JP5050267B2 - Manufacturing method of three-dimensional structure - Google Patents
Manufacturing method of three-dimensional structure Download PDFInfo
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本発明は、微細な粒状材料を、架橋体を介して結合してなり、例えば生化学的反応・分析のための核酸、酵素、抗体、抗原、プローブ等を担持する担体等として利用できる三次元構造体の製造方法に関する。 The present invention is a three-dimensional structure in which a fine particulate material is bound via a cross-linked body and can be used as a carrier for carrying a nucleic acid, enzyme, antibody, antigen, probe, etc. for biochemical reaction / analysis, for example. The present invention relates to a method for manufacturing a structure.
ポリスチレンビーズ、シリカゲルビーズ等の各種の粒状材料は、生化学的反応・分析のための核酸、酵素、抗体、抗原、プローブ等を担持する担体等として広く利用されている。試料の反応・分析系を微小領域に集約した微小分析システム（micro-total analysis systems μ-TAS）等においては、その担持担体は、微小分析システム基板上の所望の微細空間位置に配される。 Various granular materials such as polystyrene beads and silica gel beads are widely used as carriers for supporting nucleic acids, enzymes, antibodies, antigens, probes, etc. for biochemical reactions and analyses. In a micro-total analysis system (micro-total analysis system μ-TAS) in which sample reaction / analysis systems are aggregated in a micro region, the carrier is disposed in a desired micro space position on the micro analysis system substrate.
例えば、下記特許文献１には、各々単一のビーズを収容する２次元的に規則的に配列された複数のビーズ固定部位と、前記ビーズ固定部位同士を結合する微細溝とを有する微細構造パターンが表面に形成された硬質基板と、弾性基板と、前記硬質基板の前記微細構造パターンが形成された表面に前記弾性基板を接触させ加圧するための手段とを備えることを特徴とするマイクロ流路ビーズアレイデバイスの発明が開示されている。
しかしながら、上記特許文献１に記載のマイクロ流路ビーズアレイデバイスでは、基板上に微細加工を施してビーズの固定部位となる穴部を設け、この穴部にビーズを配設するので、微細穴部を加工したり、固定位置に配位しないビーズを取り除いた後、ビーズが充填された穴部に蓋をする加工をしたりしなければならず、担持担体としてのビーズの利用が、加工に煩雑な工程を要するプラットフォームに制限されてしまうという問題があった。また、１０マイクロメートル以下の直径のビーズを利用するのには困難を伴うという問題があった。更に、単位スポットとなる１の穴部あたりに単一のビーズが配設されるので、ビーズ直径サイズで規定される以上に担持担体としての有効表面積を稼ぐことができなかった。 However, in the microchannel bead array device described in Patent Document 1 described above, the micro-processing is performed on the substrate to provide a hole to be a fixed part of the bead, and the bead is disposed in the hole. Or removing the beads that are not coordinated at the fixed position, and then processing to cover the hole filled with beads, and the use of beads as a carrier is complicated to process. There is a problem that it is limited to a platform that requires a complicated process. In addition, there is a problem that it is difficult to use beads having a diameter of 10 micrometers or less. Furthermore, since a single bead is disposed per one hole serving as a unit spot, the effective surface area as a carrier could not be obtained more than specified by the bead diameter size.
したがって、本発明の目的は、粒状材料が三次元に配列してなる三次元構造体であって、機械的に安定で、担持担体として様々なプラットフォームに応用可能であって、且つ、占有空間あたりの表面積の大きな三次元構造体の製造方法を提供することにある。 Accordingly, an object of the present invention is a three-dimensional structure in which granular materials are arranged three-dimensionally, is mechanically stable, can be applied to various platforms as a carrier, and It is to provide a method for producing a large three-dimensional structure of the surface area.
本発明者らは、ポリスチレンビーズ等の粒状材料を、高分子物質を含有する液状媒質に懸濁し、その分散媒を徐々に除くと該粒状材料の各粒子が自己集合して三次元に配列し、その三次元に配列した粒状材料同士を架橋することによって、機械的に安定な三次元構造体を作製できることを見出し、本発明を完成するに至った。 The present inventors suspended a granular material such as polystyrene beads in a liquid medium containing a polymer substance, and when the dispersion medium was gradually removed, the particles of the granular material self-assembled and arranged in three dimensions. The inventors have found that a mechanically stable three-dimensional structure can be produced by crosslinking the three-dimensionally arranged granular materials, and have completed the present invention.
すなわち、本発明の三次元構造体の製造方法は、平均粒子径０．０２〜１０μｍの粒状材料を光反応性架橋剤を含む架橋体構成材料により結合してなる三次元構造体の製造方法であって、液状媒質に前記粒状材料を懸濁し、該懸濁液を支持面に液滴状にして付着させる液滴形成工程と、前記液滴形成工程によって前記支持面に付着させた前記懸濁液の液体成分を、前記光反応性架橋剤の光反応性架橋が起こらない条件で蒸発させ、前記粒状材料の各粒子を自己集合させて三次元に配列化させる粒子配列工程と、前記光反応性架橋剤を含む架橋体構成材料をＵＶ光の照射により架橋させて架橋体を形成し、該架橋体を介して、前記粒子配列工程によって三次元に配列化させた前記粒状材料の各粒子を結合する架橋工程と、を含むことを特徴とする。 That is, the method for producing a three-dimensional structure according to the present invention is a method for producing a three-dimensional structure formed by bonding a granular material having an average particle diameter of 0.02 to 10 μm with a crosslinked material constituting a photoreactive crosslinking agent. A droplet forming step of suspending the particulate material in a liquid medium and attaching the suspension to the support surface in the form of droplets, and the suspension adhered to the support surface by the droplet formation step. the liquid component of the liquid is evaporated under the conditions photoreactive crosslinking of the photoreactive crosslinking agent does not occur, the particle array step of each particle is arranged into three-dimensional by self-assembly of the particulate material, the photoreactive the crosslinked material of construction including sexual crosslinking agent to form a crosslinked body by crosslinking by irradiation with UV light, through the cross member, each particle of said particulate material are arranged into three-dimensional by the particles aligning step A cross-linking step for bonding .
本発明の三次元構造体の製造方法によれば、粒状材料を分散させた懸濁液を支持面に液滴状にして付着させた後に、懸濁液の液体成分を蒸発させることにより、粒状材料の各粒子が自己集合し三次元に配列化するので、粒状材料の各粒子が三次元に密に配位しつつ各粒子間に空隙を有している三次元構造となる。そして、その状態で架橋体構成材料を架橋させることにより、架橋体を介して、粒状材料の各粒子を結合することができる。それによって、機械的にも安定な三次元構造体を製造することができる。
また、ＵＶ光を照射して粒状材料の各粒子を結合するので、粒状材料の各粒子が自己集合し三次元に配列化するタイミングにあわせてそのＵＶ光を照射でき、各粒子が配列化する前や、配列が崩れてしまった後に結合してしまうことを防ぐことができる。
According to the method for producing a three-dimensional structure of the present invention, the suspension in which the particulate material is dispersed is deposited in the form of droplets on the support surface, and then the liquid component of the suspension is evaporated, Since the particles of the material are self-assembled and arranged in a three-dimensional manner, the particles of the granular material have a three-dimensional structure in which the particles are closely coordinated in three dimensions and have voids between the particles. And each particle | grains of a granular material can be couple | bonded through a crosslinked body by bridge | crosslinking a crosslinked body structural material in the state. As a result, a mechanically stable three-dimensional structure can be manufactured.
Moreover, since each particle | grain of a granular material is couple | bonded by irradiating UV light, the particle | grains of a granular material can irradiate the UV light according to the timing which self-assembles and arranges in three dimensions, and each particle arranges It is possible to prevent them from being combined before or after the arrangement is broken.
本発明の三次元構造体の製造方法においては、前記液滴形成工程において、架橋体構成材料の少なくとも一成分を含有する液状媒質に前記粒状材料を懸濁し、該懸濁液を支持面に液滴状にして付着させることができる。これによれば、その架橋体構成材料の少なくとも一成分を構成成分とする架橋体を介して粒状材料の各粒子を結合することができる。 In the method for producing a three-dimensional structure according to the present invention, in the droplet forming step, the granular material is suspended in a liquid medium containing at least one component of the cross-linked body constituent material, and the suspension is liquid-loaded on the support surface. It can be deposited in the form of drops. According to this, each particle | grain of granular material can be couple | bonded through the crosslinked body which uses at least 1 component of the crosslinked body constituent material as a structural component.
本発明の三次元構造体の製造方法においては、前記粒子配列工程を経て配列化した粒状材料の各粒子に、架橋体構成材料の少なくとも一成分を添加することができる。これによれば、その架橋体構成材料の少なくとも一成分を構成成分とする架橋体を介して粒状材料の各粒子を結合することができる。 In the method for producing a three-dimensional structure according to the present invention, at least one component of the crosslinked body constituting material can be added to each particle of the granular material arranged through the particle arrangement step. According to this, each particle | grain of granular material can be couple | bonded through the crosslinked body which uses at least 1 component of the crosslinked body constituent material as a structural component.
本発明の三次元構造体の製造方法においては、前記架橋体構成材料が、平均分子量１，０００〜１，０００，０００の高分子物質と、前記光反応性架橋剤として、前記高分子物質及び前記粒状材料に結合し得る架橋剤と、を含むものであり、該架橋剤は、（１）前記粒状材料の各粒子が三次元に配列化する前に添加するか、（２）前記粒状材料の各粒子が三次元に配列化する途中に添加するか、（３）前記粒状材料の各粒子が三次元に配列化した後に添加するか、又は（４）予め前記高分子物質に側鎖として導入されていることが好ましい。これによれば、添加されるか、又は予め前記高分子物質に側鎖として導入された架橋剤により、その高分子物質を構成成分とする架橋体が形成され、その架橋体を介して、粒状材料を結合させることができる。
また、前記架橋体が、平均分子量１，０００〜１，０００，０００の高分子物質を含む堅固な構造体となるので、粒状材料の各粒子間に充分な空隙を保ちつつ、機械的にもより安定である。
In the method for producing a three-dimensional structure according to the present invention, the crosslinked material constituting material includes a polymer substance having an average molecular weight of 1,000 to 1,000,000, and the polymer substance and the photoreactive crosslinking agent. wherein the cross-linking agent capable of binding to the particulate material, is intended to include a crosslinking agent, (1) or the particles of the particulate material is added prior to sequencing the three-dimensional, (2) said particulate material Or (3) added after each particle of the granular material is arranged in three dimensions, or (4) as a side chain to the polymer material in advance. It is preferably introduced. According to this, a cross-linked body containing the high-molecular substance as a constituent component is formed by the cross-linking agent that is added or previously introduced as a side chain to the high-molecular substance, and the granular material is formed through the cross-linked body. Materials can be combined.
In addition, since the crosslinked body becomes a solid structure including a high molecular weight substance having an average molecular weight of 1,000 to 1,000,000, mechanically while maintaining sufficient voids between the particles of the granular material. More stable.
本発明の三次元構造体の製造方法においては、前記架橋工程において光を照射して前記粒状材料の各粒子を結合することが好ましい。これによれば、粒状材料の各粒子が自己集合し三次元に配列化するタイミングにあわせて光照射できるので、各粒子が配列化する前や、配列が崩れてしまった後に結合してしまうことを防ぐことができる。 In the production method of the three-dimensional structure of the present invention, it is preferable to irradiate light in front Symbol crosslinking step to combine the particles of the particulate material. According to this, each particle of the granular material can irradiate light in accordance with the timing of self-assembly and three-dimensional arrangement, so that the particles are combined before or after the arrangement is broken. Can be prevented.
本発明の三次元構造体の製造方法においては、前記高分子物質が、蛋白質、ポリアミノ酸、核酸、及び樹脂からなる群より選ばれた少なくとも一種であることが好ましく、ウシ血清アルブミンであることがより好ましい。 In the method for producing a three-dimensional structure of the present invention, the polymer substance is preferably at least one selected from the group consisting of proteins, polyamino acids, nucleic acids, and resins, and is bovine serum albumin. More preferred.
本発明の三次元構造体の製造方法においては、前記液滴形成工程において、インクジェット方式により、前記懸濁液を支持面に液滴状にして付着させることが好ましい。これによれば、支持面に数十マイクロメートルの位置精度で液滴状にして付着させることができるので、前記三次元構造体を所望の場所に、所望の大きさで作製することが容易である。特に、微小分析システム（micro-total analysis systems μ-TAS）に応用する場合に、基板上の所望の微細空間位置に前記三次元構造体を配置することが容易にできる。 In the method for producing a three-dimensional structure according to the present invention, in the droplet forming step, it is preferable that the suspension is deposited in the form of droplets on a support surface by an inkjet method. According to this, since the droplet can be attached to the support surface with a positional accuracy of several tens of micrometers, it is easy to produce the three-dimensional structure at a desired location and in a desired size. is there. In particular, when applied to a micro-total analysis system (μ-TAS), the three-dimensional structure can be easily arranged at a desired fine space position on the substrate.
本発明によって得られる三次元構造体によれば、平均粒子径０．０２〜１０μｍの粒状材料が、架橋体を介して結合しているので、粒状材料の各粒子が三次元に密に配位しつつ各粒子間に空隙を有している。したがって、粒状材料の各粒子の表面を被担持物質が結合する担持表面とすることができるので、該三次元構造体の占める空間あたりの担持担体としての有効面積がきわめて大きな三次元構造体とすることができる。また、機械的にも安定である。 According to the three-dimensional structure obtained by the present invention , since the granular material having an average particle diameter of 0.02 to 10 μm is bonded via the crosslinked body, each particle of the granular material is closely coordinated in three dimensions. However, there are voids between the particles. Therefore, since the surface of each particle of the granular material can be a supporting surface to which a supported substance is bonded, a three-dimensional structure having an extremely large effective area as a supporting carrier per space occupied by the three-dimensional structure is obtained. be able to. It is also mechanically stable.
また、本発明によって得られる三次元構造体に、生体由来物質又は該生体由来物質に結合するプローブ分子を担持して生体関連分子担持体とすれば、前記三次元構造体における粒状材料の各粒子の表面に生化学的反応・分析のための生体由来物質や、これに結合するプローブ分子が担持されているので、各粒子間の空隙においてその生体由来物質の活性を発揮させることができる。そして、高度に集積した場で酵素反応や結合反応等を行わせることができるので、高感度な反応・分析系及び／又は検出系を構築することができる。 Further, the three-dimensional structure obtained by the present invention, each of the particulate material the probe molecules that bind to the biological material or biological derived material carrying the biologically relevant molecule carrying member and to lever, the three-dimensional structure Since the surface of the particle carries a biological substance for biochemical reaction / analysis and a probe molecule that binds to the biological substance, the activity of the biological substance can be exhibited in the voids between the particles. And since an enzyme reaction, a binding reaction, etc. can be performed in the highly integrated place, a highly sensitive reaction / analysis system and / or detection system can be constructed.
また、本発明の製造方法によれば、粒状材料を分散させた懸濁液を支持面に液滴状にして付着させた後に、懸濁液の液体成分を蒸発させることにより、粒状材料の各粒子が自己集合し三次元に配列化するので、粒状材料の各粒子が三次元に密に配位しつつ各粒子間に空隙を有している三次元構造となる。そして、その状態で架橋体構成材料を架橋させて形成される架橋体を介して粒状材料の各粒子同士を結合させることができるので、機械的にも安定な三次元構造体を製造することができる。 Further, according to the manufacturing method of the present invention, after depositing in the droplet shape of the suspension of the particulate material is dispersed support surface, by evaporating the liquid component of the suspension, of the particulate material Since each particle is self-assembled and arranged in three dimensions, each particle of the granular material has a three-dimensional structure in which voids are provided between the particles while being closely coordinated in three dimensions. And since each particle | grain of granular material can be couple | bonded through the crosslinked body formed by bridge | crosslinking a crosslinked body structural material in the state, it can manufacture a mechanically stable three-dimensional structure. it can.
本発明の三次元構造体は以下のようにして得ることができる。 The three-dimensional structure of the present invention can be obtained as follows.
すなわち、平均粒子径０．０２〜１０μｍの粒状材料を液状媒質に懸濁する。その懸濁液を支持面に液滴状にして付着させた後（液滴形成工程）、前記液滴形成工程によって前記支持面に付着させた懸濁液の液体成分を、光反応性架橋剤の光反応性架橋が起こらない条件で蒸発させ、前記粒状材料の各粒子を自己集合させて三次元に配列化させる（粒子配列工程）。そして、光反応性架橋剤を含む架橋体構成材料をＵＶ光の照射により架橋させて架橋体を形成し、該架橋体を介して、前記粒子配列工程によって三次元に配列化させた粒状材料の各粒子を結合する（架橋工程）。 That is, a granular material having an average particle diameter of 0.02 to 10 μm is suspended in a liquid medium. After the suspension is adhered to the support surface in the form of droplets (droplet formation step), the liquid component of the suspension adhered to the support surface by the droplet formation step is converted into a photoreactive crosslinking agent. The particles are evaporated under conditions that do not cause photoreactive crosslinking, and the particles of the granular material are self-assembled to form a three-dimensional array (particle alignment step). Then, the crosslinked structure material containing a photoreactive crosslinking agent by crosslinking by irradiation with UV light to form a crosslinked body, through the cross member, of the particulate material are arranged into three-dimensional by the particles aligning step Each particle is bonded (crosslinking step).
架橋体構成材料は、液滴形成工程において粒状材料を懸濁する液状媒質に配合しておいてもよく、粒子配列工程を経て配列化した粒状材料の各粒子に添加することもできる。なお、粒子配列工程を経て配列化した粒状材料の各粒子に添加する場合には、三次元の配列を崩さないように添加することが重要であるので、例えばキャピラリー現象を利用して、粒状材料の各粒子間に浸透させるようにして添加することが好ましい。 The crosslinked body constituting material may be blended in a liquid medium in which the particulate material is suspended in the droplet forming step, or may be added to each particle of the particulate material arranged through the particle arranging step. In addition, when adding to each particle of the granular material arranged through the particle arrangement process, it is important to add so as not to destroy the three-dimensional arrangement. For example, using the capillary phenomenon, the granular material It is preferable to add so as to penetrate between the particles.
架橋体構成材料としては、平均分子量１，０００〜１，０００，０００の高分子物質と、前記光反応性架橋剤として、この高分子物質及び前記粒状材料を結合し得る架橋剤とを含むものが好ましく用いられる。この場合、液滴形成工程において、液状媒質中に高分子物質と架橋剤とを一緒に含有させてもよく、あるいは、液滴形成工程後に架橋体構成材料を添加してもよい。更に、架橋剤を予め前記高分子物質に側鎖として導入させておくこともできる。したがって、架橋剤の添加は、（１）前記粒状材料の各粒子が三次元に配列化する前に添加するか、（２）前記粒状材料の各粒子が三次元に配列化する途中に添加するか、（３）前記粒状材料の各粒子が三次元に配列化した後に添加するか、又は（４）予め前記高分子物質に側鎖として導入されるかのいずれであってもよい。 The cross-linked body constituting material includes a high-molecular substance having an average molecular weight of 1,000 to 1,000,000, and a cross-linking agent capable of binding the high-molecular substance and the granular material as the photoreactive cross-linking agent. Is preferably used. In this case, in the droplet formation step, the polymer substance and the crosslinking agent may be contained together in the liquid medium, or the cross-linked material may be added after the droplet formation step. Furthermore, a crosslinking agent can be introduced into the polymer substance as a side chain in advance. Therefore, the addition of the crosslinking agent is (1) added before the particles of the granular material are arranged in three dimensions, or (2) added in the middle of the particles of the granular material being arranged in three dimensions. Alternatively, (3) each particle of the granular material may be added after being arranged in three dimensions, or (4) it may be previously introduced as a side chain into the polymer substance.
架橋体構成材料に用いる高分子物質としては、例えば、蛋白質、ポリアミノ酸、核酸、樹脂などが挙げられる。特にウシ血清アルブミンを好ましく例示できる。また、２種以上の高分子物質を併用して用いてもよい。高分子物質の平均分子量は、１，０００〜１，０００，０００が好ましく、１０，０００〜１５０，０００がより好ましく、６６，０００〜１７０，０００が更に好ましい。 Examples of the polymer substance used for the crosslinked body constituting material include proteins, polyamino acids, nucleic acids, resins, and the like. Particularly preferred is bovine serum albumin. Two or more kinds of polymer substances may be used in combination. The average molecular weight of the polymer substance is preferably 1,000 to 1,000,000, more preferably 10,000 to 150,000, and still more preferably 66,000 to 170,000.
上記粒状材料としては、特に制限はなく、例えばポリスチレンビーズ、シリカゲルビーズ、ポリ塩化ビニルビーズ、ゼラチンビーズ、ナイロンビーズ、ポリウレタンビーズなどを用いることができる。その粒子径は、上記粒状材料の各粒子が自己集合して三次元の配列化を生じさせることができる範囲であることが必要であり、平均粒子径０．０２〜１０μｍであることが好ましく、平均粒子径１〜７．０μｍであることがより好ましく、平均粒子径２．５〜５．０μｍであることが更により好ましい。 There is no restriction | limiting in particular as said granular material, For example, a polystyrene bead, a silica gel bead, a polyvinyl chloride bead, a gelatin bead, a nylon bead, a polyurethane bead etc. can be used. The particle diameter needs to be in a range where each particle of the granular material can self-assemble to produce a three-dimensional arrangement, and preferably has an average particle diameter of 0.02 to 10 μm. The average particle size is more preferably 1 to 7.0 μm, and even more preferably 2.5 to 5.0 μm.
上記粒状材料を懸濁させた懸濁液を支持面に液滴状にして付着させるためには、ガラス、シリコン、ＰＤＭＳ（Polydimethylsiloxane）等を材料とする基板表面を支持面として用いることができる。特に、ＰＤＭＳ基板を用いれば、ＰＤＭＳを半硬化状にしたものを上記支持面として用いた後に、完全硬化してビーズ等をＰＤＭＳ基板に接着固定化できるので、好ましい。 In order to adhere the suspension in which the particulate material is suspended to the support surface in the form of droplets, a substrate surface made of glass, silicon, PDMS (Polydimethylsiloxane), or the like can be used as the support surface. In particular, it is preferable to use a PDMS substrate because a semi-cured PDMS is used as the support surface and then completely cured to bond and fix the beads to the PDMS substrate.
上記粒状材料を懸濁させる液状媒質としては、上記粒状材料の各粒子を分散させることができる媒質であって、且つ、上記支持面を損傷しない媒質であれば特に制限はなく、水などを用いることができる。 The liquid medium for suspending the granular material is not particularly limited as long as it is a medium that can disperse each particle of the granular material and does not damage the support surface, and water or the like is used. be able to.
上記光反応性架橋剤としては、特に光反応性官能基であるアジド基（−Ｎ３基）を有する架橋剤が挙げられる。これによれば、ポリスチレンビーズなどの骨格炭素部分に直接に共有結合を形成させることができるので好ましい。 As the photoreactive crosslinking agent, a crosslinking agent having azido groups (--N3 group) is a photoreactive functional group, especially the like. According to this, good preferable in can be formed directly covalently attached to the backbone carbon moiety such as polystyrene beads.
また、架橋体構成材料として、平均分子量１，０００〜１，０００，０００の高分子物質に、光反応性官能基を有する架橋剤を予め側鎖として導入したものを用いることもできる。これによれば、側鎖に光反応性官能基が結合しているので、別途架橋剤を添加しなくても、光照射するだけで架橋させることができる。 In addition, as the cross-linked material, a material obtained by introducing a cross-linking agent having a photoreactive functional group into a polymer substance having an average molecular weight of 1,000 to 1,000,000 as a side chain in advance can be used. According to this, since the photoreactive functional group is bonded to the side chain, it can be crosslinked only by light irradiation without adding a separate crosslinking agent.
架橋は、上記粒状材料と上記高分子物質との共存下、架橋剤が架橋作用を発揮させる条件にすることにより行う。例えば、アジド基を有する架橋剤であれば、光（ＵＶ）を照射してアジド基を活性化してアルキル骨格の炭素元素等と共有結合させることができる。アジド基を有する架橋剤としては、光反応性官能基を有する「光架橋剤」、ATFB-SE(4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester)、ATFB-STP ester (4-azido-2,3,5,6-tetrafluorobenzoic acid, STP ester, sodium salt)、4-azido-2,3,5,6-tetrafluorobenzyl amine, hydrochloride、PEAS-AET (N-((2-pyridyldithio)ethyl)-4-azidosalicylamide)、benzophenone-4-maleimide、benzophenone-4-isothiocyanate、4-benzoylbenzoic acid, succinimidyl ester等を好ましく例示することができる。 Crosslinking is performed under conditions where the crosslinking agent exhibits a crosslinking action in the presence of the particulate material and the polymer substance. For example, in the case of a crosslinking agent having an azide group, the azide group can be activated by irradiation with light (UV) to be covalently bonded to a carbon element or the like of an alkyl skeleton. Examples of the crosslinking agent having an azide group include a `` photocrosslinking agent '' having a photoreactive functional group, ATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester), ATFB-STP ester ( 4-azido-2,3,5,6-tetrafluorobenzoic acid, STP ester, sodium salt), 4-azido-2,3,5,6-tetrafluorobenzyl amine, hydrochloride, PEAS-AET (N-((2-pyridyldithio ) ethyl) -4-azidosalicylamide), benzophenone-4-maleimide, benzophenone-4-isothiocyanate, 4-benzoylbenzoic acid, succinimidyl ester and the like.
本発明においては、上記懸濁液の液体成分を蒸発させて除き、上記粒状材料の各粒子を自己集合させて三次元に配列化させ、架橋剤又は高分子物質に側鎖として予め導入された架橋剤の官能基による架橋反応を行わせる。したがって、上記粒状材料の各粒子が配列化する前や、配列が崩れてしまった後に結合してしまうことがないように、上記懸濁液の液体成分を蒸発させて除くタイミングと、架橋作用を発揮させるタイミングとを図ることが重要であり、架橋は、上記粒状材料と上記高分子物質との共存下、上記粒状材料の各粒子が三次元に配列化した状態で架橋剤又は高分子物質に予め導入された官能基が架橋作用を発揮させる条件にすることにより行う。 In the present invention, the liquid component of the suspension is removed by evaporation, and the particles of the granular material are self-assembled and arranged in a three-dimensional manner. A crosslinking reaction is performed by the functional group of the crosslinking agent. Therefore, the timing for removing the liquid component of the suspension by evaporating and the cross-linking action so that the particles of the granular material are not bonded before being arranged or after the arrangement is broken. It is important to achieve the timing to exert the crosslinking, and in the coexistence of the granular material and the polymer substance, the crosslinking agent or the polymer substance is formed in a state where the particles of the granular material are arranged in three dimensions. It is carried out by setting the conditions so that the previously introduced functional group exerts a crosslinking action.
好ましい態様を挙げると、例えば、アジド基を有する架橋剤をあらかじめ上記高分子物質に共有結合させておき、その高分子物質を含有する液状媒質に粒状材料を懸濁し、その懸濁液を支持面に液滴状にして付着させ、その懸濁液の液体成分を蒸発させて除き、ＵＶを照射してアジド基を活性化して反応させることができる。更に、上記懸濁液の液体成分の蒸発の際には、湿度や温度を調節して、上記粒状材料の各粒子の自己配列化のタイミングを調節することができる。 As a preferred embodiment, for example, a crosslinking agent having an azido group is covalently bonded to the above-mentioned polymer substance in advance, the particulate material is suspended in a liquid medium containing the polymer substance, and the suspension is supported on the support surface. The liquid component of the suspension is evaporated and removed, and the azide group is activated by UV irradiation to react . Further, in case of evaporation of the liquid component of the suspension, it is possible to regulate the humidity and temperature, to adjust the timing of self-sequencing of each particle of the particulate material.
以下には、本発明において、粒状材料の各粒子を、架橋剤により高分子物質を介して結合する一態様について説明する。 Hereinafter, in the present invention, an embodiment in which each particle of the particulate material is bonded via a polymer substance with a crosslinking agent will be described.
ここでは、まず、高分子物質としてウシ血清アルブミン（ＢＳＡ）を用いる。このウシ血清アルブミン（ＢＳＡ）の側鎖アミノ基に、光架橋剤であるATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester)を用いて、光反応性官能基（Ｎ３基；アジド基）を導入する。ウシ血清アルブミン（ＢＳＡ）以外の蛋白質を用いても同様に光反応性官能基（Ｎ３基；アジド基）を導入することができることは勿論である。その反応を模式的に表すと下記のように表すことができる。また、図１（ａ）にはその模式図を示す。 Here, first, bovine serum albumin (BSA) is used as a polymer substance. A photoreactive functional group is used for the side chain amino group of bovine serum albumin (BSA) by using a photocrosslinking agent ATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester). introducing; (azide group N 3 group). It goes without saying that a photoreactive functional group (N 3 group; azide group) can be similarly introduced using a protein other than bovine serum albumin (BSA). The reaction can be schematically expressed as follows. Moreover, the schematic diagram is shown in FIG.
次に、上記のようにして光反応性官能基が導入された蛋白質をポリスチレンビーズ等の粒状材料に導入する。反応を模式的に表すと下記のように表すことができる。 Next, the protein into which the photoreactive functional group has been introduced as described above is introduced into a granular material such as polystyrene beads. The reaction can be schematically expressed as follows.
すなわち、ＵＶ照射でＮ３基（アジド基）を活性化し、ポリスチレンビーズ等のアルキル骨格の炭素部分等に共有結合させることができる。そして、蛋白質１分子中に２箇所以上にＮ３基（アジド基）を導入しておけば、架橋剤により高分子物質を介して粒状材料の各粒子同士を結合することができる。図１（ｂ）にはその架橋態様の模式図を示す。また、蛋白質同士も架橋され得るので、この場合は、蛋白質の重合体を介して粒状材料の各粒子が結合する。図１（Ｃ）にはその架橋態様の模式図を示す。 That is, the N 3 group (azido group) can be activated by UV irradiation and can be covalently bonded to a carbon portion of an alkyl skeleton such as polystyrene beads. Then, it is possible if by introducing N 3 group (azide group) in two or more locations in the protein molecule, coupling each particles of the particulate material through the polymeric material with a crosslinking agent. FIG. 1B shows a schematic diagram of the crosslinking mode. In addition, since the proteins can also be cross-linked, in this case, the particles of the particulate material are bonded via the protein polymer. FIG. 1C shows a schematic diagram of the crosslinking mode.
このようにして得られた三次元構造体では、構造体の厚さ寸法領域当たりに複数個の上記粒状材料の各粒子を含み、空隙を保ちつつ三次元に密に配列している。平均粒子径０．１〜１０μｍの粒状材料を用いた場合、その空隙はおよそ０．０５〜１０μｍの幅となる。更に、上記高分子物質を成分として含む架橋体が上記粒状材料の各粒子間に形成されているので機械的にも安定である。 The three-dimensional structure thus obtained includes a plurality of particles of the granular material per thickness dimension region of the structure, and is densely arranged in three dimensions while maintaining voids. When a granular material having an average particle size of 0.1 to 10 μm is used, the voids have a width of about 0.05 to 10 μm. Furthermore, since the crosslinked body containing the polymer substance as a component is formed between the particles of the granular material, it is mechanically stable.
上記の三次元構造体には、これに生体由来物質又は該生体由来物質に結合するプローブ分子を担持して、生体関連分子担持体とすることができる。 The three-dimensional structure of the upper SL, to which carries a probe molecule which binds to a biological material or biological derived materials can be a bio-related molecule carrier.
生体由来物質としては、核酸、酵素、抗体、抗原等が挙げられる。また、生体由来物質に結合するプローブ分子としては、抗体エピトープ等の合成ペプチド断片、ヒスチジンＴａｇ導入蛋白質精製のためのニッケルキレート官能基、ビオチン、グルタチオン、アビジン等が挙げられる。具体的には、例えば、所定の抗原に対する特異抗体を担持しておき、ＥＬＩＳＡ法に応用することができる。また、例えば、オリゴヌクレオチドを担持しておき、相補的に結合するＤＮＡのクローニングや定量などに応用することができる。
The BIOLOGICAL derived substances, nucleic acids, enzymes, antibodies, antigens and the like. Examples of probe molecules that bind to biologically derived substances include synthetic peptide fragments such as antibody epitopes, nickel chelate functional groups for purification of histidine Tag-introduced proteins, biotin, glutathione, and avidin. Specifically, for example, a specific antibody against a predetermined antigen can be carried and applied to the ELISA method. In addition, for example, it can be applied to cloning or quantification of DNA that supports oligonucleotides and binds complementarily.
これらの生体由来物質又は該生体由来物質に結合するプローブ分子を上記の三次元構造体に担持するには、通常用いられる架橋方法等で行うことができる。そのための官能基として、例えば、−ＮＨ２基、−ＣＯＯＨ基、−ＳＨ基などを有する粒状材料を用いるか、粒状材料に−ＮＨ２基、−ＣＯＯＨ基、−ＳＨ基などの官能基を導入してもよく、更には、上記高分子物質として用いたものの官能基を利用するか、上記高分子物質として用いたものに官能基を導入することができる。例えば、−ＮＨ３基対−ＣＯＯＨ基を架橋するための架橋剤、又は−ＮＨ３基対−ＳＨ基を架橋するための、いわゆる二官能性架橋剤等を用いることによって効率よく導入することができる。このような架橋剤としては、−ＮＨ２基対−ＣＯＯＨ基を架橋するための架橋剤として、１，１−カルボニルジイミダゾール、ジシクロヘキシルカルボジイミドおよび３−（３−ジメチルアミノプロピル）−１−エチルカルボジイミド・塩酸塩等、−ＮＨ２基対−ＳＨ基を架橋するための架橋剤として、N-Succinimidyl 3-(2-pyridyldithio)propionate（ＳＰＤＰ）、N-(8-Maleimidocapryloxy)sulfosuccinimide、N-(6-Maleimidocaproyloxy)sulfosuccinimide等が挙げられる。 In order to carry such a biological substance or a probe molecule that binds to the biological substance on the three-dimensional structure, it can be performed by a commonly used crosslinking method or the like. For this purpose, for example, a granular material having —NH 2 group, —COOH group, —SH group or the like is used, or a functional group such as —NH 2 group, —COOH group, —SH group is introduced into the granular material. Furthermore, the functional group of the material used as the polymer material may be used, or the functional group may be introduced into the material used as the polymer material. For example, it can be efficiently introduced by using a cross-linking agent for cross-linking —NH 3 groups to —COOH groups or a so-called bifunctional cross-linking agent for cross-linking —NH 3 groups to —SH groups. it can. Such crosslinking agents include 1,1-carbonyldiimidazole, dicyclohexylcarbodiimide and 3- (3-dimethylaminopropyl) -1-ethylcarbodiimide as crosslinking agents for crosslinking —NH 2 groups to —COOH groups. -As crosslinking agents for crosslinking -NH2 groups versus -SH groups such as hydrochlorides, N-Succinimidyl 3- (2-pyridyldithio) propionate (SPDP), N- (8-Maleimidocapryloxy) sulfosuccinimide, N- (6- Maleimidocaproyloxy) sulfosuccinimide and the like.
なお、生体由来物質又は該生体由来物質に結合するプローブ分子が、それ自体、上記架橋体構成材料として架橋体の一部を構成していてもよい。 In addition, the living body-derived substance or the probe molecule that binds to the living body-derived substance may itself constitute a part of the crosslinked body as the crosslinked body constituent material.
本発明においては、上記懸濁液を支持面に液滴状にして付着させる工程を、インクジェット方式により行うことができる。ここでインクジェット方式とは、上記高分子物質を含む液状媒質に粒状材料を懸濁した懸濁液を噴射ノズルから上記支持面に直接に吹き付けて定着させる方式をいう。従来の印刷技術では、インクジェット方式によりピコリットル単位で数十マイクロメートルの位置精度で吹き付けることも可能であるので、これを応用して、特に、微小分析システム（micro-total analysis systems μ-TAS）の基板上の所望の微細空間位置に配置される担持担体としての加工が容易に行うことができる。 In the present invention, the step of depositing the suspension on the support surface in the form of droplets can be performed by an ink jet method. Here, the ink jet method refers to a method in which a suspension in which a particulate material is suspended in a liquid medium containing the above-described polymer substance is directly blown and fixed to the support surface from an injection nozzle. With conventional printing technology, it is possible to spray with a positional accuracy of several tens of micrometers in picoliter units using the inkjet method, and this is especially applied to micro-total analysis systems (micro-total analysis systems μ-TAS). Can be easily processed as a carrier to be arranged at a desired fine space position on the substrate.
図２には、本発明の三次元構造体を、PDMS（Polydimethylsiloxane）の基板上に形成したときの模式図を示す。 FIG. 2 shows a schematic view when the three-dimensional structure of the present invention is formed on a PDMS (Polydimethylsiloxane) substrate.
以下、実施例を挙げて本発明を具体的に説明するが、これらの実施例は本発明の範囲を限定するものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, these Examples do not limit the scope of the present invention.
<実施例１>
上述した光架橋剤、ATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester)を用い、常法に従い、ウシ血清アルブミン（ＢＳＡ）の側鎖アミノ基に光反応性官能基であるアジド基を導入した。
<Example 1>
Using the photocrosslinking agent, ATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester) described above, photoreactivity to the side chain amino group of bovine serum albumin (BSA) according to a conventional method A functional azide group was introduced.
具体的には、ＤＭＳＯ中にATFB-SE を１０ｍｇ／ｍｌの濃度で溶解し、その０．１ｍｌを、ＢＳＡを１０ｍｇ／ｍｌの濃度で水に溶解したもの１ｍｌと混合し室温で１時間インキュベートした。１．５Ｍ ＮＨ２ＯＨ・ＨＣｌ水溶液０．１ｍｌを添加し１時間インキュベートして反応を完全に停止させた。 Specifically, ATFB-SE was dissolved in DMSO at a concentration of 10 mg / ml, and 0.1 ml thereof was mixed with 1 ml of BSA dissolved in water at a concentration of 10 mg / ml and incubated at room temperature for 1 hour. . The reaction was completely stopped by adding 0.1 ml of 1.5 M NH 2 OH · HCl aqueous solution and incubating for 1 hour.
上記のようにして得た光反応性官能基導入後のＢＳＡ含有反応液と、平均粒子径５μｍのポリスチレンビーズ（Duke Scientific Corporation社製）を６％（ｗ／ｖ）の濃度で水に懸濁させたものを用いて、ビーズ濃度が１％（ｗ／ｖ）であり、ＢＳＡ含有換算濃度が０．０５ｍｇ／ｍｌ、となるように水に混合してビーズ懸濁調製液とした。 The BSA-containing reaction solution after introduction of the photoreactive functional group obtained as described above and polystyrene beads having an average particle diameter of 5 μm (manufactured by Duke Scientific Corporation) are suspended in water at a concentration of 6% (w / v). The bead suspension was prepared by mixing with water so that the bead concentration was 1% (w / v) and the BSA-containing conversion concentration was 0.05 mg / ml.
６０℃で３０分間焼成したPDMS（Polydimethylsiloxane）の基板を用意し、上記ビーズ懸濁調製液をその基板上にマイクロピペットで２．５μｌ滴下した。 A substrate of PDMS (Polydimethylsiloxane) baked at 60 ° C. for 30 minutes was prepared, and 2.5 μl of the bead suspension preparation solution was dropped on the substrate with a micropipette.
湿度４０％以上の容器内で24時間以上かけてゆっくり乾燥させた後、UV (254 nm)を３０分間照射した後、水で３回洗浄して、PDMS（Polydimethylsiloxane）の基板上に、平均粒子径５μｍのポリスチレンビーズを結合してなる三次元構造体を得た。図３にはその電子顕微鏡写真を示す。 After drying slowly in a container with a humidity of 40% or more over 24 hours, UV (254 nm) is irradiated for 30 minutes, then it is washed with water three times, and the average particle is deposited on a PDMS (Polydimethylsiloxane) substrate. A three-dimensional structure formed by bonding polystyrene beads having a diameter of 5 μm was obtained. FIG. 3 shows an electron micrograph thereof.
図３に明らかなように、この三次元構造体のポリスチレンビーズは、およそ０．５〜５μｍの間隔をあけて略規則的に配列していた。また、その空隙の一部にはビーズを架橋する構造体が形成していた。また、この三次元構造体は、水で１５回洗浄しても崩れなかった。したがって、反応場として用いるのに十分な機械的安定性があることが示唆された。 As is clear from FIG. 3, the polystyrene beads of this three-dimensional structure were arranged approximately regularly with an interval of approximately 0.5 to 5 μm. In addition, a structure that cross-links the beads was formed in part of the gap. In addition, this three-dimensional structure did not collapse even after being washed with water 15 times. Therefore, it was suggested that there is sufficient mechanical stability to use as a reaction field.
<実施例２>
上記ビーズ懸濁調製液中のＢＳＡ含有換算濃度が０．５ｍｇ／ｍｌとなるように混合した以外は、実施例１と同様にして、三次元構造体を得た。図４にはその電子顕微鏡写真を示す。
<Example 2>
A three-dimensional structure was obtained in the same manner as in Example 1, except that mixing was performed so that the BSA-containing conversion concentration in the bead suspension preparation solution was 0.5 mg / ml. FIG. 4 shows an electron micrograph thereof.
図４に明らかなように、この三次元構造体のポリスチレンビーズは、およそ０．５〜５μｍの間隔をあけて略規則的に配列していた。また、この三次元構造体は、水で１５回洗浄しても崩れなかった。したがって、反応場として用いるのに十分な機械的安定性があることが示唆された。 As apparent from FIG. 4, the polystyrene beads of this three-dimensional structure were arranged approximately regularly with an interval of about 0.5 to 5 μm. In addition, this three-dimensional structure did not collapse even after being washed with water 15 times. Therefore, it was suggested that there is sufficient mechanical stability to use as a reaction field.
<実施例３>
上記ビーズ懸濁調製液中のＢＳＡ含有換算濃度が１．５ｍｇ／ｍｌとなるように混合した以外は、実施例１と同様にして、三次元構造体を得た。図５にはその電子顕微鏡写真を示す。
<Example 3>
A three-dimensional structure was obtained in the same manner as in Example 1 except that mixing was performed so that the BSA-containing conversion concentration in the bead suspension preparation solution was 1.5 mg / ml. FIG. 5 shows an electron micrograph thereof.
図５に明らかなように、この三次元構造体のポリスチレンビーズは、およそ０．５〜５μｍの間隔をあけて略規則的に配列していた。また、この三次元構造体は、水で１５回洗浄しても崩れなかった。したがって、反応場として用いるのに十分な機械的安定性があることが示唆された。 As is apparent from FIG. 5, the polystyrene beads of this three-dimensional structure were arranged approximately regularly with an interval of about 0.5 to 5 μm. In addition, this three-dimensional structure did not collapse even after being washed with water 15 times. Therefore, it was suggested that there is sufficient mechanical stability to use as a reaction field.
<実施例４>
上記ビーズ懸濁調製液中のＢＳＡ含有換算濃度が３．０ｍｇ／ｍｌとなるように混合した以外は、実施例１と同様にして、三次元構造体を得た。図６にはその電子顕微鏡写真を示す。
<Example 4>
A three-dimensional structure was obtained in the same manner as in Example 1 except that mixing was performed so that the BSA-containing conversion concentration in the bead suspension preparation solution was 3.0 mg / ml. FIG. 6 shows an electron micrograph thereof.
図６に明らかなように、この三次元構造体のポリスチレンビーズは、およそ０．５〜５μｍの間隔をあけて略規則的に配列していた。また、この三次元構造体は、水で１５回洗浄しても崩れなかった。したがって、反応場として用いるのに十分な機械的安定性があることが示唆された。 As apparent from FIG. 6, the polystyrene beads of this three-dimensional structure were arranged approximately regularly with an interval of approximately 0.5 to 5 μm. In addition, this three-dimensional structure did not collapse even after being washed with water 15 times. Therefore, it was suggested that there is sufficient mechanical stability to use as a reaction field.
<実施例５>
上記ビーズ懸濁調製液中のＢＳＡ含有換算濃度が５．０ｍｇ／ｍｌとなるように混合した以外は、実施例１と同様にして、三次元構造体を得た。図７にはその電子顕微鏡写真を示す。
<Example 5>
A three-dimensional structure was obtained in the same manner as in Example 1 except that mixing was performed so that the BSA-containing conversion concentration in the bead suspension preparation solution was 5.0 mg / ml. FIG. 7 shows an electron micrograph thereof.
図７に明らかなように、この三次元構造体のポリスチレンビーズは、およそ０．５〜５μｍの間隔をあけて略規則的に配列していた。また、この三次元構造体は、水で１５回洗浄しても崩れなかった。したがって、反応場として用いるのに十分な機械的安定性があることが示唆された。 As is clear from FIG. 7, the polystyrene beads of this three-dimensional structure were arranged approximately regularly with an interval of approximately 0.5 to 5 μm. In addition, this three-dimensional structure did not collapse even after being washed with water 15 times. Therefore, it was suggested that there is sufficient mechanical stability to use as a reaction field.
<実施例６>
上記ビーズ懸濁調製液中のＢＳＡ含有換算濃度が６．６７ｍｇ／ｍｌとなるように混合した以外は、実施例１と同様にして、三次元構造体を得た。図８にはその電子顕微鏡写真を示す。
<Example 6>
A three-dimensional structure was obtained in the same manner as in Example 1 except that the BSA-containing conversion concentration in the bead suspension preparation solution was 6.67 mg / ml. FIG. 8 shows an electron micrograph thereof.
図８に明らかなように、この三次元構造体のポリスチレンビーズは、およそ０．５〜５μｍの間隔をあけて略規則的に配列していた。また、この三次元構造体は、水で１５回洗浄しても崩れなかった。したがって、反応場として用いるのに十分な機械的安定性があることが示唆された。 As is apparent from FIG. 8, the polystyrene beads of this three-dimensional structure were arranged approximately regularly with an interval of approximately 0.5 to 5 μm. In addition, this three-dimensional structure did not collapse even after being washed with water 15 times. Therefore, it was suggested that there is sufficient mechanical stability to use as a reaction field.
なお、上記実施例４〜６で得られた三次元構造体は、支持体から剥離しても構造体を維持していた。これらの三次元構造体は、内部に大表面積を有する膜素材等として有用であると考えられた。 In addition, even if it peeled from the support body, the three-dimensional structure obtained in the said Examples 4-6 maintained the structure. These three-dimensional structures were considered useful as film materials having a large surface area inside.
<比較例１>
ＵＶ照射しない以外は実施例１と同様に三次元構造体の調製操作を行った。乾燥後、ビーズは基板上にとどまっていたが、水で洗浄操作を施すと配列がくずれ、２次元で配列したビーズの上に積層していたビーズの一部は流去してしまった。
<Comparative Example 1>
A three-dimensional structure was prepared in the same manner as in Example 1 except that UV irradiation was not performed. After drying, the beads remained on the substrate, but when washed with water, the arrangement was lost, and some of the beads stacked on the two-dimensionally arranged beads were washed away.
<実施例７>
BSAの代わりにAnti-IgAを用いてポリスチレンビーズを結合してなる三次元構造体を作成した。
<Example 7>
A three-dimensional structure formed by binding polystyrene beads using Anti-IgA instead of BSA was prepared.
ビーズ上述した光架橋剤、ATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester)を用い、常法に従い、Anti-IgAの側鎖アミノ基に光反応性官能基であるアジド基を導入した。 Beads Using the photocrosslinking agent mentioned above, ATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester), according to a conventional method, a photoreactive functional group on the side chain amino group of Anti-IgA An azide group was introduced.
具体的には、ＤＭＳＯ中にATFB-SE を１０ｍｇ／ｍｌの濃度で溶解し、その０．１ｍｌを、１ｍｇ／ｍｌのAnti-IgA１ｍｌと混合し室温で１時間インキュベートした。 Specifically, ATFB-SE was dissolved in DMSO at a concentration of 10 mg / ml, and 0.1 ml thereof was mixed with 1 ml of 1 mg / ml Anti-IgA and incubated at room temperature for 1 hour.
上記のようにして得た光反応性官能基導入後のAnti-IgA含有反応液と、平均粒子径２．５μｍのポリスチレンビーズ（Duke Scientific Corporation社製）を５％（ｗ／ｖ）の濃度で水に懸濁させたものを用いて、ビーズ濃度が１％（ｗ／ｖ）であり、Anti-IgA含有換算濃度が０．７３ｍｇ／ｍｌ、となるように混合してビーズ懸濁調製液とした。 Anti-IgA-containing reaction solution after introduction of the photoreactive functional group obtained as described above and polystyrene beads having an average particle size of 2.5 μm (Duke Scientific Corporation) at a concentration of 5% (w / v) Using a suspension in water, the bead concentration is 1% (w / v) and the equivalent concentration of Anti-IgA is 0.73 mg / ml. did.
６０℃で１時間焼成したPDMS（Polydimethylsiloxane）の基板を用意し、上記ビーズ懸濁調製液をその基板上にインクジェットで２５０滴（約１６０ｎＬ）吐出した。 A substrate of PDMS (Polydimethylsiloxane) baked at 60 ° C. for 1 hour was prepared, and 250 drops (about 160 nL) of the bead suspension preparation solution were ejected onto the substrate by inkjet.
湿度約８５％の容器内で２４時間以上かけてゆっくり乾燥させた後、UV (254 nm)を３０分間照射した後、水で３回洗浄して、PDMS（Polydimethylsiloxane）の基板上に、平均粒子径２．５μｍのポリスチレンビーズを結合してなる三次元構造体を得た。図９にはその電子顕微鏡写真を示す。 After drying slowly in a container with a humidity of about 85% over 24 hours, UV (254 nm) is irradiated for 30 minutes, and then washed three times with water, and the average particle is deposited on a PDMS (Polydimethylsiloxane) substrate. A three-dimensional structure formed by bonding polystyrene beads having a diameter of 2.5 μm was obtained. FIG. 9 shows an electron micrograph thereof.
図９に明らかなように、この三次元構造体のポリスチレンビーズは、およそ０．５〜５μｍの間隔をあけて略規則的に配列していた。また、この三次元構造体は、水で１５回洗浄しても崩れなかった。したがって、ＢＳＡのかわりにAnti-IgAを用いても、反応場として用いるのに十分な機械的安定性があることが示唆された。 As is clear from FIG. 9, the polystyrene beads of this three-dimensional structure were arranged approximately regularly with an interval of approximately 0.5 to 5 μm. In addition, this three-dimensional structure did not collapse even after being washed with water 15 times. Therefore, it was suggested that even when Anti-IgA was used instead of BSA, there was sufficient mechanical stability to be used as a reaction field.
<実施例８>
ビーズを基板上に滴下・凝集(乾燥)させた後、三次元に配列したビーズ構造体の端に架橋体構成材料を滴下し、ビーズによる三次元構造の内部に架橋体構成材料を浸透させることで、三次元構造体を作成した。
<Example 8>
After dropping and aggregating (drying) the beads on the substrate, the cross-linked component material is dropped on the end of the three-dimensionally arranged bead structure, and the cross-linked component material penetrates into the inside of the three-dimensional structure of beads. A three-dimensional structure was created.
まず、架橋体構成材料をビーズによる三次元構造の内部に浸透させることができるかどうかを確認する目的で、以下のような実験を行った。 First, the following experiment was conducted for the purpose of confirming whether or not the cross-linked body constituent material can be infiltrated into the interior of the three-dimensional structure of beads.
平均粒子径２．５μｍのポリスチレンビーズ（Duke Scientific Corporation社製）を、ビーズ濃度が５％（ｗ／ｖ）となるように水に懸濁し、ＰＤＭＳ上にインクジェットで２５０滴（約１６０ｎＬ）吐出し、乾燥させた。その顕微鏡写真を図１０に示す。 Polystyrene beads having an average particle size of 2.5 μm (manufactured by Duke Scientific Corporation) are suspended in water so that the bead concentration is 5% (w / v), and 250 drops (about 160 nL) are ejected onto PDMS by inkjet. , Dried. The micrograph is shown in FIG.
図１０に示すように、このビーズ構造体は、ビーズ粒子の配列が規則正しく、全体的な形もきれいであった。 As shown in FIG. 10, this bead structure had a regular array of bead particles and a clean overall shape.
上記ビーズ構造体の端から１００μｍ離れた位置に、モデル物質として蛍光物質(１０−４Ｍ Resorufin)を２５０滴吐出し、ビーズ構造体の中心部の蛍光強度を測定した。その結果を、図１１に示す。 250 drops of a fluorescent substance (10 −4 M Resorufin) was discharged as a model substance at a position 100 μm away from the end of the bead structure, and the fluorescence intensity at the center of the bead structure was measured. The result is shown in FIG.
図１１に示すように、吐出から数分で、ビーズ構造体の中心部まで蛍光物質が浸透した。したがって、この方法により、ビーズによる三次元構造の内部にまで有効に架橋体構成材料を浸透させることができることが明らかとなった。 As shown in FIG. 11, the fluorescent substance penetrated to the center of the bead structure within a few minutes after the discharge. Therefore, it has been clarified that the cross-linked material can be effectively infiltrated into the inside of the three-dimensional structure of beads by this method.
上述した実施例１〜６におけるＢＳＡのかわりにAnti-IgAを用いて三次元構造体を作成した。 A three-dimensional structure was created using Anti-IgA instead of BSA in Examples 1 to 6 described above.
そのために、上述した光架橋剤、ATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester)を用い、常法に従い、Anti-IgAの側鎖アミノ基に光反応性官能基であるアジド基を導入した。 For this purpose, the photocrosslinker mentioned above, ATFB-SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester) is used and photoreactive to the side chain amino group of Anti-IgA according to a conventional method. A functional azide group was introduced.
具体的には、ＤＭＳＯ中にATFB-SE を１０ｍｇ／ｍｌの濃度で溶解し、その０．１ｍｌを、１ｍｇ／ｍｌのAnti-IgA１ｍｌと混合し室温で１時間インキュベートした。 Specifically, ATFB-SE was dissolved in DMSO at a concentration of 10 mg / ml, and 0.1 ml thereof was mixed with 1 ml of 1 mg / ml Anti-IgA and incubated at room temperature for 1 hour.
他方、平均粒子径２．５μｍのポリスチレンビーズ（Duke Scientific Corporation社製）を１％（ｗ／ｖ）の濃度で水に懸濁させたものを、６０℃で１時間焼成したＰＤＭＳの基板上にインクジェットで２５０滴（約１６０ｎＬ）吐出し、湿度約８５％の容器内で２４時間以上かけてゆっくり乾燥させ、ビーズ構造体を得た。そして、上記光反応性官能基導入後のAnti-IgA含有反応液を、ビーズ構造体の端から１００μｍ離れた位置に２５０滴吐出し、湿度約８５％の容器内で24時間以上放置してビーズ構造体の内部に浸透させた後、ＵＶを照射し，水で３回洗浄した。その結果得られた三次元構造体の顕微鏡写真を図１２に示す。 On the other hand, polystyrene beads having an average particle diameter of 2.5 μm (manufactured by Duke Scientific Corporation) suspended in water at a concentration of 1% (w / v) were baked at 60 ° C. for 1 hour on a PDMS substrate. 250 drops (about 160 nL) were ejected by ink jet and slowly dried over a period of 24 hours or more in a container having a humidity of about 85% to obtain a bead structure. Then, 250 drops of the anti-IgA-containing reaction solution after the introduction of the photoreactive functional group is discharged at a position 100 μm away from the end of the bead structure and left in a container with a humidity of about 85% for 24 hours or more. After penetrating the inside of the structure, it was irradiated with UV and washed three times with water. FIG. 12 shows a micrograph of the three-dimensional structure obtained as a result.
<試験例１>
上記実施例７や実施例８で得られた三次元構造体は、架橋体構成材料としてAnti-IgAを用いているので、そのAnti-IgAは三次元構造体の架橋体の構成成分となっているとともに、それ自体でＩｇＡと結合することができる。したがって、生体関連分子担持体を構成している。そこで、実施例８で得られた三次元構造体を用いて、ヒトＩｇＡの段階希釈溶液に対するＥＬＩＳＡを行った。また、比較のために、上記実施例８において、ビーズを滴下せず、光反応性官能基導入後のAnti-IgA含有反応液のみをＰＤＭＳの基板上に滴下し、光照射したものを調製し、同様にＥＬＩＳＡを行った。その結果を図１３に示す。
<Test Example 1>
Since the three-dimensional structure obtained in Example 7 or Example 8 uses Anti-IgA as a cross-linked material, the Anti-IgA is a constituent of the cross-linked material of the three-dimensional structure. And can itself bind IgA. Therefore, it constitutes a biorelevant molecule carrier. Therefore, ELISA for serially diluted human IgA solution was performed using the three-dimensional structure obtained in Example 8. For comparison, in Example 8 above, beads were not dropped, but only an anti-IgA-containing reaction solution after introduction of a photoreactive functional group was dropped onto a PDMS substrate to prepare a photoirradiated one. In the same manner, ELISA was performed. The result is shown in FIG.
図１３に示すように、ビーズを架橋した三次元構造体では，良好な抗体と抗原の結合が見られるのに対して、Anti-IgAを直接ＰＤＭＳ基板上に固化した場合には、シグナルが弱く、良好な抗体と抗原の結合が見られなかった。 As shown in FIG. 13, in the three-dimensional structure in which beads are cross-linked, a good binding between an antibody and an antigen is observed, whereas when Anti-IgA is directly solidified on a PDMS substrate, the signal is weak. Good antibody-antigen binding was not observed.
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