならびにそれらの使用方法に関する。
デジタルマイクロ流体工学(DMF)は、流体のミクロスケール小滴の簡単かつ正確な操作のための強力な技術である。DMFは、複数の試薬に対する簡単な制御(ポンプ、弁または管が不要)、固体および液体の両方の容易な取り扱い(目詰まりするチャネルがない)および扱いにくい試薬(たとえば有機溶媒、腐食性薬品)との適合性(疎水面(一般的にはテフロンコートされている)が化学的に不活性であるため)を許すため、化学的、生物学的および医学的用途のために急速に人気を博した。しかし、DMFに関する継続的な難題は、事前の濃縮なしでは下流側の処理および検出を支えるには希薄すぎる濃度の分析対象物を含有する、DMFデバイスによって容易に収容される流体量よりも大きい流体量を一般的に含む「現実的」試料の取り扱いである。一部のグループは、オフデバイスリザーバに含まれる大きな量(数百マイクロリットル〜ミリリットル)からの小滴をDMFデバイス上に小出しすることができることを実証した。しかし、これは、難題に部分的にしか対処していない。理由は、さらなる操作に十分な分析対象物を収集するための何百または何千もの小滴のオンデバイス処理は、多くの場合、現実的な戦略ではないからである。DMFデバイスへの導入の前のオフデバイス濃縮が試薬のための良好な解決手段となることができる。理由は、概して、試薬は、何百または何千もの反応における使用のために大きなバッチで調製されるからである。しかし、試料分析対象物のオフデバイス濃縮は試料ごとに独立して実施されなければならず、かなりの手作業(またはロボットシステム)を必要とし、試料の汚染および試料への作業員の暴露の危険を招く。
概して、本明細書に記載されるものは、試料を取得し、調製し、操作し、分析するためのデバイス、システムなどを含む方法および装置である。たとえば、本明細書に記載されるものは、臨床試料、検査室試料、生物学的試料または化学的試料を取り扱い、分析するのに特に役立ち得るデジタルマイクロ流体(DMF)デバイス、流体適用・抽出装置、およびそれらの使用方法である。方法および装置は、比較的大量の流体を取り扱う場合に特に有用であり得る。装置、デバイス、システムおよび方法は、任意の濃度の分析対象物を含有する試料とともに使用され得るが、比較的希薄な試料を取り扱い、分析する場合(たとえば事前の試料濃縮なしで)に特に有用であり得る。本明細書に記載されるDMF装置、システム、デバイスおよび方法は、本明細書に記載される流体適用・抽出デバイスおよび方法とともに使用されてもよいし、単独で、または他のデバイス、たとえば他の流体適用・抽出デバイスとともに使用されてもよい。同様に、本明細書に記載される流体適用・抽出デバイスおよび方法は、本明細書に記載されるDMF装置、システム、デバイスおよび方法とともに使用されてもよいし、単独で、または他のデバイス、たとえば他のDMFデバイスとともに使用されてもよい。本明細書に記載される装置、デバイス、システムおよび方法は概して、別個の液体試料(滴/小滴)の操作を含む。いくつかの局面においては、液体(滴/小滴)を作製し、輸送するために回路が利用される。いくつかの他の局面においては、別個の液体試料を装置、デバイス、システムなどに通して動かすための正圧または負圧を選択的に加えるために、制御可能な圧力源(たとえばポンプ)が利用される。制御可能な圧力源はまた、大量の流体の効果的な試料混合のためにも利用され得る。本明細書に記載される装置、デバイスおよび方法は、分析対象物濃縮、輸送、反応または分析の任意の段階で、たとえば試料(たとえば細胞試料、血液または血漿試料を含む組織試料、生検試料、細菌、酵母、唾液試料、スワブなど)から分析対象物を抽出し、試料からの分析対象物、たとえばRNA、DNA、タンパク質(抗体を含む)、小さな化学的分子、小さな有機分子、薬物などの分析対象物を富化または精製もしくは部分的に精製し、他のハイブリダイゼーション反応、たとえばRNA−DNA、RNA−RNA、抗体−DNAハイブリダイゼーションを実行し;他の分析、たとえばPCR、酵素的タンパク質分析、イムノアッセイ、DNA配列決定における工程のいくつかまたはすべてを実行するために使用され得る。
本明細書に記載されるものは、少量〜中間量(または大量)の流体試料、たとえば臨床試料、検査室試料、生物学的試料または化学的試料を取り扱い、操作する場合に有用な試料取り扱いシステムである。システムは、(たとえば、分析を容易に実行するのに十分な物質を得るために)比較的大量の試料が望まれる液体媒体中の希薄な試料を取り扱う場合に特に有用であり得る。システムは、臨床試料、検査室試料、環境試料または他の試料から分析対象物を抽出および精製する場合に有用であり得る。システムは、複数の取り扱い工程、たとえば複数の洗浄またはインキュベーション工程を要する試料を操作する場合に有用であり得る。操作は、たとえば、洗浄バッファを加える操作、使用済みバッファを試料から除去する操作、磁気粒子を加える操作などを含み得る。
現実試料の処理における本明細書に記載されるシステムの有用性を評価するために、本発明者らは、TaqMan(登録商標)miRNA ABC精製キット(Thermo Fisher Scientific)の実現を通してヒト血漿からのmiRNAの抽出および精製に尽力した。図4A〜4Fに示すように、血漿試料(50μL)を第一の管(たとえば試料調製管)からシステムの精製モジュール(すなわちDMFプラットフォーム)に導入し、その後、溶解バッファ(100μL)およびmiRNA結合ビーズ(80μL)と混合した。いずれの場合も、試薬混合物を精製モジュールから移送導管中に吸引したのち、2つの位置の間でボーラスを高速で往復運動させて徹底的に混合した(フレーム1〜3)。ビーズを溶解物中のmiRNAに結合させたのち(35℃で40分間のインキュベーション)、インタフェース区域の下に外部磁石を係合させながら反応混合物(総量150μL)を精製モジュールと移送導管との間で3回交換して、ビーズをDMFデバイスの表面に固定化することによって反応混合物から回収した。反応混合物流体を廃棄物チャンバ中に吸引し、エレクトロウェッティングを介して精製モジュールによってインタフェースに送られる洗浄1バッファ100μLを使用してビーズを再構成した(フレーム4)。ビーズを洗浄1バッファ中に解放し(磁石を解除することにより)、流体を精製モジュールと移送チューブとの間で往復運動させて、ビーズ結合miRNAを徹底的に洗浄した。ビーズを表面上に回収し(磁石を再び係合させることにより)、流体を廃棄物チャンバに送ったのち、洗浄2バッファ100μLを使用して洗浄サイクルを3回繰り返した(フレーム5)。最後に、溶出バッファ100μLを使用してサイクルを繰り返した(フレーム6)(70℃で3分間のインキュベーション)。本発明者らは、得られた試料のRT-qPCR分析を使用して、その処理物を、血漿miRNAのベンチトップ抽出および精製によって生成されたものに対して定量することにより、本発明者らのDMFシステムの性能を評価した。qPCRの前にcDNA試料を1:20に希釈した。試料によっては、qPCRの前に、cDNA合成後の希釈が重要である。本明細書に記載されるものは、DMFにおける精密な1工程希釈法である。
本明細書に記載される装置(たとえばシステムおよび方法)のいずれも、DMF装置のためのカートリッジの一部として使用されてもよいし、DMF装置のためのカートリッジとして構成されてもよい。
Claims (42)
- 下部および上部を有し、
第一の誘電体層;
第一の誘電体層上の第一の疎水性層;
第一の面および第二の面を有するトッププレート;
該トッププレートの第一の面上の接地電極;
該接地電極を被覆する、該トッププレートの該第一の面上の第二の疎水性層;
該第一の疎水性層と該第二の疎水性層とを離隔する空隙;
該トッププレートの該第二の面上にある、第一の試料コンパートメントおよび第二の試料コンパートメント;
該第一の試料コンパートメントと該空隙との間の第一の開口および該第二の試料コンパートメントと該空隙との間の第二の開口であって、約2cm以下の範囲内で互いに隣接する、第一および第二の開口;
該第一の試料コンパートメントと連通した、第一のポンプ接続のための第一の入口;ならびに
該第二の試料コンパートメントと連通した、第二のポンプ接続のための第二の入口
を含む、
デジタルマイクロ流体工学(DMF)装置のためのカートリッジ。 - 第一の誘電体層が、第一の面および第二の面を有する誘電体材料のシートを含み、該第一の面が、カートリッジの下部の露出した下面を形成しており、第一の疎水性層が該第二の面上にある、請求項1記載のカートリッジ。
- 誘電体材料のシートが可撓性である、請求項2記載のカートリッジ。
- カートリッジの下部が誘電体材料のシートの第一の面によって形成されている、請求項2記載のカートリッジ。
- 第一の疎水性層と第二の疎水性層とを離隔する空隙が、500マイクロメートルを超える離隔距離を含む、請求項1記載のカートリッジ。
- 第一および第二の試料コンパートメントがトッププレート中に形成されている、請求項1記載のカートリッジ。
- 第一および第二の試料コンパートメントがトッププレートの第二の面に沿って延びている、請求項1記載のカートリッジ。
- 第一の試料コンパートメントを被覆するトップカバーをさらに含み、第一の入口が該トップカバーに結合されている、請求項1記載のカートリッジ。
- 第一の試料コンパートメントと空隙との間の第一の開口が第一のマイクロ流体工学チャネルを含み、第二の試料コンパートメントと該空隙との間の第二の開口が第二のマイクロ流体工学チャネルを含む、請求項1記載のカートリッジ。
- 第一および第二の試料コンパートメントがそれぞれ1mlを超える流体を収容するように構成されている、請求項1記載のカートリッジ。
- 第一および第二の試料コンパートメントがそれぞれ25mLまでの流体を収容するように構成されている、請求項1記載のカートリッジ。
- トッププレートがアクリル樹脂材料を含む、請求項1記載のカートリッジ。
- トッププレートの第二の面上に1つまたは複数の試薬リザーバをさらに含む、請求項1記載のカートリッジ。
- トッププレートの第二の面上に1つまたは複数の凍結乾燥試薬リザーバをさらに含む、請求項1記載のカートリッジ。
- 下部および上部を有し、
第一の面および第二の面を有する誘電体材料のシートであって、該第一の面は、カートリッジの該下部の露出した下面を形成している、誘電体材料のシート;
該誘電体材料のシートの該第二の面上の第一の疎水性層;
第一の面および第二の面を有するトッププレート;
該トッププレートの第一の面上の接地電極;
該接地電極を覆う、該トッププレートの該第一の面上の第二の疎水性層;
該第一の疎水性層と該第二の疎水性層とを離隔する、500マイクロメートルを超える離隔距離を含む空隙;
該トッププレートの該第二の面上にある、第一の試料コンパートメントおよび第二の試料コンパートメント;
該第一の試料コンパートメントと該空隙との間の第一の開口および該第二の試料コンパートメントと該空隙との間の第二の開口であって、約2cmの範囲内で互いに隣接する、第一および第二の開口;
該第一の試料コンパートメントと連通した、第一のポンプ接続のための第一の入口;ならびに
該第二の試料コンパートメントと連通した、第二のポンプ接続のための第二の入口
を含む、
デジタルマイクロ流体工学(DMF)装置のためのカートリッジ。 - 下部および上部を有し、
下部誘電体層;
第一の面および第二の面を有するトッププレート;
該トッププレートの第一の面上の接地電極;
該下部誘電体層と該接地電極との間の空隙;
該トッププレートの該第二の面上にある、第一の試料コンパートメントおよび第二の試料コンパートメント;
該第一の試料コンパートメントと該空隙との間の第一の開口および該第二の試料コンパートメントと該空隙との間の第二の開口であって、約2cm以下の範囲内で互いに隣接する、第一および第二の開口;
該第一の試料コンパートメントと連通した、第一のポンプ接続のための第一の入口;ならびに
該第二の試料コンパートメントと連通した、第二のポンプ接続のための第二の入口
を含む、
デジタルマイクロ流体工学(DMF)装置のためのカートリッジ。 - デジタルマイクロ流体工学(DMF)装置のための流体適用・抽出インタフェースデバイスであって、
該デバイスが直立状態に保持されるとき廃棄物チャンバの上方に延びる試料入口を含む流体トラップを含む、廃棄物リザーバ;
該デバイスが直立状態に保持されるとき該試料入口の上方で該廃棄物リザーバを貫通する開口;および
該廃棄物リザーバを貫通して延びる移送導管であって、流体が該移送導管から該廃棄物リザーバの中へと進み該廃棄物チャンバ内に閉じ込められ得るように、該試料入口が該試料入口の基端で該移送導管へ通じている、移送導管
を含み、
該移送導管が、該移送導管の末端で該DMF装置に結合するように構成されている、
デバイス。 - デジタルマイクロ流体工学装置のための流体適用・抽出インタフェースデバイスであって、
該デバイスが直立状態に保持されるとき廃棄物チャンバの上方に延びる試料入口を含む流体トラップを含む、廃棄物リザーバ;
該デバイスが直立状態に保持されるとき該試料入口の上方で該廃棄物リザーバを貫通する開口;
該開口に結合されたコネクタ導管;および
該廃棄物リザーバを貫通して延びる移送導管であって、流体が該移送導管から該廃棄物リザーバの中へと進み該廃棄物チャンバ内に閉じ込められ得るように、該試料入口が該試料入口の基端で該移送導管へ通じている、移送導管
を含み、
該移送導管が、該移送導管の末端で該デジタルマイクロ流体工学装置に結合するように構成されており、該移送導管が該廃棄物リザーバと該移送導管の該末端との間で2回以上折り返している、
デバイス。 - 移送導管と結合するように構成された、DMF装置上の連結器をさらに含む、請求項17または18記載のデバイス。
- 移送導管が廃棄物リザーバと該移送導管の末端との間で2回以上折り返している、請求項17記載のデバイス。
- 移送導管が廃棄物リザーバと該移送導管の末端との間に1つまたは複数のループを含む、請求項17または18記載のデバイス。
- 試料入口の上方で廃棄物リザーバを貫通する開口に結合されたコネクタ導管をさらに含む、請求項17記載のデバイス。
- 移送導管中に正圧または負圧を選択的に加えるためにコネクタ導管または該移送導管のいずれかに結合された制御可能な圧力源をさらに含む、請求項17または18記載のデバイス。
- 移送導管中に正圧または負圧を選択的に加えるためにコネクタ導管または該移送導管のいずれかに結合された蠕動ポンプをさらに含む、請求項17または18記載のデバイス。
- 廃棄物リザーバが、0.5ml〜50mlの容積を有するチューブを含む、請求項17または18記載のデバイス。
- 廃棄物チャンバの容積が0.4〜50mlである、請求項17または18記載のデバイス。
- 移送導管が廃棄物リザーバの底部を貫通して延びている、請求項17または18記載のデバイス。
- 移送導管の内径が約0.5mm ID〜5mmである、請求項17または18記載のデバイス。
- 移送導管が管を含む、請求項17または18記載のデバイス。
- 大きな試料量を取り扱うように構成されたデジタルマイクロ流体工学(DMF)装置であって、デバイスが、
第一の疎水性層を有する第一のプレート;
第二の疎水性層を有する第二のプレート;
該第一の疎水性層と該第二の疎水性層との間に形成された間隙であって、該第一のプレートと該第二のプレートとの間の距離は1mm以上である、間隙;
該第一の疎水性層に隣接した第一の平面中に配置された複数の作動電極;
流体を該間隙の中に適用するまたは取り出すように構成された流体適用・抽出インタフェースデバイスであって、
廃棄物チャンバの上方に延びる試料入口を含む流体トラップを含む、廃棄物リザーバ;
該試料入口の上方で該廃棄物リザーバを貫通する開口;および
該廃棄物リザーバを貫通して延びる移送導管であって、流体が該移送導管から該廃棄物リザーバの中へと進み該廃棄物チャンバ内に閉じ込められ得るように、該試料入口が該試料入口の基端で該移送導管へ通じている、移送導管
を含み、
該移送導管の末端が、該第一のプレート上、該第二のプレート上、または該第一のプレートと該第二のプレートとの間のうちの1つにある開口に結合されている、
流体適用・抽出インタフェースデバイス
を含む、装置。 - 移送導管が廃棄物リザーバと該移送導管の末端との間で2回以上折り返している、請求項30記載の装置。
- 移送導管が廃棄物リザーバと該移送導管の末端との間に1つまたは複数のループを含む、請求項30記載の装置。
- 試料入口の上方で廃棄物リザーバを貫通する開口に結合されたコネクタ導管をさらに含む、請求項30記載の装置。
- 移送導管中に正圧または負圧を選択的に加えるための制御可能な圧力源をさらに含み、該制御可能な圧力源が、試料入口の上方で廃棄物リザーバを貫通する開口に接続されたコネクタ導管、または該移送導管のいずれかに結合されている、請求項30記載の装置。
- 移送導管中に正圧または負圧を選択的に加えるように構成された蠕動ポンプをさらに含む、請求項30記載の装置。
- 廃棄物リザーバが、0.4ml〜50mlの容積を有するチューブを含む、請求項30記載の装置。
- 廃棄物チャンバの容積が0.4〜50mlである、請求項30記載の装置。
- 移送導管が廃棄物リザーバの底部を貫通して延びている、請求項30記載の装置。
- 移送導管の内径が約0.5mm ID〜5mmである、請求項30記載の装置。
- 移送導管が管を含む、請求項30記載の装置。
- デジタルマイクロ流体(DMF)装置から大量の流体を選択的に取り出す方法であって、
該DMF装置の第一のプレートと第二のプレートとの間で流体を流体抽出領域に移動させる工程であって、該第一のプレートおよび該第二のプレートは1mm以上の第一の間隙によって離隔されており、該第一のプレートは複数の作動電極を含む、工程;
該流体抽出領域に結合された、該DMF装置の該第一のプレートと該第二のプレートとの間の移送導管または該DMF装置の該第一のプレートもしくは該第二のプレートを貫通する開口への移送導管に、負圧を加える工程;
該流体の全部または一部分を抜き取って該移送導管に入れ、2回以上折り返す反転経路に沿って該移送導管中を通過させ、流体トラップの試料入口から出し、該試料入口の下方の廃棄物チャンバに入れる工程;および
該複数の作動電極のサブセットにエネルギーを供給して、小滴を、該第一の間隙の間から、1mm未満である距離によって離隔されている該第一のプレートと第二のプレートとの間の第二の間隙へと移動させる工程
を含む、方法。 - 第一の疎水性層を有する第一のプレート;
第二の疎水性層を有する第二のプレート;
第三の疎水性層を有する第三のプレート;
該第一の疎水性層と該第二の疎水性層との間に形成された、1mm以下である第一の空隙;
該第一の疎水性層と該第二の疎水性層との間に形成された、1mmを超える第二の空隙であって、該第一および第二の空隙は連続的であり、該第二および第三のプレートは互いに重なっている、第二の空隙;
該第一の空隙から該第二の空隙まで延びている、該第一の疎水性層に隣接する複数の作動電極;および
該第一第二の空隙中の流体抽出領域
を含む、エアマトリックスデジタルマイクロ流体(DMF)装置。
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2017
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WO2018126082A1 (en) | 2018-07-05 |
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