JP2018538535A - 検知のための方法及びシステム - Google Patents
検知のための方法及びシステム Download PDFInfo
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Abstract
Description
本出願は、2015年12月9日に出願された、米国特許仮出願第62/264,913号及び同第62/264,944号の両方の優先権を主張するものであり、これらの内容の全体が参照により本明細書に組み込まれる。
標的分子を検出するように構成された検知コンパートメントと、
複数のチャンバであって、それぞれが対応するマイクロチャネルと、当該チャネル上に装着された対応する弁とを介して、前記検知コンパートメントと制御可能に流体連通している複数のチャンバと、
少なくとも2つの前記チャンバから前記検知コンパートメントへの流体の流れを制御するように、それぞれの弁を選択的に操作するためのコントローラと、を具備する。
本発明のいくつかの実施形態の態様によれば、検知コンパートメントと制御可能な流体連通にある少なくとも1つのチャンバを具備したシステムが提供される。
ここで図面を参照すると、図9は、本発明のいくつかの実施形態による検知システム10の概略図である。
システム10はまた、チャネル22と、チャネル22上に装着された弁24とを介して、検知コンパートメント12と制御可能に流体連通している1つ以上のチャンバ20も備え得る。複数のチャンバが使用される場合、各チャンバは対応するチャネル22と、対応するチャネル22上に装着された対応する弁24とを介して、同じ検知コンパートメント12と制御可能に流体連通していてもよい。本発明のいくつかの実施形態では、2つ以上の検知コンパートメント12などの検知コンパートメントが使用され、この少なくとも2つのチャンバは、異なる検知コンパートメントと制御可能に流体連通している。本発明のいくつかの実施形態では、異なるチャンバにより供給される検知コンパートメント間に流体連通は存在せず、本発明のいくつかの実施形態では、異なるチャンバにより供給される検知コンパートメント間に流体連通が存在する。システムが2つ以上の検知コンパートメントを備える実施形態の代表的な例を後に提供する。
図11A〜Cは、本発明のいくつかの実施形態による、2つ以上の検知コンパートメント12を備えたシステム200の概略図である。本発明のいくつかの実施形態では検知コンパートメントは同じ基板の異なる領域上に形成され、本発明のいくつかの実施形態では検知コンパートメントは異なる基板上に形成されている。
本明細書に記載される検知システムとして、1つ以上の検知コンパートメント内にSiNW(シリコンナノワイヤー)FETアレイを使用する例を示した。本検知システムでは、シリコンナノワイヤーの少なくともいくつかが、その表面に官能基を導入することにより修飾され、官能基と酸化性物質又は還元性物質を含む試料との接触により、複数のナノワイヤーが電気特性における検出可能な変化を示す。この変化は、例えば、代謝産物等の還元性物質生成物質又は酸化性物質生成物質の存在及び/又は量の指標となる。官能基は、例えば、酸化還元反応を、好ましくは可逆的にできることが可能な基であってもよく、その酸化又は還元の結果、ナノワイヤー上の電子密度が変化する。
Xは、水素、又は、好ましくは金属原子若しくはイオンであり、追加の1つ以上の同じ又は異なってもよいフェロセン部分で更に置換されていてもよい。なお、複数のフェロセン部分が存在する場合、酸化還元反応でフェロセン部分の数に対応する電子移動が行われる。
本明細書に記載した検知システムの任意の1つ、及び検知システムの実施形態の任意の1つに関して、検知システムは、本明細書に記載されるナノ構造体を含む複数の、同じ又は異なってもよい検知コンパートメントを備えてもよい。
本明細書に記載される任意の実施形態のうちのいくつかの態様によれば、標的分子の検出方法が提供される。
ラクテートオキシダーゼの触媒する反応においてH2O2を生成する乳酸塩;
グルコースオキシダーゼの触媒する反応においてH2O2を生成するグルコース;
無機リン酸塩及び酸素の存在下でH2O2を生成するピルビン酸塩;
キサンチンオキシダーゼの触媒する反応においてH2O2を生成するヒポキサンチン;
NAD(P)Hオキシダーゼの触媒する反応において超酸化物を生成するNAD(P)H;
スーパーオキシドジスムターゼの触媒する反応においてH2O2を生成する超酸化物(O2 −)、
対応するアルデヒドオキシダーゼの触媒する反応においてH2O2を生成するアルデヒド、
コリンオキシダーゼによって触媒される反応でH2O2を生成する、コリンが挙げられる。
本明細書に記載される検知システムの任意の1つに導入される試料は、例えば、本明細書に記載するように、標的部分や、標的部分を生成する物質を含有する溶液であってもよい。
本明細書に記載される検知方法は、多様な診断及び療法の用途に使用することができる。
癌疾患としては、癌腫、リンパ腫、芽細胞腫、肉腫及び白血病が挙げられるが、これらに限定されない。癌疾患の特定の例としては、これらに限定されるものではないが、以下のものがあげられる:慢性骨髄性白血病、成熟を伴う急性骨髄性白血病、急性前骨髄球性白血病、好塩基球の増大を伴う急性非リンパ球性白血病、急性単球性白血病、好酸球増加症を有する急性骨髄単球性白血病等の骨髄性白血病;バーキット型非ホジキン悪性リンパ腫等の悪性リンパ腫;急性リンパ性(lumphoblastic)白血病、慢性リンパ性白血病等のリンパ性(Lymphoctyic)白血病;固形腫瘍良性髄膜腫、唾液腺の混合腫瘍、結腸腺腫等の骨髄増殖性疾患;小細胞肺癌、腎臓癌、子宮癌、前立腺癌、膀胱癌、卵巣癌、結腸癌、肉腫、脂肪肉腫、粘液性肉腫、滑膜肉腫、横紋筋肉腫(alveolar)、骨外性粘液型軟骨肉腫(chonodrosarcoma)、ユーイング腫瘍等の腺癌。その他には、精巣及び卵巣の未分化胚細胞腫、網膜芽細胞腫、ウィルムス腫瘍、神経芽腫、悪性黒色腫、中皮腫、乳房、皮膚、膵臓、子宮頸部、前立腺、及び卵巣の腫瘍が挙げられる。
(a)上述した方法に従って、被験者における疾病の存在を診断し;
(b)当該診断に基づいて被験者を治療する。
被験体から得た、少なくとも1種の医薬と共に細胞を含む生物学的試料について、本明細書に記載されるいずれかの関連方法(その態様も含む)から選ばれた任意の1種を用いて、試料中の代謝産物の存在及び/又は量を決定することを含み、
(本明細書に記載されるように、1種以上の代謝産物のレベルを測定して得られた)細胞の代謝活性における、同一条件下で試験された正常かつ健康な細胞試料の代謝活性に近づくようなシフトは、疾病に効果的な医薬であるという指標となる。
(a)疾病に対する少なくとも1種の医薬を被験体に投与し;
(b)投与後、被験体の細胞を含む生物学的試料を回収し;
(c)当該生物学的試料を、本明細書に記載される実施形態の任意の1種による検知システムに導入し;
試料中の1種以上の代謝産物のレベルを決定する。
(a)細胞を薬剤に暴露し;
(b)薬剤に暴露する前と後の細胞の代謝活性を測定し、1種以上の代謝産物のレベルのシフトは、薬剤が代謝活性を変更することが可能であることの指標となる。
(a)疾病に対する少なくとも1種の医薬を被験体に投与し;
(b)前記投与後に、被験体の細胞を含む生物学的試料を回収し;
(c)前記細胞の細胞外環境において酸化剤又は還元剤を生成する代謝産物の存在及び/又は量を、本明細書に記載される実施形態の任意の1種に記載される任意の検知方法及びシステムを使用して、モニタリングする。
「具備する(comprise)」、「具備している(comprising)」、「含む(include)」「含んでいる(including)」、「有している(having)」及びそれらの活用形は、「含むが、限定されない」ことを意味する。
システムの製作
代謝産物の多重リアルタイムモニタリング用のナノワイヤーバイオセンサとして使用され得る、本発明のいくつかの実施形態に係る例示的なマイクロ流体バイオ検知システム(本明細書でマイクロ流体アレイ又はチップとも称される)を、図1A及び1Bに示す。
検知コンパートメントの主要部であるSiNW−FETアレイをフォトリソグラフィーにより製作した。FETのソース電極及びドレイン電極を、LOR5A(Microchem)及びS1805(Shipley社製)からなる多層フォトレジスト構造により作成した。FETのソース電極とドレイン電極との間の間隙は、2μmであった。フォトレジストの暴露及び現像後、パターンをTi/Pd/Ti(それぞれ、5/60/10nm)の電子ビーム蒸着により金属化した。その後、80℃でのプラズマ増強化学蒸着(ICP−PECVD,Axic Inc.社製)により、蒸着したSi3N4の60nmの層と、原子層堆積(ALD)により作製した20nmのアルミナ層(Savannah 200システム、Cambridge Nanotech社製)とにより電極を絶縁した。
SiNW FETアレイの製作後、細胞代謝産物を検知するために(例えば図2に示すように)チップを化学的に修飾した。
図2の差し込み図に示すように、9,10−アントラキノン−2−スルホン酸ナトリウムのスルホネート基を、トルエン中で塩化オキサリル及びDMFを使用してスルホン酸クロリドに変換した。
作成したSiNW FETアレイチップをアセトン、イソプロピルアルコール(IPA)、及び脱イオン水(DIW)の順で洗浄し、その後、窒素乾燥した。酸素プラズマ(100W、0.2トル)で15分間処理した。その直後、チップをおよそ100μlの(3−アミノプロピル)−ジメチル−エトキシシラン(APDMES;Gelest Inc.社製のSIA0603.0)で10分間覆った。次いで、チップを65℃のホットプレート上に2時間静置した。その後、チップ表面を再度IPAで洗浄し、その後窒素乾燥した。
以下のパラメータを使用する質量分析法(Autospec M250Q,Waters社製)を用いて、表面修飾に使用した9,10−アントラキノン−2−スルホン酸クロリドの元素組成を確認した: 電子衝突の測定モード、70eVで正イオン化、溶媒はCH2Cl2。
図1A及び1Bに示すような、細胞の培養及び複数の溶液の制御のためにソレノイド作動弁を有するPDMS(ポリジメチルシロキサン)培養コンパートメントを、ソフトリソグラフィーを用いて製作した。ソレノイド作動PDMS弁の製作は、Hulme et al. Lab Chip 2009,9(1):79-86に従った。その後、この弁をPDMS培養コンパートメントに組み込んだ。
検知
実験方法:
一般的な検知設定:
検体溶液中の検体の酸化中に検出されるROS又はH2O2、又は検体溶液中の検体の還元中に検出される還元性物質のいずれかの表面電荷により誘導されるSiNW FET(Ids)の電流を、データ収集システムを使用して測定した。
さまざまな濃度のH2O2を含む溶液に関して、データ収集システムを使用して、H2O2による表面電荷により誘導されたSiNW FETの電流を測定した。
乳酸塩の検知のためには、乳酸塩がFETアレイに到達する前に、0.1単位/mlのラクテートオキシダーゼ(LOX;L0638,SIGMA製)をフェノールレッド不含有培地に加えて、(図1Dに示すように)乳酸塩をピルビン酸塩及びH2O2に変換した。
還元性物質添加溶液を使用することによって、修飾FETをpHセンサに変換した。培地に0.1%v/v DEHAを添加して修飾FET表面を還元した後、プロトン化又は脱プロトン化により変動する表面プロトン密度は、測定電流を顕著に変化させる。観察によると、培地への0.1%v/v DEHAの添加によってpHの有意な変化は生じなかった。
SDは、試験したナノワイヤー装置の平均値における変動性の指数と見なされるため、検知特性のデータは平均±SDで表した。
血清添加培地中のH2O2に反応した9,10−ジヒドロキシアントラセン修飾SiNW FETによる検知を、図3A、Bに示す。
小分子代謝産物の検知には、代謝産物をH2O2に変換するオキシダーゼ酵素を使用した。
図5Aに概略的に示されるように、上記の修飾FETは、単に還元性物質を加えることにより、pHセンサへと変換された。培地にDEHAを添加し、修飾単層を9,10−ジヒドロキシアントラセン及びH2O2含有物に還元した後は、プロトン化又は脱プロトン化により変化する表面プロトン密度が、測定電流を顕著に変化させる。
細胞の代謝活性のモニタリング
実験方法:
細胞培養、薬物処理及び生存率評価:
ヒトTリンパ球、Jurkat(TIB−152,ATCC)を、5% CO2雰囲気下にて37℃で培養及びインキュベートした。培地は、10%ウシ胎児血清(FBS;04−001−1A,Biological Industry社製)及び1%ペニシリン/ストレプトマイシン(15140,GIBCO社製)を含むRPMI 1640培地(52400,GIBCO社製)を使用した。
健康なドナーと、慢性リンパ性白血病(CLL)の患者とから末梢血(PB)細胞を得た。
フローサイトメトリー分析により、分析上、93%を超える正常な細胞又はCLL分別細胞がCD19+であることが確認された。
細胞のナノワイヤー検知との比較のために、ジクロロジヒドロフルオレセイン(DCFH)を使用した対照実験を行った。理論的には、ROSによってDCFHは酸化されて蛍光ジクロロフルオレセイン(DCF)となる。
細胞代謝産物に関するデータを、平均±標準誤差(SEM)又は標準偏差(SD)にて示す。加えて、両側2標本t検定を行って、細胞代謝産物に関するデータの有意性を統計的に分析した。
T細胞株を使用した検知:
活性酸素種(ROS)は、酸素の正常な代謝の天然副産物として形成され、多様な生物学的プロセスの調節におけるシグナリング分子としての重要な役割を有する。シグナリング分子として、ROSの1つの重要な特徴は、例えば細胞膜を横切るなど異なるコンパートメント間を移動する能力である。従って、段階的に増大する細胞内ROSは、細胞膜を通って細胞外空間に拡散し、代謝活性を表す指標となりうる。
慢性リンパ性白血病(CLL)細胞及び正常なB細胞の代謝レベルを24時間モニタリングし、代謝有意性を決定した。
電気制御型デバイス
本発明の一部の実施形態に係る酸化還元反応性ナノワイヤセンサデバイスの試作品を作製し、動作させた。
本発明の一部の実施形態に係る、排泄された細胞外代謝物質の非侵襲的モニタリングのためのナノデバイスを作製した。続くシラン処理工程に備えて表面の清浄化及び活性化を行うため、SiNW表面を酸素プラズマを使用して酸化した。その後、SiNWを収容したチップを、乾燥雰囲気下(アルゴン気流下)で(3−アミノプロピル)−ジメチル−エトキシシランで被覆した。これは、デバイ長を考慮すると、生理溶液下での検知に有利である、シラン単層を確実に形成するためである。修飾のために(3−アミノプロピル)−ジメチル−エトキシシランを使用するもう1つの理由は、他のシランに基づく修飾と比較したときの、生成物の生理条件下における長期安定性である。
化学気相蒸着(CVD)によるp型シリコンナノワイヤ(SiNW)の合成
20nm金ナノ粒子(Ted Pella社製)によって、蒸気−液体−固体(VLS)機構によるSiNWの成長を触媒した。これらのナノ粒子を最初にシリコン(100)成長基板上に蒸着させることで、SiNWを成長させる場所を画定した。金ナノ粒子のシリコン基板上への付着を補助するため、ポリ−L−リシン(Ted Pella社製)を最初にシリコンウエハに導入し、静電結合剤として作用させた。100W、0.200Torrの酸素プラズマを5分間印加することによって、有機材料を除去した。次に、ウエハを石英管炉に入れた。ここでは、シラン(SiH4)及びジボラン(B2H6、H2バランスガス中100ppm)をSiNW成長過程における反応物質として使用し、この過程は、1/4000のホウ素/ケイ素比で、ホウ素がp型ドーパントとして働くものであった。SiNW成長速度は、約1μm/minであった。
SiNW−FETアレイをフォトリソグラフィによって作製した(図12及び13A〜13Eを参照)。それぞれ、フォトリソグラフィ及びクロム/金(5/60nm)の蒸着によって外部電極(ゲートを含む)を作製した後、エタノール中への分散及び酸化物層への0.5μlの液滴の滴下により、SiNWをウエハ上に蒸着させた。3インチシリコンウエハ上にp型SiNWを蒸着させ、600nmの熱酸化物層(<0.005ohm/cm、SSP最高グレード、Silicon Quest International社製)で被覆した。FETのソース電極及びドレイン電極を、500nmのLOR5A(Microchem社製)と500nmのS1805(Shipley社製)とからなる多層フォトレジスト構造体によって画定した。ソース電極とドレイン電極との間のギャップは、2μmであった。露光及びMF319現像液中で現像の後、チップを緩衝酸化物エッチング液(1/6のフッ化水素酸/フッ化アンモニウム比)中に6秒間浸漬し、チタン/パラジウム/チタン(5/60/10nm)の電子ビーム蒸着によって即座に金属化した。続いて、電極を、80℃でのプラズマ化学気相蒸着(ICP−PECVD、Axic社製)によって蒸着させた65nmのSi3N4の層と、原子層堆積(ALD)(Cambridge Nanotech社製のSavannah 200 system)によって作った10nmのアルミナの層とで絶縁した。次いで、チップに対しPG除去剤(又はN−メチル−2−ピロリドン)中でリフトオフを行った。高速熱処理装置(RTP:rapid thermal processor)によって、380℃でのフォーミングガス(1/9の水素/窒素比)中のアニーリングを2分間行うことで、FETデバイスの作製を終えた。
図19A及び19Bは、ウォーターゲート構成下におけるp型SiNW FETナノデバイスの電気的特徴付けを示す。図19Aは、様々なゲート電圧(Vg)でのソース−ドレイン間電流対ソース−ドレイン間電圧(Vsd)のプロットである。図19Bは、0.1Vのソース−ドレイン間電圧(Vg)でのソース−ドレイン間電流対ゲート電圧(Vg)のプロットである。
合成したp型SiNW及びSiNWデバイスの品質を、Quanta 200 FEG環境制御型走査電子顕微鏡(ESEM:environmental scanning electron microscope)を使用して分析した。図20A及び20Bは、SiNWの走査電子顕微鏡(SEM)像である。図20Aは、化学気相蒸着システムによってシリコン(100)ウエハ上に合成した20nmのp型SiNWのSEM像であり、図20Bは、ソース電極及びドレイン電極に接続されたSiNWからなるSiNW FETデバイスのSEM像である。これらの像は、合成したナノワイヤの品質が良好であり(図20A)、これらのFETデバイスへの組立てが成功した(図20B)ことを示していている
SiNW FETアレイの作製の後、チップを9,10−アントラキノン−2−スルホクロリドでさらに化学的に修飾して、細胞代謝物質の検知を行った。修飾剤は、以下のように事前に合成した。
9,10−アントラキノン−2−スルホクロリド(SiNWの表面修飾に使用したもの)の質量分析(米国、Waters Corp.社製のAutospec M250Q)を行って、スルホクロリド基の形成を確認した。
X線光電子分光(XPS)測定を、超高真空(2.5×10−10Torr ベース圧力)において行った(Multi−Technique System 5600、PHI社製)。試料にAl Kα単色化光源(1486.6eV)を照射し、生じた電子を、スリット開口0.8mmの球面コンデンサによって分析した。測定中に試料がわずかに帯電していたため、285eVにおけるC1sをエネルギー参照値として、入力を数学的に補正した。すべての測定は、25°の浅い射出角で行った。低エネルギー範囲窓において高分解能で異なるピークを得るため、高分解能多重スペクトルを取得した(パスエネルギー=11.75eV、0.05eV/ステップ)。これらの測定により、結合解析に必要な、高精度のエネルギーの位置付けと、ピーク形状の決定が可能となる。
PDMSを、60℃において、質量比10:1で硬化剤と共に終夜インキュベートした。次いで、得られたデバイスを寸法が10×10×5mmの矩形片に切断した。上流のポリエチレンチューブ(PE 20、Intramedic社製)は、長さ14cm、内径0.38mmであった。下流のTygonチューブ(S−50−HL、Tygon社製)は、長さ13cmであった。
Claims (25)
- 電気特性の変化を検出するためのシステムであって、
ゲート電極と半導体ナノ構造体とを有するトランジスタを備え、前記半導体ナノ構造体は、ソース電極及びゲート電極の間を接続し、前記半導体ナノ構造体に共有結合した官能性部分によって修飾されている検知素子と、
前記ゲート電極に接続された電圧源と、
前記官能性部分が酸化還元反応剤と接触したときに生じる酸化還元反応を逆転させるために、前記電圧源によって前記ゲート電極に印加されるゲート電圧を制御するように構成された制御装置と、
前記ナノ構造体の電気特性の変化を検出する回路と
を具備するシステム。 - 前記官能性部分が酸化還元反応性部分である、請求項1に記載のシステム。
- 前記官能性部分が、酸化数又は酸化状態が可逆的に変化しうる原子を少なくとも1つ有する官能基を少なくとも1つ含む、請求項1又は2に記載のシステム。
- 前記官能性部分がキノンを含む、請求項1に記載のシステム。
- 前記官能性部分がキノンを含む、請求項2又は3に記載のシステム。
- 前記官能性部分が芳香族キノンを含む、請求項1に記載のシステム。
- 前記官能性部分が芳香族キノンを含む、請求項2〜5のいずれか一項に記載のシステム。
- 前記官能性部分が、各々が置換又は非置換である、キノン、ベンゾキノン、アンフラキノン、及びフェナントレンキノンからなる群から選択される官能基を含む、請求項1に記載のシステム。
- 前記官能性部分が、各々が置換又は非置換である、キノン、ベンゾキノン、アンフラキノン、及びフェナントレンキノンからなる群から選択される官能基を含む、請求項2〜7のいずれか一項に記載のシステム。
- 前記電気特性が、前記ナノ構造体の表面上の電子密度又は電荷密度を含む、請求項1に記載のシステム。
- 前記電気特性が、前記ナノ構造体の表面上の電子密度又は電荷密度を含む、請求項2〜9のいずれか一項に記載のシステム。
- 前記ナノ構造体がナノワイヤである、請求項1に記載のシステム。
- 前記ナノ構造体がナノワイヤである、請求項2〜11のいずれか一項に記載のシステム。
- 前記半導体ナノ構造体がケイ素を含む、請求項1に記載のシステム。
- 前記半導体ナノ構造体がケイ素を含む、請求項2〜13のいずれか一項に記載のシステム。
- 前記検知素子が、非酸化還元活性部分によって修飾されたナノ構造体を有する少なくとも1つのさらなるトランジスタを備え、前記検出する回路が、前記非酸化還元活性部分によって修飾された前記ナノ構造体から受信したシグナルから、前記酸化還元活性部分によって修飾された前記ナノ構造体から受信したシグナルを差し引くように構成されている、請求項1に記載のシステム。
- 前記検知素子が、非酸化還元活性部分によって修飾されたナノ構造体を有する少なくとも1つのさらなるトランジスタを備え、前記検出する回路が、前記非酸化還元活性部分によって修飾された前記ナノ構造体から受信したシグナルから、前記酸化還元活性部分によって修飾された前記ナノ構造体から受信したシグナルを差し引くように構成されている、請求項2〜15のいずれか一項に記載のシステム。
- 皮膚用貼付剤に収容された、請求項1に記載のシステム。
- 皮膚用貼付剤に収容された、請求項2〜16のいずれか一項に記載のシステム。
- 生体内に埋込可能な外装体に収容された、請求項1に記載のシステム。
- 生体内に埋込可能な外装体に収容された、請求項2〜16のいずれか一項に記載のシステム。
- 生体内の代謝活性を検知する方法であって、
ゲート電極と半導体ナノ構造体とを有するトランジスタを備え、前記半導体ナノ構造体は、ソース電極及びゲート電極の間を接続し、前記半導体ナノ構造体に共有結合した官能性部分によって修飾されている検知素子を、前記生体と接触させる工程と、
前記接触中に生じる酸化還元反応に起因する、前記ナノ構造体の電気特性の変化を検出する工程と、
前記酸化還元反応を逆転させるように、前記ゲート電極にゲート電圧を印加する工程と
を含む、方法。 - 前記ゲート電圧が、前記ナノ構造体上の前記官能性部分の数をほぼ一定に維持するように選択される、請求項22に記載の方法。
- 前記ゲート電圧が一定である、請求項1〜23のいずれか一項に記載の方法又はシステム。
- 前記ゲート電圧が約−3ボルト〜約+3ボルトである、請求項1〜23のいずれか一項に記載の方法又はシステム。
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