JP5270486B2 - ナノ物質集積体の製造方法、ナノ物質集積体およびそれを用いたデバイス、ならびにナノ物質の構造解析方法 - Google Patents
ナノ物質集積体の製造方法、ナノ物質集積体およびそれを用いたデバイス、ならびにナノ物質の構造解析方法 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C30—CRYSTAL GROWTH
- C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
- C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
-
- C—CHEMISTRY; METALLURGY
- C30—CRYSTAL GROWTH
- C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
- C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
- C30B29/54—Organic compounds
- C30B29/58—Macromolecular compounds
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Crystallography & Structural Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Metallurgy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Enzymes And Modification Thereof (AREA)
- Crystals, And After-Treatments Of Crystals (AREA)
Description
(1)微粒子溶液の中にゲル化剤を含ませ、微粒子が3次元規則配列構造をとった後にゲル化させ、微粒子の3次元規則配列構造をマトリックス中に固定する方法(特許文献1参照)。
(2)重力により積み上げていく方法。
(3)液体中で電界の力を利用する方法(非特許文献1参照)。
(4)微粒子表面間の斥力によって規則的な構造を形成していく方法(特許文献2参照)。
(5)特に微粒子表面間の斥力によって微粒子を配列する方法に関し、微粒子間蒸気圧および表面張力を異にする2種以上の液体混合物からなる媒質中に微粒子を分散させ、微粒子分散液を基板表面上に流延し、格子配列した液膜を形成する微粒子薄膜を形成する方法(特許文献3参照)。
(6)微粒子の懸濁液中に基板を浸漬し、これを引き上げて基板上に微粒子単層膜を移流集積する方法。
(7)微粒子溶液を平行な面間の比較的狭い間隙に挿入し、微粒子のブラウン運動の振動数より大きく、振幅が平行な2面間の間隙とほぼ同じになるようにステッピングモータと直線並進装置を利用して制御して、微粒子集積体を得る方法(特許文献4参照)。
(8)微粒子を選択的に配列するため自己組織化単分子膜をテンプレートとして利用する方法。この方法では、基板上に作製した自己組織化単分子膜(SAM:Self assembled monolayer)をテンプレートに用いることにより、SAM表面官能基の親水性、疎水性、化学反応性、ゼータ電位、分子認識現象等を利用し、微粒子の精密配置を実現する(非特許文献2参照)。
(1)タンパク質の結晶化条件において安定なナノ物質を調製する。
(2)タンパク質とナノ物質を結晶化条件の環境で共存させる。
(3)タンパク質の結晶をテンプレートとし、自己組織化で結晶の細孔中にナノ物質を取り込ませて、結晶内にナノ物質を高次元に集積化した結晶を成長させる。
後述するように、結晶化溶液にはタンパク質溶解度調整剤等が添加され、高塩濃度になる場合が多い。そこで、高塩濃度の溶液中でも安定して溶解または分散する微粒子や生体分子等のナノ物質を調製することが好ましい。例えば、微粒子の場合は、安定化剤により安定化された安定化微粒子等の安定化ナノ物質が挙げられ、クエン酸安定化微粒子、シュウ酸安定化微粒子等のカルボン酸安定化微粒子、ドデシル硫酸ナトリウム(SDS)安定化微粒子、オレイン酸安定化微粒子等の界面活性剤安定化微粒子、ポリビニルピロリドン(PVP)安定化微粒子、ポリビニルピリジン安定化微粒子等の高分子化合物安定化微粒子等が挙げられる。
タンパク質の結晶化条件の溶液に上記ナノ粒子を共存させ、結晶化溶液を調製する。タンパク質とナノ物質の存在比、結晶化溶液のpH等を調整することにより、結晶中へのナノ物質の取り込み量等を制御することができる。
タンパク質の結晶化の場合、例えば、ハンギング・ドロップ(hanging drop)法やシッティング・ドロップ(sitting drop)法等の蒸気拡散法、バッチ法等を用いることができる。これにより、タンパク質の結晶中において水等で占められている空間(細孔)にナノ物質を占有させて、ナノ物質を結晶中に高次元集積化する。
(1)金微粒子、銀微粒子、白金微粒子の調製
結晶化溶液には塩やPEG等のタンパク質溶解度調整剤が添加され、高塩濃度になる場合が多い。そこで、高塩濃度の溶液中でも分散する金属微粒子として、金微粒子の場合について検討した。安定化剤が添加されていない金微粒子、クエン酸安定化金微粒子、SDS安定化金微粒子、PVP安定化金微粒子等の高塩濃度溶液中の分散性を検討した結果、PVP(粘性特性値K:30)で安定化された金微粒子(以下、PVP金微粒子とする。)の分散性がもっとも優れていることがわかった。PVP金微粒子の合成法は、まず0.1mMのHAuCl4の溶液10mLに111mgのPVPを溶解する。これに0.1mMの濃度のNaBH4を0.1mL添加し、12時間、撹拌子で撹拌しながら25℃で放置する。遠心機を用いて金微粒子の沈殿と、純水中への再分散による金微粒子の洗浄とを3回繰り返した。さらにこれを径が0.45μmのフィルタに通して、PVP金微粒子を得た。
結晶化させるタンパク質としてリゾチームを用い、下記のリゾチーム単体の結晶化条件の下、pHを変えて、sitting drop法にて結晶成長を行い、その後、結晶の析出状態を立体顕微鏡(倍率100倍)で観察した(図1〜8)。その結果、結晶系は正方晶であり、pHが6以下のときは{001}セクタが大きくなることにより細長い結晶に、pHが7以上のときは{101}セクタが大きくなることにより丸い結晶になることがわかった。
[溶液組成]
pH3,4,5,6,7,8,9,11(50mM)
NaCl(300mM)
0.7mMのリゾチーム
溶媒:イオン交換水
[リザーバ溶液組成]
pH3,4,5,6,7,8,9,11(50mM)
NaCl(300mM)
溶媒:イオン交換水
[保温温度]
4℃
(2)の結果を踏まえ、1.05mMのリゾチームの過飽和溶液にPVP金微粒子を加え、sitting drop法にて下記の条件で保温後、結晶の析出状態を立体顕微鏡で観察した。
[溶液組成]
pH3,4,5,6,7,8,9,11(50mM)
NaCl(300mM)
0.7mMのリゾチーム
溶媒:イオン交換水
[リザーバ溶液組成]
pH3,4,5,6,7,8,9,11(50mM)
NaCl(300mM)
溶媒:イオン交換水
[保温温度]
4℃
金微粒子の高次元集積化リゾチーム結晶を金微粒子の入っていないリゾチーム結晶化用母液で2回洗浄し、そのまま光学セルに入れ、可視紫外吸収スペクトルを測定した(図25)。その結果、520nmに金微粒子の表面プラズモン共鳴が観測された。また、表面プラズモン共鳴の赤方偏移は観測されなかったことから、金微粒子はリゾチーム結晶中においても分散した状態で取り込まれていることがわかった。
(2)の結果を踏まえ、1.05mMのリゾチームの過飽和溶液にPVP銀微粒子を加え、sitting drop法にて下記の条件で保温後、結晶の析出状態を立体顕微鏡で観察した。
[溶液組成]
pH7,8,9(50mM)
NaCl(300mM)
0.7mMのリゾチーム
溶媒:イオン交換水
[リザーバ溶液組成]
pH7,8,9(50mM)
NaCl(300mM)
溶媒:イオン交換水
[保温温度]
4℃
(2)の結果を踏まえ、1.05mMのリゾチームの過飽和溶液にPVP白金微粒子を加え、sitting drop法にて下記の条件で保温後、結晶の析出状態を立体顕微鏡で観察した。
[溶液組成]
pH6,7,8(50mM)
NaCl(300mM)
0.7mMのリゾチーム
溶媒:イオン交換水
[リザーバ溶液組成]
pH6,7,8(50mM)
NaCl(300mM)
溶媒:イオン交換水
[保温温度]
4℃
Claims (5)
- タンパク質、ナノ物質を溶媒中に共存させた状態で結晶化させて、前記タンパク質の結晶の細孔中に前記ナノ物質を集積化してナノ物質集積体を得ることを特徴とするナノ物質集積体の製造方法。
- 請求項1に記載のナノ物質集積体の製造方法であって、
前記溶媒中に、前記タンパク質の溶解度を調整するタンパク質溶解度調整剤をさらに共存させることを特徴とするナノ物質集積体の製造方法。 - 請求項1または2に記載のナノ物質集積体の製造方法であって、
前記ナノ物質が、金属微粒子および生体分子のうちの少なくとも1つであることを特徴とするナノ物質集積体の製造方法。 - 請求項1〜3のいずれか1項に記載のナノ物質集積体の製造方法で得られるナノ物質集積体を含むことを特徴とするナノ物質集積体およびそれを用いたデバイス。
- 請求項1〜3のいずれか1項に記載のナノ物質集積体の製造方法で得られるナノ物質集積体についての、物質の構造を解析するための分析手法による分析値より、前記タンパク質からの前記分析手法による分析値を差し引くことにより、前記ナノ物質の分析値を得て、前記ナノ物質の構造を解析することを特徴とするナノ物質の構造解析方法。
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