JP4700345B2 - 分子で被覆された電極のアレイとその製造 - Google Patents
分子で被覆された電極のアレイとその製造 Download PDFInfo
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- JP4700345B2 JP4700345B2 JP2004542640A JP2004542640A JP4700345B2 JP 4700345 B2 JP4700345 B2 JP 4700345B2 JP 2004542640 A JP2004542640 A JP 2004542640A JP 2004542640 A JP2004542640 A JP 2004542640A JP 4700345 B2 JP4700345 B2 JP 4700345B2
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Description
本発明は、数本の異なる電極上に、被覆を形成することにより、数種類の異なる被覆分子を有するアレイ製造方法に関する。これらの方法により製造される分子のアレイにも関する。
分子を基礎とするナノテクノロジーの進展に直面する数多くの挑戦では、個々の分子対象物を直接組織化すること、ならびに制御して巨視的構造体に組み込むことが基本である。ある種の分子に特徴的で固有な、選択的自己組織化特性(例えば、相補的なDNA1本鎖の間で生じるワトソン−クリック固有塩基対形成)は、これらの挑戦に取り組むために活用される特性である。例えば、金のナノ粒子の規則的懸濁液は、まず短いDNAオリゴヌクレオチドでナノ粒子を機能化し、次いで個々の粒子を連結するために相補的DNAを導入することで、分子鎖ハイブリッド化のように組織化した(Mirkin他、「A DNA−based method for rationally assembling nanoparticles into macroscopic materials(ナノ粒子を巨視的材料に合理的に組織化するDNAを基礎とする方法)」、Nature 382、607−609(1996))。この概念は主として、金属電極のアレイなど巨視的基材へのナノスケール要素の組込みに、取り掛かるのに利用される。特異的配列のアンカーオリゴヌクレオチドで各電極を機能化した場合、ナノスケールの要素を相補的オリゴヌクレオチドで機能化するなら、適切に組織化するであろう。
本発明により、少なくとも2本の電極上に少なくとも2種類の異なる被覆分子を被覆形成する方法を提供するもので、この方法は、
(a)独立してアドレスを呼び出せる少なくとも2本の電極のアレイを準備し、
(b)全ての電極上にマスキング分子膜を吸着させ、
(c)全てではないが、少なくとも1本の電極からマスキング分子の脱着を電気化学的に起こして、第1組の露出電極を曝し、
(d)第1被覆分子を、第1組の露出電極上に吸着させ、
(e)マスキング分子に全ての電極を曝して、マスキング分子を全ての電極上に吸着させ、
(f)第2組の電極からマスキング分子の脱着を電気化学的に起こして、第2組の露出電極を曝し、
(g)第2被覆分子を第2組の露出電極上に吸着させることを含む。
図1は、清浄化電極、被覆電極および露出電極のCVトレースを示す。
図2は、マスキング分子が選択的に脱着した電極アレイを示す。
図3は、本発明の方法による2本の電極間領域のSEM画像である。
図4は、本発明の方法により被覆された、さらに2枚の電極アレイを示す。
本発明の方法は、少なくとも2本の独立してアドレスを呼び出せる電極のアレイの使用を必要とする。このようなアレイは知られていて、既知の方法で製作できる。1つの方法を以下に記述する。このアレイは、少なくとも10本の、より好ましくは少なくとも20本の、特に好ましくは少なくとも50本の、独立してアドレスを呼び出せる電極を含む。
一般的には、全ての電極が同じ材料から形成されるが、本発明では、一部の電極を1つの材料で形成し、追加する1組または複数組の電極を異なる材料で形成することも可能である。
この方法のステップ(c)では、予め決めた1組の電極から、マスキング分子を電気化学的に脱着することを含めることが欠かせない。これは、既知の方法によって、関係する電極の電位を制御することで行う。
吸着時間は、通常、第1マスキング分子の吸着に対して示した上記範囲内にある。
以下に記述するように、一連の向かい合ったサブ50nm間隔の金電極を、既知のUVリソグラフィ/リフトオフ技術を使って、Si/SiO2ウエハ上に作製した。このウエハを「ピラニア・エッチ」(H2O230%、H2SO470%)で1時間洗い、次いで脱イオン水、エタノール、再度脱イオン水中で完全にすすいだ。次に、6−メルカプト−1−ヘキサノール(MCH)保護単分子膜による電極アレイ全体の被覆は、ウエハをMCHの1mM水溶液に60分浸漬して行った。図1で、被覆電極のCVトレース(実線)を、被覆前の清浄電極のCVトレース(点線)と比較する。被覆電極に対して約−1Vで観察された還元的脱着の特徴は、MCH単分子膜の除去を示す。あらゆる電気化学的測定を、標準的な3電極構成を使い、100mMリン酸塩緩衝液中、pH10、62mV/秒の速度で行った。高純度白金線を対電極として使用した。全ての電気化学電位は、Ag/AgCl参照電極に対して記録されている。
MCH単分子膜が分子マスクとして働くことを実証するために、電極2、4、6からMCHを選択的に脱着した電極アレイを図2に示す。配列がCAGGATGGCGAACAACAAGA−チオールのチオール化オリゴヌクレオチドX(チオールは炭素C6リンカーを介してオリゴヌクレオチドに結合する)を、pH8のトリス(ヒドロキシメチル)アミノメタン10mM、EDTA1mMおよびNaCl1M溶液に溶解して、最終濃度10μMにした。それから、MCH分子マスクが電極1、3、5のみを覆っているこのアレイを、その水溶液に60分間浸漬して、オリゴヌクレオチドを露出電極に化学吸着させた。
2段階のシャドー蒸発技法(Philipp,G.,Weimann,T.,Hinze,P.,Burghard,M.&Weis,J.,“Shadow evaporation method for fabrication of sub 10 nm gaps between metal electrodes(金属電極間にサブ10nmの間隙を作成するシャドー蒸発法)”,Microelectron.Eng.46,157−160(1999))を使用して、Si/SiO2ウエハ上に電極アレイを作製した。第1段階では、標準的なUVフォトリソグラフィ、金属蒸発、およびリフト−オフ法によって、Ni/Crの10nm接着層表面上に、35nm厚のAu層を含めて、35μm間隔で向き合う一連の電極を作製した。第2段階では、このウエハを蒸発装置内で適当に傾けて、Ni/Cr5nmを、その後Au17nmを、向き合う電極間を連結する帯状部分に堆積させた。しかし、ウエハが傾いているので、蒸発源の最も近くにある既存電極のエッジが蒸発ビームから表面を遮り、向かい合う電極間にサブ50nmの大きさの間隙が形成する。
アレイの特定電極上に形成された特定オリゴヌクレオチド単分子膜を視覚化するために採用したプロトコルは、オリゴヌクレオチドハイブリッド化の比色検出法に基づく。チオール化されたオリゴヌクレオチドXおよびYに相補的な配列をもつビオチン化オリゴヌクレオチド(XはTCTTGTTGTTCGCCATCCTG−ビオチン、およびYはGGGCGGCGACCT−ビオチン)を、トリス(ヒドロキシメチル)アミノメタン10mM、EDTA1mM(TE溶液)およびNaCl1Mに溶解して、最終濃度を2.5μMとする。ビオチン化したオリゴヌクレオチドの適当な溶液を、室温で90分間電極アレイに作用させ、表面結合した相補的チオール化オリゴヌクレオチドとハイブリッド化させる。ビオチン化オリゴヌクレオチド溶液をトリス緩衝食塩水(TBS)ですすぎ、さらに数回の洗浄ステップの後、TBS/ツゥイーン20中で、アルカリホスファターゼと複合化する単一クローン性抗ビオチン抗体を1:1000に稀釈した液に、電極アレイを60分間浸漬した。この電極アレイを、5−ブロモ−4−クロロ−3−インドリルホスフェート/ニトロブルーテトラゾリウムの溶液に浸漬すると、アルカリホスファターゼが存在する箇所で、したがってビオチン化オリゴヌクレオチドが電極アレイにハイブリッド化している箇所で、色の局所的な黒ずみが起きる。全てのオリゴヌクレオチドはMWGバイオテックAG(MWG Biotech AG)から購入し、全ての試薬はシグマ(Sigma)から購入した。
図1:洗浄直後の露出したAu電極(破線)、MCH分子単分子膜で被覆したあとの同一電極(実線)、およびMCH単分子膜を脱着したあとの同一電極(点線)のサイクリックボルタモグラム。いずれのボルタモグラムも、62mV/秒、100mMリン酸塩緩衝液中、pH10で、Ag/AgCl参照電極に対して測定し、−0.4Vからスタートした。上昇および下降掃引がそれぞれの場合に示されている。
Claims (20)
- 少なくとも2本の電極上に、少なくとも2種類の異なる被覆分子を被覆形成する方法であって、前記方法が、
(a)少なくとも2本の独立してアドレスを呼び出せる電極のアレイを準備し、
(b)マスキング分子の層を全ての電極上に吸着させて、
(c)前記マスキング分子を、全てではないが少なくとも1本の電極から電気化学的な脱着を起こして、第1組の露出電極を曝し、
(d)第1被覆分子を第1組の露出電極上に吸着させ、
(e)全ての電極をマスキング分子に曝して、マスキング分子を全ての電極上に吸着させ、
(f)第2組の電極からマスキング分子の電気化学的脱着を起こして、第2組の露出電極を曝し、
(g)第2被覆分子を、第2組の露出電極上に吸着させること、
を含む方法。 - アレイが少なくとも10本の、独立してアドレスを呼び出せる電極を含む請求項1に記載の方法。
- アレイが少なくとも50本の、独立してアドレスを呼び出せる電極を含む請求項2に記載の方法。
- 少なくとも10組の異なる電極上に少なくとも10種類の異なる被覆分子を被覆形成するために、ステップ(c)から(e)を少なくとも8回繰り返すことを含む請求項1または請求項2に記載の方法。
- 各電極の直径が50μm以下である請求項1〜4のいずれかに記載の方法。
- 各電極の直径が900nm以下である請求項1〜4のいずれかに記載の方法。
- 各電極の直径が500nm以下である請求項1〜4のいずれかに記載の方法。
- 電極間の隔たりが30μm以下である請求項1〜7のいずれかに記載の方法。
- 電極間の隔たりが900nm以下である請求項1〜7のいずれかに記載の方法。
- 電極間の隔たりが100nm以下である請求項1〜7のいずれかに記載の方法。
- 電極が金属電極であり、マスキング分子および被覆分子がチオール化されている請求項1〜10のいずれかに記載の方法。
- 被覆分子が、少なくとも500の分子量を有する高分子である請求項1〜11のいずれかに記載の方法。
- 被覆分子が、少なくとも1000の分子量を有する高分子である請求項1〜11のいずれかに記載の方法。
- 被覆分子が電極に吸着できるチオール化オリゴヌクレオチドである請求項1〜12のいずれかに記載の方法。
- オリゴヌクレオチド被覆分子に相補的なオリゴヌクレオチドで機能化されたナノ粒子を用意すること、および前記相補的なオリゴヌクレオチドをハイブリッド化すること、をさらに含む請求項14に記載の方法。
- 被覆分子が電極上に吸着できるチオール化ポリペプチドである請求項1〜11のいずれかに記載の方法。
- 吸着分子の配向を起こすために、ステップ(b)および/またはステップ(d)および/またはステップ(e)および/またはステップ(g)で、マスキング分子および/または被覆分子にACもしくはDC電場を印加することを含む請求項1〜16のいずれかに記載の方法。
- 電極からの脱着を阻止するために、ステップ(c)および/または(f)で、脱着を要しない電極の電位を制御することを含む請求項1〜17のいずれかに記載の方法。
- ステップ(b)および/またはステップ(d)および/またはステップ(e)および/またはステップ(g)で、吸着が必要な電極に電気化学的ACもしくはDC電位を印加することを含む請求項1〜18のいずれかに記載の方法。
- 少なくとも3組の独立してアドレスを呼び出せる電極をもつアレイであって、各組は吸着する異なる被覆分子を有し、電極間の最小間隔が900ナノメートル以下であるアレイ。
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