JP6906542B2 - 試料の特性評価のためのデバイス及び方法 - Google Patents
試料の特性評価のためのデバイス及び方法 Download PDFInfo
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Description
本出願は、それぞれ「Devices,Methods,and Kits for Sample Characterization」と題する、2015年11月30日に出願された米国仮特許出願第62/260,944号、及び2016年5月18日に出願された米国仮特許出願第62/338,074号の非仮出願であって、これらの利益を主張するものであり、これらの各々の開示は、その全体が参照として本明細書に組み込まれる。
(デバイス)
図1は、一実施形態による、自動的に装填された試料の2次元分離及びESIのためのデバイスの概略図である。マイクロ流体ネットワーク100は、基板102によって画定される。基板は、実施される富化ステップと適合する材料から製造される。例えば、材料の選択に関連して、化学的適合性、pH安定性、温度、光の様々な波長における透明性、機械的強度等が考慮される。
チャネル124は、クロマトグラフィ富化ステップ又は電気泳動富化ステップのいずれかを行うために使用することができる。
図6は、一実施形態による、検体混合物富化の方法を示す。この方法は、20において、検体混合物をマイクロ流体デバイスに装填及び/又は導入することを含む。マイクロ流体デバイスは、図1〜図3を参照して上述したマイクロ流体デバイスと同様であり得る。幾つかの実施形態において、検体混合物は、例えば、グリカン、炭水化物、DNA、RNA、インタクトタンパク質、消化されたタンパク質、ペプチド、代謝産物、ワクチン、ウイルス及び小分子であり得る。幾つかの実施形態では、検体混合物は、培養細胞の溶解物、体細胞由来の治療剤、又は腫瘍もしくは他の組織由来の細胞などのタンパク質の混合物、バイオ医薬品を含む組み換えタンパク質、血液由来細胞、灌流又は任意の他のソースからのタンパク質混合物であり得る検体混合物は、デバイスに直接装填することができ、又は複数の混合物の連続分析のためにオートサンプラに装填することができる。
実施形態の態様は、以下の実施例に照らして更に理解することができるが、これらは決して限定するものと解釈すべきではない。
この例では、図4に示すチャネルネットワークは、標準フォトリソグラフィーエッチング技術を用いて、ソーダ石灰ガラスのプレートから作製され、280nmの光に対する非常に低い透過率を有する。富化チャネル418の深さは、ガラス層402の厚みと同じであり、すなわち富化チャネル418は、このガラス板402の上部から底部まで全体を通過している。デバイス400は、デバイス400の一方の側に配置された光源によって照射され、デバイス400の反対側に配置された検出器によって画像化することができる。基板402は不透明であるが、富化チャネル418が光学スリットを画定するので、基板402は、富化チャネル418を通過しない光を遮断し、迷光を遮断し、画像化プロセスの分解能を向上させることができる。
実施例2は実施例1と同様であり得るが、図1を参照して説明する。チャネル116は、C18で誘導体化されたゾルゲルが充填された第1の富化ゾーンであり得る。タンパク質を装填した後、一定量の溶出液(IEF両性電解質及び標準を有するMeCN/H2O)がチャネル116の中に充填されて、ゾルゲルに捕捉された最も疎水性の低いタンパク質を溶出することができる。溶出液は、実施例1に記載したように、IEF、UV吸光度モニタリング及び最後にESIが行われる第2の富化ゾーンであり得るチャネル124に向けられる。第1の溶出液のESIがいったん完了すると、次に一定量のより高い濃度のMeCNが使用され、2番目に低い疎水性を有するタンパク質画分が溶出される。
実施例3は実施例2に類似であり得るが、生物学的薬物標的誘導体化ビーズをチャネル116の中に充填し、タンパク質を捕捉するために使用することができる。反応の親和性は、溶液相標的(競合的)、塩、pH等による溶出を通して特性評価される。
実施例4は実施例2と同様であり得るが、図5を参照して説明する。タンパク質混合物は、入口521を通って充填され移動し富化ゾーン510に至ることができ、タンパク質混合物は、逆相クロマトグラフィのためにC18で誘導体化されたビーズを含有することができる。装填の間、流体はゾーン510を通過し、視認領域511を通って出口522から出て廃棄物となる。視認領域510は、280nmのUV光に対して不透明なソーダ石灰ガラスでできている内部層を横断し、上部層及び底部層は280nmの光に対して透明な溶融シリカからできている。
噴霧窒素ガスラインは、ポート508及び528でデバイスに接続され、チャネル512及び530を通って移動し、エレクトロスプレーからの材料がオリフィス520を介してデバイスから出る際に、その材料に隣接(flank)する。
この例では、図7のレイアウトによって表されるマイクロ流体チャネル層は、環状オレフィンコポリマーから作製される。同様に述べると、マイクロ流体デバイス800の基板802はチャネルネットワークを画定する。多くの用途、例えば、蛍光検出が使用される用途では、この材料が検体を検出するために必要な波長範囲の光を透過するならば、単一の材料を用いてマイクロ流体デバイス800を製造することができる。
場合によっては、質量分析計インタフェースの有無によって異なるマイクロ流体層の2つの設計を有することが有利である。いったん検体の特性評価がなされると、確認の特性評価は質量分析データなしで行うことができる。確認の特性評価を、ほとんど同じ設計のマイクロ流体で行うことにより、異常が認識されたとき、質量同定のために質量分析計インタフェースを用いてアッセイをチップに戻すことが簡単になる。これにより、さもなければ、確認データの異常が質量分析データで分析されていることを示すために必要な作業を省略することができる。
Claims (14)
- (a)検体混合物を、分離チャネルを含むマイクロ流体デバイスに導入する段階と、
(b)前記分離チャネルの間に電界を印加して、等電点電気泳動により、前記検体混合物を富化された検体のフラクションに分離する段階と、
(c)前記検体混合物の分離、及び、この後の前記分離チャネルの内部における前記富化された検体のフラクションの移動を、前記マイクロ流体デバイスの透明な部分を介して、画像化する段階と、
(d)分離された検体混合物にシース流体電解質を導入して、前記富化された検体のフラクションの実質的にすべてを、移動させ、前記分離チャネルと直線状に並ぶ単一のオリフィスからエレクトロスプレーイオン化を介して質量分析計に排出する段階と、
(e)前記(c)における前記分離チャネルの画像化により検出された特定の富化された検体のフラクションに対する吸収ピークと、該特定の富化された検体のフラクションに対する質量分析計のデータと、を関連付ける段階と、
を含む、
ことを特徴とする方法。 - 前記マイクロ流体デバイスが、第1の分離チャネル及び第2の分離チャネルを含む、請求項1に記載の方法。
- 前記電界を印加して前記第2の分離チャネルにおいて等電点電気泳動による前記検体混合物の分離を実行する前に、前記第1の分離チャネルにおいて前記検体混合物をクロマトグラフィにより分離する段階、をさらに含む、請求項2に記載の方法。
- 前記富化された検体のフラクションの実質的にすべてが連続した流れとなって前記オリフィスから排出される、請求項1に記載の方法。
- 両性電解質及び等電点(pI)マーカを、前記分離チャネルに導入して該分離チャネルにおけるpH勾配を生成しかつ該分離チャネルにおけるpI範囲をマッピングする前に、前記検体混合物と混合する段階、をさらに含む、請求項1に記載の方法。
- 前記検体混合物がインタクトタンパク質を含む、請求項1に記載の方法。
- 前記シース流体電解質の導入は、前記分離チャネルの下流にある合流領域に流体結合された電解質チャネルから、シース流体電解質溶液を流すことにより、実行される、請求項1に記載の方法。
- イオンポテンシャルの補充が、前記シース流体電解質の導入によって前記マイクロ流体デバイスに生ずる、請求項1に記載の方法。
- 前記マイクロ流体デバイスが、前記分離チャネル、前記オリフィス及び前記シース流体電解質のチャネルを含む、請求項7に記載の方法。
- 前記分離チャネルと前記シース流体電解質のチャネルとが前記合流領域において交差する、請求項9に記載の方法。
- 前記合流領域が、前記分離チャネルに印加される前記電界の内部に含まれる、請求項10に記載の方法。
- 前記合流領域が、前記分離チャネル及び前記オリフィスと直線状に並ぶ、請求項10に記載の方法。
- 前記マイクロ流体デバイスが、シース流体電解質のチャネルに沿って電界を発生させる2つの電極を含む、請求項1に記載の方法。
- 前記マイクロ流体デバイスが、検体を導入するチャネルとイオン化のための噴霧ガス搬送チャネルとをさらに含む、請求項9に記載の方法。
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JP2023139239A (ja) | 2023-10-03 |
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