JP5188228B2 - Method for detecting fluorescently labeled biologically relevant molecules - Google Patents
Method for detecting fluorescently labeled biologically relevant molecules Download PDFInfo
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- JP5188228B2 JP5188228B2 JP2008078128A JP2008078128A JP5188228B2 JP 5188228 B2 JP5188228 B2 JP 5188228B2 JP 2008078128 A JP2008078128 A JP 2008078128A JP 2008078128 A JP2008078128 A JP 2008078128A JP 5188228 B2 JP5188228 B2 JP 5188228B2
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Images
Description
本発明は、生体関連分子が固定化された担体を用いて、蛍光標識された生体関連分子を含む試料を分析する方法に関する。 The present invention relates to a method for analyzing a sample containing a fluorescently labeled biologically relevant molecule using a carrier on which the biologically relevant molecule is immobilized.
生体関連分子を担体上に固定化し、蛍光標識した生体関連分子を相互作用させてこれを検出する方法としては、担体上にレーザーを走査させて画像を構築する走査型検出器が一般的に市販されている。しかし、走査型検出器は、測定に時間がかかり、コストやメンテナンスの点でも問題がある。 As a method of detecting biological molecules by immobilizing biological molecules on a carrier and interacting with fluorescently labeled biological molecules, a scanning detector that scans the carrier and constructs an image is generally commercially available. Has been. However, the scanning detector takes time to measure and has problems in terms of cost and maintenance.
一方、レーザーを担体の全面に照射して結像光学系の検出器で蛍光を測定する方法を用いれば、走査型検出器とは異なり、操作が簡単で測定時間も短縮される（特許文献１）。 On the other hand, if a method of measuring the fluorescence with a detector of the imaging optical system by irradiating the entire surface of the carrier is used, unlike the scanning detector, the operation is simple and the measurement time is shortened (Patent Document 1). ).
生体関連分子の相互作用及び洗浄では塩を含む溶液が使用されることから、担体を乾燥させると塩による乾燥ムラが発生し、乾燥ムラによる散乱光が強く、正確な検出が困難な場合がある。 Since a solution containing a salt is used in the interaction and washing of biological molecules, drying unevenness due to salt occurs when the carrier is dried, and scattered light due to drying unevenness is strong, and accurate detection may be difficult. .
特に、結像光学系の検出器においては、乾燥ムラによる散乱光が強く、走査型検出器と比較して、正確な検出が非常に難しいことが明らかとなった。 In particular, in the detector of the imaging optical system, scattered light due to drying unevenness is strong, and it has become clear that accurate detection is very difficult compared to a scanning detector.
本発明者らは、結像光学系の検出器を用いる場合の上記問題点を見出し、さらに、相互作用後の担体を乾燥させることなく測定すれば、結像光学系の検出器においても散乱光の影響を低減でき、正確な検出が可能になることを見出した。 The present inventors have found the above-mentioned problems when using a detector of an imaging optical system, and further, if measurement is performed without drying the carrier after interaction, scattered light is also detected in the detector of the imaging optical system. It has been found that the influence of this can be reduced and accurate detection becomes possible.
すなわち、本発明は以下の発明を包含する。
（１）生体関連分子が固定化された担体を用いて、蛍光標識された生体関連分子を含む試料を分析する方法であって、担体に試料を接触させることにより、蛍光標識された生体関連分子と担体に固定化された生体関連分子とを相互作用させる相互作用工程、担体を洗浄することにより、担体に固定化された生体関連分子と相互作用しなかった生体関連分子を除去する洗浄工程、および担体を乾燥させることなく、担体に励起光を照射し、結像光学系の検出器で蛍光を検出する検出工程を含む前記方法。
（２) 前記検出器が結像光学系の検出器である、(１)の方法。
（３）洗浄工程において、潮解性物質を含む洗浄液を用いて担体を洗浄する、（１）又は(２)記載の方法。
（４）相互作用工程において、試料として、蛍光標識された生体関連分子とともに潮解性物質を含む試料を用いる、（３）記載の方法。
（５）潮解性物質が、塩化マグネシウム、水酸化マグネシウム、炭酸カリウム、臭化ナトリウム、及び塩化カルシウムからなる群から選択される、（３）又は（４）記載の方法。
（６）洗浄工程の後、励起光及び蛍光を透過するカバーで担体を覆うことにより担体の乾燥を防止する、（１）〜（５）のいずれかに記載の方法。
That is, the present invention includes the following inventions.
(1) A method for analyzing a sample containing a fluorescently labeled biologically relevant molecule using a carrier on which the biologically relevant molecule is immobilized, the fluorescently labeled biologically relevant molecule by contacting the sample with the carrier. An interaction step for allowing the biologically relevant molecule immobilized on the carrier to interact, a washing step for removing the biologically relevant molecule that did not interact with the biologically relevant molecule immobilized on the carrier by washing the carrier, And a method of detecting the fluorescence by irradiating the support with excitation light and detecting the fluorescence with a detector of the imaging optical system without drying the support.
(2) The method according to (1), wherein the detector is a detector of an imaging optical system.
(3) The method according to (1) or (2), wherein in the washing step, the carrier is washed with a washing liquid containing a deliquescent substance.
(4) The method according to (3), wherein in the interaction step, a sample containing a deliquescent substance together with a fluorescently labeled biologically relevant molecule is used as the sample.
(5) The method according to (3) or (4), wherein the deliquescent material is selected from the group consisting of magnesium chloride, magnesium hydroxide, potassium carbonate, sodium bromide, and calcium chloride.
(6) The method according to any one of (1) to (5), wherein the carrier is prevented from being dried by covering the carrier with a cover that transmits excitation light and fluorescence after the washing step.
本発明より、塩の乾燥ムラによる外乱の影響を受けることなく、担体上の生体関連分子の蛍光標識を検出することができる。 According to the present invention, a fluorescent label of a biologically relevant molecule on a carrier can be detected without being affected by disturbance due to uneven drying of the salt.
さらに、結像光学系の検出器で塩の乾燥ムラによる外乱の影響を受けることなく、担体上の生体関連分子の蛍光標識を検出することができる。従って、市販されている共焦点を用いた走査型の検出器を用いる方法よりもコストの点で有利であるとともにメンテナンス性も良い方法で提供される。そのため、本発明の方法は、研究や臨床用途に広く使用できる。 Furthermore, the fluorescent label of the bio-related molecule on the carrier can be detected without being affected by disturbance due to uneven drying of the salt by the detector of the imaging optical system. Therefore, the method is advantageous in terms of cost and has good maintainability as compared with a method using a commercially available confocal scanning type detector. Therefore, the method of the present invention can be widely used for research and clinical applications.
本発明は、生体関連分子が固定化された担体を用いて、蛍光標識された生体関連分子を含む試料を分析する方法に関し、換言すれば、生体関連分子が固定化された担体を用いて、生体関連分子間の相互作用を検出する方法である。 The present invention relates to a method for analyzing a sample containing a fluorescently labeled biologically relevant molecule using a carrier on which a biologically relevant molecule is immobilized, in other words, using a carrier on which a biologically relevant molecule is immobilized, This is a method for detecting an interaction between biologically relevant molecules.
本発明において、生体関連分子には、ＤＮＡおよびＲＮＡなどの核酸、ポリペプチド、糖鎖、これらの複合体、並びにこれらとその他の分子との複合体などが包含される。本発明において、ポリペプチドには、オリゴペプチドおよびタンパク質が包含されるものとする。担体に固定化する生体関連分子がポリペプチドである場合、通常１〜１０００ｋＤａ、好ましくは１〜２００ｋＤａのポリペプチドが好適に用いられる。また、担体に固定化する生体関連分子が核酸である場合、通常３〜５０００塩基、好ましくは１０〜１０００塩基の核酸が好適に用いられる。また、担体に固定化する生体関連分子が糖鎖である場合、通常１〜１００糖、好ましくは１〜３０糖の糖鎖が好適に用いられる。 In the present invention, biologically relevant molecules include nucleic acids such as DNA and RNA, polypeptides, sugar chains, complexes thereof, and complexes of these with other molecules. In the present invention, polypeptides include oligopeptides and proteins. When the biologically relevant molecule immobilized on the carrier is a polypeptide, a polypeptide of usually 1 to 1000 kDa, preferably 1 to 200 kDa is suitably used. Moreover, when the biologically relevant molecule | numerator fix | immobilized to a support | carrier is a nucleic acid, normally 3-5000 bases, Preferably a 10-1000 bases nucleic acid is used suitably. When the biologically relevant molecule immobilized on the carrier is a sugar chain, a sugar chain of 1 to 100 sugars, preferably 1 to 30 sugars, is preferably used.
生体関連分子間の相互作用には、例えば、タンパク質間の相互作用、タンパク質とポリペプチドの相互作用、核酸間の相互作用、タンパク質と核酸の相互作用、タンパク質と化合物との相互作用などが包含され、好ましくは生体関連分子間の特異的相互作用である。より具体的には、核酸相補鎖間のハイブリダイゼーション、抗原と抗体またはその断片との反応、酵素と基質または阻害剤の結合反応、リガンドとレセプターの結合反応、アビジンとビオチンの結合反応、核酸と転写因子の結合反応、細胞接着因子の結合反応、糖鎖とタンパク質の結合反応、脂肪鎖とタンパク質の結合反応、リン酸基とタンパク質の結合反応、補欠因子とタンパク質の結合反応などが挙げられる。本発明の方法は、担体に固定化されたＤＮＡと、試料中の蛍光標識されたＤＮＡとのハイブリダイゼーションの検出に特に好適に用いられる。 Interactions between biological molecules include, for example, interactions between proteins, interactions between proteins and polypeptides, interactions between nucleic acids, interactions between proteins and nucleic acids, and interactions between proteins and compounds. Preferably a specific interaction between biologically relevant molecules. More specifically, hybridization between nucleic acid complementary strands, reaction between antigen and antibody or fragment thereof, binding reaction between enzyme and substrate or inhibitor, binding reaction between ligand and receptor, binding reaction between avidin and biotin, nucleic acid and Examples include transcription factor binding reaction, cell adhesion factor binding reaction, sugar chain-protein binding reaction, fatty chain-protein binding reaction, phosphate group-protein binding reaction, prosthetic factor-protein binding reaction, and the like. The method of the present invention is particularly preferably used for detection of hybridization between DNA immobilized on a carrier and fluorescently labeled DNA in a sample.
試料中の生体関連分子に付加する蛍光標識は、特に制限されないが、例えば、Ｃｙ３およびＣｙ５などのＣｙＤｙｅ、ＦＩＴＣ、ＲＩＴＣ、ローダミン、テキサスレッド、ＴＥＴ、ＴＡＭＲＡ、ＦＡＭ、ＨＥＸ、ＲＯＸなどが挙げられる。 The fluorescent label added to the biologically relevant molecule in the sample is not particularly limited, and examples thereof include CyDye such as Cy3 and Cy5, FITC, RITC, rhodamine, Texas red, TET, TAMRA, FAM, HEX, ROX and the like.
本発明の方法では、担体に試料を接触させることにより、試料中の蛍光標識された生体関連分子と担体に固定化された生体関連分子とを相互作用させる。続いて、担体を洗浄すると、担体上の生体関連分子と相互作用しなかった生体関連分子は除去され、相互作用した生体関連分子のみが担体上に残ることになる。担体に残った生体関連分子の蛍光標識を検出器、好ましくは結像光学系の検出器で検出することにより、相互作用を検出することができる。 In the method of the present invention, by bringing a sample into contact with a carrier, the fluorescently labeled biologically relevant molecule in the sample is allowed to interact with the biologically relevant molecule immobilized on the carrier. Subsequently, when the carrier is washed, the biologically relevant molecules that have not interacted with the biologically relevant molecules on the carrier are removed, and only the biologically relevant molecules that have interacted remain on the carrier. The interaction can be detected by detecting the fluorescent label of the biologically relevant molecule remaining on the carrier with a detector, preferably a detector of an imaging optical system.
本発明の方法は、上記の担体を洗浄する工程の後で、担体を乾燥させることなく、検出器で蛍光を検出することを特徴とする。生体関連分子を相互作用させた後に用いる洗浄液は、通常、食塩などの塩を含むが、潮解性物質をさらに含む洗浄液を用いて担体を洗浄することにより、担体を乾燥させることなく、蛍光を検出することができる。 The method of the present invention is characterized in that after the step of washing the carrier, fluorescence is detected with a detector without drying the carrier. The washing solution used after the interaction with biologically relevant molecules usually contains a salt such as salt, but by washing the carrier with a washing solution further containing a deliquescent substance, fluorescence can be detected without drying the carrier. can do.
潮解性物質は、生体関連分子の相互作用を阻害しないものであれば、特に制限されないが、例えば、塩化マグネシウム、塩化カルシウム、水酸化マグネシウムなどのアルカリ土類金属塩、炭酸カリウム、臭化ナトリウムなどのアルカリ金属塩などが挙げられる。洗浄液における潮解性物質の濃度は、通常０．０１〜３．０ｍｏｌ／ｌ、好ましくは０．０５〜１．０ｍｏｌ／ｌ、さらに好ましくは０．２〜０．５ｍｏｌ／ｌである。 The deliquescent substance is not particularly limited as long as it does not inhibit the interaction of biologically relevant molecules. For example, alkaline earth metal salts such as magnesium chloride, calcium chloride, magnesium hydroxide, potassium carbonate, sodium bromide, etc. And alkali metal salts of the above. The concentration of the deliquescent substance in the cleaning liquid is usually 0.01 to 3.0 mol / l, preferably 0.05 to 1.0 mol / l, more preferably 0.2 to 0.5 mol / l.
洗浄液として、さらに界面活性剤を含むものを用いるのが好ましい。界面活性剤としては、非イオン界面活性剤、アニオン界面活性剤、カチオン界面活性剤のいずれも使用できるが、非イオン界面活性剤が好ましく用いられる。非イオン界面活性剤としては、例えば、Ｔｗｅｅｎ（登録商標）、エパン（登録商標）、ノイゲン（登録商標）などが挙げられる。洗浄液における界面活性剤の濃度は、通常、０．０１〜１０ｗｔ％、好ましくは０．３〜２．０ｗｔ％である。 It is preferable to use a cleaning liquid that further contains a surfactant. As the surfactant, any of a nonionic surfactant, an anionic surfactant, and a cationic surfactant can be used, and a nonionic surfactant is preferably used. Examples of the nonionic surfactant include Tween (registered trademark), Epan (registered trademark), and Neugen (registered trademark). The concentration of the surfactant in the cleaning liquid is usually 0.01 to 10 wt%, preferably 0.3 to 2.0 wt%.
潮解性物質を含む洗浄液を用いるのに加えて、蛍光標識された生体関連分子を含む試料として、さらに潮解性物質を含む試料を担体に接触させてもよい。試料に添加する潮解性物質としては、洗浄液に添加するものと同様のものを使用できる。試料における潮解性物質の濃度は、通常０．０１〜３．０ｍｏｌ／ｌ、好ましくは０．０５〜１．０ｍｏｌ／ｌ、さらに好ましくは０．２〜０．５ｍｏｌ／ｌである。洗浄液に添加する潮解性物質と試料に添加する潮解性物質は同じものを使用することが好ましい。 In addition to using a cleaning solution containing a deliquescent substance, a sample containing a deliquescent substance may be brought into contact with a carrier as a sample containing a fluorescently labeled biologically relevant molecule. As the deliquescent substance added to the sample, the same substances as those added to the cleaning liquid can be used. The concentration of the deliquescent substance in the sample is usually 0.01 to 3.0 mol / l, preferably 0.05 to 1.0 mol / l, more preferably 0.2 to 0.5 mol / l. It is preferable to use the same deliquescence substance added to the cleaning liquid and deliquescence substance added to the sample.
洗浄工程の後、担体を、励起光を透過するカバーで覆うことにより担体の乾燥を防止することもできる。カバーで覆ったまま励起光を照射して蛍光を検出することにより、担体の乾燥を防止し、乾燥ムラによる散乱光を低減することができる。カバーは、励起光を透過するものであれば特に制限されず、板状のもの、シート状のもの、及びフィルム状のものなどを使用できる。シート状のもの、及びフィルム状のものとしては、アクリル、ＣＯＣ、ＣＯＰ、ポリエチレン、ポリスチレン、ポリカーボネートが好ましい。また、ガラス製のもの、プラスチック製のものなどを使用できる。好ましくは、カバーガラスが用いられる。これらの例としては、石英ガラスや、「武藤化学株式会社製 カバーガラス」、「ＶＷＲ Ｉｎｔｅｒｎａｔｉｏｎａｌ社製 ＶＷＲ Ｍｉｃｒｏ Ｃｏｖｅｒ Ｇｌａｓｓｅｓ」を用いることができる。カバーの厚みは、特に制限されず、用いる材料によって適宜決定されるが、通常０．０２〜３ｍｍ、好ましくは０．０５〜０．２ｍｍである。 After the washing step, the carrier can be prevented from drying by covering the carrier with a cover that transmits excitation light. By detecting the fluorescence by irradiating excitation light while being covered with the cover, drying of the carrier can be prevented and scattered light due to drying unevenness can be reduced. The cover is not particularly limited as long as it transmits the excitation light, and a plate-shaped member, a sheet-shaped member, a film-shaped member, or the like can be used. As the sheet-like material and the film-like material, acrylic, COC, COP, polyethylene, polystyrene, and polycarbonate are preferable. Moreover, the thing made from glass, the thing made from a plastics, etc. can be used. Preferably, a cover glass is used. As these examples, quartz glass, “cover glass made by Muto Chemical Co., Ltd.”, and “VWR Micro Cover Glasses made by VWR International” can be used. The thickness of the cover is not particularly limited and is appropriately determined depending on the material to be used, but is usually 0.02 to 3 mm, preferably 0.05 to 0.2 mm.
担体を乾燥させることなく、検出器で蛍光を検出するための方法として、上記潮解性物質を用いる方法とカバーで覆う方法は、単独で行ってもよいし、組み合わせて行ってもよい。 As a method for detecting fluorescence with a detector without drying the carrier, the method using the deliquescent substance and the method of covering with a cover may be performed alone or in combination.
本発明で蛍光の検出に好ましく用いられる結像光学系の検出器は、励起光を担体に照射し、得られる蛍光の強度を検出するものである。本発明の方法においては、通常、レーザーで一度に担体全体に斜めに励起光を照射し、担体の正面から蛍光を検出する。励起光を担体に対して斜めに照射するとは、担体表面とレーザー光のなす角度が、９０°未満であることをさし、通常、３０〜７０°、好ましくは４０〜６０°の角度で照射する。結像光学系の検出器では、担体の全面にレーザーを走査させる必要がなく、検出を短時間で実施することができる。一度に担体全体に励起光を照射するため、生体関連分子を固定化する担体としては、比較的サイズの小さいもの、例えば、寸法が１０ｍｍ以下、好ましくは５ｍｍ以下、さらに好ましくは３ｍｍ以下の担体、最も好ましくは１〜５ｍｍ四方の担体を用いる。 The detector of the imaging optical system that is preferably used for detecting fluorescence in the present invention irradiates the support with excitation light and detects the intensity of the fluorescence obtained. In the method of the present invention, usually, the entire support is irradiated obliquely with excitation light at once with a laser, and fluorescence is detected from the front of the support. To irradiate the excitation light obliquely with respect to the carrier means that the angle formed between the carrier surface and the laser beam is less than 90 °, and is usually irradiated at an angle of 30 to 70 °, preferably 40 to 60 °. To do. In the detector of the imaging optical system, it is not necessary to scan the entire surface of the carrier with a laser, and detection can be performed in a short time. In order to irradiate the entire carrier at once with excitation light, the carrier for immobilizing the biological molecule is a relatively small one, for example, a carrier having a size of 10 mm or less, preferably 5 mm or less, more preferably 3 mm or less, Most preferably, a 1 to 5 mm square carrier is used.
本発明における結像光学系の検出器は、通常、励起光を照射するためのレーザー、目的の波長の蛍光のみを透過させる蛍光フィルター、蛍光フィルターを透過した蛍光を検出するための光検出部（例えば、ＣＣＤカメラ）を有する。走査型の検出器では、励起光を担体表面に垂直に照射することから、散乱光による外乱が少ない。一方、結像光学系の検出器を用いる場合は、励起光を垂直に照射すると正反射が強すぎて正確な検出が難しいため、担体に対して斜めに励起光を照射する。しかし、励起光を斜めに照射すると散乱光が生じ、目的の波長以外の波長を有する散乱光が蛍光フィルターを透過してしまい、正確な検出ができなくなってしまう。これに対し、本発明の方法では、乾燥ムラによる散乱光が抑制されることから、結像光学系の検出器を用いる場合でも、目的の波長以外の波長を有する散乱光が蛍光フィルターを透過することがなく、正確な検出が可能になる。 The detector of the imaging optical system in the present invention usually includes a laser for irradiating excitation light, a fluorescence filter that transmits only fluorescence of a target wavelength, and a light detection unit for detecting fluorescence that has passed through the fluorescence filter ( For example, it has a CCD camera. In the scanning detector, the excitation light is irradiated perpendicularly to the surface of the carrier, so that there is little disturbance due to scattered light. On the other hand, in the case of using an imaging optical system detector, the excitation light is obliquely applied to the carrier because the regular reflection is too strong and accurate detection is difficult if the excitation light is irradiated vertically. However, when the excitation light is irradiated obliquely, scattered light is generated, and scattered light having a wavelength other than the target wavelength is transmitted through the fluorescent filter, so that accurate detection cannot be performed. On the other hand, in the method of the present invention, since scattered light due to drying unevenness is suppressed, even when an imaging optical system detector is used, scattered light having a wavelength other than the target wavelength passes through the fluorescent filter. And accurate detection is possible.
本発明における結像光学系の検出器の一実施形態を図１に示す。本実施形態は、光検出部にＣＣＤカメラ（１１）を用いており、１ショットで１担体を検出できる。励起光を照射する時間が長くなる（１ｓ以上）場合は、冷却ＣＣＤカメラを用いることが好ましい。励起光を照射するためのレーザー（１２）としては、担体全体を照射できるように、φの大きなレーザーを用いることが好ましい。φの大きなレーザー光は、スリット（１６）を介して担体全体に照射されるように調整してもよい。また、レーザー光を担体（１４）に対して斜めに照射することで、正反射光をカットしつつ、蛍光標識からの微弱な光を捉えることができ、より高いＳ／Ｎ値を実現することができる。蛍光（１７）は蛍光フィルター（１８）を透過してＣＣＤ素子（１９）において検出される。目的の波長と異なる波長を有する光は、蛍光フィルターにおいてカットされる。正反射光は吸収ホーン（１５）で吸収される。 One embodiment of the detector of the imaging optical system in the present invention is shown in FIG. In this embodiment, a CCD camera (11) is used for the light detection unit, and one carrier can be detected in one shot. When the time for irradiating the excitation light becomes long (1 s or longer), it is preferable to use a cooled CCD camera. As the laser (12) for irradiating the excitation light, it is preferable to use a laser having a large φ so that the entire carrier can be irradiated. You may adjust so that a laser beam with a large φ may be irradiated to the entire carrier through the slit (16). In addition, by irradiating the laser beam obliquely with respect to the carrier (14), it is possible to capture weak light from the fluorescent label while cutting specular reflection light, and realize a higher S / N value. Can do. The fluorescence (17) passes through the fluorescence filter (18) and is detected by the CCD element (19). Light having a wavelength different from the target wavelength is cut in the fluorescent filter. The specularly reflected light is absorbed by the absorption horn (15).
生体関連分子を固定化する担体の材料としては、当技術分野で公知のものを使用でき、特に制限されない。例えば、白金、白金黒、金、パラジウム、ロジウム、銀、水銀、タングステンおよびそれらの化合物などの貴金属、およびグラファイト、カ−ボンファイバ−に代表される炭素などの導電体材料；単結晶シリコン、アモルファスシリコン、炭化ケイ素、酸化ケイ素、窒化ケイ素などに代表されるシリコン材料、ＳＯＩ（シリコン・オン・インシュレータ）などに代表されるこれらシリコン材料の複合素材；ガラス、石英ガラス、アルミナ、サファイア、セラミクス、フォルステライト、感光性ガラスなどの無機材料；ポリエチレン、エチレン、ポリプロビレン、環状ポリオレフィン、ポリイソブチレン、ポリエチレンテレフタレート、不飽和ポリエステル、含フッ素樹脂、ポリ塩化ビニル、ポリ塩化ビニリデン、ポリ酢酸ビニル、ポリビニルアルコール、ポリビニルアセタール、アクリル樹脂、ポリアクリロニトリル、ポリスチレン、アセタール樹脂、ポリカーボネート、ポリアミド、フェノール樹脂、ユリア樹脂、エポキシ樹脂、メラミン樹脂、スチレン・アクリロニトリル共重合体、アクリロニトリル・ブタジエンスチレン共重合体、ポリフェニレンオキサイドおよびポリスルホンなどの有機材料等が挙げられる。 As the material for the carrier for immobilizing the bio-related molecule, those known in the art can be used and are not particularly limited. For example, noble metals such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, tungsten and their compounds, and conductor materials such as graphite and carbon typified by carbon fiber; single crystal silicon, amorphous Silicon materials represented by silicon, silicon carbide, silicon oxide, silicon nitride, etc., composite materials of these silicon materials represented by SOI (silicon on insulator), etc .; glass, quartz glass, alumina, sapphire, ceramics, Inorganic materials such as stellite and photosensitive glass; polyethylene, ethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl Lucol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide and Examples thereof include organic materials such as polysulfone.
本発明においては、担体として、好ましくは表面にカーボン層と化学修飾基とを有する担体を用いる。表面にカーボン層と化学修飾基とを有する担体には、基板の表面にカーボン層と化学修飾基とを有するもの、およびカーボン層からなる基板の表面に化学修飾基を有するものが包含される。基板の材料としては、当技術分野で公知のものを使用でき、特に制限されず、上述の担体材料と同様のものを使用できる。 In the present invention, a carrier having a carbon layer and a chemical modification group on the surface is preferably used as the carrier. Carriers having a carbon layer and a chemical modification group on the surface include those having a carbon layer and a chemical modification group on the surface of the substrate, and those having a chemical modification group on the surface of the substrate made of the carbon layer. As the material for the substrate, those known in the art can be used, and there are no particular restrictions, and the same materials as those described above can be used.
本発明は、微細な平板状の構造を有する担体に対し好適に用いられる。微細な平板状の構造の担体を製造しやすいことから、シリコン材料や樹脂材料からなる基板を用いるのが好ましく、特に単結晶シリコンからなる基板の表面にカーボン層および化学修飾基を有する担体がより好ましい。単結晶シリコンには、部分部分でごくわずかに結晶軸の向きが変わっているものや（モザイク結晶と称される場合もある）、原子的尺度での乱れ（格子欠陥）が含まれているものも包含される。 The present invention is suitably used for a carrier having a fine flat plate structure. Since it is easy to produce a carrier having a fine flat plate structure, it is preferable to use a substrate made of a silicon material or a resin material. In particular, a carrier having a carbon layer and a chemical modification group on the surface of a substrate made of single crystal silicon is more preferable. preferable. Single crystal silicon has a slightly different orientation of the crystal axis in some parts (sometimes referred to as mosaic crystal), or includes disorder on the atomic scale (lattice defects) Are also included.
基板上に形成させるカーボン層としては、特に制限されないが、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、また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。カーボン層は、化学的安定性に優れておりその後の化学修飾基の導入や生体関連分子との結合における反応に耐えることができる点、生体関連分子と静電結合によって結合できるためその結合が柔軟性を持っている点において有利である。また、生体関連分子との結合反応において、非特異的吸着が少ない点においても有利である。前記のとおり基板自体がカーボン層からなる担体を用いてもよい。 The carbon layer formed on the substrate is not particularly limited, but synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, graphite, fullerene, carbon nanotube) ), A mixture thereof, or a laminate of them is preferably used. 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 incomplete 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. The carbon layer is excellent in chemical stability and can withstand subsequent reactions in the introduction of chemical modification groups and binding to biologically relevant molecules, and the binding is flexible because it can be bound to biologically relevant molecules by electrostatic binding. It is advantageous in that it has sex. It is also advantageous in that non-specific adsorption is small in the binding reaction with biologically relevant molecules. As described above, a carrier whose substrate itself is made of a carbon layer may be used.
本発明においてカーボン層の形成は公知の方法で行うことができる。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、イオン化蒸着法、アーク式蒸着法、レーザー蒸着法、ＥＢ（Ｅｌｅｃｔｒｏｎ ｂｅａｍ）蒸着法、抵抗加熱蒸着法などが挙げられる。 In the present invention, the carbon layer can be formed by a known method. For example, a microwave plasma CVD (Chemical vapor deposition) method, an ECRCVD (Electrical cyclotron resonance) method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, a direct current sputtering method. Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.
高周波プラズマＣＶＤ法では、高周波によって電極間に生じるグロー放電により原料ガス（メタン）を分解し、基板上にＤＬＣ（ダイヤモンドライクカーボン）層を合成する。イオン化蒸着法では、タングステンフィラメントで生成される熱電子を利用して、原料ガス（ベンゼン）を分解・イオン化し、バイアス電圧によって基板上にカーボン層を形成する。水素ガス１〜９９体積％と残りメタンガス９９〜１体積％からなる混合ガス中で、イオン化蒸着法によりＤＬＣ層を形成してもよい。 In the high-frequency plasma CVD method, a source gas (methane) is decomposed by glow discharge generated between electrodes by a high frequency to synthesize a DLC (diamond-like carbon) layer on a substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on the substrate by a bias voltage. The DLC layer may be formed by ionized vapor deposition in a mixed gas composed of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of remaining methane gas.
アーク式蒸着法では、固体のグラファイト材料（陰極蒸発源）と真空容器（陽極）の間に直流電圧を印加することにより真空中でアーク放電を起こして陰極から炭素原子のプラズマを発生させ蒸発源よりもさらに負のバイアス電圧を基板に印加することにより基板に向かってプラズマ中の炭素イオンを加速しカーボン層を形成することができる。 In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), and a plasma of carbon atoms is generated from the cathode to generate an evaporation source. Further, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.
レーザー蒸着法では、例えばＮｄ：ＹＡＧレーザー（パルス発振）光をグラファイトのターゲット板に照射して溶融させ、ガラス基板上に炭素原子を堆積させることによりカーボン層を形成することができる。 In the laser vapor deposition method, for example, a carbon target layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light and melting it, and depositing carbon atoms on a glass substrate.
基板の表面にカーボン層を形成する場合、カーボン層の厚さは、通常、単分子層〜１００μｍ程度であり、薄すぎると下地基板の表面が局部的に露出する可能性があり、逆に厚くなると生産性が悪くなるので、好ましくは２ｎｍ〜１μｍ、より好ましくは５ｎｍ〜５００ｎｍである。 When the carbon layer is formed on the surface of the substrate, the thickness of the carbon layer is usually a monomolecular layer to about 100 μm, and if it is too thin, the surface of the base substrate may be locally exposed, and conversely thick. Since productivity will worsen in this case, it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm.
カーボン層が形成された基板の表面に化学修飾基を導入することにより、生体関連分子を担体に強固に固定化できる。導入する化学修飾基は、当業者であれば適宜選択することができ、特に制限されないが、例えば、アミノ基、カルボキシル基、エポキシ基、ホルミル基、ヒドロキシル基、金属キレート、及び活性エステル基が挙げられる。 By introducing a chemical modification group on the surface of the substrate on which the carbon layer is formed, the biologically relevant molecule can be firmly immobilized on the carrier. The chemical modification group to be introduced can be appropriately selected by those skilled in the art and is not particularly limited. Examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, a metal chelate, and an active ester group. It is done.
アミノ基の導入は、例えば、カーボン層をアンモニアガス中で紫外線照射することによりまたはプラズマ処理することにより実施できる。または、カーボン層を塩素ガス中で紫外線を照射して塩素化し、さらにアンモニアガス中で紫外線照射することにより実施できる。または、メチレンジアミン、エチレンジアミンで等の多価アミン類ガス中を、塩素化したカーボン層と反応させることによって実施することもできる。 An amino group can be introduced, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas or by plasma treatment. Alternatively, the carbon layer can be chlorinated by irradiation with ultraviolet rays in chlorine gas, and further irradiated with ultraviolet rays in ammonia gas. Alternatively, it can also be carried out by reacting a polyvalent amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.
カルボキシル基の導入は、例えば、前記のようにアミノ化したカーボン層に適当な化合物を反応させることにより実施できる。カルボキシル基を導入するために用いられる化合物としては、例えば、式：Ｘ−Ｒ１−ＣＯＯＨ（式中、Ｘはハロゲン原子、Ｒ１は炭素数１０〜１２の２価の炭化水素基を表す。）で示されるハロカルボン酸、例えばクロロ酢酸、フルオロ酢酸、ブロモ酢酸、ヨード酢酸、２−クロロプロピオン酸、３−クロロプロピオン酸、３−クロロアクリル酸、４−クロロ安息香酸；式：ＨＯＯＣ−Ｒ２−ＣＯＯＨ（式中、Ｒ２は単結合または炭素数１〜１２の２価の炭化水素基を表す。）で示されるジカルボン酸、例えばシュウ酸、マロン酸、コハク酸、マレイン酸、フマル酸、フタル酸；ポリアクリル酸、ポリメタクリル酸、トリメリット酸、ブタンテトラカルボン酸などの多価カルボン酸；式：Ｒ３−ＣＯ−Ｒ４−ＣＯＯＨ（式中、Ｒ３は水素原子または炭素数１〜１２の２価の炭化水素基、Ｒ４は炭素数１〜１２の２価の炭化水素基を表す。）で示されるケト酸またはアルデヒド酸；式：Ｘ−ＯＣ−Ｒ５−ＣＯＯＨ（式中、Ｘはハロゲン原子、Ｒ５は単結合または炭素数１〜１２の２価の炭化水素基を表す。）で示されるジカルボン酸のモノハライド、例えばコハク酸モノクロリド、マロン酸モノクロリド；無水フタル酸、無水コハク酸、無水シュウ酸、無水マレイン酸、無水ブタンテトラカルボン酸などの酸無水物が挙げられる。 The introduction of the carboxyl group can be carried out, for example, by reacting an appropriate compound with the carbon layer aminated as described above. The compound used to introduce a carboxyl group, for example, the formula: X-R 1 -COOH (wherein, X is a halogen atom, R 1 represents a divalent hydrocarbon group having 10 to 12 carbon atoms. ), For example, chloroacetic acid, fluoroacetic acid, bromoacetic acid, iodoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid, 3-chloroacrylic acid, 4-chlorobenzoic acid; formula: HOOC-R 2 -COOH (wherein R 2 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, phthalic acid; polyacrylic acid, polymethacrylic acid, trimellitic acid, polycarboxylic acids such as butane tetracarboxylic acid; formula: R 3 -CO-R 4 -COOH ( wherein, R 3 Divalent hydrocarbon group of a hydrogen atom or 1 to 12 carbon atoms, R 4 is keto acid or aldehyde acids represented by representing) a divalent hydrocarbon group having 1 to 12 carbon atoms; wherein:. X-OC- A monohalide of a dicarboxylic acid represented by R 5 —COOH (wherein X represents a halogen atom, R 5 represents a single bond or a divalent hydrocarbon group having 1 to 12 carbon atoms), such as succinic acid monochloride, Malonic acid monochloride; acid anhydrides such as phthalic anhydride, succinic anhydride, oxalic anhydride, maleic anhydride, and butanetetracarboxylic anhydride.
エポキシ基の導入は、例えば、前記のようにアミノ化したカーボン層に適当な多価エポキシ化合物を反応させることによって実施できる。あるいは、カーボン層が含有する炭素＝炭素２重結合に有機過酸を反応させることにより得ることができる。有機過酸としては、過酢酸、過安息香酸、ジペルオキシフタル酸、過ギ酸、トリフルオロ過酢酸などが挙げられる。 Introduction of an epoxy group can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Or it can obtain by making an organic peracid react with the carbon = carbon double bond which a carbon layer contains. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.
ホルミル基の導入は、例えば、前記のようにアミノ化したカーボン層に、グルタルアルデヒドを反応させることにより実施できる。 Introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.
ヒドロキシル基の導入は、例えば、前記のように塩素化したカーボン層に、水を反応させることにより実施できる。 Introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.
活性エステル基は、エステル基のアルコール側に酸性度の高い電子求引性基を有して求核反応を活性化するエステル群、すなわち反応活性の高いエステル基を意味する。エステル基のアルコール側に、電子求引性の基を有し、アルキルエステルよりも活性化されたエステル基である。活性エステル基は、アミノ基、チオール基、水酸基等の基に対する反応性を有する。さらに具体的には、フェノールエステル類、チオフェノールエステル類、Ｎ−ヒドロキシアミンエステル類、シアノメチルエステル、複素環ヒドロキシ化合物のエステル類等がアルキルエステル等に比べてはるかに高い活性を有する活性エステル基として知られている。より具体的には、活性エステル基としては、たとえばｐ−ニトロフェニル基、Ｎ−ヒドロキシスクシンイミド基、コハク酸イミド基、フタル酸イミド基、５−ノルボルネン−２，３−ジカルボキシイミド基等が挙げられ、特に、Ｎ−ヒドロキシスクシンイミド基が好ましく用いられる。 The active ester group means an ester group having an electron-withdrawing group having high acidity on the alcohol side of the ester group and activating the nucleophilic reaction, that is, an ester group having high reaction activity. The ester group has an electron-withdrawing group on the alcohol side of the ester group and is activated more than the alkyl ester. The active ester group has reactivity with groups such as amino group, thiol group, and hydroxyl group. More specifically, an active ester group in which phenol esters, thiophenol esters, N-hydroxyamine esters, cyanomethyl esters, esters of heterocyclic hydroxy compounds, etc. have much higher activity than alkyl esters etc. Known as. More specifically, examples of the active ester group include a p-nitrophenyl group, an N-hydroxysuccinimide group, a succinimide group, a phthalimide group, and a 5-norbornene-2,3-dicarboximide group. In particular, an N-hydroxysuccinimide group is preferably used.
活性エステル基の導入は、例えば、前記のように導入したカルボキシル基を、シアナミドやカルボジイミド（例えば、１−［３−（ジメチルアミノ）プロピル］−３−エチルカルボジイミド）などの脱水縮合剤とＮ−ヒドロキシスクシンイミドなどの化合物で活性エステル化することにより実施できる。この処理により、アミド結合を介して炭化水素基の末端に、Ｎ−ヒドロキシスクシンイミド基等の活性エステル基が結合した基を形成することができる（特開２００１−１３９５３２）。 The introduction of the active ester group is performed by, for example, converting the carboxyl group introduced as described above into a dehydrating condensing agent such as cyanamide or carbodiimide (for example, 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide) and N- It can be carried out by active esterification with a compound such as hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide group is bonded to the end of the hydrocarbon group via an amide bond can be formed (Japanese Patent Laid-Open No. 2001-139532).
ＤＮＡおよびＲＮＡ等の核酸を固定化する場合は、アミノ基、エポキシ基、カルボジイミド基、ホルミル基または活性エステル基を導入するのが好ましい。ポリペプチドを固定化する場合は、アミノ基、カルボジイミド基、エポキシ基、ホルミル基、金属キレートまたは活性エステル基を導入するのが好ましい。金属キレートを導入した担体を使用すると、ポリヒスチジン配列等の金属イオンと親和性のある標識を有するポリペプチドを効果的かつ安定に固定化することができる。 When immobilizing nucleic acids such as DNA and RNA, it is preferable to introduce an amino group, an epoxy group, a carbodiimide group, a formyl group or an active ester group. When immobilizing a polypeptide, it is preferable to introduce an amino group, carbodiimide group, epoxy group, formyl group, metal chelate or active ester group. When a carrier into which a metal chelate is introduced is used, a polypeptide having a label having an affinity for metal ions such as a polyhistidine sequence can be immobilized effectively and stably.
本発明の担体に生体関連分子を固定化する方法は、特に制限されない。例えば、生体関連分子をバッファーに溶解して溶液を作成し、これに上記のような担体を浸漬することによって、担体表面に生体関連分子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。この場合、一定時間浸漬した後、担体を洗浄することによって、固定化されていない生体関連分子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、多種類の生体関連分子を担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、スポッターで生体関連分子溶液を担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分子を除去する。スポッター装置を用いることにより他種類の生体関連分子を担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る。 The method for immobilizing the bio-related molecule on the carrier of the present invention is not particularly limited. For example, the biologically relevant molecule can be immobilized on the surface of the carrier by dissolving the biologically relevant molecule in a buffer to prepare a solution and immersing the carrier as described above. The immersion is usually performed at 0 to 98 ° C., preferably 4 to 50 ° C., and usually 1 minute to 24 hours, preferably 10 minutes to 1 hour. In this case, the biologically relevant molecules that are not immobilized can be removed by immersing the carrier for a certain period of time and then washing the carrier. Also, by using a device called a spotter, many types of biologically relevant molecules can be immobilized on the surface of the carrier. In the case of using a spotter, for example, a biologically relevant molecule solution is spotted on a carrier with a spotter, followed by baking in a heated oven for a certain period of time, and then unfixed molecules are removed by washing. By using a spotter device, other types of bio-related molecules can be immobilized at different positions on the carrier, so a large number of tests can be performed at once.
以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲は以下の実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.
実施例１ 担体の調製
３ｍｍ角のシリコン基板にイオン化蒸着法を用いて、下記の条件で２層のＤＬＣ層の製膜を行った。
Example 1 Preparation of Support A two-layer DLC layer was formed on a 3 mm square silicon substrate using the ionized vapor deposition method under the following conditions.
得られた表面にＤＬＣ層を有するシリコン基板上に、下記の条件でアンモニアプラズマを用いて、アミノ基を導入した。 An amino group was introduced onto a silicon substrate having a DLC layer on the obtained surface using ammonia plasma under the following conditions.
１４０ｍＭ 無水コハク酸及び０．１Ｍ ホウ酸ナトリウムを含む１−メチル−２−ピロリドン溶液に３０分間浸漬し、カルボキシル基を導入した。０．１Ｍ リン酸カリウムバッファー、０．１Ｍ １−［３−（ジメチルアミノ）プロピル］−３−エチルカルボジイミド、２０ｍＭ Ｎ−ヒドロキシスクシイミドを含む溶液に３０分間浸漬し、活性化を行い、シリコン基板表面にＤＬＣ層及び化学修飾基としてのＮ−ヒドロキシスクシイミド基を有する担体を得た。 It was immersed in a 1-methyl-2-pyrrolidone solution containing 140 mM succinic anhydride and 0.1 M sodium borate for 30 minutes to introduce carboxyl groups. It is activated by immersing in a solution containing 0.1 M potassium phosphate buffer, 0.1 M 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide, 20 mM N-hydroxysuccinimide for 30 minutes. A carrier having a DLC layer and an N-hydroxysuccinimide group as a chemical modification group on the substrate surface was obtained.
実施例２ 生体関連分子の担体への固定化
以下の５種のＤＮＡプローブをＳｏｌ．６で１０μＭに溶解し、前記で得られた担体上に横一行に４スポットずつスポットした。
ＴＴＡＴＧＡＣＴＣＴＧＣＣＧＣＣＧＴＣＡ（配列番号１）
ＣＴＧＡＡＡＴＴＧＡＧＡＡＣＧＡＡＡＡＧ（配列番号２）
ＡＣＧＣＡＣＡＧＧＡＡＣＴＧＡＡＧＡＡＴ（配列番号３）
ＣＴＧＡＧＴＧＡＡＴＡＴＡＴＣＧＡＡＣＡ（配列番号４）
ＴＴＧＴＣＣＧＣＧＣＣＧＧＧＣＴＴＣＧＣＴＣ（配列番号５）
８０℃で１時間、ベーキングを行い、２×ＳＳＣ／０．２％ＳＤＳで洗浄した後、超純水で洗浄し、遠心乾燥を行うことにより、担体にＤＮＡプローブを固定化した。
Example 2 Immobilization of biologically relevant molecules on a carrier The following five types of DNA probes were prepared as Sol. 6 was dissolved in 10 μM, and 4 spots were spotted in a horizontal row on the carrier obtained above.
TTATGACTCTGCCGCCGTCA (SEQ ID NO: 1)
CTGAAATTGAGAACGAAAAG (SEQ ID NO: 2)
ACGCACAGGAACTGAAGAAT (SEQ ID NO: 3)
CTGAGGTGAATATATCGAACA (SEQ ID NO: 4)
TTGTCCGCGCCGGGGCTTCGCTC (SEQ ID NO: 5)
After baking at 80 ° C. for 1 hour, washing with 2 × SSC / 0.2% SDS, washing with ultrapure water, and centrifugal drying, the DNA probe was immobilized on the carrier.
実施例３ 生体関連分子の相互作用
ラムダファージのゲノムＤＮＡを鋳型とし、上記５種のプローブとハイブリダイズする領域を以下のプライマーセットを用いてＰＣＲで増幅した。
プライマー１：ＡＣＡＧＧＧＡＡＴＧＣＣＣＧＴＴＣＴＧＣ（配列番号６）
プライマー２：ＡＡＴＡＡＣＣＧＡＣＡＣＧＧＧＣＡＧＡＣ（配列番号７）
標識は、ＣｙＤｙｅを用いて行った。ＰＣＲ溶液の組成は以下のとおりとした。
Example 3 Interaction of Biological Related Molecules Genomic DNA of lambda phage was used as a template, and the region hybridized with the above five probes was amplified by PCR using the following primer set.
Primer 1: ACAGGGAATGCCCGTTCTGC (SEQ ID NO: 6)
Primer 2: AATAACCGACACGGGCAGAC (SEQ ID NO: 7)
Labeling was performed using CyDye. The composition of the PCR solution was as follows.
得られたＰＣＲ産物３０μｌを、ハイブリダイズ溶液（１×ＳＳＣ／０．１％ＳＤＳ溶液）３０μｌに溶かして試料を調製した。試料を、ＤＮＡプローブを固定化した担体に滴下し、５０℃で３０分間静置し、湿箱に入れて反応を行った。２×ＳＳＣ／０．２％ＳＤＳ、続いて２×ＳＳＣで洗浄した。 A sample was prepared by dissolving 30 μl of the obtained PCR product in 30 μl of a hybridization solution (1 × SSC / 0.1% SDS solution). The sample was dropped onto a carrier on which a DNA probe was immobilized, left at 50 ° C. for 30 minutes, and reacted in a wet box. Washed with 2 × SSC / 0.2% SDS followed by 2 × SSC.
実施例４ カバーガラスによる効果
実施例３で洗浄を行った後、乾かさない状態でカバーガラス（１８ｍｍ角、０．１５ｍｍ厚）を上から被せた。そして、図１に示される結像光学系の検出器を用いて、蛍光を検出した。
Example 4 Effect of Cover Glass After washing in Example 3, a cover glass (18 mm square, 0.15 mm thickness) was covered from above without being dried. And fluorescence was detected using the detector of the imaging optical system shown in FIG.
図１の検出器において、光検出部には冷却ＣＣＤカメラを用い、蛍光フィルター（Ｃｙ５用、エドモンド社製）を介して、担体上の相互作用した生体関連分子の蛍光標識を検出した。φ５ｍｍのレーザー（６４０ｎｍ波長）を用いて、担体表面に対して５０°の角度（図１の角度ａ）で励起光を照射した。正反射光をレーザーの吸収ホーンで吸収し、ノイズの低減を図った。 In the detector shown in FIG. 1, a cooled CCD camera was used as the light detection unit, and a fluorescent label of interacting biological molecules on the carrier was detected via a fluorescent filter (for Cy5, manufactured by Edmond). Using a φ5 mm laser (640 nm wavelength), excitation light was irradiated at an angle of 50 ° (angle a in FIG. 1) with respect to the carrier surface. The specularly reflected light was absorbed by a laser absorption horn to reduce noise.
比較として、ハイブリダイズ後洗浄し、エアーで吹きつけ乾燥したものと遠心乾燥機にて遠心乾燥（１５００ｒｐｍ、３分）したものについても同様に結像光学系の検出器を用いて蛍光を検出した。結果を図２に示す。これより、洗浄後、乾燥したものは塩の残留による散乱光が大きく影響し、正確な検出が難しいのに対し、カバーガラスで乾燥せず測定すると塩の残量の影響がなく、良好な測定ができることがわかった。 For comparison, fluorescence was similarly detected using a detector of the imaging optical system after washing after hybridization and drying by blowing with air and centrifugal drying (1500 rpm, 3 minutes) using a centrifugal dryer. . The results are shown in FIG. As a result, the dried product after washing is greatly affected by scattered light due to the residual salt, and accurate detection is difficult. I found out that
実施例５ 潮解性物質による効果
実施例３において、得られたＰＣＲ産物３０μｌを、ハイブリダイズ溶液（２×ＳＳＣ／０．２％ＳＤＳ）３０μｌに溶かして試料を調製した。試料を、ＤＮＡプローブを固定化した担体に滴下し、５０℃で３０分間静置し、湿箱に入れて反応を行った。続いて、以下の条件で洗浄を行った。
Example 5 Effect of deliquescent substance In Example 3, 30 μl of the obtained PCR product was dissolved in 30 μl of a hybridization solution (2 × SSC / 0.2% SDS) to prepare a sample. The sample was dropped onto a carrier on which a DNA probe was immobilized, left at 50 ° C. for 30 minutes, and reacted in a wet box. Subsequently, cleaning was performed under the following conditions.
洗浄液１（１Ｎ ＮａＡｃ／０．５％ Ｔｗｅｅｎ２０）２８０μｌ×１分×４回、揺動
洗浄液２（１Ｎ ＭｇＣｌ２／０．５％ Ｔｗｅｅｎ２０）２８０μｌ×１分×１回、揺動
Washing solution 1 (1N NaAc / 0.5% Tween20) 280 μl × 1 minute × 4 times, shaking Washing solution 2 (1N MgCl 2 /0.5% Tween20) 280 μl × 1 minute × 1 time, shaking
洗浄後、乾燥せずに３分後に、実施例４と同様に結像光学系の検出器を用いて蛍光を検出した。結果を図３に示す。また、比較として、洗浄液２として、塩化マグネシウムを含まないものを用い、洗浄後乾燥したものについても同様に蛍光を検出した。 After washing and without drying, fluorescence was detected using the imaging optical system detector in the same manner as in Example 4. The results are shown in FIG. For comparison, the cleaning liquid 2 containing no magnesium chloride was used, and fluorescence was also detected in the case of the cleaning liquid dried after cleaning.
これより、洗浄後、乾燥したものは塩の残留による散乱光が大きく影響し、正確な検出が難しいのに対し、潮解性物質である塩化マグネシウムを含む洗浄液で洗浄することにより乾燥させず測定すると塩の残量の影響がなく、良好な測定ができることがわかった。 As a result, the dried product after washing is greatly affected by scattered light due to residual salt, and it is difficult to detect accurately, but it is measured without washing by washing with a cleaning solution containing magnesium chloride, a deliquescent substance. It was found that there was no influence of the remaining amount of salt, and good measurement could be performed.
１１ ＣＣＤカメラ
１２ レーザー
１３ レンズ
１４ 担体
１５ 吸収ホーン
１６ スリット
１７ 蛍光
１８ 蛍光フィルター
１９ ＣＣＤ素子
11
Claims (5)
担体に試料を接触させることにより、蛍光標識された生体関連分子と担体に固定化された生体関連分子とを相互作用させる相互作用工程、
担体を、０．０５〜１．０ｍｏｌ／ｌの濃度で潮解性物質を含む洗浄液を用いて洗浄することにより、担体に固定化された生体関連分子と相互作用しなかった生体関連分子を除去する洗浄工程、および
担体を乾燥させることなく、担体に励起光を照射し、結像光学系の検出器で蛍光を検出する検出工程
を含む前記方法。 A method for analyzing a sample containing a fluorescently labeled biologically relevant molecule using a carrier on which the biologically relevant molecule is immobilized, comprising:
An interaction step in which a fluorescently labeled biologically relevant molecule interacts with a biologically relevant molecule immobilized on the carrier by contacting the sample with the carrier;
By washing the carrier with a cleaning solution containing a deliquescent substance at a concentration of 0.05 to 1.0 mol / l, the biologically relevant molecules that did not interact with the biologically relevant molecules immobilized on the carrier are removed. The method comprising: a washing step; and a detection step of irradiating the support with excitation light without drying the support and detecting fluorescence with a detector of an imaging optical system.
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