JP4706533B2 - Solid phase carrier for immobilizing physiologically active substance and method of using the same - Google Patents
Solid phase carrier for immobilizing physiologically active substance and method of using the same Download PDFInfo
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Description
本発明は、生理活性物質を固相表面に配置・固定する為の固相担体およびその使用方法に関する。 The present invention relates to a solid phase carrier for arranging and fixing a physiologically active substance on a solid phase surface and a method for using the same.
近年、様々な機能を有する生理活性物質として、糖鎖が注目されてきている。糖鎖とは,グルコース,ガラクトース,マンノース,フコース,キシロース,N−アセチルグルコサミン,N−アセチルガラクトサミン,シアル酸などの単糖およびこれらの誘導体がグリコシド結合によって鎖状に結合した分子の総称である。
糖鎖は、非常に多様性に富んでおり、天然に存在する生物が有する様々な機能に関与する物質である。糖鎖は生体内でタンパク質や脂質などに結合した複合糖質として存在することが多く,生体内の重要な構成成分の一つである。生体内の糖鎖は細胞間情報伝達,タンパク質の機能や相互作用の調整などに深く関わっていることが明らかになりつつある。
In recent years, sugar chains have attracted attention as physiologically active substances having various functions. A sugar chain is a general term for molecules in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and derivatives thereof are linked in a chain form by glycosidic bonds.
Sugar chains are very diverse and are substances that are involved in various functions of naturally occurring organisms. Sugar chains often exist as complex carbohydrates bound to proteins and lipids in vivo, and are one of the important components in vivo. It is becoming clear that sugar chains in living organisms are deeply involved in cell-to-cell information transmission, protein functions, and coordination of interactions.
糖鎖を固定化した糖鎖チップは,糖鎖と糖鎖レセプター,糖鎖と細胞,糖鎖とウイルスなどの相互作用の研究に大いに寄与すると予想されている(たとえば,非特許文献1)。さらには,糖鎖チップを感染性疾患や糖鎖異常関連疾患の診断用デバイスとして利用することも期待されている。 A sugar chain chip on which sugar chains are immobilized is expected to greatly contribute to the study of interactions between sugar chains and sugar chain receptors, sugar chains and cells, sugar chains and viruses, etc. (for example, Non-Patent Document 1). Furthermore, it is expected that the sugar chain chip will be used as a diagnostic device for infectious diseases and sugar chain abnormality-related diseases.
また、遺伝子活性の評価や、薬物効果の分子レベルでの生理的プロセスを解読するための試みは、伝統的にゲノミクスに焦点が当てられてきたが、プロテオミクスは、細胞の生物学的機能についてより詳細な情報を提供する。プロテオミクスは、遺伝子レベルというよりもむしろ、タンパク質レベルでの発現を検出しそして定量することによる、遺伝子活性の定性的かつ定量的な測定を含む。また、タンパク質の翻訳後修飾、タンパク質間の相互作用など遺伝子にコードされない事象の研究を含む。 Attempts to assess gene activity and decipher the physiological processes at the molecular level of drug effects have traditionally focused on genomics, but proteomics is more about the biological function of cells. Provide detailed information. Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantifying expression at the protein level rather than at the gene level. It also includes studies of non-gene-encoded events such as post-translational modification of proteins and protein-protein interactions.
「生命の設計図」であるゲノムの構造が明らかにされ、膨大なゲノム情報の入手が可能となった今日、プロテオミクス研究はますます盛んになっており、それに伴って生理活性物質検出の迅速高効率(ハイスループット)化が求められている。この目的の分子アレイ(バイオチップ)として、DNAチップが開発され、実用化されつつある。一方、生体機能において最も複雑で多様性の高いタンパク質の検出に関してはプロテインチップが提唱され、近年研究が進められている。プロテインチップとは、タンパク質、またはそれを捕捉する分子をチップ(微小な基板)表面に固定化したものを総称する。 Now that the structure of the genome, which is the “blueprint of life”, has been clarified and a large amount of genome information has become available, proteomics research has become increasingly popular, and as a result, rapid detection of bioactive substances has been accelerated. There is a need for higher efficiency (high throughput). As a molecular array (biochip) for this purpose, a DNA chip has been developed and put into practical use. On the other hand, protein chips have been proposed for the detection of the most complex and highly diverse proteins in biological functions, and research has been underway in recent years. A protein chip is a generic term for a protein or a molecule that captures it immobilized on a chip (micro substrate) surface.
しかし、現状のプロテインチップは一般にDNAチップの延長線上に位置付けられて開発がなされているため、ガラス基板上にタンパク質、又はそれを捕捉する分子をチップ表面に固定化する検討がなされている(例えば特許文献1参照)。 However, since the current protein chip is generally developed by being positioned on the extension line of the DNA chip, studies have been made to immobilize a protein or a molecule that captures it on a glass substrate on the surface of the chip (for example, Patent Document 1).
本発明は、
(1)プラスチックからなる固相担体の固相表面にホスホリルコリン基及び生理活性物質を固定化する為の分子を有する高分子物質を有し、該分子がビオチン又はビオチン誘導体と反応しうるアビジン又はストレプトアビジン又はニュートラアビジンであることを特徴とする生理活性物質固定化用固相担体。
(2)固相担体の形態が、平板状基板、マイクロウェルプレート、微細流路を有する基板、又はマイクロビーズである(1)記載の生理活性物質固定化用固相担体。
(3)前記ホスホリルコリン基が2−メタクリロイルオキシエチルホスホリルコリン基である(1)又は(2)記載の生理活性物質固定化用固相担体。
(4)前記高分子物質がホスホリルコリン基を有する単量体及び生理活性物質を固定化する為の官能基を有する単量体を共重合して得られる高分子物質である(1)〜(3)いずれか記載の生理活性物質固定化用固相担体。
(5)前記高分子物質が更にブチルメタクリレート基を含む単量体との共重合体である(4)記載の生理活性物質固定化用固相担体。
(6)前記プラスチックがポリカーボネート、ポリエチレン、ポリプロピレン、ポリスチレン、飽和環状ポリオレフィン、ポリペンテン、ポリアミド、及びそれらの共重合体よりなる群より選択された少なくとも1 種である(1)〜(5)いずれか記載の生理活性物質固定化用固相担体。
(7)(1)〜(6)いずれか記載の生理活性物質固定化用固相担体に生理活性物質を固定化した固相担体。
(8)前記生理活性物質が、糖鎖、複合糖質、核酸、アプタマー、タンパク質、抗体、ペプチド、及び脂質から選択された少なくとも1種である請求項7記載の固相担体。
(9)前記生理活性物質がビオチン又はビオチン誘導体が導入されたものである(7)または(8)記載の固相担体。
(10)(1)〜(6)いずれか記載の生理活性物質固定化用固相担体の使用方法であって、
(a)固相表面に生理活性物質を固定化する工程、
(b)該生理活性物質と特異的に反応する別の物質を作用させる工程、及び
(c)該生理活性物質と特異的に反応した別の物質又は特異的に反応した該生理活性物質を蛍光、発光又は発色を用いて検出する工程、を含む生理活性物質固定化用固相担体の使用方法。
(11)前記生理活性物質と特異的に反応する別の物質が、核酸、アプタマー、タンパク質、抗体、ペプチド、糖鎖、複合糖質、及び脂質から選択された少なくとも1種である(10)記載の生理活性物質固定化用固相担体の使用方法。
(12)前記生理活性物質と特異的に反応する別の物質が、蛍光、発光、発色で検出可能な分子である(10)又は(11)記載の生理活性物質固定化用固相担体の使用方法。
(13)前記生理活性物質と特異的に反応する別の物質が、蛍光、発光、発色で検出可能な分子と反応しうる物質である(10)〜(12)いずれか記載の生理活性物質固定化用固相担体の使用方法。
The present invention
(1) Avidin or streptoid having a polymer substance having a molecule for immobilizing a phosphorylcholine group and a physiologically active substance on the solid phase surface of a solid support made of plastic, which can react with biotin or a biotin derivative A solid phase carrier for immobilizing a physiologically active substance, which is avidin or neutravidin .
(2) The solid phase carrier for immobilizing a physiologically active substance according to (1), wherein the form of the solid phase carrier is a flat substrate, a microwell plate, a substrate having a fine flow path, or a microbead.
(3) The solid phase carrier for immobilizing a physiologically active substance according to (1) or (2), wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group.
( 4 ) The polymer substance is a polymer substance obtained by copolymerizing a monomer having a phosphorylcholine group and a monomer having a functional group for immobilizing a physiologically active substance (1) to ( 3 ) A solid phase carrier for immobilizing a physiologically active substance according to any one of the above.
( 5 ) The solid phase carrier for immobilizing a physiologically active substance according to ( 4 ), wherein the polymer substance is a copolymer with a monomer further containing a butyl methacrylate group.
( 6 ) Any one of (1) to ( 5 ), wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, and copolymers thereof. Solid phase carrier for immobilizing physiologically active substances.
( 7 ) A solid phase carrier in which a physiologically active substance is immobilized on the solid phase carrier for immobilizing a physiologically active substance according to any one of (1) to ( 6 ).
( 8 ) The solid phase carrier according to claim 7 , wherein the physiologically active substance is at least one selected from sugar chains, complex carbohydrates, nucleic acids, aptamers, proteins, antibodies, peptides, and lipids.
( 9 ) The solid phase carrier according to ( 7) or (8 ), wherein the physiologically active substance has biotin or a biotin derivative introduced therein.
( 10 ) A method of using the solid phase carrier for immobilizing a physiologically active substance according to any one of (1) to ( 6 ),
(A) a step of immobilizing a physiologically active substance on the solid surface;
(B) a step of allowing another substance that specifically reacts with the physiologically active substance to act; and (c) another substance that specifically reacts with the physiologically active substance or the physiologically active substance that specifically reacts. A method of using a solid phase carrier for immobilizing a physiologically active substance, comprising a step of detecting using luminescence or color development.
( 11 ) The other substance that specifically reacts with the physiologically active substance is at least one selected from nucleic acids, aptamers, proteins, antibodies, peptides, sugar chains, glycoconjugates, and lipids ( 10 ) Of using a solid phase carrier for immobilizing a physiologically active substance.
( 12 ) Use of the solid phase carrier for immobilizing a physiologically active substance according to (10) or (11) , wherein another substance that specifically reacts with the physiologically active substance is a molecule that can be detected by fluorescence, luminescence, or color development. Method.
( 13 ) The physiologically active substance immobilization according to any one of (10) to (12 ), wherein another substance that specifically reacts with the physiologically active substance is a substance that can react with a molecule that can be detected by fluorescence, luminescence, or color development. Method of using solid phase carrier for crystallization.
非常に有用であるバイオチップだが、ペプチドや糖鎖のように担体表面に物理化学吸着させることが難しい場合もある。この問題を解決する為の方法として、固定化したい分子と反応しうる分子や官能基を担体表面に導入する方法がある。 Although it is a very useful biochip, it may be difficult to physicochemically adsorb on the surface of a carrier, such as peptides and sugar chains. As a method for solving this problem, there is a method of introducing a molecule or functional group capable of reacting with a molecule to be immobilized on the surface of the carrier.
担体表面に導入した反応性官能基を用いて生理活性物質を共有結合で固定化する場合、生理活性物質がどの部位で結合したかを明確にする事が困難になる場合や、担体表面との結合部位と生理活性に重要な部位とが重なる事で、生理活性物質が本来有する活性が減衰する、もしくは消失する場合がある、などの問題点があった。また、固定化する生理活性物質の構造によって、担体表面に導入した反応性官能基との反応性に差が生じ、複数の生理活性物質を固定化する際に固定化量にばらつきが生じるという問題もあった。 When a physiologically active substance is immobilized by covalent bond using a reactive functional group introduced on the surface of the carrier, it is difficult to clarify at which site the physiologically active substance is bound, The overlapping of the binding site and the site important for physiological activity has a problem that the activity inherent to the physiologically active substance may be attenuated or lost. In addition, depending on the structure of the physiologically active substance to be immobilized, there is a difference in reactivity with the reactive functional group introduced on the surface of the carrier, and the amount of immobilization varies when immobilizing multiple physiologically active substances. There was also.
一方、生理活性物質を基板上に固定化した後に該表面上で他の物質と反応させ、最終的に検出機等で検出する場合、生理活性物質が固定されていない部分に他の物質が固定されると、検出時にノイズとなる為に信号対雑音比(S/N比)を低下させる原因となり、検出精度を低下させる(例えば非特許文献3参照)。 On the other hand, when a physiologically active substance is immobilized on a substrate and then reacted with another substance on the surface and finally detected by a detector or the like, the other substance is immobilized on a portion where the physiologically active substance is not immobilized. If this is done, it becomes a noise at the time of detection, which causes a reduction in the signal-to-noise ratio (S / N ratio) and lowers the detection accuracy (see, for example, Non-Patent Document 3).
このため、生理活性物質と反応させる物質の非特異吸着を防止するため、ブロッキング剤のコーティングが必要となるが、これらの非特異吸着防止能は十分でない。また、生理活性物質を固定化した後にブロッキング剤をコーティングするため、固定化した生理活性物質の上にコーティングされてしまう場合があり、生理活性物質と反応させる物質が反応しにくいという問題があった。このため、生理活性物質固定化後のブロッキング工程がなく、かつ生理活性物質と反応させる物質の非特異吸着量の少ないバイオチップが求められている。 For this reason, in order to prevent nonspecific adsorption | suction of the substance made to react with a physiologically active substance, the coating of a blocking agent is needed, However, These nonspecific adsorption | suction prevention ability is not enough. In addition, since the blocking agent is coated after immobilizing the physiologically active substance, it may be coated on the immobilized physiologically active substance, which causes a problem that the substance that reacts with the physiologically active substance is difficult to react. . For this reason, there is a need for a biochip that does not have a blocking step after immobilization of a physiologically active substance and has a small amount of nonspecific adsorption of a substance that reacts with the physiologically active substance.
また、すべてのタンパク質(プロテオーム)や糖鎖(グライコーム)の変動をプロファイリングする技術面では、超微量の生理活性物質や数ナノリットルというような超微量の溶液の操作を可能とするマイクロフルイディクスの技術や、チップ上での前処理、分離、検出を目標とする「ラボ・オン・チップ」の概念が重要となってくる。この技術においては、サンプルであるタンパク質や複合糖質などの生理活性物質が、流路内に固定化されたキャプチャーと特異的に反応し、かつキャプチャー部以外の流路の内壁への非特異吸着を抑制することが必要となる。
本発明は、吸着防止剤をコーティングすることなく、非特異的な吸着・結合を抑制し、検出精度の高い生理活性物質固定化用固相担体を提供することを目的とする。 An object of the present invention is to provide a solid support for immobilizing a physiologically active substance with high detection accuracy by suppressing nonspecific adsorption / binding without coating with an adsorption inhibitor.
本発明は、以下のとおりである。
(1)プラスチックからなる固相担体の固相表面にホスホリルコリン基及び生理活性物質を固定化する為の分子を有する高分子物質を有し、該分子がビオチン又はビオチン誘導体と反応しうる分子であることを特徴とする生理活性物質固定化用固相担体。
(2)固相担体の形態が、平板状基板、マイクロウェルプレート、微細流路を有する基板、又はマイクロビーズである(1)記載の生理活性物質固定化用固相担体。
(3)前記ホスホリルコリン基が2−メタクリロイルオキシエチルホスホリルコリン基である(1)又は(2)記載の生理活性物質固定化用固相担体。
(4)生理活性物質を固定化する為の分子がアビジン又はストレプトアビジン又はニュートラアビジンである(1)〜(3)いずれか記載の生理活性物質固定化用固相担体。
(5)前記高分子物質がホスホリルコリン基を有する単量体及び生理活性物質を固定化する為の官能基を有する単量体を共重合して得られる高分子物質である(1)〜(4)いずれか記載の生理活性物質固定化用固相担体。
(6)前記高分子物質が更にブチルメタクリレート基を含む単量体との共重合体である(5)記載の生理活性物質固定化用固相担体。
(7)前記プラスチックがポリカーボネート、ポリエチレン、ポリプロピレン、ポリスチレン、飽和環状ポリオレフィン、ポリペンテン、ポリアミド、又はそれらの共重合体よりなる群より選択された少なくとも1種である(1)〜(6)いずれか記載の生理活性物質固定化用固相担体。
(8)(1)〜(7)いずれか記載の生理活性物質固定化用固相担体に生理活性物質を固定化した固相担体。
(9)前記生理活性物質が、糖鎖、複合糖質、核酸、アプタマー、タンパク質、抗体、ペプチド、及び脂質から選択された少なくとも1種である(8)記載の固相担体。
(10)前記生理活性物質がビオチン又はビオチン誘導体が導入されたものである(8)又は(9)記載の固相担体。
(11)(1)〜(8)いずれか記載の生理活性物質固定化用固相担体の使用方法であって、
(a)固相表面に生理活性物質を固定化する工程、
(b)該生理活性物質と特異的に反応する別の物質を作用させる工程、及び
(c)該生理活性物質と特異的に反応した別の物質又は特異的に反応した該生理活性物質を蛍光、発光又は発色を用いて検出する工程、を含む生理活性物質固定化用固相担体の使用方法。
(12)前記生理活性物質と特異的に反応する別の物質が、核酸、アプタマー、タンパク質、抗体、ペプチド、糖鎖、複合糖質、及び脂質から選択された少なくとも1種である(11)記載の生理活性物質固定化用固相担体の使用方法。
(13)前記生理活性物質と特異的に反応する別の物質が、蛍光、発光、発色で検出可能な分子である(11)又は(12)記載の生理活性物質固定化用固相担体の使用方法。
(14)前記生理活性物質と特異的に反応する別の物質が、蛍光、発光、発色で検出可能な分子と反応しうる物質である(11)〜(13)いずれか記載の生理活性物質固定化用固相担体の使用方法。
The present invention is as follows.
(1) A polymer substance having a molecule for immobilizing a phosphorylcholine group and a physiologically active substance on a solid phase surface of a solid support made of plastic, and the molecule can react with biotin or a biotin derivative A solid phase carrier for immobilizing a physiologically active substance.
(2) The solid phase carrier for immobilizing a physiologically active substance according to (1), wherein the form of the solid phase carrier is a flat substrate, a microwell plate, a substrate having a fine flow path, or a microbead.
(3) The solid phase carrier for immobilizing a physiologically active substance according to (1) or (2), wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group.
(4) The solid phase carrier for immobilizing a physiologically active substance according to any one of (1) to (3), wherein the molecule for immobilizing the physiologically active substance is avidin, streptavidin, or neutravidin.
(5) The polymer substance is a polymer substance obtained by copolymerizing a monomer having a phosphorylcholine group and a monomer having a functional group for immobilizing a physiologically active substance (1) to (4) ) A solid phase carrier for immobilizing a physiologically active substance according to any one of the above.
(6) The solid phase carrier for immobilizing a physiologically active substance according to (5), wherein the polymer substance is a copolymer with a monomer further containing a butyl methacrylate group.
(7) Any one of (1) to (6), wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, or a copolymer thereof. Solid phase carrier for immobilizing physiologically active substances.
(8) A solid phase carrier in which a physiologically active substance is immobilized on the solid phase carrier for immobilizing a physiologically active substance according to any one of (1) to (7).
(9) The solid phase carrier according to (8), wherein the physiologically active substance is at least one selected from sugar chains, complex carbohydrates, nucleic acids, aptamers, proteins, antibodies, peptides, and lipids.
(10) The solid phase carrier according to (8) or (9), wherein the physiologically active substance has biotin or a biotin derivative introduced therein.
(11) A method of using the solid phase carrier for immobilizing a physiologically active substance according to any one of (1) to (8),
(A) a step of immobilizing a physiologically active substance on the solid surface;
(B) a step of allowing another substance that specifically reacts with the physiologically active substance to act; and (c) another substance that specifically reacts with the physiologically active substance or the physiologically active substance that specifically reacts. A method of using a solid phase carrier for immobilizing a physiologically active substance, comprising a step of detecting using luminescence or color development.
(12) The other substance that specifically reacts with the physiologically active substance is at least one selected from nucleic acids, aptamers, proteins, antibodies, peptides, sugar chains, complex carbohydrates, and lipids. Of using a solid phase carrier for immobilizing a physiologically active substance.
(13) Use of the solid phase carrier for immobilizing a physiologically active substance according to (11) or (12), wherein another substance that specifically reacts with the physiologically active substance is a molecule that can be detected by fluorescence, luminescence, or color development. Method.
(14) The physiologically active substance immobilization according to any one of (11) to (13), wherein another substance that specifically reacts with the physiologically active substance is a substance that can react with a molecule that can be detected by fluorescence, luminescence, or color development. Method of using solid phase carrier for crystallization.
本発明の固相担体によれば、生理活性物質を効率よく簡単に固相担体に結合する事が可能となり、また、該生理活性物質と反応する別の物質の非特異吸着を効果的に抑制する事から、簡便かつハイスループットなバイオチップの作製と評価が可能となる。 According to the solid phase carrier of the present invention, a physiologically active substance can be efficiently and easily bound to the solid phase carrier, and nonspecific adsorption of another substance that reacts with the physiologically active substance is effectively suppressed. Therefore, it is possible to produce and evaluate a biochip with a simple and high throughput.
本発明の担体は、固相表面にホスホリルコリン基及び生理活性物質を固定化する為の分子を有する高分子物質を有することを特徴とする。ホスホリルコリン基と該分子とを有する高分子物質は、固定化した生理活性物質と反応し得る別の生理活性物質や蛍光物質等の非特異的吸着を抑制する性質と生理活性物質を固定化する性質とを併せ持つポリマーである。ホスホリルコリン基を有するポリマーは、生体膜(リン脂質二重層膜)類似の構造を有しているポリマーであって、生理活性物質と反応し得る物質の吸着を抑制する効果を有する。また、該分子は生理活性物質を固定化する役割を果たす。上記ポリマーの特性により、固相担体表面への生理活性物質や蛍光物質の非特異的吸着を効果的に抑制する事が可能となり、その結果、固相担体表面に固定化した生理活性物質と特異的に反応した物質のみを捕獲する事が可能となる。 The carrier of the present invention is characterized by having a polymer substance having a molecule for immobilizing a phosphorylcholine group and a physiologically active substance on the solid surface. A polymer substance having a phosphorylcholine group and the molecule has a property of suppressing non-specific adsorption of another physiologically active substance or a fluorescent substance capable of reacting with the immobilized physiologically active substance and a property of immobilizing the physiologically active substance. It is a polymer that has both. The polymer having a phosphorylcholine group is a polymer having a structure similar to a biological membrane (phospholipid bilayer membrane), and has an effect of suppressing adsorption of a substance that can react with a physiologically active substance. The molecule also plays a role in immobilizing physiologically active substances. Due to the characteristics of the polymer, it is possible to effectively suppress nonspecific adsorption of physiologically active substances and fluorescent substances on the surface of the solid phase carrier. It is possible to capture only the substances that have reacted automatically.
本発明に使用するホスホリルコリン基としては、例えば2−メタクリロイルオキシエチルホスホリルコリン、2−メタクリロイルオキシエトキシエチルホスホリルコリン、6−メタクリロイルオキシヘキシルホスホリルコリン、10−メタクリロイルオキシエトキシノニルホスホリルコリン、アリルホスホリルコリン、ブテニルホスホリルコリン、ヘキセニルホスホリルコリン、オクテニルホスホリルコリン、デセニルホスホリルコリン等を挙げられるが、2−メタクリロイルオキシエチルホスホリルコリンがより好ましい。 Examples of the phosphorylcholine group used in the present invention include 2-methacryloyloxyethyl phosphorylcholine, 2-methacryloyloxyethoxyethylphosphorylcholine, 6-methacryloyloxyhexylphosphorylcholine, 10-methacryloyloxyethoxynonylphosphorylcholine, allylphosphorylcholine, butenylphosphorylcholine, hexenylphosphorylcholine. Octenyl phosphorylcholine, decenyl phosphorylcholine and the like, and 2-methacryloyloxyethyl phosphorylcholine is more preferable.
本発明に使用する生理活性物質を固定化する為の分子としては、生理活性物質に結合したビオチン又はビオチン誘導体と特異的に反応する分子が好ましく、特にアビジン、ストレプトアビジン、ニュートラアビジンが好ましい。ビオチンとアビジンとの相互作用は非常に強く、特異性も高い。また、低分子であるビオチンを生理活性物質へ導入する事も容易な為、物質固定化の為の強力なツールとなる。
本発明に使用する高分子物質は、ホスホリルコリン基を有する単量体及び該分子を有する単量体を共重合して得られる高分子物質であることが好ましい。更にホスホリルコリン基及び該分子以外に他の官能基や分子を含んでもよく、ブチルメタクリレート基を含む単量体との共重合体が好ましい。
生理活性物質を固定化する為の分子と高分子物質との結合はどのような形式でもよいが、共有結合や、高分子物質に導入したビオチンとの相互作用を用いる事が望ましい。
As the molecule for immobilizing the physiologically active substance used in the present invention, a molecule that specifically reacts with biotin or a biotin derivative bound to the physiologically active substance is preferable, and avidin, streptavidin, or neutravidin is particularly preferable. The interaction between biotin and avidin is very strong and highly specific. In addition, since biotin, which is a low molecule, can be easily introduced into a physiologically active substance, it is a powerful tool for immobilizing substances.
The polymer substance used in the present invention is preferably a polymer substance obtained by copolymerizing a monomer having a phosphorylcholine group and a monomer having the molecule. Furthermore, other functional groups and molecules may be included in addition to the phosphorylcholine group and the molecule, and a copolymer with a monomer containing a butyl methacrylate group is preferable.
The bond between the molecule for immobilizing the physiologically active substance and the polymer substance may be in any form, but it is desirable to use a covalent bond or an interaction with biotin introduced into the polymer substance.
(固相担体の素材)
本発明の固相担体は、固相材質がプラスチックであることを特徴とする。
プラスチックとしては、熱可塑性樹脂、熱硬化性樹脂を用いることができるが、熱可塑性樹脂の方が製造効率の観点から好ましい。熱可塑性樹脂としては、蛍光発生量の少ないものが好ましく、例えばポリエチレンやポリプロピレン等の直鎖状ポリオレフィン、ポリカーボネート、ポリスチレン、ポリアミド、環状ポリオレフィン、含フッ素樹脂等が挙げられる。耐熱性、耐薬品性、低蛍光性、成形性に特に優れる環状ポリオレフィンを用いることがより好ましい。ここで環状ポリオレフィンとは、環状オレフィン構造を有する重合体単独または環状オレフィンとα―オレフィンとの共重合体を水素添加した飽和重合体をさす。
(Material of solid support)
The solid phase carrier of the present invention is characterized in that the solid phase material is plastic.
As the plastic, a thermoplastic resin or a thermosetting resin can be used, but the thermoplastic resin is preferable from the viewpoint of production efficiency. As the thermoplastic resin, those that generate a small amount of fluorescence are preferable, and examples thereof include linear polyolefins such as polyethylene and polypropylene, polycarbonates, polystyrenes, polyamides, cyclic polyolefins, and fluorine-containing resins. It is more preferable to use a cyclic polyolefin that is particularly excellent in heat resistance, chemical resistance, low fluorescence, and moldability. Here, the cyclic polyolefin refers to a saturated polymer obtained by hydrogenating a polymer having a cyclic olefin structure or a copolymer of a cyclic olefin and an α-olefin.
前者の例としては、例えばノルボルネン、ジシクロペンタジエン、テトラシクロドデセンに代表されるノルボルネン系モノマー、および、これらのアルキル置換体を開環重合して得られる重合体を水素添加して製造される飽和重合体である。 Examples of the former are produced by hydrogenating norbornene monomers represented by, for example, norbornene, dicyclopentadiene, and tetracyclododecene, and polymers obtained by ring-opening polymerization of these alkyl-substituted products. It is a saturated polymer.
後者の共重合体はエチレンやプロピレン、イソプロピル、1−ブテン、3−メチル−1−ブテン、1−ペンテン、3−メチル−1−ペンテン、1−ヘキセン、1−オクテン等のα―オレフィンと環状オレフィン系モノマーのランダム共重合体を水素添加することにより製造される飽和重合体である。共重合体では、エチレンとの共重合体が最も好ましい。
これら樹脂は単独で用いてもよく、2種類またはそれ以上の共重合体あるいは混合物であってもよい。また、樹脂成分以外に繊維状、球状その他の形状を有する無機物あるいは有機物充填材、または各種添加剤成分を含んでもよい。
The latter copolymer is cyclic with α-olefins such as ethylene, propylene, isopropyl, 1-butene, 3-methyl-1-butene, 1-pentene, 3-methyl-1-pentene, 1-hexene and 1-octene. It is a saturated polymer produced by hydrogenating a random copolymer of olefinic monomers. As the copolymer, a copolymer with ethylene is most preferable.
These resins may be used alone, or two or more copolymers or a mixture may be used. Further, in addition to the resin component, an inorganic or organic filler having a fibrous shape, a spherical shape, or other shapes, or various additive components may be included.
(固相担体の形状)
固相担体の形状としては、平板状基板、マイクロウェルプレート、マイクロビーズ、微細流路形状を有した基板等が挙げられる。
マイクロビーズ表面に生理活性物質を固定化させた担体の場合、基板やプレートに固定化させた場合に比べ生理活性物質を固定化できる表面積が大幅に増加するため、結果的に多くの検出目的となる、生理活性物質と特異的に反応する物質を捕獲でき、S/N比の増加が可能となる。
また微細流路内に生理活性物質を固定化した場合、生理活性物質が検出目的となる物質を捕獲できる頻度が相対的に増加するため、反応時間の短縮ができる。
さらには、生理活性物質を固定化したマイクロビーズを微細流路内に封入した場合、前記の利点の両方が達成できる。
(Shape of solid support)
Examples of the shape of the solid phase carrier include a flat substrate, a microwell plate, microbeads, and a substrate having a fine channel shape.
In the case of a carrier in which a physiologically active substance is immobilized on the surface of a microbead, the surface area on which the physiologically active substance can be immobilized is greatly increased as compared with the case of immobilization on a substrate or plate. The substance that specifically reacts with the physiologically active substance can be captured, and the S / N ratio can be increased.
In addition, when a physiologically active substance is immobilized in the fine channel, the frequency at which the physiologically active substance can capture the substance to be detected increases relatively, so that the reaction time can be shortened.
Furthermore, when the microbeads on which the physiologically active substance is immobilized are encapsulated in the fine channel, both of the advantages described above can be achieved.
(生理活性物質の固定化)
本発明において生理活性物質を固相上に固定化する際には、生理活性物質を溶解又は分散させた液体を付着する方法が好ましい。生理活性物質を溶解又は分散した液体のpHは6.0〜8.0であることが好ましく、pH6.5〜7.5がより好ましい。この範囲外だと、生理活性物質や生理活性物質を固定化する分子が変性・分解する恐れがある。
生理活性物質付着後は、固相表面のホスホリルコリン基の特性により、界面活性剤を含む水や緩衝液で洗浄することで、生理活性物質と反応しうる別の物質の固相表面への非特異吸着を抑制することが可能となる。しかし、生理活性物質と反応しうる別の物質に固相表面の分子と反応しうるビオチン又はビオチン誘導体が含まれる場合、生理活性物質を固定化した以外の固相表面に残存する分子の不活性化処理を、ビオチン又はビオチン誘導体を有する他の化合物で行うことが好ましい。
(Immobilization of physiologically active substances)
In the present invention, when the physiologically active substance is immobilized on the solid phase, a method of attaching a liquid in which the physiologically active substance is dissolved or dispersed is preferable. The pH of the liquid in which the physiologically active substance is dissolved or dispersed is preferably 6.0 to 8.0, more preferably 6.5 to 7.5. Outside this range, the physiologically active substance and the molecule that immobilizes the physiologically active substance may be denatured and decomposed.
After the bioactive substance is attached, due to the properties of the phosphorylcholine group on the surface of the solid phase, non-specificity of another substance that can react with the bioactive substance on the solid surface by washing with water or a buffer containing a surfactant. Adsorption can be suppressed. However, when another substance that can react with a physiologically active substance contains biotin or a biotin derivative that can react with a molecule on the solid phase surface, the inactivity of the molecule remaining on the solid phase surface other than where the physiologically active substance is immobilized It is preferable to perform the conversion treatment with other compounds having biotin or a biotin derivative.
(固定化する生理活性物質の構造)
固定化する生理活性物質にビオチン又はビオチン誘導体を導入する事で、基材表面に固定化された分子によって、効果的に目的の生理活性物質を固定化する事が可能となる。より反応性を高める為に、ビオチン又はビオチン誘導体の導入位置は生理活性物質の末端や外側であることが望ましい。生理活性物質の合成法が進歩してきた事で、任意の位置にビオチン又はビオチン誘導体を導入できるようになってきており、生理活性を発現する為に重要な部位に影響を及ぼさないような位置で生理活性物質を担体表面に固定化することが可能である。
(Structure of bioactive substance to be immobilized)
By introducing biotin or a biotin derivative into the physiologically active substance to be immobilized, the target physiologically active substance can be effectively immobilized by the molecules immobilized on the substrate surface. In order to enhance the reactivity, it is desirable that the biotin or biotin derivative is introduced at the end or outside of the physiologically active substance. Advances in the synthesis of bioactive substances have made it possible to introduce biotin or biotin derivatives at any position, and at positions that do not affect the important sites for developing bioactivity. The physiologically active substance can be immobilized on the surface of the carrier.
以下、実施例を挙げて本発明を更に具体的に説明するが、この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。
(実施例)
ポリスチレン樹脂製の96ウェルマイクロプレート(住友ベークライト製 ELISA用プレートS MS-8496F)の各ウェルを2−メタクリロイルオキシエチルホスホリルコリン−ブチルメタクリレート−p−ニトロフェニルカルボニルオキシエチルメタクリレート共重合体の0.3重量%エタノール溶液100mlに浸漬することにより、基材表面にホスホリルコリン基を有するポリマーを導入した。
次に、リン酸緩衝液を用いて1mg/mlに希釈し、pHが9.5に調整されたビオチンヒトラジド溶液100mlを添加したウェルと、pH9.5のリン酸緩衝液100mlのみを添加したウェルを準備し、37℃で一晩静置してビオチンをポリマーに固定化させた後に、純水で洗浄を行った。その後、ビオチンを固定化したウェルと、リン酸緩衝液のみを加えたウェルとに、リン酸緩衝液を用いて5.0mg/mlに希釈したストレプトアビジン(ケミコン社製、SA101)100mlを添加して30分静置してストレプトアビジンを固定化した。0.05%Tween20含有リン酸緩衝液で3回洗浄した後に、市販のビオチン標識ペルオキシダーゼ(ZYMED Laboratories社製、43−2040)をリン酸緩衝液を用いて0.5mg/mlに希釈し、100mlをウェルに添加して30分静置して固定化し酵素標識した。0.05%Tween20含有リン酸緩衝液で3回洗浄した後に、処理した各ウェルにおいて、ペルオキシダーゼ用発色キットT(住友ベークライト社製 ML−1120T)を用いて、過酸化水素を基質とする発色反応を行い、発色基質として用いたTMBZ(3,3‘,5,5’テトラメチルベンチジン)の吸光度を測定した。ビオチンヒドラジド溶液を加えたウェルの吸光度をシグナル値、リン酸緩衝液のみを入れたウェルの吸光度をバックグラウンド値(非特異吸着無し)として、その際のシグナル値、バックグラウンド値を表1に示す。
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
(Example)
Each well of a polystyrene resin 96-well microplate (Sumitomo Bakelite ELISA plate S MS-8496F) was treated with 0.3 weight of 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate-p-nitrophenylcarbonyloxyethyl methacrylate copolymer. A polymer having a phosphorylcholine group was introduced on the surface of the substrate by immersing in 100 ml of a% ethanol solution.
Next, the well was diluted with phosphate buffer to 1 mg / ml, and 100 ml of biotin human razide solution adjusted to pH 9.5 was added, and only 100 ml of phosphate buffer with pH 9.5 was added. Wells were prepared and allowed to stand overnight at 37 ° C. to immobilize biotin on the polymer, followed by washing with pure water. Thereafter, 100 ml of streptavidin (SA101, manufactured by Chemicon, Inc.) diluted to 5.0 mg / ml using a phosphate buffer was added to the well in which biotin was immobilized and the well to which only the phosphate buffer was added. Streptavidin was immobilized by allowing to stand for 30 minutes. After washing 3 times with 0.05% Tween20-containing phosphate buffer, commercially available biotin-labeled peroxidase (ZYMED Laboratories, 43-2040) was diluted to 0.5 mg / ml with phosphate buffer, and 100 ml was diluted. It was added to the well and allowed to stand for 30 minutes for immobilization and enzyme labeling. After washing 3 times with 0.05% Tween20-containing phosphate buffer, in each well treated, color development reaction using hydrogen peroxide as a substrate using peroxidase color development kit T (ML-1120T manufactured by Sumitomo Bakelite Co., Ltd.) And the absorbance of TMBZ (3,3 ′, 5,5 ′ tetramethylbenzidine) used as a chromogenic substrate was measured. Table 1 shows the signal value and background value at that time, with the absorbance of the well containing the biotin hydrazide solution as the signal value and the absorbance of the well containing only the phosphate buffer as the background value (no non-specific adsorption). .
(比較例)
表面にカルボキシル基が直接結合した、ポリスチレン樹脂製の96ウェルマイクロプレート(住友ベークライト製 ELISA用プレートカルボ MS-8796F)を用いた。pH5.8のリン酸緩衝液を用いて調製した20mg/mlのWSC溶液100mlを各ウェルに添加し、37℃で2時間反応させた。純水でウェルを洗浄し、リン酸緩衝液を用いて1mg/mlに希釈し、pHが5.8に調整されたビオチンヒトラジド溶液100mlをウェルに添加して、37℃で一晩静置してビオチンを担体表面に固定化させた後に、純水で洗浄を行った。その後、ビオチンを固定化したウェルと、何も処理していないウェルとに、2.5%ウシ血清アルブミン溶液を100ml加え、1時間静置した。純水で洗浄した後に、ビオチンを固定化したウェルと、ウシ血清アルブミンのみを入れたウェルと、何も処理していないウェルとに、5.0mg/mlに希釈したストレプトアビジン(ケミコン社製、SA101)100mlをウェルに添加して1時間静置してストレプトアビジンを固定化した。0.05%Tween20含有リン酸緩衝液で3回洗浄した後に、市販のビオチン標識ペルオキシダーゼ(ZYMED Laboratories社製、43−2040)を0.5mg/mlに希釈し、100mlをウェルに添加して30分静置して固定化し酵素標識した。0.05%Tween20含有リン酸緩衝液で3回洗浄した後に、ペルオキシダーゼ用発色キット(住友ベークライト社製 ML−1120T)を用いて、過酸化水素を基質とする発色反応を行い、発色基質として用いたTMBZ(3,3‘,5,5’テトラメチルベンチジン)の吸光度を測定した。ビオチンヒドラジド溶液を加えたウェルの吸光度をシグナル値、ウシ血清アルブミンを加えたウェルの吸光度をバックグラウンド値(非特異吸着無し)、何も処理していないウェルの吸光度をバックグラウンド値(非特異吸着有り)として、その際のシグナル値、バックグラウンド値を表1に示す。
(Comparative example)
A 96-well microplate made of polystyrene resin (Sumitomo Bakelite ELISA plate carbo MS-8796F) having a carboxyl group directly bonded to its surface was used. 100 ml of a 20 mg / ml WSC solution prepared using a phosphate buffer having a pH of 5.8 was added to each well and reacted at 37 ° C. for 2 hours. The wells were washed with pure water, diluted to 1 mg / ml with a phosphate buffer, 100 ml of biotin human razide solution adjusted to pH 5.8 was added to the wells, and allowed to stand at 37 ° C. overnight. After fixing biotin on the surface of the carrier, washing was performed with pure water. Thereafter, 100 ml of a 2.5% bovine serum albumin solution was added to a well in which biotin was immobilized and a well not subjected to any treatment, and allowed to stand for 1 hour. After washing with pure water, streptavidin (SA101, manufactured by Chemicon, Inc.) diluted to 5.0 mg / ml into a well in which biotin was immobilized, a well containing only bovine serum albumin, and a well not subjected to any treatment. ) 100 ml was added to the well and allowed to stand for 1 hour to immobilize streptavidin. After washing three times with 0.05% Tween20-containing phosphate buffer, commercially available biotin-labeled peroxidase (ZYMED Laboratories, 43-2040) was diluted to 0.5 mg / ml, and 100 ml was added to the well for 30 minutes. It was allowed to stand, immobilized and enzyme-labeled. After washing three times with 0.05% Tween20-containing phosphate buffer, a color development reaction using hydrogen peroxide as a substrate is performed using a color kit for peroxidase (ML-1120T manufactured by Sumitomo Bakelite Co., Ltd.) and used as a color substrate. The absorbance of TMBZ (3,3 ′, 5,5 ′ tetramethylbenzidine) was measured. Absorbance of wells to which biotin hydrazide solution was added was the signal value, absorbance of wells to which bovine serum albumin was added was the background value (no nonspecific adsorption), and the absorbance of wells that had not been treated was the background value (nonspecific adsorption) Table 1 shows the signal values and background values at that time.
実施例および比較例における吸光度の測定には、TECAN社製SPECTRAFLUORを用い、測定波長450nm、参照波長620nmで測定した。
実施例は、シグナル値がブランク値に比べて高く、ブロッキング操作を行っていないにもかかわらず、タンパク質の非特異吸着を抑制する表面効果によって、ブランク値は有意に低かった。
比較例はBSAによるブロッキング操作を行わなかった場合はブランク値の上昇が見られ、タンパク質の非特異吸着が起こっていた。
The absorbance in Examples and Comparative Examples was measured using a SPECTRAFLUOR manufactured by TECAN at a measurement wavelength of 450 nm and a reference wavelength of 620 nm.
In Examples, the blank value was significantly lower due to the surface effect of suppressing non-specific adsorption of the protein even though the signal value was higher than the blank value and the blocking operation was not performed.
In the comparative example, when the blocking operation with BSA was not performed, the blank value increased and non-specific adsorption of the protein occurred.
本発明の生理活性物質固定化用固相担体を用いることで、ブロッキング剤をコーティングすることなしに、生理活性物質を固相担体の任意の位置に固定化し、それ以外の部分への不要な生理活性物質や蛍光物質の吸着および結合を抑制することでき、高感度でハイスループットなバイオチップの評価が可能となる。また形状の自由度も高く、マイクロビーズやマイクロフルイディクスを含む各種バイオチップの検出方法に適用できる。
By using the solid phase carrier for immobilizing a physiologically active substance of the present invention, the physiologically active substance can be immobilized at an arbitrary position of the solid phase carrier without coating with a blocking agent, and unnecessary physiology to other portions can be performed. Adsorption and binding of active substances and fluorescent substances can be suppressed, and high-sensitivity and high-throughput biochips can be evaluated. Moreover, it has a high degree of freedom in shape, and can be applied to various biochip detection methods including microbeads and microfluidics.
Claims (13)
(a)固相表面に生理活性物質を固定化する工程、
(b)該生理活性物質と特異的に反応する別の物質を作用させる工程、及び
(c)該生理活性物質と特異的に反応した別の物質又は特異的に反応した該生理活性物質を蛍光、発光又は発色を用いて検出する工程、を含む生理活性物質固定化用固相担体の使用方法。 A method of using the solid phase carrier for immobilizing a physiologically active substance according to any one of claims 1 to 6 ,
(A) a step of immobilizing a physiologically active substance on the solid surface;
(B) a step of causing another substance that specifically reacts with the physiologically active substance to act; and (c) another substance that specifically reacts with the physiologically active substance or the physiologically active substance that specifically reacts with fluorescence. A method of using a solid phase carrier for immobilizing a physiologically active substance, comprising a step of detecting using luminescence or color development.
The solid substance carrier for immobilizing a physiologically active substance according to any one of claims 10 to 12, wherein the another substance that specifically reacts with the physiologically active substance is a substance that can react with a molecule that can be detected by fluorescence, luminescence, or color development. How to use.
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US9186674B2 (en) | 2013-01-24 | 2015-11-17 | Sabic Global Technologies B.V. | Polycarbonate microfluidic articles |
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US9168532B2 (en) | 2013-01-24 | 2015-10-27 | Sabic Global Technologies B.V. | Microwell plate |
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