JP2005195465A - Solid support having electrostatic layer and its use - Google Patents
Solid support having electrostatic layer and its use Download PDFInfo
- Publication number
- JP2005195465A JP2005195465A JP2004002168A JP2004002168A JP2005195465A JP 2005195465 A JP2005195465 A JP 2005195465A JP 2004002168 A JP2004002168 A JP 2004002168A JP 2004002168 A JP2004002168 A JP 2004002168A JP 2005195465 A JP2005195465 A JP 2005195465A
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- Prior art keywords
- solid support
- nucleic acid
- primer
- substrate
- acid molecule
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Abstract
Description
本発明は、ＤＮＡ等を固定化するための支持体及び固定化核酸分子に関する。 The present invention relates to a support for immobilizing DNA or the like and an immobilized nucleic acid molecule.
従来、既存の配列から核酸配列を合成する方法として、ポリメラーゼ連鎖反応（ＰＣＲ）がある（例えば、特許文献１及び２参照）。 Conventionally, there is a polymerase chain reaction (PCR) as a method for synthesizing a nucleic acid sequence from an existing sequence (see, for example, Patent Documents 1 and 2).
ポリメラーゼ連鎖反応（ＰＣＲ）とは、目的とするＤＮＡを１組のプライマーで挟み、ＤＮＡポリメラーゼを作用させることを繰り返し、プライマーで挟んだ領域を無限に増幅させることができる方法である。 Polymerase chain reaction (PCR) is a method in which a target DNA is sandwiched between a pair of primers and a DNA polymerase is repeatedly actuated to amplify the region sandwiched between the primers indefinitely.
ＰＣＲによれば、目的とする配列のみをかなり正確に多数増幅させることができ、しかも短時間で効率よく増幅することができるので、現在、生化学、医療分野等の各種研究、試験、検査等に広く用いられている。 According to PCR, it is possible to amplify a large number of target sequences fairly accurately and efficiently in a short time, so various researches, tests, examinations, etc. in biochemistry, medical fields, etc. Widely used in
ＰＣＲ反応は加熱及び冷却の繰り返しにより進められている（サーマルサイクル）。すなわち、増幅対象である二本鎖ＤＮＡ分子を相補的一本鎖に高温変性させた後、冷却して該ＤＮＡの一部に相補するように選択されたプライマーを鎖にアニールさせ、再び加熱してＤＮＡポリメラーゼによりプライマーの後ろにＤＮＡを伸長させるという風に、変性、アニール、伸長のプロセスを１サイクルとして複数サイクル繰り返すことにより二本鎖ＤＮＡを多数増幅することができる。 The PCR reaction proceeds by repeated heating and cooling (thermal cycle). That is, a double-stranded DNA molecule to be amplified is denatured at a high temperature into a complementary single strand, and then cooled and annealed with a primer selected to complement a part of the DNA and heated again. Thus, a large number of double-stranded DNAs can be amplified by repeating the cycles of denaturation, annealing, and extension as one cycle, such that DNA is extended behind the primer by DNA polymerase.
具体的には、１）二本鎖ＤＮＡの水素結合をほどくために試料の温度を９５℃に上昇させる、２）次いでＤＮＡを複製するためのプライマーと再結合させるために試料の温度を４５℃に下降させる、３）更に耐熱性ポリメラーゼによりプライマーを伸長させてＤＮＡを複製させるために試料の温度を７４℃に上昇させる、といった１）〜３）のサーマルサイクルを幾度も繰り返す必要があった。このようなＤＮＡの増幅反応では、試料を合成樹脂の容器などに入れ、この容器をアルミニウムブロックに収容し前記サーマルサイクルを行っていた。 Specifically, 1) the temperature of the sample is raised to 95 ° C. to undo the hydrogen bonding of the double-stranded DNA, and 2) the temperature of the sample is then changed to 45 ° C. in order to recombine with the primer for replicating the DNA. 3) Further, it was necessary to repeat the thermal cycle 1) to 3) several times, such as increasing the temperature of the sample to 74 ° C. in order to replicate the DNA by extending the primer with a thermostable polymerase. In such a DNA amplification reaction, a sample is put in a synthetic resin container or the like, and the container is accommodated in an aluminum block to perform the thermal cycle.
しかし、前記サーマルサイクルは多大な時間がかかり、目的とする量のＤＮＡを得るには数時間を要していた。また、加熱、冷却による温度制御により反応を進めると、一瞬にして温度を変化させるには限界があり、各段階への切り替えがスムーズにいかず、増幅される核酸の配列の正確性に影響が出たり、目的とする以外のＤＮＡも複製される場合も考えられた。また、迅速な温度変化をさせるためには、特別の装置や技術が必要となるため、設備投資等の経済的な問題や技術的な問題があった。 However, the thermal cycle takes a lot of time, and it takes several hours to obtain the target amount of DNA. In addition, when the reaction is advanced by temperature control by heating and cooling, there is a limit to changing the temperature in an instant, and switching to each stage does not go smoothly, and the accuracy of the sequence of the nucleic acid to be amplified is affected. In some cases, DNA other than the target DNA may be replicated. Moreover, in order to change temperature rapidly, special equipment and technology are required, so there have been economic and technical problems such as capital investment.
このような問題に鑑み、ＤＮＡを容易に固定化できて、ＤＮＡ増幅反応によりＤＮＡを複製するために適する支持体として、基板の表面に、表面処理層、及び核酸分子と共有結合しうる官能基を有する化学修飾層を順次設けてなる固体支持体が開発されている（例えば、特許文献３〜５参照）。 In view of such a problem, as a support suitable for replicating DNA by a DNA amplification reaction, which can easily fix DNA, a surface treatment layer, and a functional group capable of covalently bonding with a nucleic acid molecule A solid support in which chemical modification layers having sequentially are provided has been developed (see, for example, Patent Documents 3 to 5).
しかしながら、前記固体支持体のＤＮＡの固定化量及びＤＮＡの結合強度は、必ずしも充分とはいえない。 However, the amount of DNA immobilized and the DNA binding strength of the solid support are not necessarily sufficient.
また、ＤＮＡアレイ技術を、タンパク質解析のツールとして用いたものとしてプロテインチップが開発されている。プロテインチップの原理はＤＮＡチップと同じで、スライドガラスや膜の上にタンパク質を高密度に固定し、それらと相互作用するタンパク質や核酸などを検出するものである。従来開発されたプロテインチップは、スライドガラス又はシリコン基板表面にポリリジン等の高分子を塗布し、その後にタンパク質を固定化するものであるが、この方法では、タンパク質の固定化状態が不安定であり、洗浄工程において剥離するといった問題が生じるとともに、固定化されたタンパク質を長期間保存することも不可能であった。また、生体物質の非特異吸着や、タンパク質の機能失活、検出系ＵＶ不透明性等の問題もあった。従って、ＤＮＡまたはタンパク質の固定化量がより高く結合強度がより高い固体支持体の出現が望まれている。 A protein chip has been developed using the DNA array technology as a protein analysis tool. The principle of a protein chip is the same as that of a DNA chip. Proteins are immobilized on a slide glass or membrane at a high density, and proteins or nucleic acids that interact with them are detected. Conventionally developed protein chips are those in which a polymer such as polylysine is applied to the surface of a glass slide or silicon substrate, and then the protein is immobilized. However, with this method, the protein immobilization state is unstable. In addition, problems such as peeling in the washing process occur, and it is impossible to store the immobilized protein for a long period of time. In addition, there are problems such as non-specific adsorption of biological substances, protein function inactivation, and detection system UV opacity. Therefore, the appearance of a solid support having a higher amount of DNA or protein immobilized and higher binding strength is desired.
この出願の発明に関する先行技術文献情報として次のものがある。
本発明の課題は、生体分子の固定化量がより高く、生体分子の結合強度がより高い固体支持体を提供することである。 An object of the present invention is to provide a solid support having a higher amount of immobilized biomolecules and a higher binding strength of biomolecules.
本発明者らは、前記課題を解決すべく鋭意検討の結果、基板上に、生体分子を静電的に引き寄せるための静電層を設けることにより、生体分子の固定化量及び生体分子の結合強度が著しく向上することを見出し本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have provided an electrostatic layer on the substrate for electrostatically attracting biomolecules, thereby immobilizing the amount of biomolecules and binding of biomolecules. The inventors found that the strength was remarkably improved and completed the present invention.
即ち、本発明は以下の発明を包含する。
（１）基板上に、生体分子を静電的に引き寄せるための静電層を有する固体支持体。
（２）基板の表面がダイヤモンド、軟ダイヤモンド、炭素系物質及び炭化物から選ばれる少なくとも１種で表面処理されている（１）に記載の固体支持体。
（３）静電層が、基板と共有結合していないアミノ基含有化合物を含む（１）又は（２）に記載の固体支持体。
（４）静電層が、基板と共有結合しているアミノ基含有化合物で構成され、該アミノ基含有化合物が、基板と結合していない側の末端にアミノ基を有する（１）又は（２）に記載の固体支持体。
That is, the present invention includes the following inventions.
(1) A solid support having an electrostatic layer for electrostatically attracting biomolecules on a substrate.
(2) The solid support according to (1), wherein the surface of the substrate is surface-treated with at least one selected from diamond, soft diamond, carbonaceous material and carbide.
(3) The solid support according to (1) or (2), wherein the electrostatic layer contains an amino group-containing compound that is not covalently bonded to the substrate.
(4) The electrostatic layer is composed of an amino group-containing compound covalently bonded to the substrate, and the amino group-containing compound has an amino group at the terminal not bonded to the substrate (1) or (2 ).
（５）基板上に、非置換又は一置換されたアミノ基を有する化合物及び炭素化合物を蒸着させて得られる（１）〜（３）のいずれかに記載の固体支持体。
（６）基板を、非置換又は一置換されたアミノ基を有する化合物を含有する溶液中に浸漬して得られる（１）〜（４）のいずれかに記載の固体支持体。
（７）非置換又は一置換されたアミノ基を有する化合物がポリアリルアミンである（６）に記載の固体支持体。
(5) The solid support according to any one of (1) to (3) obtained by vapor-depositing a compound having an unsubstituted or monosubstituted amino group and a carbon compound on a substrate.
(6) The solid support according to any one of (1) to (4), obtained by immersing the substrate in a solution containing a compound having an unsubstituted or monosubstituted amino group.
(7) The solid support according to (6), wherein the compound having an unsubstituted or monosubstituted amino group is polyallylamine.
（８）生体分子がＤＮＡである（１）〜（７）のいずれかに記載の固体支持体。
（９）（１）〜（８）のいずれかに記載の固体支持体に生体分子が固定化されてなる固定化生体分子。
(8) The solid support according to any one of (1) to (7), wherein the biomolecule is DNA.
(9) An immobilized biomolecule obtained by immobilizing a biomolecule on the solid support according to any one of (1) to (8).
（１０）基板上に、非置換又は一置換されたアミノ基を有する化合物及び炭素化合物を蒸着させることを特徴とする固体支持体の製造方法。
（１１）基板を、非置換又は一置換されたアミノ基を有する化合物を含有する溶液中に浸漬することを特徴とする固体支持体の製造方法。
（１２）（１）〜（８）のいずれかに記載の固体支持体上にプライマーを固定化し、該プライマーに核酸分子をハイブリダイズさせて、該核酸分子に相補的な核酸分子を伸長する方法。
(10) A method for producing a solid support, comprising depositing a compound having an unsubstituted or monosubstituted amino group and a carbon compound on a substrate.
(11) A method for producing a solid support, wherein the substrate is immersed in a solution containing a compound having an unsubstituted or monosubstituted amino group.
(12) A method in which a primer is immobilized on the solid support according to any one of (1) to (8), a nucleic acid molecule is hybridized to the primer, and a nucleic acid molecule complementary to the nucleic acid molecule is extended. .
（１３）（１）〜（８）のいずれかに記載の固体支持体上にプライマーを固定化し、該プライマーに核酸分子をハイブリダイズさせ、標識した核酸の存在下で該核酸分子に相補的な核酸分子を伸長させ、該相補的な核酸分子に取り込まれた標識核酸に由来するシグナルを読みとることを含む、核酸分子の検出方法。
（１４）（１）〜（８）のいずれかに記載の固体支持体上にプライマーを固定化し、該プライマーに核酸分子をハイブリダイズさせ、これをＰＣＲ反応に付すことにより、核酸分子を増幅する方法。
(13) A primer is immobilized on the solid support according to any one of (1) to (8), a nucleic acid molecule is hybridized to the primer, and complementary to the nucleic acid molecule in the presence of a labeled nucleic acid. A method for detecting a nucleic acid molecule, comprising extending a nucleic acid molecule and reading a signal derived from a labeled nucleic acid incorporated into the complementary nucleic acid molecule.
(14) A primer is immobilized on the solid support according to any one of (1) to (8), a nucleic acid molecule is hybridized to the primer, and subjected to PCR reaction to amplify the nucleic acid molecule. Method.
（１５）（１）〜（８）のいずれかに記載の固体支持体上にプライマーを固定化し、該プライマーにＤＮＡをハイブリダイズさせて、鎖置換型ＤＮＡポリメラーゼと反応させることにより、該ＤＮＡを増幅する方法。
（１６）プライマーに核酸分子をハイブリダイズさせた後に、該核酸分子を増幅する工程をさらに含む、（１３）に記載の方法。
(15) Immobilizing a primer on the solid support according to any one of (1) to (8), hybridizing the DNA to the primer, and reacting with a strand displacement DNA polymerase, How to amplify.
(16) The method according to (13), further comprising a step of amplifying the nucleic acid molecule after hybridizing the nucleic acid molecule to the primer.
本発明の固体支持体は、従来の固体支持体よりも核酸分子等の生体分子を大量に固定化することができることから、従来ＤＮＡアレイの課題であった検出感度と信頼性を改良することができ、また、核酸分子を固定化した状態で伸長反応を行ったり、ＰＣＲ反応を実施して核酸分子を増幅することもできるので、広くＤＮＡアレイの普及を図ることが可能となる。 Since the solid support of the present invention can immobilize a larger amount of biomolecules such as nucleic acid molecules than the conventional solid support, it can improve the detection sensitivity and reliability that have been the problems of conventional DNA arrays. In addition, since the extension reaction can be performed in a state where the nucleic acid molecule is immobilized, or the nucleic acid molecule can be amplified by performing the PCR reaction, the DNA array can be widely spread.
本発明に用いる基板の材料としては、例えば、シリコン、ガラス、繊維、木材、紙、セラミックス、プラスチック（例えば、ポリエステル樹脂、ポリエチレン樹脂、ポリプロピレン樹脂、ＡＢＳ樹脂(Acrylonitrile Butadiene Styrene 樹脂)、ナイロン、アクリル樹脂、フッ素樹脂、ポリカーボネート樹脂、ポリウレタン樹脂、メチルペンテン樹脂、フェノール樹脂、メラミン樹脂、エポキシ樹脂、塩化ビニル樹脂）が挙げられる。 Examples of the substrate material used in the present invention include silicon, glass, fiber, wood, paper, ceramics, plastic (for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), nylon, and acrylic resin. , Fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin).
基板の材料として前記のものを用いる場合には、表面処理層を施さなくてもよいが、生体分子を基板上に強固に固定化するために、表面処理を施すことがより好ましい。 When the above-mentioned materials are used as the substrate material, the surface treatment layer may not be provided, but it is more preferable to perform the surface treatment in order to firmly fix the biomolecule on the substrate.
表面処理には、合成ダイヤモンド、高圧合成ダイヤモンド、天然ダイヤモンド、軟ダイヤモンド（例えば、ダイヤモンドライクカーボン）、アモルファスカーボン、炭素系物質（例えば、グラファイト、フラーレン、カーボンナノチューブ）のいずれか、それらの混合物、又はそれらを積層させたものを用いることが好ましい。また、炭化ハフニウム、炭化ニオブ、炭化珪素、炭化タンタル、炭化トリウム、炭化チタン、炭化ウラン、炭化タングステン、炭化ジルコニウム、炭化モリブデン、炭化クロム、炭化バナジウム等の炭化物を用いてもよい。ここで、軟ダイヤモンドとは、いわゆるダイヤモンドライクカーボン（ＤＬＣ：Diamond Like Carbon）等の、ダイヤモンドとカーボンとの混合体である不完全ダイヤモンド構造体を総称し、その混合割合は、特に限定されない。 For the surface treatment, synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (eg, diamond-like carbon), amorphous carbon, carbon-based material (eg, graphite, fullerene, carbon nanotube), a mixture thereof, or It is preferable to use a laminate of them. Further, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide may be used. Here, the soft diamond is a generic term for an imperfect diamond structure that is a mixture of diamond and carbon, such as so-called diamond-like carbon (DLC), and the mixing ratio is not particularly limited.
表面処理された基板の一例としては、スライドガラスに軟ダイヤモンドを製膜した基板が挙げられる。このような基板は、ダイヤモンドライクカーボンが、水素ガス０〜９９体積％、残りメタンガス１００〜１体積％を含んだ混合ガス中で、イオン化蒸着法により作成したものであることが好ましい。
表面処理層の厚みは、１ｎｍ〜１００μｍであることが好ましい。
As an example of the surface-treated substrate, a substrate obtained by forming a soft diamond film on a slide glass can be given. Such a substrate is preferably prepared by ionized vapor deposition of diamond-like carbon in a mixed gas containing 0 to 99% by volume of hydrogen gas and 100 to 1% by volume of the remaining methane gas.
The thickness of the surface treatment layer is preferably 1 nm to 100 μm.
基板の表面処理層の形成は、公知の方法、例えば、マイクロ波プラズマＣＶＤ(Chemical Vapor Deposit)法、ＥＣＲＣＶＤ(Electric Cyclotron Resonance Chemical Vapor Deposit)法、ＩＣＰ(Inductive Coupled Plasma)法、直流スパッタリング法、ＥＣＲ(Electric Cyclotron Resonance)スパッタリング法、イオンプレーティング法、アークイオンプレーティング法、ＥＢ(Electron Beam)蒸着法、抵抗加熱蒸着法、イオン化蒸着法、アーク蒸着法、レーザ蒸着法などにより行うことができる。 The surface treatment layer of the substrate is formed by a known method, for example, microwave plasma CVD (Chemical Vapor Deposit) method, ECRCVD (Electric Cyclotron Resonance Chemical Vapor Deposit) method, ICP (Inductive Coupled Plasma) method, DC sputtering method, ECR (Electric Cyclotron Resonance) A sputtering method, an ion plating method, an arc ion plating method, an EB (Electron Beam) vapor deposition method, a resistance heating vapor deposition method, an ionization vapor deposition method, an arc vapor deposition method, a laser vapor deposition method, or the like.
本発明に用いる基板としては、前記のように表面処理層を形成した構造だけでなく、合成ダイヤモンド、高圧合成ダイヤモンド、天然ダイヤモンド、軟ダイヤモンド（例えば、ダイヤモンドライクカーボン）、アモルファスカーボン；金、銀、銅、アルミニウム、タングステン、モリブデン等の金属；プラスチック（例えば、ポリエステル樹脂、ポリエチレン樹脂、ポリプロピレン樹脂、ＡＢＳ樹脂、ナイロン、アクリル樹脂、フッ素樹脂、ポリカーボネート樹脂、ポリウレタン樹脂、メチルペンテン樹脂、フェノール樹脂、メラミン樹脂、エポキシ樹脂、塩化ビニル樹脂）；前記金属粉末、セラミック粉末等に、前記樹脂をバインダーとして混合、結合形成したもの；前記金属粉末やセラミックス粉末等の原料をプレス成形機で圧粉したものを高温で焼結したものが挙げられ、また、前記の材料の積層体や複合体（例えば、ダイヤモンドと他の物質との複合体、（例えば２相体））であってもよい。 As the substrate used in the present invention, not only the structure in which the surface treatment layer is formed as described above, but also synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon; gold, silver, Metals such as copper, aluminum, tungsten, molybdenum; plastics (for example, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin) , Epoxy resin, vinyl chloride resin); the above-mentioned metal powder, ceramic powder, etc. mixed with the above resin as a binder, and formed by bonding; The include those sintered at high temperatures, also, the laminate or composite of said materials (e.g., complex with diamond and other materials, (eg 2-phase body)) may be used.
基板の形状及びサイズは特に限定されないが、形状としては、平板状、糸状、球状、多角形状、粉末状などが挙げられ、サイズは、平板状のものを用いる場合、通常は、幅０．１〜１００ｍｍ、長さ０．１〜１００ｍｍ、厚み０．０１〜１０ｍｍ程度である。 Although the shape and size of the substrate are not particularly limited, examples of the shape include a flat plate shape, a thread shape, a spherical shape, a polygonal shape, a powder shape, and the size is usually 0.1 mm in width when a flat plate shape is used. ˜100 mm, length 0.1˜100 mm, thickness 0.01˜10 mm.
また、基板の表面又は裏面に、反射層としてＴｉ、Ａｕ、Ｐｔ、Ｎｂ、Ｃｒ、ＴｉＣ、ＴｉＮ等の単層又はこれらの複合膜を製膜してもよい。反射層の厚みは、全体に均一であることが必要なため、好ましくは１０ｎｍ以上、更に好ましくは１００ｎｍ以上である。 Further, a single layer of Ti, Au, Pt, Nb, Cr, TiC, TiN, or a composite film thereof may be formed on the front surface or the back surface of the substrate as a reflective layer. Since the thickness of the reflective layer needs to be uniform throughout, it is preferably 10 nm or more, more preferably 100 nm or more.
基板としてガラスを用いる場合、その表面は、Ｒａ（ＪＩＳ Ｂ ０６０１）で１ｎｍ〜１０００ｎｍの範囲で意図的に粗面化されていることも好ましい。このような粗面化表面は基板の表面積が増えて、多量のＤＮＡプローブ等を高密度で固定化できる点で好都合である。 When glass is used as the substrate, the surface thereof is preferably intentionally roughened in the range of 1 nm to 1000 nm with Ra (JIS B 0601). Such a roughened surface is advantageous in that the surface area of the substrate is increased and a large amount of DNA probes and the like can be immobilized at a high density.
本発明の固体支持体には、核酸分子等の生体分子を静電的に引き寄せるために静電層が設けられている。 The solid support of the present invention is provided with an electrostatic layer for electrostatically attracting biomolecules such as nucleic acid molecules.
静電層としては、核酸分子等の生体分子を静電的に引き寄せ、生体分子の固定化量を向上させるものであれば、特に制限はないが、例えば、アミノ基含有化合物など正荷電を有する化合物を用いて形成することができる。 The electrostatic layer is not particularly limited as long as it electrostatically attracts biomolecules such as nucleic acid molecules and improves the amount of immobilized biomolecules. For example, the electrostatic layer has a positive charge such as an amino group-containing compound. It can be formed using a compound.
前記アミノ基含有化合物としては、非置換のアミノ基（−ＮＨ２）、又は炭素数１〜６のアルキル基等で一置換されたアミノ基（−ＮＨＲ；Ｒは置換基）を有する化合物、例えばエチレンジアミン、ヘキサメチレンジアミン、ｎ−プロピルアミン、モノメチルアミン、ジメチルアミン、モノエチルアミン、ジエチルアミン、アリルアミン、アミノアゾベンゼン、アミノアルコール（例えば、エタノールアミン）、アクリノール、アミノ安息香酸、アミノアントラキノン、アミノ酸（グリシン、アラニン、バリン、ロイシン、セリン、トレオニン、システイン、メチオニン、フェニルアラニン、トリプトファン、チロシン、プロリン、シスチン、グルタミン酸、アスパラギン酸、グルタミン、アスパラギン、リシン、アルギニン、ヒスチジン）、アニリン、又はこれらの重合体（例えば、ポリアリルアミン、ポリリシン）や共重合体；４,４’,４”-トリアミノトリフェニルメタン、トリアムテレン、スペルミジン、スペルミン、プトレシンなどのポリアミン（多価アミン）が挙げられる。 Examples of the amino group-containing compound include an unsubstituted amino group (—NH 2 ), or an amino group monosubstituted by an alkyl group having 1 to 6 carbon atoms (—NHR; R is a substituent), for example, Ethylenediamine, hexamethylenediamine, n-propylamine, monomethylamine, dimethylamine, monoethylamine, diethylamine, allylamine, aminoazobenzene, aminoalcohol (eg, ethanolamine), acrinol, aminobenzoic acid, aminoanthraquinone, amino acid (glycine, alanine) , Valine, leucine, serine, threonine, cysteine, methionine, phenylalanine, tryptophan, tyrosine, proline, cystine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, histidine , Aniline, or a polymer (for example, polyallylamine, polylysine) or a copolymer thereof; 4,4 ′, 4 ″ -triaminotriphenylmethane, triamterene, spermidine, spermine, putrescine, and other polyamines (polyvalent amines) Is mentioned.
静電層は、基板又は表面処理層と共有結合させずに形成してもよく、基板又は表面処理層と共有結合させて形成してもよい。 The electrostatic layer may be formed without being covalently bonded to the substrate or the surface treatment layer, or may be formed to be covalently bonded to the substrate or the surface treatment layer.
静電層を基板又は表面処理層と共有結合させずに形成する場合には、例えば、表面処理層を製膜する際に前記アミノ基含有化合物を製膜装置内に導入することによって、アミノ基を含有する炭素系皮膜を製膜する。製膜装置内に導入する化合物として、アンモニアガスを用いてもよい。また、表面処理層は、密着層を形成した後にアミノ基を含有する皮膜を形成するといった、複層であってもよく、この場合もアンモニアガスを含んだ雰囲気で行ってもよい。製膜は、例えばプラズマ法によって実施できる。 In the case where the electrostatic layer is formed without being covalently bonded to the substrate or the surface treatment layer, for example, when the surface treatment layer is formed, the amino group-containing compound is introduced into the film forming apparatus to thereby form an amino group. A carbon-based film containing is formed. Ammonia gas may be used as the compound introduced into the film forming apparatus. Further, the surface treatment layer may be a multi-layer structure in which a film containing an amino group is formed after the adhesion layer is formed, and in this case, it may be performed in an atmosphere containing ammonia gas. Film formation can be performed by, for example, a plasma method.
また、静電層を基板又は表面処理層と共有結合させずに形成する場合には、静電層と基板又は表面処理層との親和性、即ち密着性を高める点で、基板上に、前記の非置換又は一置換されたアミノ基を有する化合物及び炭素化合物を蒸着させることが好ましい。ここで用いる炭素化合物としては、気体として供給することができれば特に制限はないが、例えば常温で気体であるメタン、エタン、プロパンが好ましい。蒸着の方法としては、イオン化蒸着法が好ましく、イオン化蒸着法の条件としては、作動圧が０．１〜５０Ｐａ、そして加速電圧が２００〜１０００Ｖの範囲であることが好ましい。 Further, when the electrostatic layer is formed without being covalently bonded to the substrate or the surface treatment layer, the affinity between the electrostatic layer and the substrate or the surface treatment layer, that is, the adhesion is improved on the substrate. It is preferable to vapor-deposit a compound having an unsubstituted or monosubstituted amino group and a carbon compound. The carbon compound used here is not particularly limited as long as it can be supplied as a gas. For example, methane, ethane, and propane which are gases at normal temperature are preferable. As a method of vapor deposition, ionized vapor deposition is preferable, and as conditions for ionized vapor deposition, it is preferable that an operating pressure is 0.1 to 50 Pa and an acceleration voltage is in a range of 200 to 1000 V.
静電層を基板又は表面処理層と共有結合させて形成する場合には、例えば、基板又は表面処理層を施した基板に、塩素ガス中で紫外線照射して表面を塩素化し、次いで前記アミノ基含有化合物のうち、例えば、ポリアリルアミン、ポリリシン、４,４',４”-トリアミノトリフェニルメタン、トリアムテレン等の多価アミンを反応させて、基板と結合していない側の末端にアミノ基を導入することにより、静電層を形成することができる。 When the electrostatic layer is formed by covalently bonding to the substrate or the surface treatment layer, for example, the surface of the substrate or the substrate to which the surface treatment layer is applied is chlorinated by irradiating with ultraviolet rays in chlorine gas, and then the amino group. Among the contained compounds, for example, a polyamine such as polyallylamine, polylysine, 4,4 ′, 4 ″ -triaminotriphenylmethane, triamterene, etc. is reacted to form an amino group at the terminal not bonded to the substrate. By introducing, an electrostatic layer can be formed.
基板を、非置換又は一置換されたアミノ基を有する化合物を含有する溶液中に浸漬することにより、静電層を形成する場合に、アミノ基含有化合物としてポリアリルアミンを用いると、基板との密着性に優れ、生体分子の固定化量がより向上する。アミノ基含有化合物とともにシランカップリング剤が共存する溶液に基板を浸漬することにより、静電層を形成することもできる。
静電層の厚みは、１ｎｍ〜５００μｍであることが好ましい。
When forming an electrostatic layer by immersing the substrate in a solution containing a compound having an unsubstituted or monosubstituted amino group, when polyallylamine is used as the amino group-containing compound, the substrate is in close contact with the substrate. This improves the amount of immobilized biomolecules. The electrostatic layer can also be formed by immersing the substrate in a solution in which the silane coupling agent coexists with the amino group-containing compound.
The thickness of the electrostatic layer is preferably 1 nm to 500 μm.
本発明において生体分子とは、生体内に存在する分子を意味し、本発明の固体支持体に固定化できる生体分子としては、ＤＮＡ及びＲＮＡ等の核酸分子、ペプチド、オリゴペプチド、ポリペプチド及びタンパク質、ならびに糖鎖などが挙げられる。本発明の固体支持体は、核酸分子、特にＤＮＡの固定化に好適に用いられる。 In the present invention, the biomolecule means a molecule existing in the living body, and the biomolecule that can be immobilized on the solid support of the present invention includes nucleic acid molecules such as DNA and RNA, peptides, oligopeptides, polypeptides, and proteins. As well as sugar chains. The solid support of the present invention is suitably used for immobilizing nucleic acid molecules, particularly DNA.
ＤＮＡ及びＲＮＡ等の核酸分子を固定化する場合、その塩基数は、通常１〜１０億、好ましくは５〜１００万である。また、ＤＮＡは一本鎖、二本鎖のいずれも固定化することができる。 When immobilizing nucleic acid molecules such as DNA and RNA, the number of bases is usually 1 to 1 billion, preferably 5 to 1 million. In addition, DNA can be immobilized either single-stranded or double-stranded.
本発明の固体支持体は、核酸分子、例えば、ＤＮＡの伸長反応に用いることができる。この場合、まず、固体支持体上にプライマーを固定化して、一本鎖又は二本鎖ＤＮＡをハイブリダイズさせる。その後、ＤＮＡ伸長反応によりプライマーにハイブリダイズしたＤＮＡと相補的なＤＮＡを伸長させる。 The solid support of the present invention can be used for an elongation reaction of a nucleic acid molecule such as DNA. In this case, first, a primer is immobilized on a solid support, and single-stranded or double-stranded DNA is hybridized. Thereafter, DNA complementary to the DNA hybridized to the primer is extended by a DNA extension reaction.
プライマーとしては、長さ及び配列が明らかな一本鎖又は二本鎖の核酸分子を使用する。長さは特に限定されないが、好ましくは５〜２００塩基、さらに好ましくは１０〜１００塩基である。プライマーの固定化方法としては、特に限定されないが、例えば、核酸分子をバッファーに溶解してプライマー溶液を作成し、これに本発明の固定化支持体を浸漬することによって、固体支持体表面にプライマーを固定化することができる。浸漬は、通常、０〜９８℃、好ましくは４℃〜５０℃で、通常、１分〜２４時間、好ましくは１０分〜１時間行うことによってプライマーを固定化できる。この場合、一定時間浸漬した後、固体支持体を洗浄することによって、固定化されていないプライマーを除去することができる。また、スポッターといわれる装置を使用することによって、多種類のプライマー溶液を固体支持体の表面に固定化できる。スポッターを用いる場合には、例えば、スポッターでプライマー溶液を固体支持体上にスポットした後、加熱したオーブン中で一定時間ベーキングを行い、その後洗浄によって固定していないプライマーを除去する。スポッター装置を用いることにより他種類のプライマーを固体支持体上の異なる位置に固定化できるため一度に多数の試験を実施することができ、膨大な試験を要する核酸検出の分野において有利である。 As a primer, a single-stranded or double-stranded nucleic acid molecule having a clear length and sequence is used. Although length is not specifically limited, Preferably it is 5-200 bases, More preferably, it is 10-100 bases. The primer immobilization method is not particularly limited. For example, a primer solution is prepared by dissolving a nucleic acid molecule in a buffer, and the immobilization support of the present invention is immersed in the primer solution. Can be immobilized. The primer can be immobilized by performing the dipping usually at 0 to 98 ° C., preferably at 4 to 50 ° C. and usually for 1 minute to 24 hours, preferably 10 minutes to 1 hour. In this case, after being immersed for a certain time, the non-immobilized primer can be removed by washing the solid support. Further, by using an apparatus called a spotter, various types of primer solutions can be immobilized on the surface of the solid support. When using a spotter, for example, after spotting the primer solution on the solid support with a spotter, the primer solution is baked in a heated oven for a certain period of time, and then the unfixed primer is removed by washing. By using a spotter device, other types of primers can be immobilized at different positions on the solid support, so that a large number of tests can be carried out at one time, which is advantageous in the field of nucleic acid detection requiring an enormous number of tests.
従来の固体支持体では、伸長反応における熱処理によってプライマーが剥離することがあるが、本発明の固体支持体では、熱を加えてもプライマーが剥離せず、プライマーを固体支持体に固定化した状態で伸長反応を進行させることができる。 In the conventional solid support, the primer may be peeled off by the heat treatment in the extension reaction, but in the solid support of the present invention, the primer does not peel off even when heat is applied, and the primer is immobilized on the solid support. Allows the extension reaction to proceed.
この伸長反応の時に、標識した核酸を取り込ませ、伸長反応後、標識に由来するシグナルを読みとることによって、プライマーに特定のＤＮＡがハイブリダイズして伸長反応が進行したか否かを検出することができる。従って、試験した試料中に、固定化支持体上のプライマーにハイブリダイズしうるＤＮＡが含まれているかどうかを判定することができ、研究及び医療における有用な検出手段となりうる。 During this extension reaction, the labeled nucleic acid is incorporated, and after the extension reaction, the signal derived from the label is read to detect whether the extension reaction has progressed by hybridization of specific DNA to the primer. it can. Therefore, it can be determined whether or not the tested sample contains DNA that can hybridize to the primer on the immobilization support, which can be a useful detection means in research and medicine.
標識としては、核酸分子に取り込むことが可能なものであれば特に限定されないが、例えば、蛍光標識（Ｃｙ３及びＣｙ５などのＣｙＤｙｅ、ＦＩＴＣ、ＲＩＴＣ、ローダミン、テキサスレッド、ＴＥＴ、ＴＡＭＲＡ、ＦＡＭ、ＨＥＸ、ＲＯＸなど）、放射能標識（α−３２Ｐ、γ−３２Ｐ、３５Ｓなど）などが挙げられる。蛍光標識核酸を用いた場合は、伸長反応の後の固体支持体を蛍光撮影することにより、検出することができる。 The label is not particularly limited as long as it can be incorporated into a nucleic acid molecule. For example, fluorescent labels (CyDye such as Cy3 and Cy5, FITC, RITC, rhodamine, Texas red, TET, TAMRA, FAM, HEX, ROX etc.), radioactivity labels (α- 32 P, γ- 32 P, 35 S etc.) and the like. When a fluorescently labeled nucleic acid is used, it can be detected by fluorescence imaging of the solid support after the extension reaction.
本発明の固体支持体は、ＤＮＡの増幅反応に用いることができる。ＰＣＲ反応により増幅させる場合には、例えば、まず、固体支持体上にフォワードプライマーを固定化して、一本鎖又は二本鎖ＤＮＡをハイブリダイズさせ、その後酵素反応で相補鎖ＤＮＡを伸長する。更にi)アニーリング、ii)ハイブリダイゼーション、iii)伸長反応という工程を連続で行うことによって、いわゆるＰＣＲ反応が進行する。 The solid support of the present invention can be used for a DNA amplification reaction. In the case of amplification by PCR reaction, for example, first, a forward primer is immobilized on a solid support, single-stranded or double-stranded DNA is hybridized, and then complementary strand DNA is extended by enzymatic reaction. Furthermore, a so-called PCR reaction proceeds by continuously performing steps i) annealing, ii) hybridization, and iii) extension reaction.
従来の固体支持体では、ＰＣＲ反応における熱処理によってプライマーが剥離したり、サーマルサイクルの制御がうまくいかないといった問題があったが、本発明の固体支持体では、熱を加えてもプライマーが剥離せず、さらに、反応を容器の中ではなく固体支持体上にＤＮＡを固定化した状態で行うため、ＰＣＲ反応における温度制御が正確で、増幅される核酸の配列の正確性に影響が出たり、目的外のＤＮＡが複製される可能性も低く、効率的にＤＮＡを増幅することができる。 In the conventional solid support, there was a problem that the primer was peeled off by the heat treatment in the PCR reaction, or the thermal cycle was not well controlled, but in the solid support of the present invention, the primer was not peeled even when heat was applied. Furthermore, since the reaction is carried out with DNA immobilized on a solid support rather than in a container, temperature control in the PCR reaction is accurate, and the accuracy of the sequence of the nucleic acid to be amplified is affected. The DNA is less likely to be replicated and can be amplified efficiently.
本発明の固体支持体を上記のＰＣＲによるＤＮＡの増幅に使用する場合は、固体支持体の熱伝導率が０．１Ｗ／ｃｍ・Ｋ以上であることが好ましい。さらに好ましくは熱伝導率が０．５Ｗ／ｃｍ・Ｋ以上、最も好ましくは熱伝導率が１Ｗ／ｃｍ・Ｋ以上である。固体支持体の熱伝導率が高いと、ＰＣＲ反応を行う場合等において、加熱、冷却の追随性が優れているからである。 When the solid support of the present invention is used for the amplification of DNA by the above PCR, the solid support preferably has a thermal conductivity of 0.1 W / cm · K or more. More preferably, the thermal conductivity is 0.5 W / cm · K or more, and most preferably the thermal conductivity is 1 W / cm · K or more. This is because, when the thermal conductivity of the solid support is high, the followability of heating and cooling is excellent when performing a PCR reaction or the like.
具体的には、固体支持体を製造するための基板として、熱伝導率の点からはダイヤモンド又は各種基板に表面処理層としてダイヤモンドを被覆した物等を使用するのが好ましい。 Specifically, from the viewpoint of thermal conductivity, it is preferable to use diamond or various substrates coated with diamond as a surface treatment layer as a substrate for producing a solid support.
さらに、上記の標識核酸を用いた検出とＰＣＲによる増幅とを組み合わせることにより、固体支持体上のプライマーにハイブリダイズしうるＤＮＡが試料中に少量しか含まれていない場合であっても、上記のようにＤＮＡが複製され、結果的に多量のＤＮＡが固体支持体上のプライマーにハイブリダイズし、その相補鎖が伸長されるため、検出感度を増大させることが可能になる。 Furthermore, by combining detection using the above labeled nucleic acid and amplification by PCR, even if the sample contains only a small amount of DNA that can hybridize to the primer on the solid support, Thus, the DNA is replicated, and as a result, a large amount of DNA is hybridized to the primer on the solid support and its complementary strand is extended, so that the detection sensitivity can be increased.
あるいは、伸長反応に使用する酵素として、鎖置換型ＤＮＡポリメラーゼを選択し、リバースプライマーを加えることによって、サーマルサイクルを経ることなく定温で、ＤＮＡを固体支持体上で増幅することができる。鎖置換型ＤＮＡポリメラーゼとは、鋳型ＤＮＡに相補的なＤＮＡ鎖を合成していく過程で、伸長方向に二本鎖領域があった場合その鎖を解離しながら、相補鎖合成を継続できるＤＮＡ合成酵素である。 Alternatively, by selecting a strand displacement type DNA polymerase as an enzyme used for the extension reaction and adding a reverse primer, DNA can be amplified on a solid support at a constant temperature without going through a thermal cycle. Strand displacement DNA polymerase is a process of synthesizing a complementary DNA strand to the template DNA. If there is a double-stranded region in the extension direction, DNA synthesis that can continue complementary strand synthesis while dissociating the strand It is an enzyme.
鎖置換型ＤＮＡポリメラーゼとしては、特に限定されないが、例えば、ＢｃａＢＥＳＴ ＤＮＡポリメラーゼ(タカラバイオ)、Ｐｈｉ２９ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ(アマシャムバイオサイエンス)等が挙げられる。 The strand displacement type DNA polymerase is not particularly limited, and examples thereof include BcaBEST DNA polymerase (Takara Bio), Phi29 DNA Polymerase (Amersham Bioscience) and the like.
本発明の別の態様においては、ｍＲＮＡから合成したｃＤＮＡを対象とすることにより、間接的ながらＲＮＡも対象となりうる。 In another embodiment of the present invention, by targeting cDNA synthesized from mRNA, RNA can be targeted indirectly.
この場合には、ｍＲＮＡから逆転写反応を利用してｃＤＮＡを得るが、ｃＤＮＡを得ると同時に固体支持体に固定化させることができる。まず、固体支持体の化学修飾部分に逆転写プライマーを結合させる。プライマーとしては、一般にオリゴｄＴプライマー、特定塩基配列に相補的なプライマー、ランダム６塩基プライマーが用いられるが、中でもＲＮＡの５’末端のｐｏｌｙ（Ａ）＋配列に対応させてＴ（チミン塩基）が１０〜２０個程度連なった配列からなるオリゴｄＴプライマーを用いることが望ましい。 In this case, cDNA is obtained from mRNA using reverse transcription reaction, and can be immobilized on a solid support simultaneously with obtaining cDNA. First, a reverse transcription primer is bound to the chemically modified portion of the solid support. As the primer, an oligo dT primer, a primer complementary to a specific base sequence, and a random 6 base primer are generally used. Among them, T (thymine base) is associated with the poly (A) + sequence at the 5 ′ end of RNA. It is desirable to use an oligo dT primer having a sequence of about 10 to 20 sequences.
オリゴｄＴプライマーを用いる場合には、鋳型となるＲＮＡの５’末端のｐｏｌｙ（Ａ）部分をアニーリングさせる。これに逆転写酵素を作用させ、鋳型ＲＮＡに対し相補的なｄＮＴＰをプライマーの３’末端に順々に重合させることで、５’から３’の方向にｃＤＮＡを合成する。この逆転写反応のプライマーの結合、アニーリング、逆転写酵素による相補鎖重合は、定法に従い温度制御（サーマルサイクル）を行うことによって実施できる。 When an oligo dT primer is used, the poly (A) portion at the 5 'end of the template RNA is annealed. A reverse transcriptase is allowed to act on this, and dNTPs complementary to the template RNA are sequentially polymerized at the 3 'end of the primer to synthesize cDNA in the 5' to 3 'direction. Primer binding, annealing, and complementary strand polymerization by reverse transcriptase in this reverse transcription reaction can be performed by performing temperature control (thermal cycle) according to a conventional method.
このように逆転写反応を行うと同時に固体支持体への固定も可能であることから、本発明の方法によればいわゆるＲＴ−ＰＣＲ（reverse transcript-PCR）を効率よく行うことができ、ｍＲＮＡの定量用としても有用である。 As described above, since the reverse transcription reaction can be performed and the solid support can be fixed at the same time, according to the method of the present invention, so-called RT-PCR (reverse transcript-PCR) can be efficiently performed, It is also useful for quantitative purposes.
更に、本発明の支持体を用い、オリゴ核酸の末端塩基を固定化し、更に、このオリゴ核酸と相補的塩基配列を有するＤＮＡを固定して、ＤＮＡライブラリーチップとして用いることもできる。また、ＤＮＡの代わりに、ヌクレオチド、オリゴヌクレオチド、ＤＮＡフラグメント等を固定化して、ライブラリーとすることもできる。 Furthermore, using the support of the present invention, the terminal base of the oligonucleic acid can be immobilized, and DNA having a complementary base sequence with this oligonucleic acid can be immobilized and used as a DNA library chip. Further, instead of DNA, nucleotides, oligonucleotides, DNA fragments and the like can be immobilized to form a library.
本発明の固体支持体を用いて上記のような検出を行うことにより、疾患の診断を行うこともできる。 Diseases can also be diagnosed by performing the above detection using the solid support of the present invention.
以下、実施例により本発明を説明するが、本発明はこれらに限定されるものではない。
(実施例１）アンモニアプラズマによる静電層の導入
スライドグラスにイオン化蒸着法によって、メタンガス９５体積％と水素５体積％を混合したガスを原料として、加速電圧０．５ｋＶでＤＬＣ層を１０ｎｍの厚みに形成した。その後、メタンガスと水素ガスの代わりにアンモニアガス雰囲気とし、プラズマ法により１０分間アミノ化し、固体支持体を作成した。
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these.
(Example 1) Introduction of electrostatic layer by ammonia plasma By using ionized vapor deposition on a slide glass, a mixture of 95% by volume of methane gas and 5% by volume of hydrogen is used as a raw material, and the DLC layer is 10 nm thick at an acceleration voltage of 0.5 kV. Formed. Thereafter, an atmosphere of ammonia gas was used instead of methane gas and hydrogen gas, and amination was performed by a plasma method for 10 minutes to prepare a solid support.
この固体支持体に、マイクロアレイ用スポッターＳＰＢＩＯ２０００（日立ソフト社）を使用して、λＤＮＡから増幅した５００ｂｐのＣｙ３標識二本鎖ＤＮＡ約１ｎｌをスポットした。ＤＮＡ濃度は１μｇ／μｌ、スポット溶液は３×ＳＳＣである。 On this solid support, about 1 nl of 500 bp Cy3-labeled double-stranded DNA amplified from λDNA was spotted using a spotter for microarray SPBIO2000 (Hitachi Soft). The DNA concentration is 1 μg / μl and the spot solution is 3 × SSC.
この固体支持体を８０℃のオーブンに１時間入れた後に、室温の２×ＳＳＣ／０．２％ＳＤＳ溶液で１５分間洗浄を行った。その後、９５℃に加熱した２×ＳＳＣ／０．２％ＳＤＳ溶液で５分間洗浄した後に、超純水で数回洗浄して、遠心乾燥させた。 The solid support was placed in an oven at 80 ° C. for 1 hour, and then washed with a 2 × SSC / 0.2% SDS solution at room temperature for 15 minutes. Thereafter, the plate was washed with a 2 × SSC / 0.2% SDS solution heated to 95 ° C. for 5 minutes, then washed several times with ultrapure water, and dried by centrifugation.
その後、蛍光スキャナーＦＬＡ−８０００（富士写真フィルム社）によって蛍光画像を観察し、蛍光強度を測定した。結果として、比較例１として用いた市販の静電結合型基板(コーニング社製ＧＡＰＳコートスライド)と比較して高い蛍光強度が得られた。 Then, the fluorescence image was observed with the fluorescence scanner FLA-8000 (Fuji Photo Film company), and the fluorescence intensity was measured. As a result, high fluorescence intensity was obtained as compared with the commercially available electrostatic coupling type substrate (Corning GAPS coated slide) used as Comparative Example 1.
（実施例２）シランカップリング剤／ポリアリルアミンによる静電層の導入
スライドグラスを、シランカップリング剤とポリアリルアミンを９５％エタノールに溶解した溶液に１０分間浸漬した。このとき、シランカップリング剤は２％、ポリアリルアミンは０．１％になるように添加した。その後、エタノールで２回洗浄した後に遠心乾燥し、１００℃に設定したオーブンで１０分間加熱し、固体支持体を作成した。
(Example 2) Introduction of electrostatic layer by silane coupling agent / polyallylamine The slide glass was immersed for 10 minutes in a solution of silane coupling agent and polyallylamine dissolved in 95% ethanol. At this time, 2% of the silane coupling agent and 0.1% of polyallylamine were added. Then, after washing twice with ethanol, it was centrifuged and heated in an oven set at 100 ° C. for 10 minutes to prepare a solid support.
この固体支持体に、マイクロアレイ用スポッターＳＰＢＩＯ２０００（日立ソフト社）を使用して、λＤＮＡから増幅した５００ｂｐのＣｙ３標識二本鎖ＤＮＡ約１ｎｌをスポットした。ＤＮＡ濃度は１μｇ／μｌ、スポット溶液は３×ＳＳＣである。 On this solid support, about 1 nl of 500 bp Cy3-labeled double-stranded DNA amplified from λDNA was spotted using a spotter for microarray SPBIO2000 (Hitachi Soft). The DNA concentration is 1 μg / μl and the spot solution is 3 × SSC.
この固体支持体を８０℃のオーブンに１時間入れた後に、室温の２×ＳＳＣ／０．２％ＳＤＳ溶液で１５分間洗浄を行った。その後、９５℃に加熱した２×ＳＳＣ／０．２％ＳＤＳ溶液で５分間洗浄した後に、超純水で数回洗浄して、遠心乾燥させた。 The solid support was placed in an oven at 80 ° C. for 1 hour, and then washed with a 2 × SSC / 0.2% SDS solution at room temperature for 15 minutes. Thereafter, the plate was washed with a 2 × SSC / 0.2% SDS solution heated to 95 ° C. for 5 minutes, then washed several times with ultrapure water, and dried by centrifugation.
その後、蛍光スキャナーＦＬＡ−８０００（富士写真フィルム社）によって蛍光画像を観察し、蛍光強度を測定した。結果として、比較例１として用いた市販の静電結合型基板(コーニング社製ＧＡＰＳコートスライド)と比較して高い蛍光強度が得られた。 Then, the fluorescence image was observed with the fluorescence scanner FLA-8000 (Fuji Photo Film company), and the fluorescence intensity was measured. As a result, high fluorescence intensity was obtained as compared with the commercially available electrostatic coupling type substrate (Corning GAPS coated slide) used as Comparative Example 1.
また、X線光電子分光分析法(ＸＰＳ)により導入されたアミノ基由来のＮピーク強度を測定したところ、シランカップリング剤単独で処理した場合にはピーク強度が４０００、ポリアリルアミン単独で処理した場合にはピーク強度が７１００、本実施例のようにシランカップリング剤とポリアリルアミンが共存した場合にはピーク強度が１５２００となった。従って、本実施例の処理方法は、アミノ基を多く導入するのに有効である。 In addition, when the N peak intensity derived from the amino group introduced by X-ray photoelectron spectroscopy (XPS) was measured, the peak intensity was 4000 when treated with the silane coupling agent alone, and the polyarylamine alone was treated. The peak intensity was 7100, and the peak intensity was 15200 when the silane coupling agent and polyallylamine coexisted as in this example. Therefore, the treatment method of this example is effective for introducing many amino groups.
（実施例３）ポリアリルアミンによる静電層の導入
スライドグラスを、０．１％ポリアリルアミン水溶液に１０分間浸漬した。その後、超純水で２回洗浄した後に遠心乾燥し、１００℃に設定したオーブンで１０分間加熱して、固体支持体を作成した。
(Example 3) Introduction of electrostatic layer with polyallylamine A slide glass was immersed in a 0.1% polyallylamine aqueous solution for 10 minutes. Then, after washing twice with ultrapure water, it was centrifuged and heated in an oven set at 100 ° C. for 10 minutes to produce a solid support.
この固体支持体に、マイクロアレイ用スポッターＳＰＢＩＯ２０００（日立ソフト社）を使用して、λＤＮＡから増幅した５００ｂｐのＣｙ３標識二本鎖ＤＮＡ約１ｎｌをスポットした。ＤＮＡ濃度は１μｇ／μｌ、スポット溶液は３×ＳＳＣである。 On this solid support, about 1 nl of 500 bp Cy3-labeled double-stranded DNA amplified from λDNA was spotted using a spotter for microarray SPBIO2000 (Hitachi Soft). The DNA concentration is 1 μg / μl and the spot solution is 3 × SSC.
この固体支持体を８０℃のオーブンに１時間入れた後に、室温の２×ＳＳＣ／０．２％ＳＤＳ溶液で１５分間洗浄を行った。その後、９５℃に加熱した２×ＳＳＣ／０．２％ＳＤＳ溶液で５分間洗浄した後に、超純水で数回洗浄して、遠心乾燥させた。 The solid support was placed in an oven at 80 ° C. for 1 hour, and then washed with a 2 × SSC / 0.2% SDS solution at room temperature for 15 minutes. Thereafter, the plate was washed with a 2 × SSC / 0.2% SDS solution heated to 95 ° C. for 5 minutes, then washed several times with ultrapure water, and dried by centrifugation.
その後、蛍光スキャナーＦＬＡ−８０００（富士写真フィルム社）によって蛍光画像を観察し、蛍光強度を測定した。結果として、比較例１として用いた市販の静電結合型基板(コーニング社製ＧＡＰＳコートスライド)と比較して高い蛍光強度が得られた。 Then, the fluorescence image was observed with the fluorescence scanner FLA-8000 (Fuji Photo Film company), and the fluorescence intensity was measured. As a result, high fluorescence intensity was obtained as compared with the commercially available electrostatic coupling type substrate (Corning GAPS coated slide) used as Comparative Example 1.
（実施例４）ＤＬＣ層を有する基板へ静電層の導入
２５ｍｍ（幅）×７５ｍｍ（長さ）×１ｍｍ（厚み）のスライドガラスに、イオン化蒸着法によって、メタンガス９５体積％と水素５体積％を混合したガスを原料として、加速電圧０．５ｋＶでＤＬＣ層を１０ｎｍの厚みに形成した。ＤＬＣ層を形成したスライドガラスを０．１％のポリアリルアミン溶液中に浸漬し、洗浄後乾燥することにより、固体支持体を作成した。
(Example 4) Introduction of electrostatic layer to substrate having DLC layer 95% by volume of methane gas and 5% by volume of hydrogen by ionized vapor deposition on a slide glass of 25 mm (width) x 75 mm (length) x 1 mm (thickness) A DLC layer having a thickness of 10 nm was formed at a accelerating voltage of 0.5 kV using a mixed gas as a raw material. The slide glass on which the DLC layer was formed was immersed in a 0.1% polyallylamine solution, washed and dried to prepare a solid support.
この固体支持体に、マイクロアレイ用スポッターＳＰＢＩＯ２０００（日立ソフト社）を使用して、λＤＮＡから増幅した５００ｂｐのＣｙ３標識二本鎖ＤＮＡ約１ｎｌをスポットした。ＤＮＡ濃度は１μｇ／μｌ、スポット溶液は２０％ＰＥＧである。 On this solid support, about 1 nl of 500 bp Cy3-labeled double-stranded DNA amplified from λDNA was spotted using a spotter for microarray SPBIO2000 (Hitachi Soft). The DNA concentration is 1 μg / μl, and the spot solution is 20% PEG.
この固体支持体を８０℃のオーブンに１時間入れた後に、室温の２×ＳＳＣ／０．２％ＳＤＳ溶液で１５分間洗浄を行った。その後、９５℃に加熱した２×ＳＳＣ／０．２％ＳＤＳ溶液で５分間洗浄した後に、超純水で数回洗浄して、遠心乾燥させた。 The solid support was placed in an oven at 80 ° C. for 1 hour, and then washed with a 2 × SSC / 0.2% SDS solution at room temperature for 15 minutes. Thereafter, the plate was washed with a 2 × SSC / 0.2% SDS solution heated to 95 ° C. for 5 minutes, then washed several times with ultrapure water, and dried by centrifugation.
その後、蛍光スキャナーＦＬＡ−８０００（富士写真フィルム社）によって蛍光画像を観察し、蛍光強度を測定した。結果として、比較例１として用いた市販の静電結合型基板(コーニング社製ＧＡＰＳコートスライド)と比較して高い蛍光強度が得られた。 Then, the fluorescence image was observed with the fluorescence scanner FLA-8000 (Fuji Photo Film company), and the fluorescence intensity was measured. As a result, high fluorescence intensity was obtained as compared with the commercially available electrostatic coupling type substrate (Corning GAPS coated slide) used as Comparative Example 1.
（実施例５）シランカップリング剤、ポリアクリル酸、ポリアリルアミンの積層による静電層の導入
スライドグラスを、シランカップリング剤を９５％エタノールに溶解した溶液に１０分間浸漬した。このとき、シランカップリング剤は２％になるように添加した。その後、エタノールで２回洗浄した後に遠心乾燥し、１００℃に設定したオーブンで１０分間加熱した。その後、０．２５％に調整したポリアクリル酸水溶液に１０分間浸漬し、超純水で３回洗浄した。更に、ポリアリルアミンを０．１％になるようにエタノールに溶解した溶液に１０分間浸漬して、エタノール洗浄を行った後に遠心乾燥して、固体支持体を作成した。
(Example 5) Introduction of electrostatic layer by laminating silane coupling agent, polyacrylic acid, and polyallylamine A slide glass was immersed in a solution of silane coupling agent in 95% ethanol for 10 minutes. At this time, the silane coupling agent was added to 2%. Then, after washing twice with ethanol, it was centrifuged and heated in an oven set at 100 ° C. for 10 minutes. Then, it was immersed in the polyacrylic acid aqueous solution adjusted to 0.25% for 10 minutes, and washed 3 times with ultrapure water. Furthermore, it was immersed in a solution in which polyallylamine was dissolved in ethanol to a concentration of 0.1% for 10 minutes, washed with ethanol, and then centrifuged to prepare a solid support.
この固体支持体に、マイクロアレイ用スポッターＳＰＢＩＯ２０００（日立ソフト社）を使用して、λＤＮＡから増幅した５００ｂｐのＣｙ３標識二本鎖ＤＮＡ約１ｎｌをスポットした。ＤＮＡ濃度は１μｇ／μｌ、スポット溶液は３×ＳＳＣである。 On this solid support, about 1 nl of 500 bp Cy3-labeled double-stranded DNA amplified from λDNA was spotted using a spotter for microarray SPBIO2000 (Hitachi Soft). The DNA concentration is 1 μg / μl and the spot solution is 3 × SSC.
この固体支持体を８０℃のオーブンに１時間入れた後に、室温の２×ＳＳＣ／０．２％ＳＤＳ溶液で１５分間洗浄を行った。その後、９５℃に加熱した２×ＳＳＣ／０．２％ＳＤＳ溶液で５分間洗浄した後に、超純水で数回洗浄して、遠心乾燥させた。 The solid support was placed in an oven at 80 ° C. for 1 hour, and then washed with a 2 × SSC / 0.2% SDS solution at room temperature for 15 minutes. Thereafter, the plate was washed with a 2 × SSC / 0.2% SDS solution heated to 95 ° C. for 5 minutes, then washed several times with ultrapure water, and dried by centrifugation.
その後、蛍光スキャナーＦＬＡ−８０００（富士写真フィルム社）によって蛍光画像を観察し、蛍光強度を測定した。結果として、比較例１として用いた市販の静電結合型基板(コーニング社製ＧＡＰＳコートスライド)と比較して高い蛍光強度が得られた。
実施例１〜５及び比較例１で測定した蛍光強度の結果を以下の表１に示す。
Then, the fluorescence image was observed with the fluorescence scanner FLA-8000 (Fuji Photo Film company), and the fluorescence intensity was measured. As a result, high fluorescence intensity was obtained as compared with the commercially available electrostatic coupling type substrate (Corning GAPS coated slide) used as Comparative Example 1.
The results of fluorescence intensity measured in Examples 1 to 5 and Comparative Example 1 are shown in Table 1 below.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2008169080A (en) * | 2007-01-11 | 2008-07-24 | Research Organization Of Information & Systems | Method for producing coated substrate, coated substrate produced by using the method, and its use |
| JP2009106220A (en) * | 2007-10-31 | 2009-05-21 | Genodive Pharma Kk | Method for catching and detecting target base sequence by isothermal amplification reaction on substrate |
| WO2010001876A1 (en) * | 2008-06-30 | 2010-01-07 | 株式会社ハイペップ研究所 | Biochip substratum and method for production thereof |
| WO2019207669A1 (en) * | 2018-04-25 | 2019-10-31 | 株式会社 日立ハイテクノロジーズ | Substrate for nucleic acid analysis and flow cell for nucleic acid analysis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2008169080A (en) * | 2007-01-11 | 2008-07-24 | Research Organization Of Information & Systems | Method for producing coated substrate, coated substrate produced by using the method, and its use |
| JP2009106220A (en) * | 2007-10-31 | 2009-05-21 | Genodive Pharma Kk | Method for catching and detecting target base sequence by isothermal amplification reaction on substrate |
| WO2010001876A1 (en) * | 2008-06-30 | 2010-01-07 | 株式会社ハイペップ研究所 | Biochip substratum and method for production thereof |
| JP2010008378A (en) * | 2008-06-30 | 2010-01-14 | Hipep Laboratories | Biochip substratum and method for production thereof |
| US8822607B2 (en) | 2008-06-30 | 2014-09-02 | Hipep Laboratories | Biochip substratum and method for production thereof |
| US9857369B2 (en) | 2008-06-30 | 2018-01-02 | Hipep Laboratories | Biochip substratum and method for production thereof |
| WO2019207669A1 (en) * | 2018-04-25 | 2019-10-31 | 株式会社 日立ハイテクノロジーズ | Substrate for nucleic acid analysis and flow cell for nucleic acid analysis |
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