JP5124769B2 - Electrochemical immobilization of particles - Google Patents
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本発明は、金属イオン配位子を固定化タグとして用いる電気化学的方法を利用した、導電性材料への粒子の可逆的な電気化学的固定化法、及び該方法で得られる粒子固定化導電性材料等に関する。 The present invention relates to a reversible electrochemical immobilization method of particles to a conductive material using an electrochemical method using a metal ion ligand as an immobilization tag, and the particle-immobilized conductivity obtained by the method. Related to sex materials.
本発明者等は、金属イオン配位子に配位した金属を、電極電位により０価まで還元すると電極表面に析出し、かつ配位子との結合を保つことを見出し、この発見に基づき、「生体物質固定化チップおよびその利用」に関する国際特許出願（特許文献１）を行った。 The present inventors have found that when the metal coordinated to the metal ion ligand is reduced to zero valence by the electrode potential, it is deposited on the electrode surface and keeps the bond with the ligand. An international patent application (Patent Document 1) relating to “biological substance-immobilized chip and use thereof” was filed.
上記国際特許出願に記載されている発明は、タンパク質及び核酸のような生体物質にポリヒスチジンのようなペプチドから成る金属イオン配位結合部位を遺伝子工学的に結合させた上で、該ペプチドを導入した融合タンパク質等の固定化を行う技術である。
本発明が解決しようとする課題は、上記特許文献１に記載されたペプチドやタンパク質等の生体物質よりも大きなマクロな粒子を電気化学的反応を利用して可逆的に固定化する方法を提供することである。
The problem to be solved by the present invention is to provide a method for reversibly immobilizing macro particles larger than biological substances such as peptides and proteins described in
本発明者は、金属イオン配位結合部位及び粒子結合部位を夫々一端に含むタグを開発し、それを用いることによって上記課題を解決することに成功し、本発明を完成させた。 The present inventor has developed a tag including a metal ion coordination binding site and a particle binding site at one end, and succeeded in solving the above problems by using the tag, thereby completing the present invention.
即ち、本方法は以下の態様を有する。
［態様１］導電性材料への粒子の電気化学的固定化法であって、
（１）金属イオン配位結合部位及び粒子結合部位を夫々一端に含むタグと粒子を反応させて、粒子結合部位を介して該タグを該粒子の表面に結合させる工程、
（２）工程（１）で得られたタグ修飾粒子に該金属イオンを配位させる工程、及び
（３）工程（２）で得られた金属イオン配位粒子を導電性材料に近接させ、該導電性材料に還元電位を印加することにより、該金属イオン配位粒子を該金属イオンの還元により生じる金属原子を介して該導電性材料に固定化する工程、
を含むことを特徴とする前記電気化学的固定化法。
［態様２］導電性材料への粒子の電気化学的固定化法であって、
（１）金属イオン配位結合部位及び粒子結合部位を夫々一端に含むタグに金属イオンを配位させる工程、
（２）工程（１）で得られた金属イオン配位タグを導電性材料に近接させ、該導電性材料に還元電位を印加することにより、該タグを該金属イオンの還元により生じる金属原子を介して該導電性材料に固定化させる工程、及び
（３）工程（２）で得られたタグ修飾材料と粒子を反応させて、該粒子を該導電性材料に固定化する工程、
を含むことを特徴とする前記電気化学的固定化法。
［態様３］金属イオン配位結合部位及び粒子結合部位を夫々一端に含むタグを介して粒子が導電性材料の表面に結合されていることを特徴とする、粒子固定化導電性材料。
［態様４］本発明の粒子固定化導電性材料が担持されて成る基材。
That is, this method has the following aspects.
[Aspect 1] A method for electrochemically fixing particles to a conductive material,
(1) reacting a particle containing a metal ion coordination binding site and a particle binding site at one end with the particle, and binding the tag to the surface of the particle through the particle binding site;
(2) the step of coordinating the metal ions to the tag-modified particles obtained in step (1), and (3) the metal ion coordination particles obtained in step (2) are brought close to a conductive material, Immobilizing the metal ion coordination particles to the conductive material through metal atoms generated by reduction of the metal ion by applying a reduction potential to the conductive material;
The electrochemical immobilization method comprising the steps of:
[Aspect 2] A method for electrochemically fixing particles to a conductive material,
(1) A step of coordinating metal ions to a tag that includes a metal ion coordination binding site and a particle binding site at one end,
(2) The metal ion coordination tag obtained in the step (1) is brought close to the conductive material, and a reduction potential is applied to the conductive material, whereby the metal atoms generated by reduction of the metal ion are removed from the tag. A step of fixing the conductive material to the conductive material, and (3) a step of reacting the tag modifying material obtained in step (2) with the particles to fix the particles to the conductive material,
The electrochemical immobilization method comprising the steps of:
[Aspect 3] A particle-immobilized conductive material, wherein the particle is bound to the surface of the conductive material through a tag including a metal ion coordination binding site and a particle binding site at one end.
[Aspect 4] A substrate on which the particle-immobilized conductive material of the present invention is supported.
本発明の固定化法においては、固定化する粒子に予めタグを導入しておくことにより、導電性材料の表面へ電気化学的（還元反応）に粒子を固定化することができる。或いは、別方として、タグを予め導電性材料へ固定化し、その固定化されたタグを介して導電性材料へ粒子を固定化させることも可能である。こうして固定化された粒子は導電性材料に強固に保持される。更に、粒子を固定化した材料を、再度電池系へ接続し、逆反応（酸化反応）を行うことにより、該粒子を導電性材料から容易に脱離させることも可能である。 In the immobilization method of the present invention, the particles can be immobilized electrochemically (reduction reaction) on the surface of the conductive material by introducing a tag into the particles to be immobilized in advance. Alternatively, the tag can be fixed to the conductive material in advance, and the particles can be fixed to the conductive material via the fixed tag. The particles thus fixed are firmly held by the conductive material. Furthermore, it is also possible to easily desorb the particles from the conductive material by reconnecting the material on which the particles are immobilized to the battery system and performing a reverse reaction (oxidation reaction).
本発明の第一の態様は、（第１工程）金属イオン配位結合部位及び粒子結合部位を夫々一端に含むタグが表面に結合して成るタグ修飾粒子を調製し、（第２工程）該タグ修飾粒子に該金属イオンを配位させ、（第３工程）得られた金属イオン配位粒子を該金属イオンの還元により生じる金属原子を介して該導電性材料に固定化する、ことによって導電性材料へ粒子を電気化学的に固定化する方法に係る。かかる第一の態様の一具体例として、本明細書中の実施例１を挙げることができる。 The first aspect of the present invention is to prepare a tag-modified particle in which a tag containing a metal ion coordination binding site and a particle binding site at one end is bonded to the surface (first step) (second step) Conducting by coordinating the metal ions to the tag-modified particles (third step) and immobilizing the obtained metal ion coordination particles to the conductive material via metal atoms generated by reduction of the metal ions The present invention relates to a method for electrochemically immobilizing particles on a conductive material. As a specific example of the first aspect, Example 1 in this specification can be given.
本発明の第二の態様は、（第１工程）上記タグに金属イオンを配位させて金属イオン配位タグを調整し、（第２工程）得られた金属イオン配位タグを該金属イオンの還元により生じる金属原子を介して該導電性材料に固定化し、（第３工程）得られたタグ修飾材料と粒子を反応させて、該粒子を該導電性材料に固定化する、ことによって導電性材料へ粒子を電気化学的に固定化する方法に係る。かかる第二の態様の一具体例として、本明細書中の実施例２を挙げることができる。 In the second aspect of the present invention, (first step) a metal ion is coordinated by coordinating a metal ion to the tag, and (second step) the obtained metal ion coordination tag is used as the metal ion. By immobilizing the conductive material by immobilizing the conductive material via metal atoms generated by the reduction of (3), reacting the obtained tag modifying material with the particles, and immobilizing the particles to the conductive material. The present invention relates to a method for electrochemically immobilizing particles on a conductive material. As a specific example of the second aspect, Example 2 in this specification can be given.
本発明の第三の態様は、金属イオン配位結合部位及び粒子結合部位を夫々一端に含むタグを介して粒子が導電性材料の表面に結合されていることを特徴とする、粒子固定化導電性材料に係る。尚、タグは粒子又は導電性材料のいずれと最初に結合してもかまわない。いずれの場合でも、製造される粒子固定化導電性材料は上記タグを介して粒子が導電性材料の表面に結合された構造を有することを特徴とする。このような粒子固定化導電性材料の製造方法に特に制限はないが、好ましくは上記本発明の固定化法によって製造することができる。 According to a third aspect of the present invention, there is provided a particle-immobilized conductive material, wherein the particle is bonded to the surface of the conductive material through a tag including a metal ion coordination binding site and a particle binding site at one end. Related to sexual materials. Note that the tag may be initially bonded to either the particle or the conductive material. In any case, the produced particle-immobilized conductive material has a structure in which particles are bonded to the surface of the conductive material through the tag. Although there is no restriction | limiting in particular in the manufacturing method of such a particle fixed electroconductive material, Preferably it can manufacture by the fixing method of the said invention.
更に、本発明の第四の態様は、粒子固定化導電性材料が担持されて成る基材に係る。 Furthermore, a fourth aspect of the present invention relates to a base material on which a particle-immobilized conductive material is supported.
本発明において、「粒子」とは、ペプチドやタンパク質等の生体物質に比べて数オーダー大きいマクロな粒子を意味し、例えば、粒径が数ｎｍ〜数十ｎｍの程度の当業者に公知の任意のものを含む。尚、粒子の形態は、必ずしも球状に限定されず、棒状等の任意の形状をとることが出来る。粒子の材質に特に制限はないが、粒子の代表的な例として、例えば金コロイドのような金属コロイド、フラーレン、カーボンナノチューブ及びセラミック粒子を挙げることが出来る。又、粒子が、タグの粒子結合部位と結合することができる適当な官能基で予め活性化されたものでも良い。 In the present invention, “particle” means a macro particle several orders of magnitude larger than a biological substance such as a peptide or protein. For example, any particle size known to those skilled in the art having a particle size of several nanometers to several tens of nanometers. Including The form of the particles is not necessarily limited to a spherical shape, and can take any shape such as a rod. There are no particular restrictions on the material of the particles, but typical examples of the particles include metal colloids such as gold colloid, fullerenes, carbon nanotubes, and ceramic particles. Alternatively, the particles may be previously activated with an appropriate functional group capable of binding to the particle binding site of the tag.
このような粒子は、夫々の物理的特性に応じて、当業者に公知の任意の方法で容易に作製することが可能である。 Such particles can be easily prepared by any method known to those skilled in the art depending on the respective physical properties.
本発明方法で使用する「タグ」の各端は、夫々、金属イオン配位結合部位及び粒子結合部位から成っている。 Each end of the “tag” used in the method of the present invention consists of a metal ion coordination binding site and a particle binding site, respectively.
「金属イオン配位結合部位」は当業者に公知の任意の構造を有することが出来、例えば、金属に配位結合可能な原子（窒素原子、硫黄原子、酸素原子など）を含む分子鎖・分子骨格などが挙げられる。このような分子鎖・分子骨格はペプチドであってもよいし、それ以外の化合物であってもよい。具体的には、ポリヒスチジンを含むペプチド（ポリヒスチジン）、ポルフィリン、チオール基を含むペプチドや化合物などが挙げられる。この中で、特に好適には、ポリヒスチジンが挙げられる。ここで、ポリヒスチジンとはヒスチジンを２個以上含むポリペプチドをいう。ヒスチジンの個数としては、２個以上であれば、固定化されるタンパク質の機能に影響を及ぼさない範囲の個数でよいが、好ましくは５〜８個である。なお、ポリヒスチジンは必ずしも連続している必要はなく、各ヒスチジンの間に他のアミノ酸又はその他の分子が１〜数個挿入されたものであってもよい。 The “metal ion coordination bond site” can have any structure known to those skilled in the art, for example, a molecular chain / molecule containing an atom (nitrogen atom, sulfur atom, oxygen atom, etc.) capable of coordinating bond to a metal. Examples include skeletons. Such molecular chains / molecular skeletons may be peptides or other compounds. Specific examples include peptides containing polyhistidine (polyhistidine), porphyrins, peptides and compounds containing thiol groups, and the like. Among these, polyhistidine is particularly preferable. Here, polyhistidine refers to a polypeptide containing two or more histidines. The number of histidines may be in the range that does not affect the function of the protein to be immobilized as long as it is 2 or more, but is preferably 5 to 8. In addition, the polyhistidine does not necessarily need to be continuous, and one to several other amino acids or other molecules may be inserted between the histidines.
「粒子結合部位」は、粒子の物理化学的な性質に応じて、当業者に公知の任意の構造とすることができる。例えば、粒子が金属コロイド等である場合には、チオール基のような金属結合性基である。 The “particle binding site” can have any structure known to those skilled in the art depending on the physicochemical properties of the particle. For example, when the particle is a metal colloid or the like, it is a metal binding group such as a thiol group.
更に、本発明方法で使用するタグでは、金属イオン配位結合部位と粒子結合部位とがリンカーを介して結合されていることが好ましい。このリンカーは当業者に公知の適当な構造、例えば、数個の炭素数を有するアルキル基のような、適当な長さを有する不活性な有機基とすることが出来る。このようにタグの中央に介在させたリンカーは、チオール基等の金属結合性基において不要な電気化学反応が生起するのを防ぐのに有効である。その一方で、リンカーが長すぎるとリンカー分子に揺らぎが生じ、その結果、固定化反応が阻害される懸念がある。このような点を考慮して、本明細書の実施例においては、リンカーは７個の炭素数から成るアルキル基とした。 Furthermore, in the tag used in the method of the present invention, the metal ion coordination binding site and the particle binding site are preferably bound via a linker. The linker can be any suitable structure known to those skilled in the art, for example, an inert organic group having a suitable length, such as an alkyl group having several carbon atoms. Thus, the linker interposed in the center of the tag is effective in preventing an unnecessary electrochemical reaction from occurring in a metal-binding group such as a thiol group. On the other hand, if the linker is too long, the linker molecule fluctuates, and as a result, there is a concern that the immobilization reaction is inhibited. In view of this point, in the examples of the present specification, the linker is an alkyl group having 7 carbon atoms.
以上のような構造を有するタグは、各部位、及びリンカーのいずれか一つ又は２つを含む適当な化合物を用いて当業者に公知の任意の方法で合成することが出来る。例えば、本明細書に実施例に記載されているように、粒子結合部位（チオール基）とリンカー（ヘプチル基）を含む化合物（7-Carboxy-1-heptanethiol）を金属イオン配位結合部位（ポリヒスチジン）とを反応させることによってタグを合成することが出来る。 The tag having the structure as described above can be synthesized by any method known to those skilled in the art using an appropriate compound containing any one or two of each site and linker. For example, as described in Examples in the present specification, a compound (7-Carboxy-1-heptanethiol) containing a particle binding site (thiol group) and a linker (heptyl group) is bonded to a metal ion coordination binding site (poly A tag can be synthesized by reacting with histidine.
本発明の「導電性材料」は電位を印加し得る材料である、金属化合物又は金属から成る電極であることが好ましい。金属化合物の例としては、ＩＴＯ（インジウムスズオキサイド）等の金属酸化物、ＧａＡｓ、シリコン等の半導体、及びカーボン等を挙げることが出来る。又、金属の例としては、金、銀、銅、アルミニウム、又は白金を挙げることが出来る。例えば、合金のように、これらの二種以上を適当に混合して使用することも可能である。 The “conductive material” of the present invention is preferably an electrode made of a metal compound or metal, which is a material to which a potential can be applied. Examples of metal compounds include metal oxides such as ITO (indium tin oxide), semiconductors such as GaAs and silicon, and carbon. Examples of metals include gold, silver, copper, aluminum, and platinum. For example, it is also possible to appropriately mix and use two or more of these materials, such as an alloy.
本発明において、金属イオンの還元により生ずる金属原子はタグの金属イオン配位結合部位と配位結合を形成することが可能な金属イオンが還元されて生ずる金属が好ましい。例えば、金属イオン配位結合部位としてポリヒスチジンを用いた場合には、２価の陽イオン、すなわち、Ｃｕ２＋、Ｚｎ２＋、Ｎｉ２＋、Ｃａ２＋、Ｃｏ２＋、及びＭｇ２＋等が還元されて生ずる金属が好ましい。 In the present invention, the metal atom generated by reduction of the metal ion is preferably a metal generated by reduction of a metal ion capable of forming a coordinate bond with the metal ion coordination bond site of the tag. For example, when polyhistidine is used as a metal ion coordination binding site, divalent cations, that is, Cu 2+ , Zn 2+ , Ni 2+ , Ca 2+ , Co 2+ , Mg 2+ , and the like are reduced. Metal is preferred.
タグ（第一の態様の第１工程）又はタグ修飾材料（第二の態様の第３工程）と粒子とを結合させる反応は、両者を適当な緩衝液中で所定時間反応させたり、粒子を含む緩衝液をタグ修飾材料に滴下することによって行うことができる。その際の各種反応条件（緩衝液の種類・濃度、温度、反応時間等）は、粒子の物理化学的性質及びタグにおける粒子結合部位の化学構造等に応じて、当業者が出来適宜選択することが出来る。 The reaction of binding the tag (the first step of the first embodiment) or the tag modifying material (the third step of the second embodiment) and the particles is carried out by reacting both in a suitable buffer for a predetermined time, It can be carried out by dropping the buffer solution containing it onto the tag modifying material. Various reaction conditions (buffer type / concentration, temperature, reaction time, etc.) at that time can be appropriately selected by those skilled in the art depending on the physicochemical properties of the particles and the chemical structure of the particle binding site in the tag. I can do it.
上記金属イオン配位結合部位を有するタグ修飾粒子（第一の態様の第２工程）又はタグ（第二の態様の第１工程）に該金属イオンを配位結合させる反応は、これらを含む適当な緩衝液中にＮｉＣｌ２等の上記金属イオンに対応する塩を加えることによって実施することできる。その際の各種反応条件（緩衝液の種類・濃度、温度、反応時間等）は、粒子の物理化学的性質及びタグにおける金属イオン配位結合の化学構造等に応じて、当業者が出来適宜選択することが出来る。尚、遊離の金属イオンを除くために、配位結合反応を行った後に、金属イオンとタグ修飾粒子等との配位結合体を金属イオンを含まない緩衝液に対して透析することが好ましい。 The reaction for coordinating the metal ion to the tag-modified particle having the metal ion coordination binding site (the second step of the first embodiment) or the tag (the first step of the second embodiment) includes these. Can be carried out by adding a salt corresponding to the metal ion such as NiCl 2 in a simple buffer. Various reaction conditions (buffer type / concentration, temperature, reaction time, etc.) at that time can be appropriately selected by those skilled in the art depending on the physicochemical properties of the particles and the chemical structure of the metal ion coordination bond in the tag. I can do it. In order to remove free metal ions, it is preferable to dialyze a coordination complex of metal ions and tag-modified particles or the like against a buffer solution that does not contain metal ions after performing a coordination bond reaction.
更に、金属イオン配位粒子（第一の態様の第３工程）又は金属イオン配位タグ（第一の態様の第２工程）を該金属イオンの還元により生じる金属原子を介して該導電性材料に固定化する反応において、「導電性材料に近接させ」とは、金属イオンとの配位複合体である金属イオン配位粒子又は金属イオン配位タグを、導電性材料の近傍に配置することをいう。好ましくは、上記配位複合体を含む溶液中に導電性材料を浸すことによって容易に実施することができる。導電性材料を浸す溶液は、電位の印加が可能な限り特に限定されないが、例えば、リン酸緩衝液等を用いることができる。 Furthermore, the conductive material is formed through a metal atom produced by reduction of the metal ion with a metal ion coordination particle (third step of the first embodiment) or a metal ion coordination tag (second step of the first embodiment). In the reaction of immobilizing to metal, “close to conductive material” means that metal ion coordination particles or metal ion coordination tags that are coordination complexes with metal ions are arranged in the vicinity of the conductive material. Say. Preferably, it can be easily carried out by immersing a conductive material in a solution containing the coordination complex. The solution in which the conductive material is immersed is not particularly limited as long as potential can be applied. For example, a phosphate buffer solution or the like can be used.
上記固定化法において、導電性材料への電位の印加は、例えば、金属イオン配位粒子又は金属イオン配位タグのような配位結合体を含む電解液中に、導電性材料である作用極と参照電極とを浸し、通常の電源を用いて電位を印加することにより行うことができる。ここで、参照電極としては、例えば、銀塩化銀電極等が挙げられる。印加する電位は還元電位であるが、還元電位とは、参照電極に対してマイナスとなる電位をいう。還元電位として、好ましい電位は、導電性材料の種類により異なるが、導電性材料として白金を用いた場合には、参照電極に対して−１０ｍＶ以下の電位が好ましく、参照電極に対して−１００ｍＶ以下の電位がより好ましく、参照電極に対して−２００ｍＶ以下の電位が特に好ましい。 In the immobilization method, the potential is applied to the conductive material by, for example, working electrode which is a conductive material in an electrolyte solution including a coordination bond such as a metal ion coordination particle or a metal ion coordination tag. And a reference electrode, and a potential is applied using a normal power source. Here, as a reference electrode, a silver-silver chloride electrode etc. are mentioned, for example. The applied potential is a reduction potential, and the reduction potential is a potential that is negative with respect to the reference electrode. As the reduction potential, a preferable potential varies depending on the type of the conductive material, but when platinum is used as the conductive material, a potential of −10 mV or less is preferable with respect to the reference electrode, and −100 mV or less with respect to the reference electrode. Is more preferable, and a potential of −200 mV or less is particularly preferable with respect to the reference electrode.
このようにして製造される本発明の粒子固定化導電性材料は、更に当業者に公知の適当な基材に担持させることも出来る。このような基材として用いることのできる素材の例として、プラスチック、無機高分子、天然高分子及びセラミック等を挙げることが出来る。プラスチックとして具体的には、ポリエチレン、ポリスチレン、ポリカーボネート、ポリプロピレン、ポリアミド、フェノール樹脂、エポキシ樹脂、ポリカルボジイミド樹脂、ポリ塩化ビニル、ポリフッ化ビニリデン、ポリフッ化エチレン、ポリイミド及びアクリル樹脂等が、無機高分子としては、ガラス、水晶、カーボン、シリカゲル、及びグラファイト等が、天然高分子としては、セルロース、セルロース誘導体、キチン、キトサン、アルギン酸及びアルギン酸塩等が、セラミックとしては、アルミナ、シリカ、炭化ケイ素、窒化ケイ素及び炭化ホウ素等を例示することができる。 The particle-immobilized conductive material of the present invention thus produced can be further supported on an appropriate base material known to those skilled in the art. Examples of materials that can be used as such a substrate include plastics, inorganic polymers, natural polymers, and ceramics. Specific examples of plastics include polyethylene, polystyrene, polycarbonate, polypropylene, polyamide, phenolic resin, epoxy resin, polycarbodiimide resin, polyvinyl chloride, polyvinylidene fluoride, polyfluorinated ethylene, polyimide, and acrylic resin as inorganic polymers. Is glass, quartz, carbon, silica gel, graphite, etc., natural polymers are cellulose, cellulose derivatives, chitin, chitosan, alginic acid, alginates, etc., and ceramics are alumina, silica, silicon carbide, silicon nitride And boron carbide and the like.
尚、上記基材は、例えば、フィルム、平板、成型品（ビーズ、ストリップ、マルチウェルプレートのウェルまたはストリップ、チューブ、メッシュ、連続発砲フォーム、膜、紙、針、ファイバー、プレート、スライドおよび細胞培養容器）、ラテックス、カンチレバーの探針等の当業者に公知の任意の適当な形状をとることができ、またその大きさについては特に制限はない。又、本発明の粒子固定化導電性材料は、当業者に公知の適当な方法で基材に担持させることができる。 Examples of the substrate include films, flat plates, molded products (beads, strips, wells or strips of multi-well plates, tubes, meshes, continuous foamed forms, membranes, paper, needles, fibers, plates, slides, and cell cultures. Container), latex, cantilever probe, etc., and any suitable shape known to those skilled in the art can be used, and the size is not particularly limited. Further, the particle-immobilized conductive material of the present invention can be supported on a substrate by an appropriate method known to those skilled in the art.
更に、こうして製造される粒子固定化導電性材料の粒子の表面に、当業者に公知の任意の方法で、例えば、タンパク質若しくは核酸等の生体物質、蛍光物質等の検出用物質、その他の各種化合物を結合させることもできる。このような粒子への各種化合物の結合は、該粒子が導電性材料上に固定化される前後を問わず実施することができる。従って、このような生体物質が結合した粒子を使用して得られた粒子固定化導電性材料は、上記特許文献１に記載された各種の分析方法及び精製方法に応用することができる。
Further, on the surface of the particles of the particle-immobilized conductive material thus produced, for example, biological substances such as proteins or nucleic acids, detection substances such as fluorescent substances, and other various compounds by any method known to those skilled in the art Can also be combined. Bonding of various compounds to such particles can be performed before or after the particles are immobilized on the conductive material. Therefore, the particle-immobilized conductive material obtained by using such particles to which biological substances are bonded can be applied to various analysis methods and purification methods described in
本発明の第一及び第二の態様で示される２通りの固定化法はいずれも金属錯体の還元反応による金属析出を介した固定化である。よって、固定化後の電極に対し、適当な電圧、例えば、＋500mVの電位を印加し酸化反応を行うことで、容易に粒子を脱固定化することができる。 Both of the two immobilization methods shown in the first and second aspects of the present invention are immobilization via metal precipitation by reduction reaction of a metal complex. Therefore, the particles can be easily defixed by applying an appropriate voltage, for example, a potential of +500 mV to the electrode after immobilization to carry out the oxidation reaction.
以下、実施例に則して本発明を詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail according to an Example, the technical scope of this invention is not limited to these Examples.
実施例１：本発明の第一の態様
（１）ポリヒスチジン修飾金コロイド粒子の作製
金コロイドは塩化金酸(HAuCl4)をクエン酸とタンニン酸で還元合成した。即ち、1%塩化金酸1ml、純水79ml、及び、還元剤として1%クエン酸4mlと1%タンニン酸を混合し60℃に加熱した。色が赤くなった後、5-10 分程沸騰させることにより直径8.3nmの金コロイド粒子を得た。
Example 1: First aspect of the present invention (1) Preparation of polyhistidine-modified gold colloid particles Gold colloid was synthesized by reducing chloroauric acid (HAuCl 4 ) with citric acid and tannic acid. That is, 1 ml of 1% chloroauric acid, 79 ml of pure water, and 4 ml of 1% citric acid as a reducing agent and 1% tannic acid were mixed and heated to 60 ° C. After the color turned red, gold colloid particles having a diameter of 8.3 nm were obtained by boiling for about 5-10 minutes.
一方、常法により、あらかじめ固相合成によってポリヒスチジン（６個のヒスチジンから構成）を得た。得られたポリヒスチジンを、N,N-ジメチルホルムアミド (N,N-Dimethylformamide, DMF)中で、Ｎ，Ｎ−ジイソプロピルエチルアミン（Ｎ,Ｎ−diisopropylethylamine；DIEA）4モル等量を縮合剤として、7-Carboxy-1-heptanethiol(同仁化学)4モル等量と一晩室温で撹拌することによって、チオール基修飾ポリヒスチジンを得た。 On the other hand, polyhistidine (composed of 6 histidines) was obtained in advance by solid-phase synthesis by a conventional method. The obtained polyhistidine was used in a N, N-dimethylformamide (DMF) as a condensing agent with 4 mole equivalents of N, N-diisopropylethylamine (DIEA) as a condensing agent. -Carboxy-1-heptanethiol (Dojindo Chemical Co., Ltd.) 4 molar equivalents were stirred overnight at room temperature to obtain thiol group-modified polyhistidine.
金コロイド粒子へのポリヒスチジン修飾はAu-Sのメルカプト結合を利用した。即ち、10nM金コロイド粒子と100μMチオール基修飾ポリヒスチジンを10mMクエン酸緩衝液(pH6.0)中で12時間反応させ、その後、緩衝液を交換していくことによって未反応ペプチドを除去することにより、ポリヒスチジン修飾金コロイド粒子（タグ修飾粒子）を得た。 Polyhistidine modification on gold colloidal particles used Au-S mercapto bond. That is, by reacting 10 nM colloidal gold particles and 100 μM thiol group-modified polyhistidine in 10 mM citrate buffer (pH 6.0) for 12 hours, and then removing the unreacted peptide by exchanging the buffer. Polyhistidine modified gold colloidal particles (tag modified particles) were obtained.
（２）Ni2+配位金コロイド粒子の作製
上記（１）で得たポリヒスチジン修飾金コロイド粒子を0.1Mグリシン緩衝液(pH9)中で、最終濃度10μMとなるようにNiCl2を添加し、２時間反応させ、Ni2+をポリヒスチジン修飾金コロイド粒子に配位させた。未配位のNi2+を除去するために、透析カセット(スライドAライダー、Pierce社)を用いてNi2+配位金コロイド粒子（金属イオン配位粒子）を得た。
(2) Preparation of Ni 2+ coordinated gold colloidal particles NiCl 2 was added to the polyhistidine-modified gold colloidal particles obtained in (1) above in a 0.1 M glycine buffer (pH 9) to a final concentration of 10 μM. Reaction was performed for 2 hours to coordinate Ni 2+ to the polyhistidine-modified gold colloidal particles. In order to remove uncoordinated Ni 2+ , Ni 2+ coordinated gold colloidal particles (metal ion coordinated particles) were obtained using a dialysis cassette (Slide A lidar, Pierce).
（３）ポリヒスチジン修飾金コロイド粒子ニッケル錯体の電気化学固定化
このNi2+配位金コロイド粒子（コロイド粒子の濃度：0.25Ａ520（520 nmの吸光度が0.25になる濃度））を含むトリシン緩衝液（0.1M、ｐH8.0、0.1M KCl含有）中に、導電性材料として、ITO(Indium-Tin Oxide)電極を挿入し、このITO電極を作用極とし、対極として白金電極、参照極として銀塩化銀電極をもちいた３電極系により、Ni2+の還元電位である−100mVを10分間印加し、金コロイド粒子の固定化反応を実施して、本発明の粒子固定化導電性材料を製造した。
(3) Electro-immobilization of nickel complex with polyhistidine modified gold colloidal particles Tricine buffer containing this Ni 2+ coordinated gold colloidal particles (concentration of colloidal particles: 0.25A 520 (concentration at which the absorbance at 520 nm is 0.25)) An ITO (Indium-Tin Oxide) electrode is inserted as a conductive material in the liquid (containing 0.1M, pH8.0, 0.1M KCl), this ITO electrode is used as a working electrode, a counter electrode as a platinum electrode, and a reference electrode Using a three-electrode system using a silver-silver chloride electrode, −100 mV, the reduction potential of Ni 2+ , is applied for 10 minutes to carry out an immobilization reaction of colloidal gold particles. Manufactured.
（４）固定化状態の評価
こうして得られた材料を、JEOL EM-2000FXを用いて透過型顕微鏡（TEM）で測定した。その結果、コロイドの固定化を示唆するTEM像が観察された。又、NanoScope IIIa（Veeco metrology group/Digital Instruments）を用いた原子間力顕微鏡（AFM）（観察環境：気中、観察モード：タッピング）で測定した。その結果、ポリヒスチジン修飾金コロイド粒子が導電性材料の表面に非常に密に固定化されたことが確認された（図１・右）。尚、AFM測定用の試料としてITO電極の代わりに白金電極を用い同条件下で導電性材料を製造した。
(4) Evaluation of Immobilization State The material thus obtained was measured with a transmission microscope (TEM) using JEOL EM-2000FX. As a result, a TEM image suggesting the fixation of the colloid was observed. In addition, the measurement was performed with an atomic force microscope (AFM) (observation environment: air, observation mode: tapping) using NanoScope IIIa (Veeco metrology group / Digital Instruments). As a result, it was confirmed that the polyhistidine-modified gold colloid particles were fixed very densely on the surface of the conductive material (FIG. 1, right). A conductive material was produced under the same conditions using a platinum electrode instead of the ITO electrode as a sample for AFM measurement.
実施例２：本発明の第二の態様
本実施例では、まず、導電性材料上にチオール修飾ポリヒスチジン分子（金属イオン配位タグ）から成る層を電気化学固定化法により構築し、その後、金コロイド微粒子を自己集積させことにより、本発明の粒子固定化導電性材料を製造した。
Example 2: Second aspect of the present invention In this example, first, a layer composed of a thiol-modified polyhistidine molecule (metal ion coordination tag) is formed on a conductive material by an electrochemical immobilization method. Then, the particle-immobilized conductive material of the present invention was manufactured by self-assembling gold colloidal fine particles.
（１）金属イオン配位タグの作製
実施例１と同様な方法でチオール基修飾ポリヒスチジンを得た。尚、アルキル鎖のような直線的な構造を持った分子は導電性材料のような固相へ固定された後に分子の揺らぎが起き、その結果、揺らぎによる固定化反応阻害が懸念される。そこで、チオール基とポリヒスチジン配列のリンカーとして機能するアルキル鎖は短い方が望ましい。一方で、適度なリンカーがなければチオール基が電気化学反応を起こしてしまう可能性もあるので、本実施例で使用するタグ中のリンカーのアルキル鎖の炭素数は７とした。次に、実施例１（２）に準じて、こうして得られたチオール基修飾ポリヒスチジンペプチドにNi2+を加えることで錯体を（金属イオン配位タグ）形成させ、未配位のNi2+を透析によって除去した。
(1) Preparation of metal ion coordination tag A thiol group-modified polyhistidine was obtained in the same manner as in Example 1. In addition, molecules having a linear structure such as an alkyl chain undergo molecular fluctuation after being fixed to a solid phase such as a conductive material, and as a result, there is a concern that the immobilization reaction may be inhibited by the fluctuation. Therefore, it is desirable that the alkyl chain functioning as a linker between the thiol group and the polyhistidine sequence is short. On the other hand, if there is no appropriate linker, the thiol group may cause an electrochemical reaction, so the alkyl chain of the linker in the tag used in this example has 7 carbon atoms. Next, according to Example 1 (2), Ni 2+ is added to the thiol group-modified polyhistidine peptide thus obtained to form a complex (metal ion coordination tag), and uncoordinated Ni 2+ is formed. Was removed by dialysis.
（２）金属イオン配位タグの電気化学固定化
実施例１（３）と同様に、ITO電極を作用極、白金電極を対極、銀塩化銀電極を参照極として3電極系において還元反応を行うことによって、チオール基標識ポリヒスチジンペプチド層を電極上に形成させ、タグ修飾材料を得た。
(2) Electrochemical immobilization of metal ion coordination tag As in Example 1 (3), a reduction reaction is performed in a three-electrode system using an ITO electrode as a working electrode, a platinum electrode as a counter electrode, and a silver-silver chloride electrode as a reference electrode. Thus, a thiol group-labeled polyhistidine peptide layer was formed on the electrode to obtain a tag-modified material.
（３）蛍光コロイド粒子（ビーズ）の電気化学固定化
こうして得られたタグ修飾材料（電極）に対し、蛍光コロイド粒子（ナノマグ社製「nanomagR-D-spio 50nmコロイド」：アミノ基活性化物）（0.25 Ａ520（520 nmの吸光度が0.25になる濃度））を含有する0.1Mグリシン緩衝液(pH9)を滴下し、２５℃で３時間ゆるやかに攪拌しながら該蛍光コロイド粒子と架橋剤（ピアス社製 SMCC：製品番号22360）とを反応させ、蛍光コロイド粒子のアミノ基とポリヒスチジンペプチド層末端チオール基とを架橋結合することにより、コロイド粒子をポリヒスチジンペプチド層上へ自己集積させ、本発明の粒子固定化導電性材料を製造した。
(3) Electrochemical immobilization of fluorescent colloid particles (beads) For the tag-modified material (electrode) thus obtained, fluorescent colloid particles ("nanomagR-D-spio 50nm colloid" manufactured by Nanomag: activated amino group) ( 0.1M glycine buffer solution (pH 9) containing 0.25 A 520 (concentration at which the absorbance at 520 nm becomes 0.25)) was dropped, and the fluorescent colloid particles and the crosslinking agent (Pierce) were gently stirred at 25 ° C. for 3 hours. SMCC (Product No. 22360) is reacted, and the colloidal particles are self-assembled onto the polyhistidine peptide layer by cross-linking the amino groups of the fluorescent colloidal particles and the terminal thiol groups of the polyhistidine peptide layer. A particle-immobilized conductive material was produced.
実施例１と同様に、TEM及びAFMで測定した。その結果、蛍光コロイド粒子が導電性材料の表面に密に固定化されたことが確認された。 In the same manner as in Example 1, measurement was performed with TEM and AFM. As a result, it was confirmed that the fluorescent colloidal particles were densely fixed on the surface of the conductive material.
実施例３：固定化したポリヒスチジン導入蛍光コロイド粒子の脱固定化
以上の２通りの固定化法はいずれも金属錯体の還元反応による金属析出を介した固定化である。よって、固定化後の電極に対し、＋500mVの電位を印加し酸化反応を行うことで、粒子を脱固定化することができる。実施例２で得られた粒子固定化導電性材料を用いて得られた脱固定化の結果を以下の表１に示す。尚、蛍光コロイド粒子の蛍光性により固定化量を蛍光測定した。具体的には、ニコン社製落射蛍光顕微鏡で固定化表面を観察し、その蛍光画像を浜松ホトニクス製高感度カメラおよび画像解析装置AQUA COSMOSシステムにより定量評価した。得られた結果を表１に示す。
Example 3: Deimmobilization of immobilized polyhistidine-introduced fluorescent colloidal particles Both of the above two immobilization methods are immobilization via metal precipitation by reduction reaction of a metal complex. Therefore, the particles can be de-immobilized by applying a potential of +500 mV to the electrode after immobilization to carry out an oxidation reaction. Table 1 below shows the results of defixation obtained using the particle-immobilized conductive material obtained in Example 2. The amount of immobilization was measured by fluorescence based on the fluorescence of the fluorescent colloid particles. Specifically, the immobilized surface was observed with a Nikon epi-illumination fluorescence microscope, and the fluorescence image was quantitatively evaluated with a high-sensitivity camera made by Hamamatsu Photonics and an image analysis device AQUA COSMOS system. The obtained results are shown in Table 1.
本発明の固定化方法で製造される粒子固定化導電性材料、又は、該材料が担持されて成る基材は、センサ材料、磁気記録デバイス、及び光学材料等の様々な分野に利用することができる。 The particle-immobilized conductive material produced by the immobilization method of the present invention or the substrate on which the material is supported can be used in various fields such as sensor materials, magnetic recording devices, and optical materials. it can.
Claims (11)
（１）リンカーを介して金属イオン配位結合部位及び粒子結合部位を夫々一端に含むタグと該粒子を反応させて、粒子結合部位を介して該タグを該粒子の表面に結合させる工程、
（２）工程（１）で得られたタグ修飾粒子に該金属イオンを配位させる工程、及び
（３）工程（２）で得られた金属イオン配位粒子を導電性材料に近接させ、該導電性材料に還元電位を印加することにより、該金属イオン配位粒子を該金属イオンの還元により生じる金属原子を介して該導電性材料に固定化する工程、
を含むことを特徴とする前記電気化学的固定化法。 A method of electrochemically immobilizing particles selected from the group consisting of metal colloids, fullerenes, carbon nanotubes and ceramic particles to a conductive material, comprising :
(1) reacting a tag containing a metal ion coordination binding site and a particle binding site at one end with a linker via a linker, and binding the tag to the surface of the particle through the particle binding site;
(2) the step of coordinating the metal ions to the tag-modified particles obtained in step (1), and (3) the metal ion coordination particles obtained in step (2) are brought close to a conductive material, Immobilizing the metal ion coordination particles to the conductive material through metal atoms generated by reduction of the metal ion by applying a reduction potential to the conductive material;
The electrochemical immobilization method comprising the steps of:
（１）リンカーを介して金属イオン配位結合部位及び粒子結合部位を夫々一端に含むタグに金属イオンを配位させる工程、
（２）工程（１）で得られた金属イオン配位タグを導電性材料に近接させ、該導電性材料に還元電位を印加することにより、該タグを該金属イオンの還元により生じる金属原子を介して該導電性材料に固定化させる工程、及び
（３）工程（２）で得られたタグ修飾材料と該粒子を反応させて、該粒子を該導電性材料に固定化する工程、
を含むことを特徴とする前記電気化学的固定化法。 A method of electrochemically immobilizing particles selected from the group consisting of metal colloids, fullerenes, carbon nanotubes and ceramic particles to a conductive material, comprising :
(1) A step of coordinating a metal ion to a tag that includes a metal ion coordination binding site and a particle binding site at one end via a linker,
(2) The metal ion coordination tag obtained in the step (1) is brought close to the conductive material, and a reduction potential is applied to the conductive material, whereby the metal atoms generated by reduction of the metal ion are removed from the tag. A step of immobilizing the particles on the conductive material, and (3) a step of reacting the particle with the tag modifying material obtained in step (2) to immobilize the particles on the conductive material.
The electrochemical immobilization method comprising the steps of:
A metal colloid, a fullerene, and a carbon produced by a tag each including a metal ion coordination binding site and a particle binding site at one end via a linker , produced by the immobilization method according to claim 1. A particle-immobilized conductive material, wherein particles selected from the group consisting of nanotubes and ceramic particles are bonded to the surface of the conductive material.
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