JP2017227657A - 生体分子を特徴付けるためのナノ細孔センサー - Google Patents
生体分子を特徴付けるためのナノ細孔センサー Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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Abstract
【解決手段】本発明において、ナノ細孔を含む膜により生体分子パラメーターを特徴付けるための方法及び装置、並びにまた本明細書で示された方法及び装置で用いることができる装置の製造方法が提供される。ナノ細孔膜は、導電層及び誘電体層の多層スタックであり、組込まれた導電層又は導電層ゲートは、生体分子が移動するナノ細孔中及びその周辺によく制御された測定可能な電界を付与する。一態様において、導電層はグラフェンである。
【選択図】 図22
Description
で示される回転半径で高次コイル状のボール形であると推測される。ナノ細孔中に捕捉されると、(ii)パートで示されるように伸長及びねじ切り過程が起こる。図3bは、それに対応する、1MのKCl、10mMトリス、1mMのEDTA(pH10.4)中で、400mVの印加電圧でλ−DNAが直径11.3nmの細孔を通過するときにλ−DNAにより誘導された電流遮断を説明する。これらの実験で用いられるλ−DNA濃度は、100ng/μlである。高いpHの緩衝液を用いて、ナノ細孔底部のグラフェン表面と負電荷を有するdsDNA分子との静電的相互作用を最小化する。さらに、Al2O3はこのpH値では負電荷を有するため(Al2O3の等電点は8〜9である)、DNAとは静電的に結合しないと予想されることに留意することも重要である。従って、これらの実験条件により、繰り返し可能なグラフェン−Al2O3ナノ細孔を通るDNAの移動が起こる。
Claims (46)
- 生体分子パラメーターを特徴付けるための方法であって、
導体−誘電体スタックを含む膜中にナノ細孔を設けるステップであって、前記膜が、第一の流体区画と第二の流体区画とを隔てており、前記ナノ細孔が、前記第一の流体区画と前記第二の流体区画とを流体接続しており、前記導体が、グラフェン、又は二硫化モリブデン(MoS2)、ドープシリコン、シリセン若しくは極薄金属の原子レベルの薄さの導電層を含む、ステップと、
前記第一の流体区画に生体分子を供給するステップと、
前記膜全域に電界を印加するステップと、
前記印加された電界下で、前記生体分子を前記ナノ細孔を通って前記第二の流体区画に推進させるステップと、
生体分子がナノ細孔を通過するときに、膜全域で、又は膜によって形成された面で電気的パラメーターをモニターし、それにより前記生体分子パラメーターを特徴付けるステップと、
を含む、方法。 - 前記生体分子パラメーターが、
ポリヌクレオチド配列、
改変されたヌクレオチドの存在、
ポリヌクレオチドのメチル化状態、
ポリヌクレオチドのヒドロキシメチル化状態、
ポリヌクレオチド配列上の1つ又は複数のメチル化又はヒドロキシメチル化部位に結合した、メチル依存性又はヒドロキシメチル依存性の結合タンパク質、
タンパク質−ポリヌクレオチド結合イベントの存在、
ポリペプチド配列、
生体分子の2次構造、及び
アミノ酸配列
からなる群より選択される、請求項1に記載の方法。 - 前記ポリヌクレオチドが、有機又は合成核酸を含む、請求項2に記載の方法。
- 前記導体−誘電体スタックが、隣接するグラフェン層が誘電体層で隔てられている複数のグラフェン層を含む、請求項1に記載の方法。
- 前記グラフェン層の1つ又は複数が、グラフェンマイクロリボン、グラフェンナノリボン、又はグラフェンナノギャップを含み、これを前記ナノ細孔が、前記グラフェンマイクロリボン、グラフェンナノリボン、又はグラフェンナノギャップの長軸方向に対して横方向に通っており、前記方法が、
前記生体分子が前記ナノ細孔を通過する間の、前記グラフェンマイクロリボン若しくはグラフェンナノリボンの、又は前記グラフェンナノギャップの電位又は横方向電流の経時変化を測定し、それにより前記生体分子の配列、組成、流動、持続時間又は長さを特徴付けるステップをさらに含み、前記測定ステップが、移動する生体分子の異なる位置での同時多重測定である、請求項4に記載の方法。 - 前記導電層の1つ又は複数に独立して電気的にバイアスをかけて、前記ナノ細孔の電気的なゲーティングを行うステップをさらに含む、請求項1〜5のいずれか一項に記載の方法。
- 前記バイアスをかけるステップが、グラフェン−誘電体スタックに組込まれた個々のグラフェン層に電極を電気的に接続することによってなされ、前記バイアスをかけるステップが、膜全域に印加された電界によって生じたナノ細孔中の電界を改変する、請求項6に記載の方法。
- 前記誘電体層が、酸化アルミニウム、酸化タンタル、酸化チタン、二酸化ケイ素、酸化ハフニウム、酸化ジルコニウム、窒化ホウ素、窒化ケイ素、それらのナノ積層体、又はそれらの任意の組み合わせを含む、請求項1〜5又は請求項7のいずれか一項に記載の方法。
- 前記電気的パラメーターが、
ナノ細孔を通る電流又は電流遮断、
ナノ細孔のトンネル電流、
横方向の電極を通る電気化学的電流、
コンダクタンス、
抵抗、
インピーダンス、
電位、
前記ナノ細孔を通る前記生体分子の移動時間、及び
生体分子の流動又は移動頻度
からなる群のうち1つ又は複数から選択される、請求項1に記載の方法。 - 露出したナノ細孔グラフェン縁に化学成分を取り付けることによって、ナノ細孔中のグラフェンの露出した縁を機能化するステップをさらに含み、前記化学成分が、生体分子の一部への親和性を有し、前記生体分子の前記一部と相互作用する前記化学成分が、前記生体分子が前記ナノ細孔を通過するときにモニターされる電気的パラメーターを変化させる、請求項1に記載の方法。
- 前記化学成分が、
前記生体分子中の配列に結合する配列を有するタンパク質、ポリペプチド、及びポリヌクレオチド、並びに
ポリヌクレオチドである前記生体分子中の特異的なヌクレオチドへの結合親和性を有する化学的構築物
からなる群より選択される、請求項10に記載の方法。 - 前記生体分子中の前記特異的なヌクレオチドが、前記化学成分と前記特異的なヌクレオチドとの親和性を強化する重原子、化学官能基、又はタグで標識されている、請求項11に記載の方法。
- ポリヌクレオチド配列を有する生体分子と、グラフェン−誘電体スタックに固定されたエキソヌクレアーゼとを接触させることによって、前記生体分子を消化して複数のより小さい配列にし、それにより、消化による配列決定を行うステップをさらに含む、請求項1に記載の方法。
- 前記複数のより小さい配列の少なくとも一部が、個々の塩基に相当する、請求項13に記載の方法。
- 前記ナノ細孔を通過している前記生体分子にヌクレオチドを付加することによってポリヌクレオチド配列を合成し、それにより、合成による配列決定を行うステップをさらに含む、請求項1に記載の方法。
- 前記合成による配列決定が、前記グラフェン−誘電体スタックに固定されたポリメラーゼによってなされ、前記付加されるヌクレオチド及び試薬が、前記第一の流体区画又は前記第二の流体区画におけるヌクレオチド源のものである、請求項15に記載の方法。
- 前記合成による配列決定が、前記細孔を通過している生体分子にヌクレオチドが付加される間に放出されたH+又はピロリン酸を検出するステップをさらに含む、請求項16に記載の方法。
- 前記放出されたH+又はピロリン酸を検出するステップが、ナノ細孔電流の変化を測定することによってなされる、請求項17に記載の方法。
- 前記ナノ細孔が、前記ナノ細孔である内部チャネルを有するタンパク質複合体を含む生物学的なナノ細孔を含み、前記タンパク質が、転写因子、ポリメラーゼ、ヌクレアーゼ、及びヒストンを含むDNA結合タンパク質、アルファ溶血素、マイコバクテリウム・スメグマチスのポリンA及びGP10からなる群より選択される、請求項1に記載の方法。
- 最上部のグラフェン層が、前記第一の流体区画及び/又は前記第二の流体区画中で流体中にあり流体と電気的に接触しており、前記電気的パラメーターが、生体分子が前記細孔を通過するときに、前記第一の流体区画及び/又は前記第二の流体区画中で流体中にあり前記流体と電気的に接触しているグラフェン層によって電気化学的に測定されるものである、請求項1〜5のいずれか一項に記載の方法。
- 前記電気的パラメーターが、前記流体区画中及び前記ナノ細孔中の流体から電気的に絶縁されたグラフェン層による電界効果ゲーティング又は電界効果感知によって測定される、請求項1〜5のいずれか一項に記載の方法。
- 前記生体分子が、二本鎖ポリヌクレオチド配列であり、前記方法が、二本鎖ポリヌクレオチド配列をほどき、二本鎖ポリヌクレオチド配列の一本鎖を前記ナノ細孔を通して推進させ、それにより、前記生体分子の配列決定を行うステップをさらに含む、請求項1に記載の方法。
- 前記ほどくステップが、グラフェン−誘電体スタックに固定されたヘリカーゼによってなされる、請求項22に記載の方法。
- 埋込みグラフェン層が、別のグラフェン層を通る電気化学的電流を測定する、請求項4に記載の方法。
- 膜であって、
第一の表面及び前記第一の表面と反対側の第二の表面であり、前記膜が、前記第一の表面を備える第一の流体区画を前記第二の表面を備える第二の流体区画から隔てている、第一の表面及び第二の表面、
前記第一の表面と前記第二の表面との間に配置されたグラフェン/誘電体/グラフェン/誘電体スタック、並びに
前記第一の区画と前記第二の区画とを流体接続する、前記膜を通るナノ細孔
を備える膜と、
前記膜と電気的に接触して、前記第一の流体区画と前記第二の流体区画との間に電位差をもたらす電源と、
前記第一と第二の流体区画との間の印加される電位差の下で生体分子が前記ナノ細孔を通過するときに前記ナノ細孔を通る電流を検出するための検出器と
を具備する、生体分子パラメーターを特徴付けるための装置。 - 1つ又は複数のゲート電極をさらに具備し、前記1つ又は複数のゲート電極がそれぞれ、前記スタック中のグラフェン層である、請求項25に記載の装置。
- 前記グラフェン層が、前記ナノ細孔のところで3nm以下の厚さを有し、前記電気的な接触部が、前記グラフェン層と電気的に接触した金属パッドを備え、前記金属パッドが、前記第一及び第二の流体区画のいずれもから電気的に隔離されている、請求項26に記載の装置。
- 前記ゲート電極が、前記電源によって電力が供給されるソース電極に電気的に接続されている、請求項27に記載の装置。
- 前記グラフェン層の1つ又は複数がナノリボンを含み、これを前記ナノ細孔が、前記ナノリボンの長軸に対して横方向に通過する、請求項25〜28のいずれか一項に記載の装置。
- 前記ナノリボンが、生体分子が前記ナノ細孔を通過する間に前記ナノリボンの横方向電流を測定するための電気的な接触部をさらに備え、前記グラフェン層の別の層が、前記グラフェン層に電気的にバイアスをかけるためのゲート電極に接続されている、請求項29に記載の装置。
- 前記ナノ細孔が、前記ナノリボンの幅の5%より大きい、又は前記ナノリボンの幅の5%〜95%の範囲から選択される直径を有する、請求項29に記載の装置。
- 前記検出器が、互いに向かい合い、前記ナノ細孔中の生体分子の通過方向に対して横方向に前記ナノ細孔内の中心に配置されたグラフェン電極対を備えるトンネル検出器であり、生体分子が前記電極対の間を通過する、請求項25に記載の装置。
- 生体分子パラメーターを特徴付けるためのナノ細孔を含む膜を製造する方法であって、
前記自立型誘電体膜中に通路を形成するステップと、
前記化学蒸着法でグラフェン層を成長させるステップと、
前記自立型誘電体膜に前記グラフェン層の一部を移動させるステップと、
前記移動したグラフェン層上に誘電体層を形成するステップと、
前記グラフェン層のステップを繰り返して、第二のグラフェン層を作製するステップと、
前記誘電体層を形成するステップを繰り返して、第二の誘電体層を作製するステップと、
前記グラフェン層及び誘電体層それぞれを通る、前記膜の第一の表面から前記膜の第二の表面に伸長するナノ細孔を形成し、それによりグラフェン/誘電体/グラフェン/誘電体スタック中にナノ細孔を含む膜を作製するステップと、
を含む、方法。 - 前記第一のグラフェン層、前記第二のグラフェン層又は前記第一のグラフェン層及び前記第二のグラフェン層の両方を、電気的な接触部と独立して電気的に接触させて、電気的にゲーティングされたナノ細孔を形成するステップをさらに含む、請求項33に記載の方法。
- 前記繰り返しステップを繰り返して、隣接するグラフェン層が誘電体層で隔てられた3つ以上のグラフェン層を作製する、請求項33に記載の方法。
- 前記グラフェン層の1つ又は複数を電気的な接触部と電気的に接触させて、電気的にゲーティングされたナノ細孔を形成するステップをさらに含む、請求項35に記載の方法。
- 前記ナノ細孔膜中にゲート電極を組込んで、前記ナノ細孔中の又は前記ナノ細孔に隣接する局所電界を改変するステップをさらに含む、請求項33に記載の方法。
- 前記組込まれたゲート電極が、前記膜のグラフェン層のいずれかを含む、請求項37に記載の方法。
- 前記誘電体層が、原子層堆積により堆積させた誘電体を含む、請求項33に記載の方法。
- 前記誘電体層が、酸化アルミニウム、酸化タンタル、酸化ハフニウム、酸化ジルコニウム、二酸化ケイ素、若しくは窒化ケイ素、又はそれらの組み合わせを含む、請求項33又は39に記載の方法。
- 前記ナノ細孔で電気的パラメーターを測定するためにホイートストンブリッジ配置で前記グラフェン層を電気的に接続するステップをさらに含み、前記電気的パラメーターが、差動インピーダンス、トンネル電流、抵抗、キャパシタンス、電流又は電圧のうち1つ又は複数である、請求項33に記載の方法。
- AC電圧シグナルで1つ又は複数のグラフェン層に電気的にバイアスをかけるステップをさらに含む、請求項33に記載の方法。
- 中央のグラフェン層と外部グラフェン層との間のインピーダンスをモニターするステップ、又はグラフェン層間のインピーダンス電流又は電圧を測定するステップをさらに含む、請求項42に記載の方法。
- 生体分子のメチル化又はヒドロメチル化の状態を同定する、特徴付ける、又は定量するための方法であって、
第一の流体区画を第二の流体区画から隔てている浮遊膜中にナノ細孔を設けるステップであり、前記膜が、酸化アルミニウム、酸化タンタル、酸化チタン、二酸化ケイ素、酸化ハフニウム、酸化ジルコニウム、窒化ホウ素、窒化ケイ素、グラフェン若しくはそれらのナノ積層体、又はそれらの任意の組み合わせを含む、ステップ、
特異的なタンパク質、オリゴヌクレオチド又は化学的タグを、標的生体分子上のメチル化又はヒドロキシメチル化部位に結合させるステップ、及び
前記第一の流体区画から前記第二の流体区画まで前記膜全域に電界を印加して、生体分子を前記ナノ細孔を通して推進させるステップ
を含む方法。 - 前記生体分子上の前記結合したタンパク質又はタグが、イオン電流、トンネル電流、電圧、コンダクタンス又はインピーダンスの変化をモニターすることによって検出される、請求項44に記載の方法。
- 生体分子が前記ナノ細孔を通過するときに、結合したタンパク質又はタグを生体分子から連続的に切り離すステップをさらに含む、請求項44に記載の方法。
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US20190178840A1 (en) | 2019-06-13 |
JP6679106B2 (ja) | 2020-04-15 |
WO2013016486A1 (en) | 2013-01-31 |
JP2014521956A (ja) | 2014-08-28 |
KR102023754B1 (ko) | 2019-09-20 |
CN104011866A (zh) | 2014-08-27 |
KR20140046471A (ko) | 2014-04-18 |
US20140174927A1 (en) | 2014-06-26 |
EP2737536A4 (en) | 2015-03-18 |
CN104011866B (zh) | 2017-06-09 |
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