JP2013113735A - Biosensing method using fine particle containing functional material - Google Patents
Biosensing method using fine particle containing functional material Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
Description
本発明は、バイオセンシング方法に関し、より詳細には、ポリマー微粒子を用いたバイオセンシング方法に関する。 The present invention relates to a biosensing method, and more particularly to a biosensing method using polymer fine particles.
現在、広く利用されている生体分子検出法の１つに、ELISA(Enzyme-Linked ImmunoSorbent Assay)法がある。ELISA法においては、目的タンパク質（抗原）に対して酵素で標識された抗体を作用させた後、当該酵素と発光試薬の反応による発光を光学的に測定することによって目的タンパク質を検出する。しかしながら、ELISA法に代表される従来のバイオアッセイは、シグナル検出までに多くの工程（時間）を要していた。 One of the biomolecule detection methods that are currently widely used is the ELISA (Enzyme-Linked ImmunoSorbent Assay) method. In the ELISA method, an antibody labeled with an enzyme is allowed to act on a target protein (antigen), and then the target protein is detected by optically measuring light emission due to the reaction between the enzyme and a luminescent reagent. However, the conventional bioassay represented by the ELISA method requires many steps (time) until signal detection.
この点につき、特開２００８−１２７４５４号公報（特許文献１）は、磁性粒子を被覆するポリマー層に蛍光物質を封入することによって、蛍光標識としての機能と磁気応答性とを併せ持つ多機能型ポリマー磁性粒子を開示する。さらに、国際公開第２００９／０７２４５７号（特許文献２）は、そのような蛍光物質含有ポリマー磁性粒子の表面に抗体を固定化し、これを反応場に向けて強制的に磁気誘導することによって抗体抗原反応を促進するとともに、未反応抗体を迅速に磁気回収することによって、シグナルを検出するまでに要する時間を大幅に短縮する方法を開示する。 In this regard, Japanese Patent Application Laid-Open No. 2008-127454 (Patent Document 1) discloses a multifunctional polymer having both a function as a fluorescent label and a magnetic response by encapsulating a fluorescent substance in a polymer layer covering magnetic particles. Magnetic particles are disclosed. Further, International Publication No. 2009/072457 (Patent Document 2) discloses an antibody antigen by immobilizing an antibody on the surface of such fluorescent substance-containing polymer magnetic particles and forcibly magnetically inducing it toward the reaction field. Disclosed is a method for greatly reducing the time required to detect a signal by accelerating the reaction and rapidly magnetically recovering the unreacted antibody.
ただし、蛍光物質含有ポリマー磁性粒子を使用する従来のスキームにおいては、ポリマー層にある蛍光を測定することを前提とするため、一定以上の蛍光強度を有する蛍光物質を封入する必要があり、加えて、ポリマー内部に共存する磁性粒子（フェライト）の影響についても考慮しなければならなかった。 However, in the conventional scheme using the fluorescent substance-containing polymer magnetic particles, since it is assumed that the fluorescence in the polymer layer is measured, it is necessary to enclose a fluorescent substance having a certain level of fluorescence intensity. The influence of magnetic particles (ferrite) coexisting in the polymer had to be taken into consideration.
これらの事情から、これまでは、ポリマー層にある蛍光物質として、フェライトによって吸収されない波長で励起され、高い強度で蛍光発光する希土類金属錯体（例えば、ユーロピウム錯体）を利用する以外に選択肢がなかったが、希土類金属錯体の励起光が紫外領域の際には、専用の紫外線照射装置が必須となり、装置コストが過大となる。 Under these circumstances, until now, there has been no choice but to use a rare earth metal complex (for example, a europium complex) that is excited at a wavelength that is not absorbed by ferrite and emits fluorescence with high intensity as a fluorescent material in the polymer layer. However, when the excitation light of the rare earth metal complex is in the ultraviolet region, a dedicated ultraviolet irradiation device is indispensable, and the device cost is excessive.
本発明者らは、上記従来技術における課題について鋭意検討する中で、ポリマー層にある蛍光物質の蛍光を測定するという前提について今一度見直した結果、蛍光物質をポリマー磁性粒子から溶出させて直接的に定量する着想に至った。 As a result of reviewing the premise that the fluorescence of the fluorescent substance in the polymer layer is measured once again, the present inventors have made a direct examination by eluting the fluorescent substance from the polymer magnetic particles. It came to the idea to quantify.
さらに、本発明者らは、上記着想をさらに発展させ、任意の測定系に対応した機能性物質が封入されたポリマー磁性粒子を用いる新規なバイオセンシング方法の構成に想到し、本発明に至ったのである。 Furthermore, the present inventors have further developed the above idea and have come up with a novel biosensing method configuration using polymer magnetic particles in which a functional substance corresponding to an arbitrary measurement system is encapsulated. It is.
すなわち、本発明によれば、アフィニティ反応を利用したバイオセンシング方法であって、ポリマー微粒子の内部に機能性物質を封入する工程と、前記ポリマー微粒子の表面にリガンドを固定化する工程と、前記リガンドを介して前記ポリマー微粒子とターゲットを結合させる工程と、前記ターゲットに結合した前記ポリマー微粒子から前記機能性物質を溶出させる工程と、溶出した前記機能性物質の物性を定量的に測定する工程とを含み、前記物性の測定結果に基づいて前記ターゲットを定量化することを特徴とするバイオセンシング方法が提供される。 That is, according to the present invention, there is provided a biosensing method using an affinity reaction, the step of encapsulating a functional substance inside polymer fine particles, the step of immobilizing a ligand on the surface of the polymer fine particles, and the ligand The step of binding the polymer fine particles and the target via the step, the step of eluting the functional substance from the polymer fine particles bound to the target, and the step of quantitatively measuring the physical properties of the eluted functional substance And a biosensing method characterized in that the target is quantified based on the measurement result of the physical properties.
以下、本発明を図面に示した実施の形態をもって説明するが、本発明は、図面に示した実施の形態に限定されるものではない。なお、以下に参照する各図においては、共通する要素について同じ符号を用い、適宜、その説明を省略するものとする。 Hereinafter, the present invention will be described with reference to embodiments shown in the drawings, but the present invention is not limited to the embodiments shown in the drawings. In the drawings referred to below, the same reference numerals are used for common elements, and the description thereof is omitted as appropriate.
図１は、本発明のバイオセンシング方法の手順を示すフローチャートであり、図２および図３は、各手順を概念的に表した図である。以下、図１〜図３に基づいて、本発明のバイオセンシング方法について説明する。 FIG. 1 is a flowchart showing the procedure of the biosensing method of the present invention, and FIGS. 2 and 3 are diagrams conceptually showing each procedure. Hereinafter, the biosensing method of the present invention will be described with reference to FIGS.
（工程１）
工程１においては、まず、ナノサイズの粒径を持つポリマー微粒子を準備する。本発明では、溶液中の分散性に優れ、且つ、タンパク質に非特異的吸着しないポリマー微粒子を使用することが好ましい。さらに、本発明においては、内部にフェライトなどの磁性粒子を包含するポリマー微粒子を使用することが好ましい。以下、説明の便宜上、磁性粒子を包含するポリマー微粒子（以下、磁性ビーズとして参照する）を使用する実施形態をもって本発明を説明する。
(Process 1)
In step 1, first, polymer fine particles having a nano-sized particle diameter are prepared. In the present invention, it is preferable to use fine polymer particles that are excellent in dispersibility in a solution and that do not adsorb nonspecifically to proteins. Furthermore, in the present invention, it is preferable to use polymer fine particles including magnetic particles such as ferrite inside. Hereinafter, for convenience of explanation, the present invention will be described with an embodiment using polymer fine particles including magnetic particles (hereinafter referred to as magnetic beads).
本発明の方法に使用する磁性ビーズは、例えば、本出願人が先に出願した特開２００６−８８１３１号公報に開示される下記の方法によって作製することができる。すなわち、フェライトに界面活性物質を吸着させて疎水化させた後、これにスチレンやグリシジルメタクリレート（GMA）などのラジカル付加重合が可能なモノマー液と非イオン性の親水基を有する界面活性剤とを加え、さらに適量の水を添加して混合し、ソニケーション処理等により乳化させる。このようにして調整された乳化液を６０〜８０℃に加熱したのち、親水性の開始剤を添加して乳化重合によりモノマーを重合させる。重合終了後、乳化粒子から界面活性剤を洗浄除去することにより、磁性ビーズの水懸濁液を得ることができる。 The magnetic beads used in the method of the present invention can be produced, for example, by the following method disclosed in Japanese Patent Application Laid-Open No. 2006-88131 filed earlier by the present applicant. That is, after a surfactant is adsorbed on ferrite to be hydrophobized, a monomer liquid capable of radical addition polymerization such as styrene and glycidyl methacrylate (GMA) and a surfactant having a nonionic hydrophilic group are added thereto. In addition, an appropriate amount of water is added and mixed, and emulsified by sonication or the like. After the thus prepared emulsion is heated to 60 to 80 ° C., a hydrophilic initiator is added and the monomer is polymerized by emulsion polymerization. After completion of the polymerization, an aqueous suspension of magnetic beads can be obtained by washing away the surfactant from the emulsified particles.
なお、市販品で言えば、多摩川精機株式会社製のＦＧビーズ（フェライト粒子をポリＧＭＡで被覆した粒径約２００ｎｍのポリマー磁性微粒子）を用いることができる。 In addition, as a commercial product, FG beads (polymer magnetic fine particles having a particle diameter of about 200 nm in which ferrite particles are coated with poly GMA) manufactured by Tamagawa Seiki Co., Ltd. can be used.
工程１においては、準備したポリマー磁性微粒子（以下、磁性ビーズとして参照する）のポリマー層に対して、以下の手順で機能性物質を封入する。本発明における機能性物質とは、定量的に測定可能な物性を持つ物質全般を意味する。 In step 1, a functional substance is encapsulated in the following procedure with respect to the polymer layer of the prepared polymer magnetic fine particles (hereinafter referred to as magnetic beads). The functional substance in the present invention means all substances having physical properties that can be quantitatively measured.
磁性ビーズの水懸濁液を遠心分離して水を除去し、残ったペレットにメタノール等のアルコール性有機溶媒を加え分散させた後、遠心分離するという操作を数回繰り返す。こうして有機溶媒中で磁性ビーズのポリマー層を膨潤させる。続いて、膨潤した磁性ビーズを遠心分離して得られたペレットに対し、機能性物質の有機溶媒溶液を添加して分散させることで、磁性ビーズのポリマー層の内部に機能性物質が取り込まれる。なお、上記有機溶媒としてはアセトンを用いることが好ましい。 The operation of centrifuging the aqueous suspension of magnetic beads to remove water, dispersing and adding an alcoholic organic solvent such as methanol to the remaining pellets, and then centrifuging are repeated several times. Thus, the polymer layer of the magnetic beads is swollen in the organic solvent. Subsequently, the functional substance is taken into the polymer layer of the magnetic beads by adding and dispersing an organic solvent solution of the functional substance to the pellet obtained by centrifuging the swollen magnetic beads. Note that acetone is preferably used as the organic solvent.
ポリマー層への機能性物質の移行が飽和状態になった後、有機溶媒中に分散された磁性ビーズ懸濁液に対して、等量程度の純水を添加する。その後、この混合溶液から有機溶媒のみを蒸発除去すると、その内部から有機溶媒が除去され、機能性物質のみが封入された状態で磁性ビーズが収縮する。最終的に、機能性物質含有磁性ビーズが水中に分散した状態で得られる。 After the transfer of the functional substance to the polymer layer becomes saturated, about the same amount of pure water is added to the magnetic bead suspension dispersed in the organic solvent. Thereafter, when only the organic solvent is evaporated and removed from the mixed solution, the organic solvent is removed from the inside thereof, and the magnetic beads contract in a state where only the functional substance is sealed. Finally, the functional substance-containing magnetic beads are obtained in a state dispersed in water.
（工程２）
工程２においては、機能性物質が封入されたリンカー導入済磁性ビーズの表面に対してリガンドを固定化する。本発明におけるリガンドとは、バイオセンシングに利用するアフィニティ反応において、標的分子（ターゲット）に特異的に相互作用することができる分子全般を意味する。なお、先に例示した多摩川精機株式会社製のＦＧビーズは、各種リンカーによって表面修飾された状態で販売されているので、このような磁性ビーズを用いれば、リンカー導入処理を省略することができる。
(Process 2)
In step 2, the ligand is immobilized on the surface of the linker-introduced magnetic beads in which the functional substance is encapsulated. The ligand in the present invention means all molecules that can specifically interact with a target molecule (target) in an affinity reaction used for biosensing. In addition, since the FG beads manufactured by Tamagawa Seiki Co., Ltd. exemplified above are sold with their surface modified by various linkers, the linker introduction process can be omitted by using such magnetic beads.
（工程３）
工程３においては、標的分子（ターゲット）を含むバッファに対して、工程２でリガンドを固定化した機能性物質包含磁性ビーズを添加し、アフィニティ反応を起こさせる。図３は、本発明の方法を、抗原抗体反応を利用したサンドイッチイムノアッセイに適用した例を示す。
(Process 3)
In step 3, the functional substance-containing magnetic beads having the ligand immobilized in step 2 are added to the buffer containing the target molecule (target) to cause an affinity reaction. FIG. 3 shows an example in which the method of the present invention is applied to a sandwich immunoassay utilizing an antigen-antibody reaction.
機能性物質包含ビーズを用いたサンドイッチイムノアッセイの場合、抗原１０（ターゲット）を含むバッファ内に抗原１０に対する抗体１２を固定した基板１４を静置した後、当該バッファに対して、工程２でリガンドを固定化した機能性物質包含ビーズ１６を添加する。この場合、機能性物質包含ビーズ１６に固定化されるリガンドは抗原１０に対する抗体１７（抗体１２とは別のエピトープを認識するもの）である。 In the case of a sandwich immunoassay using functional substance-containing beads, the substrate 14 on which the antibody 12 against the antigen 10 is immobilized is placed in a buffer containing the antigen 10 (target), and then the ligand is applied to the buffer in step 2. The immobilized functional substance-containing beads 16 are added. In this case, the ligand immobilized on the functional substance-containing bead 16 is the antibody 17 against the antigen 10 (recognizes an epitope different from the antibody 12).
特に、機能性物質包含磁性ビーズを用いたサンドイッチイムノアッセイの場合、基板１４の裏側に磁石２０を配設し、バッファ内に磁界を発生させることで、機能性物質包含磁性ビーズ１６は、抗体抗原反応の反応場となる基板１４の表面近傍まで強制的に磁気誘導され、抗体１２−抗原１０−抗体１７のサンドイッチ反応が迅速化される。その後、基板１４をバッファで洗浄し、サンドイッチ反応に寄与しなかった機能性物質包含磁性ビーズ１６を磁気回収すると、抗体１７を介して抗原１０（ターゲット）と特異的に結合した機能性物質包含磁性ビーズ１６だけが基板１４上に残る。 In particular, in the case of a sandwich immunoassay using functional substance-containing magnetic beads, the functional substance-containing magnetic beads 16 can be used for antibody antigen reaction by disposing a magnet 20 on the back side of the substrate 14 and generating a magnetic field in the buffer. The magnetic induction is forced to the vicinity of the surface of the substrate 14 as a reaction field of the above, and the sandwich reaction of antibody 12-antigen 10-antibody 17 is accelerated. Thereafter, the substrate 14 is washed with a buffer, and when the functional substance-containing magnetic beads 16 that have not contributed to the sandwich reaction are magnetically recovered, the functional substance-containing magnetism specifically bound to the antigen 10 (target) via the antibody 17 is obtained. Only the beads 16 remain on the substrate 14.
以上、工程３について、サンドイッチイムノアッセイを例にとって説明してきたが、本発明の適用範囲は抗原抗体反応に限定されるものではなく、アフィニティ反応を利用するバイオセンシング全般に適用可能である。本発明におけるアフィニティ反応とは、上述した抗原抗体反応の他にも、核酸（DNAやRNAなど）の相補的結合、核酸とたんぱく質の特異的結合、シグナル伝達系に関わるタンパク質とその受容体タンパク質との結合やProtein A若しくはProtein Gと抗体のFc部位との結合のようなたんぱく質同士の特異的結合、酵素とその基質の特異的結合、ホルモン分子とその受容体の特異的結合、糖鎖とレクチンの特異的結合など、生体分子間（人工的に改変した生体分子との反応も含む）の特異的な反応を含み、さらに、アプタマー等を用いた人工抗体様分子と生体分子の特異的結合、薬剤若しくは薬剤候補物質と生体分子の特異的結合、アビジンとビオチンの特異的結合など、低分子化合物と生体分子（人工的に改変した生体分子との反応も含む）の間の特異的な反応を含む概念である。 As described above, the step 3 has been described by taking a sandwich immunoassay as an example. However, the scope of the present invention is not limited to an antigen-antibody reaction, and can be applied to all biosensing utilizing an affinity reaction. In addition to the antigen-antibody reaction described above, the affinity reaction in the present invention includes complementary binding of nucleic acids (DNA, RNA, etc.), specific binding of nucleic acids and proteins, a protein involved in a signal transduction system and its receptor protein Specific binding of proteins such as binding of protein A or protein G to the Fc site of an antibody, specific binding of an enzyme and its substrate, specific binding of a hormone molecule and its receptor, sugar chain and lectin Specific binding between biomolecules (including reactions with artificially modified biomolecules) such as specific binding, and also specific binding of artificial antibody-like molecules and biomolecules using aptamers, Specificity between low molecular weight compounds and biomolecules (including reactions with artificially modified biomolecules) such as specific binding of drugs or drug candidates to biomolecules, and specific binding of avidin and biotin Is a concept that includes the Do reaction.
（工程４）
工程４においては、抗原１０（ターゲット）に特異的に結合した機能性物質包含磁性ビーズ１６に含まれる機能性物質１８を溶出させる。図３に示す例の場合には、機能性物質包含磁性ビーズ１６が結合した基板１４を有機溶媒に浸漬し、振盪する。その結果、機能性物質包含磁性ビーズ１６のポリマー層が膨潤し、内部に封入されていた機能性物質１８が有機溶媒中に溶出される。
(Process 4)
In step 4, the functional substance 18 contained in the functional substance-containing magnetic beads 16 specifically bound to the antigen 10 (target) is eluted. In the case of the example shown in FIG. 3, the substrate 14 to which the functional substance-containing magnetic beads 16 are bonded is immersed in an organic solvent and shaken. As a result, the polymer layer of the functional substance-containing magnetic beads 16 swells, and the functional substance 18 enclosed inside is eluted in the organic solvent.
（工程５）
工程５においては、機能性物質包含磁性ビーズ１６から溶出した機能性物質１８を分離・回収する。
(Process 5)
In step 5, the functional substance 18 eluted from the functional substance-containing magnetic beads 16 is separated and recovered.
（工程６）
工程６においては、回収した機能性物質１８の物性を適切な測定装置によって定量的に測定する。
(Step 6)
In step 6, the physical properties of the recovered functional substance 18 are quantitatively measured by an appropriate measuring device.
（工程７）
最後に、工程６における測定結果を「ターゲット量−物性値」の検量線に照らし合わせて抗原１０（ターゲット）を定量する。上記検量線は、既知量のターゲットについて上述した工程３〜工程６の手順を実施した結果に基づいて事前に作成しておく。
(Step 7)
Finally, the antigen 10 (target) is quantified by comparing the measurement result in step 6 with a calibration curve of “target amount—physical property value”. The calibration curve is prepared in advance based on the results of performing the steps 3 to 6 described above for a known amount of target.
以上、本発明のバイオセンシング方法の手順について説明してきたが、ここで、磁性ビーズに封入する機能性物質について説明する。本発明における機能性物質は、磁性ビーズに封入することができる適切な物性を有し、且つ、定量的に測定可能な物性を持つ物質であればどのようなものであってもよく、本発明の方法は、機能性物質の種類に限定されるものではない。本発明における代表的な機能性物質として、蛍光発光の物性を持つ物質（蛍光物質）を挙げることができる。 The procedure of the biosensing method of the present invention has been described above. Here, the functional substance enclosed in the magnetic beads will be described. The functional substance in the present invention may be any substance as long as it has appropriate physical properties that can be enclosed in magnetic beads and has physical properties that can be quantitatively measured. This method is not limited to the type of functional substance. As a typical functional substance in the present invention, a substance having a fluorescent property (fluorescent substance) can be exemplified.
蛍光物質包含磁性ビーズを利用する従来のバイオセンシング法においては、ポリマー層を透過する蛍光を測定することが前提となっていたため、磁性ビーズに封入する蛍光物質の選択肢は、一定以上の高い蛍光強度を有するものに限定され、加えて、ポリマー内部に共存する磁性粒子（フェライト）によって吸収されない波長の蛍光を発光するものに限定されざるを得なかった。 In the conventional biosensing method using magnetic substance-containing magnetic beads, it is assumed that the fluorescence transmitted through the polymer layer is measured, so the choice of the fluorescent substance to be enclosed in the magnetic beads is higher than a certain level. In addition, it must be limited to those that emit fluorescence having a wavelength that is not absorbed by the magnetic particles (ferrite) that coexist in the polymer.
この点に関し、本発明の方法によれば、蛍光物質の選択肢の範囲は格段に広がる。すなわち、本発明では、磁性ビーズから取り出した蛍光物質の発光を直接測定するので、磁性粒子（フェライト）の影響を考慮する必要がなく、定量的な測定に必要十分な蛍光強度を有する物質であれば、如何なる蛍光物質でも使用することができる。具体的には、現在、一般的な蛍光測定装置で用いられているBODIPYなどを使用することが可能になる。 In this regard, according to the method of the present invention, the range of options for the fluorescent material is greatly expanded. That is, in the present invention, since the light emission of the fluorescent material taken out from the magnetic beads is directly measured, there is no need to consider the influence of the magnetic particles (ferrite), and any material having sufficient fluorescence intensity necessary for quantitative measurement. Any fluorescent material can be used. Specifically, it is possible to use BODIPY or the like currently used in general fluorescence measurement devices.
なお、ターゲットに結合したリガンドの標識マーカーをバッファ内で検出するこれまでのスキームにおいては、標識マーカーの選択肢はある程度限られたものにならざるを得なかった。そのため、多くの場合、反応系に与える影響が少ない蛍光物質などを標識マーカーとして使用していたが、蛍光の消光などの影響を考慮して測定系を設計する必要があるため、その構成が複雑なものにならざるを得なかった。 In the previous schemes in which the marker marker of the ligand bound to the target is detected in the buffer, the choice of the marker marker has to be limited to some extent. Therefore, in many cases, fluorescent substances that have little influence on the reaction system were used as labeling markers. However, the measurement system must be designed in consideration of the effects of fluorescence quenching, so the configuration is complicated. I had to become something.
この点につき、本発明の方法によれば、任意の機能性物質を標識マーカーとして利用することが可能になり、これまでバイオセンシングにおいて利用されることのなかった導電性物質（物性として導電性を示す物質）などをマーカーとして利用することが可能となる。すなわち、本発明においては、磁性ビーズに対して導電性物質を封入してターゲットとアフィニティ反応を起こさせた後、磁性ビーズから溶出させた導電性物質の導電性（電流値）を測定することによって、ターゲットを定量化することができる。 In this regard, according to the method of the present invention, any functional substance can be used as a marker marker, and a conductive substance that has not been used in biosensing until now (conductivity as a physical property). It is possible to use a substance to be used as a marker. That is, in the present invention, after conducting an affinity reaction with a target by encapsulating a conductive substance in the magnetic beads, the conductivity (current value) of the conductive substance eluted from the magnetic beads is measured. The target can be quantified.
導電性物質は電気化学測定によって簡便に検出・定量化することができるため、蛍光物質などを利用する光学測定に比べれば、少ない手順と短い時間をもってアッセイ結果を得ることができる。また、導電性物質の導電性は蛍光物質の蛍光のように短時間に減衰する心配がないことから、高い精度をもって検出・測定することができる。 Since a conductive substance can be easily detected and quantified by electrochemical measurement, an assay result can be obtained with fewer procedures and a shorter time compared to optical measurement using a fluorescent substance or the like. In addition, the conductivity of the conductive material can be detected and measured with high accuracy since there is no fear of decay in a short time unlike the fluorescence of the fluorescent material.
さらに、電気化学測定に用いられるサイクリックボルタンメトリー（ＣＶ）装置は、蛍光測定装置に比べて小型且つ安価であり、ＣＶ測定システムを独自に開発する場合であっても、その開発コストは、蛍光などの光学測定を利用するシステムのそれに比べて格段に小さいとされている。したがって、本発明の方法において、導電性物質を採用することは、測定精度および経済面の両方において大きなメリットがあると言える。 Furthermore, the cyclic voltammetry (CV) apparatus used for electrochemical measurement is smaller and less expensive than the fluorescence measurement apparatus, and even if the CV measurement system is independently developed, its development cost is such as fluorescence. It is said that it is much smaller than that of the system using the optical measurement. Therefore, it can be said that adopting a conductive substance in the method of the present invention has a great merit in both measurement accuracy and economy.
本発明において使用することができる導電性物質としては、フェロセン、テトラチアフルバレン(TTF)、テトラシアノキノジメタン(TCNQ)、金属錯体等を挙げることができる。 Examples of the conductive substance that can be used in the present invention include ferrocene, tetrathiafulvalene (TTF), tetracyanoquinodimethane (TCNQ), and a metal complex.
さらに、本発明の方法に用いることができるその他の機能性物質としては、ポルフィリン、フルオレン、ペンタセン、カロチン等を挙げることができる。 Furthermore, examples of other functional substances that can be used in the method of the present invention include porphyrin, fluorene, pentacene, and carotene.
以上、本発明のバイオセンシング方法について、実施形態をもって説明してきたが、本発明は上述した実施形態に限定されるものではなく、その他、当業者が推考しうる実施態様の範囲内において、本発明の作用・効果を奏する限り、本発明の範囲に含まれるものである。 As described above, the biosensing method of the present invention has been described with the embodiment. However, the present invention is not limited to the above-described embodiment, and the present invention is within the scope of embodiments that can be considered by those skilled in the art. As long as the effects and effects of the above are exhibited, the scope of the present invention is included.
以下、本発明のバイオセンシング方法について、実施例を用いてより具体的に説明を行なうが、本発明は、後述する実施例に限定されるものではない。 Hereinafter, the biosensing method of the present invention will be described more specifically using examples, but the present invention is not limited to the examples described below.
（実施例１）
蛍光分子を封入した磁性ビーズを用いて既知量の前立腺特異抗原（PSA）を検出するサンドイッチイムノアッセイを実施した。
Example 1
A sandwich immunoassay was performed to detect a known amount of prostate specific antigen (PSA) using magnetic beads encapsulating fluorescent molecules.
（１）磁性ビーズへの蛍光分子の封入
磁性ビーズ（粒子径：約２００ｎｍ、ＦＧビーズ（ＣＯＯＨビーズ）、品番：TA8848 N1140、多摩川精機株式会社製）1.0 mgをメタノール（500 μl）、メタノール/アセトン＝1/1（500 μl）、アセトン（500 μl）を順次用いて洗浄した後、蛍光分子であるDiIC18(5) solid (invitrogen製、D7757、以下Cy5として参照する)の10 mMアセトン溶液（100 μl）中に分散させた。
(1) Encapsulation of fluorescent molecules in magnetic beads Magnetic beads (particle size: about 200 nm, FG beads (COOH beads), product number: TA8848 N1140, manufactured by Tamagawa Seiki Co., Ltd.) 1.0 mg in methanol (500 μl), methanol / acetone = 1/1 (500 μl) and acetone (500 μl) in order, then washed with a 10 mM acetone solution of fluorescent molecule DiIC 18 (5) solid (invitrogen, D7757, hereinafter referred to as Cy5) 100 μl).
上記分散液を室温にて1時間混合した後、これに超純水（900 μl）を加えてさらに混合したものを遠心分離（20℃、15,000 rpm、3分）した。その後、上澄みを除去したものに超純水（500 μl）を加えて磁性ビーズを分散させた後、遠心分離（20℃、15,000rpm、3分）するといった操作を３回繰り返した。その後、上澄みを除去し、0.1％ Tween20含有50 mM HEPES溶液（500μl）を加えて磁性ビーズを分散させた後、遠心分離（20℃、15,000 rpm、３分）するといった操作を上澄みにCy5（青色）が視認できなくなるまで繰り返した。最終的に得られたCy5封入磁性ビーズを超純水（500 μl）に分散させ、４℃で保存した。 The above dispersion was mixed at room temperature for 1 hour, and then ultrapure water (900 μl) was added thereto and further mixed, followed by centrifugation (20 ° C., 15,000 rpm, 3 minutes). Then, after removing the supernatant, ultrapure water (500 μl) was added to disperse the magnetic beads, and then the centrifugation (20 ° C., 15,000 rpm, 3 minutes) was repeated three times. After removing the supernatant, add 50 mM HEPES solution (500 μl) containing 0.1% Tween20 to disperse the magnetic beads, and then centrifuge (20 ° C, 15,000 rpm, 3 minutes). ) Until no longer visible. The finally obtained Cy5-encapsulated magnetic beads were dispersed in ultrapure water (500 μl) and stored at 4 ° C.
（２）磁性ビーズ１個あたりのCy5の封入量の確認
予め、Cy5のアセトニトリル溶液を用いてCy5の量と吸光度に関する検量線を作成した。次に、上述した手順で作成したCy5封入磁性ビーズ１mgをアセトニトリル中に分散させて、磁性ビーズに封入された全てのCy5を溶出させた。具体的には、Cy5封入磁性ビーズ１mgをアセトン中に分散させてCy5を溶出させ、磁性ビーズを遠心分離後、上澄みを取るといった工程を、アセトニトリルを入れ替えて合計３回繰り返した。
(2) Confirmation of Cy5 Encapsulation Amount per Magnetic Bead A calibration curve regarding the amount of Cy5 and absorbance was prepared in advance using an acetonitrile solution of Cy5. Next, 1 mg of Cy5-encapsulated magnetic beads prepared by the above procedure was dispersed in acetonitrile to elute all Cy5 encapsulated in the magnetic beads. Specifically, the process of dispersing 1 mg of Cy5-encapsulated magnetic beads in acetone to elute Cy5, centrifuging the magnetic beads, and then removing the supernatant was repeated a total of 3 times with the replacement of acetonitrile.
３回目に取得した上澄み液においてCy5が検出されなかったため、１、２回目の上澄み液を合わせた溶液についてCy5の吸光度を測定した。吸光度の測定値を上記検量線に照らした結果、磁性ビーズ１個あたり最大で105分子のCy5が封入されていることが分かった。 Since Cy5 was not detected in the supernatant obtained at the third time, the absorbance of Cy5 was measured for the solution obtained by combining the first and second supernatants. As a result of illuminating the measured value of the absorbance with the calibration curve, it was found that 10 5 molecules of Cy5 were encapsulated at the maximum per magnetic bead.
（３）サンドイッチイムノアッセイの実施
上述した手順で作製したCy5封入磁性ビーズの表面に抗PSA抗体（Hytest製、5A6）を固定化し、これをアッセイバッファ（25 mM Tris-HCl [pH 8.0]、150 mM KCl、0.1％ Tween20、1％ skim milk）中に分散させて４℃で保存した。一方で、抗PSA抗体（Hytest製、1H12）を固定化した96穴マイクロプレートを用意し、アッセイバッファでブロッキング処理（４℃）した後、各ウェルに濃度[ng/ml]（0、0.0006、0.006、0.6、6.0、20、60）のPSA溶液（30 μl）をセットした。続いてアッセイバッファ中に分散させた抗PSA抗体固定化Cy5封入磁性ビーズを1.0 μg（20 μl）投入した。プレートを振盪（４℃、１分）させた後、96穴対応磁気プレートをマイクロプレートの下部に置き、１分間静置した。その後、アッセイバッファ（100 μl）を用いてプレートを振盪（４℃、30秒）し、非特異的に吸着した磁性ビーズを洗浄・除去した。
(3) Implementation of sandwich immunoassay An anti-PSA antibody (Hytest, 5A6) was immobilized on the surface of Cy5-encapsulated magnetic beads prepared by the above-described procedure, and this was assayed with assay buffer (25 mM Tris-HCl [pH 8.0], 150 mM). (KCl, 0.1% Tween 20, 1% skim milk) and stored at 4 ° C. On the other hand, after preparing a 96-well microplate on which an anti-PSA antibody (Hytest, 1H12) is immobilized, blocking with an assay buffer (4 ° C.), each well has a concentration [ng / ml] (0, 0.0006, 0.006, 0.6, 6.0, 20, 60) PSA solution (30 μl) was set. Subsequently, 1.0 μg (20 μl) of anti-PSA antibody-immobilized Cy5-encapsulated magnetic beads dispersed in the assay buffer was added. After the plate was shaken (4 ° C., 1 minute), a 96-well magnetic plate was placed at the bottom of the microplate and allowed to stand for 1 minute. Thereafter, the plate was shaken (4 ° C., 30 seconds) using assay buffer (100 μl), and the non-specifically adsorbed magnetic beads were washed and removed.
（４）蛍光分子の溶出および蛍光測定
96穴マイクロプレートにアセトニトリル（100 μl）を加えて振盪（室温、５分）し、Cy5封入磁性ビーズからCy5を溶出させて、その上澄み液をウェルごとに回収した。続いて回収した各上澄み液について蛍光強度を測定（PerkinElmer製、Wallac ARVO SX 1420）した。
(4) Elution and fluorescence measurement of fluorescent molecules
Acetonitrile (100 μl) was added to a 96-well microplate and shaken (room temperature, 5 minutes) to elute Cy5 from the Cy5-encapsulated magnetic beads, and the supernatant was collected for each well. Subsequently, the fluorescence intensity of each collected supernatant was measured (PerkinElmer, Wallac ARVO SX 1420).
図４は、各ウェルにセットしたPSA溶液の濃度[ng/ml]と、サンドイッチイムノアッセイ後に当該ウェルから回収した上澄み液（Cy5のアセトニトリル溶液）について測定された蛍光強度[cps]の関係を示す。図４の結果から、本発明の方法によれば、0.06 ng/mlから6.0 ng/mlまでの濃度範囲でPSAを高精度に検出しうることが示された。
（実施例２）
FIG. 4 shows the relationship between the concentration [ng / ml] of the PSA solution set in each well and the fluorescence intensity [cps] measured for the supernatant (Cy5 acetonitrile solution) collected from the well after the sandwich immunoassay. From the results of FIG. 4, it was shown that according to the method of the present invention, PSA can be detected with high accuracy in the concentration range from 0.06 ng / ml to 6.0 ng / ml.
(Example 2)
以下の手順で、磁性ポリマー粒子に対してフェロセンを封入した後、磁性ポリマー粒子からフェロセンを溶出させる実験を行った。 In the following procedure, after ferrocene was encapsulated in the magnetic polymer particles, an experiment was conducted to elute ferrocene from the magnetic polymer particles.
（１）磁性ビーズへのフェロセンの封入
磁性ビーズ（粒子径：約２００ｎｍ、ＦＧビーズ（ＣＯＯＨビーズ）、品番：TA8848 N1140、多摩川精機株式会社製）1.0 mgをメタノール（500 μl）で２回、続いてアセトン（500 μl）で３回洗浄し、フェロセン (東京化成工業製、D0444)の100 mMアセトン溶液（60 μl）中に分散させた。
(1) Encapsulation of ferrocene in magnetic beads Magnetic beads (particle size: about 200 nm, FG beads (COOH beads), product number: TA8848 N1140, manufactured by Tamagawa Seiki Co., Ltd.) 1.0 mg twice in methanol (500 μl), then The resultant was washed three times with acetone (500 μl) and dispersed in a 100 mM acetone solution (60 μl) of ferrocene (manufactured by Tokyo Chemical Industry Co., Ltd., D0444).
上記分散液を40℃にて20分混合した後、磁性ビーズを遠心分離（20℃、15,000rpm、3分）した。上澄みを除去し、超純水（500 μl）を加えてビーズを分散させた後、遠心分離（20℃、15,000rpm、３分）した。その後、上澄みを除去し、0.3％ Tween20含有50 mM HEPES溶液（500 μl）を加えて磁性ビーズを分散させた後、遠心分離（20℃、15,000rpm、3分）するといった操作を３回繰り返した。続いて上澄みを除去し、超純水（500 μl）を加えてビーズを分散させた後、遠心分離（20℃、15,000rpm、3分）するといった操作を３回繰り返した。最終的に得られたフェロセン封入磁性ビーズを超純水（500 μl）に分散させ、４℃で保存した。 After the dispersion was mixed at 40 ° C. for 20 minutes, the magnetic beads were centrifuged (20 ° C., 15,000 rpm, 3 minutes). The supernatant was removed, and ultrapure water (500 μl) was added to disperse the beads, followed by centrifugation (20 ° C., 15,000 rpm, 3 minutes). Thereafter, the supernatant was removed, and after adding a 50 mM HEPES solution (500 μl) containing 0.3% Tween 20 to disperse the magnetic beads, centrifugation (20 ° C., 15,000 rpm, 3 minutes) was repeated three times. . Subsequently, the supernatant was removed, and ultrapure water (500 μl) was added to disperse the beads, followed by centrifugation (20 ° C., 15,000 rpm, 3 minutes) three times. The finally obtained ferrocene-encapsulated magnetic beads were dispersed in ultrapure water (500 μl) and stored at 4 ° C.
（２）磁性ビーズ１個あたりのフェロセンの封入量の確認
予め、フェロセンのアセトン溶液を用いてフェロセンの量と吸光度に関する検量線を作成した。次に、上述した手順で作成したフェロセン封入磁性ビーズ１mgをアセトン中に分散させて、磁性ビーズに封入された全てのフェロセンを溶出した。具体的には、フェロセン封入磁性ビーズ１mgをアセトン中に分散させてフェロセンを溶出させ、磁性ビーズを遠心分離後、上澄みを取るといった工程を、アセトンを入れ替えて合計４回繰り返した。
(2) Confirmation of encapsulated amount of ferrocene per magnetic bead A calibration curve regarding the amount of ferrocene and the absorbance was prepared in advance using an acetone solution of ferrocene. Next, 1 mg of the ferrocene-encapsulated magnetic beads prepared in the above-described procedure was dispersed in acetone to elute all the ferrocene encapsulated in the magnetic beads. Specifically, the process of dispersing 1 mg of ferrocene-encapsulated magnetic beads in acetone to elute ferrocene, centrifuging the magnetic beads, and then removing the supernatant was repeated a total of 4 times with acetone being replaced.
４回目に取得した上澄み液においてフェロセンが検出されなかったため、１〜３回目の上澄み液を合わせた溶液についてフェロセンの吸光度を測定した。吸光度の測定値を上記検量線に照らした結果、ビーズ１mgあたり約6μmol（すなわち約1018分子（(6x10-6)x(6x1023)＝3.6x1018））のフェロセンがビーズに封入されていたことが分かった。ここで、フェロセン封入磁性ビーズ１mgが1011個のビーズに相当することから、磁性ビーズ１個あたりおよそ107分子のフェロセンが封入されていたことが分かった。 Since ferrocene was not detected in the supernatant obtained in the fourth time, the absorbance of ferrocene was measured for the solution obtained by combining the first to third supernatants. As a result of illuminating the measured value of the absorbance with the above calibration curve, about 6 μmol (ie, about 10 18 molecules (( 6 × 10 −6 ) × ( 6 × 10 23 ) = 3.6 × 10 18 )) of ferrocene was enclosed in the beads. I understood that. Here, since 1 mg of ferrocene-encapsulated magnetic beads corresponds to 10 11 beads, it was found that approximately 10 7 molecules of ferrocene were encapsulated per magnetic bead.
（３）フェロセンの溶出およびＣＶ測定
上述した手順で作成したフェロセン封入磁性ビーズ１mgを過塩素酸テトラブチルアンモニウムの0.1 Mアセトニトリル溶液に分散させた後、静置（室温、５分）してフェロセンを溶出させ、その後に磁性ビーズを遠心分離後、上澄みを取るといった工程を合計４回繰り返した。４回目に取得した上澄み液においてフェロセンが検出されなかったことから、１〜３回目の上澄み液を合わせた溶液をＣＶ測定用サンプルとして得た。
(3) Elution of ferrocene and CV measurement After dispersing 1 mg of ferrocene-encapsulated magnetic beads prepared in the above procedure in a 0.1 M acetonitrile solution of tetrabutylammonium perchlorate, the ferrocene was allowed to stand (room temperature, 5 minutes). The process of elution, and then centrifuging the magnetic beads and then removing the supernatant was repeated a total of 4 times. Since ferrocene was not detected in the supernatant obtained in the fourth time, a solution in which the first to third supernatants were combined was obtained as a sample for CV measurement.
上述した手順で得たサンプルにつき、Modulab（東陽テクニカ製）を用いてＣＶ測定を行った。なお、作用電極にはG0225白金マイクロ電極（10 μmφ、東陽テクニカ製）を、カウンター電極にはVC-3用白金カウンター電極（5 cm、BAS製）を、参照電極にはRE-7非水溶媒計参照電極（BAS製）をそれぞれ用いた。ＣＶ測定は10 mV/secの掃引速度で-0.5 V〜+0.5 Vの電圧範囲で２サイクル行った。 The sample obtained by the above-described procedure was subjected to CV measurement using Modulab (manufactured by Toyo Technica). The working electrode is a G0225 platinum microelectrode (10 μmφ, manufactured by Toyo Technica), the counter electrode is a VC-3 platinum counter electrode (5 cm, manufactured by BAS), and the reference electrode is a RE-7 nonaqueous solvent. A total reference electrode (manufactured by BAS) was used. CV measurement was performed for 2 cycles in a voltage range of -0.5 V to +0.5 V at a sweep rate of 10 mV / sec.
過塩素酸テトラブチルアンモニウムの0.1 Mアセトニトリル溶液に溶解させたフェロセンのＣＶ測定から、フェロセン濃度が1〜30 μMの範囲内で検量線を得た。1.0 μMのフェロセン溶液100 μlにおいて10-12Aの電流値が得られたことから、（(1x10-6)x(100x10-6)x(6x1023)≒）1013分子のフェロセン≒10-12Aとなる。 From a CV measurement of ferrocene dissolved in a 0.1 M acetonitrile solution of tetrabutylammonium perchlorate, a calibration curve was obtained within a ferrocene concentration range of 1 to 30 μM. Since the current value of 10 -12 A was obtained in 1.0 [mu] M of ferrocene solution 100 μl, ((1x10 -6) x (100x10 -6) x (6x10 23) ≒) 10 13 molecules ferrocene ≒ 10 -12 A.
１０…抗原
１２…抗体
１４…基板
１６…機能性物質包含磁性ビーズ
１７…抗体
１８…機能性物質
２０…磁石
DESCRIPTION OF SYMBOLS 10 ... Antigen 12 ... Antibody 14 ... Substrate 16 ... Magnetic substance inclusion magnetic bead 17 ... Antibody 18 ... Functional substance 20 ... Magnet
Claims (5)
ポリマー微粒子の内部に機能性物質を封入する工程と、
前記ポリマー微粒子の表面にリガンドを固定化する工程と、
前記リガンドを介して前記ポリマー微粒子とターゲットを結合させる工程と、
前記ターゲットに結合した前記ポリマー微粒子から前記機能性物質を溶出させる工程と、
溶出した前記機能性物質の物性を定量的に測定する工程と
を含み、
前記物性の測定結果に基づいて前記ターゲットを定量化することを特徴とするバイオセンシング方法。 A biosensing method using an affinity reaction,
A step of encapsulating a functional substance inside the polymer particles;
Immobilizing a ligand on the surface of the polymer fine particle;
Binding the polymer microparticles to a target via the ligand;
Eluting the functional substance from the polymer microparticles bound to the target;
And quantitatively measuring the physical properties of the eluted functional substance,
A biosensing method characterized in that the target is quantified based on the measurement result of the physical property.
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| PCT/JP2012/007475 WO2013080491A1 (en) | 2011-11-29 | 2012-11-21 | Biosensing method in which functional substance-containing microparticles are used |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005010043A (en) * | 2003-06-19 | 2005-01-13 | Sekisui Chem Co Ltd | Measuring reagent and measurement method |
| JP2008127454A (en) * | 2006-11-20 | 2008-06-05 | Tokyo Institute Of Technology | Magnetic polymer particle with fluorescent function, and method for producing the same |
| WO2009072457A1 (en) * | 2007-12-03 | 2009-06-11 | Tokyo Institute Of Technology | Biosensing method using coated magnetic microparticles and biosensing device to be used in the method |
| JP2011069646A (en) * | 2009-09-24 | 2011-04-07 | Sekisui Chem Co Ltd | Quantitative analysis method |
| JP2011185874A (en) * | 2010-03-10 | 2011-09-22 | Nippon Telegr & Teleph Corp <Ntt> | Kit for analyzing biomolecules and method for analyzing biomolecules using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005010043A (en) * | 2003-06-19 | 2005-01-13 | Sekisui Chem Co Ltd | Measuring reagent and measurement method |
| JP2008127454A (en) * | 2006-11-20 | 2008-06-05 | Tokyo Institute Of Technology | Magnetic polymer particle with fluorescent function, and method for producing the same |
| WO2009072457A1 (en) * | 2007-12-03 | 2009-06-11 | Tokyo Institute Of Technology | Biosensing method using coated magnetic microparticles and biosensing device to be used in the method |
| JP2011069646A (en) * | 2009-09-24 | 2011-04-07 | Sekisui Chem Co Ltd | Quantitative analysis method |
| JP2011185874A (en) * | 2010-03-10 | 2011-09-22 | Nippon Telegr & Teleph Corp <Ntt> | Kit for analyzing biomolecules and method for analyzing biomolecules using the same |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2015155813A (en) * | 2014-02-20 | 2015-08-27 | 国立大学法人東京工業大学 | Fluorescent polymer fine particle and fluorescent marker set |
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