WO2016178350A1 - Detection system and modifying substance - Google Patents

Detection system and modifying substance Download PDF

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Publication number
WO2016178350A1
WO2016178350A1 PCT/JP2016/061173 JP2016061173W WO2016178350A1 WO 2016178350 A1 WO2016178350 A1 WO 2016178350A1 JP 2016061173 W JP2016061173 W JP 2016061173W WO 2016178350 A1 WO2016178350 A1 WO 2016178350A1
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growth
specimen
modifying substance
substance
starting point
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PCT/JP2016/061173
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French (fr)
Japanese (ja)
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川口　裕一
菊川　隆
祥生 坪池
春希 柚賀
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Tdk株式会社
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    • C—CHEMISTRY; METALLURGY
    • C23—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; CHEMICAL SURFACE TREATMENT; DIFFUSION TREATMENT OF METALLIC MATERIAL; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL; INHIBITING CORROSION OF METALLIC MATERIAL OR INCRUSTATION IN GENERAL
    • C23C—COATING METALLIC MATERIAL; COATING MATERIAL WITH METALLIC MATERIAL; SURFACE TREATMENT OF METALLIC MATERIAL BY DIFFUSION INTO THE SURFACE, BY CHEMICAL CONVERSION OR SUBSTITUTION; COATING BY VACUUM EVAPORATION, BY SPUTTERING, BY ION IMPLANTATION OR BY CHEMICAL VAPOUR DEPOSITION, IN GENERAL
    • C23C18/00—Chemical coating by decomposition of either liquid compounds or solutions of the coating forming compounds, without leaving reaction products of surface material in the coating; Contact plating
    • C23C18/16—Chemical coating by decomposition of either liquid compounds or solutions of the coating forming compounds, without leaving reaction products of surface material in the coating; Contact plating by reduction or substitution, e.g. electroless plating
    • C23C18/18—Pretreatment of the material to be coated
    • C23C18/20—Pretreatment of the material to be coated of organic surfaces, e.g. resins
    • C23C18/28—Sensitising or activating
    • 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
    • G01N27/72—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating magnetic variables
    • 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

Definitions

  • the detection system of the present invention is characterized in that the growth starting point is a metal catalyst, and a metal is grown as the growth material by metal deposition by an electroless plating method in the growth step.
  • the growth starting point is a hydroxyl group
  • a metal salt aqueous solution and an oxidizing agent are supplied to the modifying substance, and a metal oxide is deposited as the growing substance to grow. It is characterized by.
  • Pd is well known as a metal catalyst for electroless plating, but may be, for example, Fe, Co, Ni, Cu, Ru, Rh, Os, Ir, Pt, Ag, Au, or an alloy combining them. .
  • a metal as the growth material 6 for example, Co, Ni, Cu, Ru, Rh, Pd, Pt, Ag, Au, or an alloy thereof can be used.
  • the electroless plating solution is prepared by combining a water-soluble halide, sulfate and sulfamate aqueous solution of the above metal with a reducing agent such as an amine borane compound, a hydrazine compound, phosphorous acid, hypophosphorous acid or a salt thereof.
  • a metal oxide can be used as the growth material 6.
  • the growth starting point 4 of the modifying substance 5 is a hydroxyl group, and an aqueous metal salt solution and an oxidizing agent are supplied to the modifying substance 5 (support 2 on which the specimen 1 bound to the modifying substance 5 is fixed) in the growth step 10, thereby oxidizing the metal.
  • the material growth material 6 can be deposited and grown.
  • the aqueous metal salt solution and the oxidizing agent may be mixed before being supplied to the support 2, but in order to prevent unnecessary reaction, it is preferable to supply the aqueous solution and the oxidizing agent to the support 2 separately. Can be alternately supplied to the support 2.
  • Detection by an optical microscope has a large system such as a lens configuration, an illumination device, and an image processing device, and resolution is limited by the wavelength of light, but magnetic detection has no wavelength limitation and is highly sensitive.
  • the system can be miniaturized.
  • the growth substance 6 can be detected with higher sensitivity by applying an external magnetic field.

Abstract

This detection system is characterized by having: a fixing step 7 for fixing a specimen to a support body; a binding step 8 for binding, to the specimen, a modifying substance having a growth starting point and a selective binding group for the specimen; a removing step 9 for removing the modifying substance not bound to the specimen; and a growing step 10 for growing a growth substance at the growth starting point, and by detecting the grown growth substance.

Description

検出システムおよび修飾物質Detection systems and modifiers
　本発明は、簡便な方法で高感度の検体検出を行うことのできる検出システムおよび、この検出システムで使用可能な修飾物質に関する。 The present invention relates to a detection system capable of detecting a highly sensitive specimen by a simple method and a modifying substance usable in this detection system.
　近年、疾患の同定や予防を遣伝子に基づいて診断する方法（遣伝子診断）が、遣伝子工学の進歩に伴って可能になってきた。また、各種の抗原、例えば、癌マーカーとして用いられるＡＦＰ、ＣＥＡ等の抗原や、ホルモンの検出が免疫学的な手法を用いて行われており、癌等の疾患の診断に役立てられている。　 In recent years, methods for diagnosing diseases based on gene transfer (transmission diagnosis) have become possible with the progress of gene engineering. In addition, various antigens, for example, antigens such as AFP and CEA used as cancer markers, and hormones are detected using an immunological technique, which is useful for diagnosis of diseases such as cancer. *
癌マーカーやホルモン等の蛋白質は生体内に微量しか存在しないことから、必然的にその検出には高感度化が要求されている。さらに、患者への負担を軽減するために、採取するサンプルの微量化が進行しており、血液電解質測定等では既に数マイクロリットルのサンプルから検出が行われている。 Since proteins such as cancer markers and hormones are present in a very small amount in the living body, high sensitivity is inevitably required for their detection. Furthermore, in order to reduce the burden on the patient, the amount of sample to be collected has been reduced, and detection has already been performed from a sample of several microliters in blood electrolyte measurement and the like.
同様にＨＩＶおよびＨＢＶ等のウイルスや細菌の検出あるいは癌遺伝子の検出等においても高い検出感度が求められており、さらなる高感度化が求められている。従来、上記物質は、例えば、化学発光物質、蛍光物質および放射性同位元素等により標識を行って該標識からの信号を獲得したり、ポリメラーゼチェーンリアクション（ＰＣＲ法）のように酵素反応によって物質を増幅することにより検出されてきた。 Similarly, high detection sensitivity is required for detection of viruses and bacteria such as HIV and HBV, and detection of oncogenes, and further enhancement of sensitivity is required. Conventionally, the above substances are labeled with, for example, a chemiluminescent substance, a fluorescent substance and a radioisotope to acquire a signal from the label or amplify the substance by an enzymatic reaction such as a polymerase chain reaction (PCR method). Has been detected.
さらなる高感度化を目的として、特許文献１では、癌マーカー等の特定タンパク質と結合する蛍光試薬およびタンパク質の検出方法が提案され、また、特許文献２では、ポリメラーゼを用いた標的核酸の増幅方法が提案されている。 For the purpose of further increasing the sensitivity, Patent Document 1 proposes a fluorescent reagent that binds to a specific protein such as a cancer marker and a method for detecting the protein, and Patent Document 2 discloses a method for amplifying a target nucleic acid using a polymerase. Proposed.
特開２００９－１８６３５７号公報JP 2009-186357 A 特表２０１３－５４２７１３号公報Special table 2013-542713 gazette
　しかしながら、化学発光物質、蛍光物質等により標識を行って該標識からの信号を獲得する方法は感度が不十分であり、要求される高感度化に対応することが困難であり、放射性同位元素を使用するためには、特定の施設が必要という問題があった。また、ＰＣＲ法のように酵素反応によって物質を増幅する方法では、Ｔａｑポリメラーゼ等の高価な酵素を使用しなければならず、検出に要する経済的な負担が大きいという問題がある。 However, the method of obtaining a signal from the label by labeling with a chemiluminescent substance, a fluorescent substance or the like is insufficient in sensitivity, and it is difficult to cope with the required high sensitivity. There was a problem that a specific facility was necessary to use it. In addition, in a method of amplifying a substance by an enzymatic reaction such as the PCR method, an expensive enzyme such as Taq polymerase must be used, and there is a problem that an economic burden required for detection is large.
本発明は、上記事情に鑑みてなされたものであり、簡便な方法で高感度の検体検出を行うことのできる検出システムおよび、この検出システムで使用可能な修飾物質を提供することを目的とする。 The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a detection system capable of detecting a highly sensitive specimen by a simple method and a modifying substance usable in the detection system. .
上記目的を達成する本発明の検出システムは、検体を支持体に固定する固定工程と、前記検体への選択性結合基と成長起点を有する修飾物質を前記検体に結合させる結合工程と、前記検体と結合していない前記修飾物質を除去する除去工程と、前記成長起点に成長物質を成長させる成長工程を有し、成長後の前記成長物質を検出することを特徴とする。 The detection system of the present invention that achieves the above object comprises a fixing step of fixing a specimen to a support, a binding step of binding a modifying substance having a selective binding group and a growth starting point to the specimen, and the specimen. And a growth step of growing a growth material at the growth starting point, and detecting the growth material after growth.
これによれば、検体に固定された修飾物質に成長物質を成長させ、成長後の成長物質を検出することで、簡便な方法で高感度の検体検出を行うことができる。 According to this, the growth substance is grown on the modifying substance fixed to the specimen, and the growth substance after the growth is detected, so that it is possible to detect the specimen with high sensitivity by a simple method.
さらに本発明の検出システムは、前記修飾物質が水溶性であることを特徴とする。 Furthermore, the detection system of the present invention is characterized in that the modifying substance is water-soluble.
これによれば、修飾物質が水溶性であることで、修飾物質の濃度分布の低減、検体との結合反応性の向上ができ、安定した検体検出が可能な検出システムを提供できる。 According to this, since the modifying substance is water-soluble, the concentration distribution of the modifying substance can be reduced and the binding reactivity with the specimen can be improved, and a detection system capable of stable specimen detection can be provided.
さらに本発明の検出システムは、前記成長起点が金属触媒であり、前記成長工程において無電解めっき法による金属析出により前記成長物質として金属を成長させることを特徴とする。 Furthermore, the detection system of the present invention is characterized in that the growth starting point is a metal catalyst, and a metal is grown as the growth material by metal deposition by an electroless plating method in the growth step.
これによれば、簡便な方法で、成長物質を成長させることができる。 According to this, the growth material can be grown by a simple method.
さらに本発明の検出システムは、前記成長起点が水酸基であり、前記成長工程において、金属塩水溶液と酸化剤を前記修飾物質に供給して、前記成長物質として金属酸化物を析出させて成長させることを特徴とする。 Furthermore, in the detection system of the present invention, the growth starting point is a hydroxyl group, and in the growth step, a metal salt aqueous solution and an oxidizing agent are supplied to the modifying substance, and a metal oxide is deposited as the growing substance to grow. It is characterized by.
これによれば、希少金属を用いなくても、簡便な方法で成長物質を成長させることができる。 According to this, the growth material can be grown by a simple method without using a rare metal.
さらに本発明の検出システムは、前記成長物質が強磁性体であり、磁気検出により成長後の前記成長物質を検出することを特徴とする。 Furthermore, in the detection system of the present invention, the growth material is a ferromagnetic material, and the growth material after growth is detected by magnetic detection.
これによれば、磁気検出にすることで、検体検出の高感度化、装置の小型化ができる。 According to this, by using magnetic detection, it is possible to increase the sensitivity of specimen detection and reduce the size of the apparatus.
また、本発明の修飾物質は、検体への選択性結合基と成長起点とを有するものであることを特徴とする。 The modifying substance of the present invention is characterized by having a selective binding group for a specimen and a growth starting point.
これによれば、上記した検出システムに用いられることで、簡便な方法で高感度の検体検出を行うことができる。 According to this, by being used in the above-described detection system, highly sensitive specimen detection can be performed by a simple method.
さらに本発明の修飾物質は、水溶性であることを特徴とする。 Furthermore, the modifying substance of the present invention is water-soluble.
これによれば、修飾物質が水溶性であることで、修飾物質の濃度分布の低減、検体との結合反応性の向上ができる。 According to this, since the modifying substance is water-soluble, the concentration distribution of the modifying substance can be reduced and the binding reactivity with the specimen can be improved.
さらに本発明の修飾物質は、前記成長起点が金属触媒であることを特徴とする。 Furthermore, the modifying substance of the present invention is characterized in that the growth starting point is a metal catalyst.
さらに本発明の修飾物質は、前記成長起点が水酸基であることを特徴とする。 Furthermore, the modifying substance of the present invention is characterized in that the growth starting point is a hydroxyl group.
さらに本発明の修飾物質は、前記選択性結合基が抗体または核酸であることを特徴とする。 Furthermore, the modifying substance of the present invention is characterized in that the selective binding group is an antibody or a nucleic acid.
本発明により、簡便な方法で高感度の検体検出を行うことのできる検出システムおよび、この検出システムで使用可能な修飾物質を提供することができる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a detection system that can perform highly sensitive specimen detection by a simple method and a modifying substance that can be used in this detection system.
図１は、検出システムの概要を説明する工程図である。FIG. 1 is a process diagram for explaining the outline of the detection system. 図２は、固定工程の模式図である。FIG. 2 is a schematic diagram of the fixing process. 図３は、修飾物質の概念図である。FIG. 3 is a conceptual diagram of the modifying substance. 図４は、結合工程の模式図である。FIG. 4 is a schematic diagram of the bonding process. 図５は、除去工程の模式図である。FIG. 5 is a schematic diagram of the removal process. 図６は、成長工程の模式図である。FIG. 6 is a schematic diagram of the growth process.
以下、本発明を実施するための好適な形態を説明する。本実施形態は本発明を実施する一例であって、本発明は本実施形態に限定されるものではない。 Hereinafter, preferred embodiments for carrying out the present invention will be described. This embodiment is an example for carrying out the present invention, and the present invention is not limited to this embodiment.
（検出システム基本構成）
本実施形態の検出システムは、図１に示すように、検体１を支持体２に固定する固定工程７と、検体１への選択性結合基３と成長起点４を有する修飾物質５を検体１に結合させる結合工程８と、検体１と結合していない修飾物質５を除去する除去工程９と、成長起点４に成長物質６を成長させる成長工程１０を有し、検出工程１１において成長後の成長物質６を検出する。 (Detection system basic configuration)
As shown in FIG. 1, the detection system of this embodiment includes a fixing step 7 for fixing the specimen 1 to the support 2, and a modifying substance 5 having a selective binding group 3 and a growth starting point 4 for the specimen 1. A growth step 10 for growing the growth material 6 at the growth starting point 4, and a growth step 10 after the growth in the detection step 11. Growing material 6 is detected.
（検体）
本実施形態の検体１は、例えば、結合可能なリガンドを有する受容体タンパク質、接着タンパク質、抗原または抗体などのように、タンパク質間相互作用が可能なタンパク質であり、疾患と関連するものである。そのようなものには、その存在量の増減が疾患の存在を示唆するタンパク質のように、疾患の診断に使用可能なタンパク質が含まれ、上皮増殖因子（ＥＧＦ）、血小板由来増殖因子（ＰＤＧＦ）、脳由来増殖因子（ＢＤＧＦ）または血管内皮増殖因子（ＶＥＧＦ）などの増殖因子、フィブロネクチン、ラミニンまたはビトロネクチンなどの細胞接着因子、インスリン、ソマトスタチン、ソマトトロンビンまたは性腺刺激ホルモン放出因子などのホルモン、ＬＤＬなどのリポタンパク質、種々の腫瘍マーカーまたは抗体などが挙げられる。また、ＨＩＶおよびＨＢＶ等のウイルスや細菌あるいは癌遺伝子等のＤＮＡ／ＲＮＡを検体１とすることもできる。また、検体１は、上に例示した検体分子だけではなく、上に例示したような検体分子に結合している分子または、上に例示したような検体分子近傍に偏在している分子とすることもできる。 (Sample)
The specimen 1 of the present embodiment is a protein capable of protein-protein interaction, such as a receptor protein having a bindable ligand, an adhesion protein, an antigen, or an antibody, and is associated with a disease. Such proteins include proteins that can be used to diagnose disease, such as proteins whose increase or decrease in their amount suggests the presence of a disease, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) Growth factors such as brain-derived growth factor (BDGF) or vascular endothelial growth factor (VEGF), cell adhesion factors such as fibronectin, laminin or vitronectin, hormones such as insulin, somatostatin, somatothrombin or gonadotropin releasing factor, LDL, etc. Lipoproteins, various tumor markers or antibodies. In addition, a virus such as HIV and HBV, or a DNA / RNA such as a bacterium or an oncogene can be used as the specimen 1. In addition, the sample 1 is not limited to the sample molecule exemplified above, but a molecule that is bound to the sample molecule exemplified above or a molecule that is unevenly distributed in the vicinity of the sample molecule exemplified above. You can also.
（支持体）
支持体２は、石英ガラスやシリコン、ポリカーボネート又はポリスチレンその他合成樹脂で形成される。また、支持体２は、不純物が少なく、化学的に安定であることが好ましく、石英ガラスやシリコンが好ましい。さらに検出工程１１において光学検出を行なう場合は、光を透過しないシリコンが好ましい。通常これらの材料表面は化学的な活性が弱く、図２に示すように、検体１を固定するために表面修飾２ａを施しても良い。表面修飾２ａとしては、アルデヒド基又はアミノ基などの活性な官能基を導入することができる。例えば、アミノ基を導入することにより、支持体２全体が正電荷を帯びるため、負電荷をもつ検体１を静電的相互作用により支持体２に固定することができる。アミノ基の導入方法として、アミノアルキルシランによる処理、窒素雰囲気下でのプラズマ処理、アミノ基含有高分子物質のコーティング又はポリ‐Ｌ－リジンなどのコーティングが挙げられる。支持体２の大きさ、形状に制限はなく、例えば、支持体２に流路を形成して溶液を流通することができる。さらに支持体２に、検出工程１１における検出用の磁気センサー素子や配線が形成されていても良い。 (Support)
The support 2 is made of quartz glass, silicon, polycarbonate, polystyrene, or other synthetic resin. The support 2 is preferably chemically stable with few impurities and is preferably quartz glass or silicon. Further, when optical detection is performed in the detection step 11, silicon that does not transmit light is preferable. Usually, the surface of these materials has weak chemical activity, and surface modification 2a may be applied to fix the specimen 1 as shown in FIG. As the surface modification 2a, an active functional group such as an aldehyde group or an amino group can be introduced. For example, since the entire support 2 is positively charged by introducing an amino group, the specimen 1 having a negative charge can be fixed to the support 2 by electrostatic interaction. Examples of the amino group introduction method include treatment with aminoalkylsilane, plasma treatment in a nitrogen atmosphere, coating of an amino group-containing polymer substance, or coating of poly-L-lysine. There is no restriction | limiting in the magnitude | size and shape of the support body 2, For example, a flow path can be formed in the support body 2 and a solution can be distribute | circulated. Furthermore, a magnetic sensor element or wiring for detection in the detection step 11 may be formed on the support 2.
（固定工程）
固定工程７では、図２に示すように、検体１は支持体２の表面に固定される。固定方法は特に限定されないが、生体由来の検体１は、一般的に負電荷をもっており、例えば上述したように支持体２にアミノ基による表面修飾２ａを施しておくことにより、検体１を支持体２の表面に固定することができる。具体的には、支持体２の表面に検体１の溶液を滴下後、３７℃程度の恒温下で２０時間程度静置することで、検体１を支持体２の表面に固定化できる。 (Fixing process)
In the fixing step 7, the specimen 1 is fixed to the surface of the support 2 as shown in FIG. Although the immobilization method is not particularly limited, the specimen 1 derived from a living body generally has a negative charge. For example, the specimen 1 is supported on the support 2 by subjecting the support 2 to surface modification 2a with an amino group as described above. 2 can be fixed on the surface. Specifically, the sample 1 can be immobilized on the surface of the support 2 by dropping the solution of the sample 1 on the surface of the support 2 and then allowing it to stand at a constant temperature of about 37 ° C. for about 20 hours.
（修飾物質）
修飾物質５は、図３のように検体１への選択性結合基３と成長起点４とをそれぞれ１つ以上有する物質であり、選択結合基３は検体１に対して適宜選択できる。検体１と選択性結合基３との結合作用としては、特に制限されないが、例えば、蛋白質間の相互作用、蛋白質とペプチドの相互作用、核酸間の相互作用、蛋白質と核酸の相互作用などが包含される。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、糖鎖と蛋白質の結合反応、脂肪鎖と蛋白質の結合反応、リン酸基と蛋白質の結合反応、補欠因子と蛋白質の結合反応などが挙げられる。 (Modified substance)
The modifying substance 5 is a substance having at least one selective binding group 3 and one growth starting point 4 for the specimen 1 as shown in FIG. 3, and the selective binding group 3 can be appropriately selected for the specimen 1. The binding action between the specimen 1 and the selective binding group 3 is not particularly limited, but includes, for example, protein-protein interaction, protein-peptide interaction, nucleic acid interaction, protein-nucleic acid interaction, and the like. Is done. 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 and protein binding reaction, fatty chain and protein binding reaction, phosphate group and protein binding reaction, prosthetic factor and protein binding reaction.
例えば、検体１がウィルス等の抗原の場合、選択性結合基３は抗体である。抗体の作製方法としては、ウサギ・ラット・ウマなど実験動物に作製したい抗体に対応する抗原を投与し、免疫によって体の中で目的の抗体を作製させた後、採血して目的の抗体の入った血液から血清を分離し、イオンクロマトグラフィーなどで精製をして目的の抗体のみを単離することができる。単離した抗体に成長起点４を発生させることで修飾物質５を作製することができる。 For example, when the specimen 1 is an antigen such as a virus, the selective binding group 3 is an antibody. The antibody can be produced by administering an antigen corresponding to the antibody to be produced to a laboratory animal such as a rabbit, rat, or horse, producing the desired antibody in the body by immunization, and then collecting blood to contain the desired antibody. Serum can be separated from the collected blood and purified by ion chromatography or the like to isolate only the target antibody. The modifying substance 5 can be produced by generating the growth starting point 4 in the isolated antibody.
また、検体１が核酸である場合は、選択性結合基３も核酸とすることができる。二本鎖である核酸は、熱を加えることにより、二本鎖構造が解きほぐされて、一本鎖となる。この時の温度を融解温度と呼び、核酸の塩基配列等により異なる。温度を融解温度より下げることで、核酸は再び結合して二本鎖となる。核酸の一本鎖の一方を選択結合基３とすることができる。 Further, when the specimen 1 is a nucleic acid, the selective binding group 3 can also be a nucleic acid. A double-stranded nucleic acid is unwound from a double-stranded structure by applying heat to become single-stranded. The temperature at this time is called the melting temperature, and varies depending on the nucleotide sequence of the nucleic acid. By lowering the temperature below the melting temperature, the nucleic acids bind again and become double stranded. One of the single strands of the nucleic acid can be a selective binding group 3.
成長起点４は成長物質６によって適宜選択されるが、例えば、金属触媒であるＰｄである。例えば、選択性結合基３をＰｄ（ＣＨ３ＣＯＯ）２　水溶液で処理した後、還元剤としてジメチルアミンボラン（ＤＭＡＢ）が加えて、Ｐｄを析出させることで、選択性結合基３に成長起点４を形成できる。 The growth starting point 4 is appropriately selected depending on the growth material 6, and is, for example, Pd which is a metal catalyst. For example, after the selective bonding group 3 is treated with an aqueous solution of Pd (CH 3 COO) 2 , dimethylamine borane (DMAB) is added as a reducing agent to precipitate Pd, whereby the selective bonding group 3 has a growth starting point 4. Can be formed.
成長起点４は水酸基とすることもできる。抗体や核酸等の選択性結合基３が水酸基を有する場合は、それを成長起点４とすることができる。選択性結合基３が水酸基を持たない場合、例えば選択性結合基３を酸化処理することで、成長起点４としての水酸基を形成することができる。 The growth starting point 4 can also be a hydroxyl group. When the selective binding group 3 such as an antibody or nucleic acid has a hydroxyl group, it can be used as the growth starting point 4. When the selective bonding group 3 does not have a hydroxyl group, for example, the hydroxyl group as the growth starting point 4 can be formed by oxidizing the selective bonding group 3.
（結合工程）
結合工程８では、図４に示すように、修飾物質５を含む溶液を検体１が固定された支持体２へ供給し、修飾物質５を検体１へ結合させる。例えば、固定工程７で検体１が固定された支持体２と修飾物質５を含む溶液をビーカーに入れ、スターラー等で良く攪拌して修飾物質５を検体１へ結合させる。検体１と選択性結合基３が核酸である場合、融解温度より５℃低く温度調節を行なうことで効率よく両者を結合させることができる。また、修飾物質５を検体１の量に対して過剰に供給することで、検体１と修飾物質５との結合確率をあげることができる。溶液の溶媒は、有機溶媒、純水等を検体１に応じて、適宜使用することができるが、血液等の生体由来の検体１等に対しては、変性をさけるため、純水を使用することが好ましい。 (Joining process)
In the binding step 8, as shown in FIG. 4, a solution containing the modifying substance 5 is supplied to the support 2 on which the specimen 1 is fixed, and the modifying substance 5 is bound to the specimen 1. For example, a solution containing the support 2 and the modifying substance 5 on which the sample 1 is fixed in the fixing step 7 is put in a beaker and stirred well with a stirrer or the like to bind the modifying substance 5 to the sample 1. When the specimen 1 and the selective binding group 3 are nucleic acids, they can be efficiently bound by adjusting the temperature 5 ° C. below the melting temperature. In addition, by supplying the modifying substance 5 in excess of the amount of the specimen 1, the binding probability between the specimen 1 and the modifying substance 5 can be increased. As the solvent of the solution, an organic solvent, pure water, or the like can be used as appropriate according to the specimen 1, but pure water is used for the specimen 1 or the like derived from a living body such as blood in order to avoid denaturation. It is preferable.
結合工程８で使用される修飾物質５は溶液中に均一に存在していることで、検体１との結合反応性の向上ができ、安定した検出が可能となる。また、検体１の多くは生体由来であり、検体１の変性を防止するため、本発明のシステムで使用される溶液の溶媒は純水であることが好ましく、修飾物質５は水溶性であることが好ましい。 Since the modifying substance 5 used in the binding step 8 is uniformly present in the solution, the binding reactivity with the specimen 1 can be improved, and stable detection is possible. Further, most of the specimen 1 is derived from a living body, and in order to prevent denaturation of the specimen 1, the solvent of the solution used in the system of the present invention is preferably pure water, and the modifying substance 5 is water-soluble. Is preferred.
（除去工程）
除去工程９では、図５に示すように、検体１と結合しなかった余剰の修飾物質５を洗浄等により除去する。例えば、支持体２と純水をビーカーに入れ、超音波洗浄を行なうことで余剰の修飾物質５を除去することができる。また、支持体２へ流路を形成しておき、ポンプ等で純水を流して洗浄しても良い。 (Removal process)
In the removal step 9, as shown in FIG. 5, excess modifying substance 5 that has not bound to the specimen 1 is removed by washing or the like. For example, the surplus modifier 5 can be removed by placing the support 2 and pure water in a beaker and performing ultrasonic cleaning. Alternatively, a flow path may be formed in the support 2 and the water may be washed by flowing pure water with a pump or the like.
（成長工程および成長物質）
成長工程１０では、図６に示すように、検体１と結合した修飾物質５の成長起点４に作用する薬液を、除去工程９を行なった支持体２に供給して成長物質６を物理的に成長させる。例えば、除去工程９を行なった支持体２と薬液をビーカーに入れ、成長起点４から成長物質６を発生させる。発生した成長物質６は、その表面が薬液と反応し、さらに成長物質６が発生することで成長する。任意の時間で薬液から支持体２を取り出すことで成長を止め、成長物質６を所望の大きさにすることができる。 (Growth process and growth material)
In the growth step 10, as shown in FIG. 6, a chemical solution that acts on the growth starting point 4 of the modifying substance 5 bound to the specimen 1 is supplied to the support 2 that has been subjected to the removal step 9, so that the growth substance 6 is physically supplied. Grow. For example, the support 2 and the chemical solution that have been subjected to the removing step 9 are placed in a beaker, and the growth material 6 is generated from the growth starting point 4. The generated growth material 6 grows when the surface thereof reacts with the chemical solution and the growth material 6 is generated. By removing the support 2 from the chemical solution at an arbitrary time, the growth can be stopped and the growth material 6 can be made to have a desired size.
成長工程１０においては、無電解めっき法による金属析出を用いることができる。ここで無電解めっきとは、外部電源を用いることなく、溶液中の金属を堆積させることをいう。その過程は、金属イオンと還元剤とを含む無電解めっき液が触媒表面で化学的に還元されて金属原子となる。そして、新しく堆積した金属層が次に堆積する金属の触媒となる自己触媒反応が起こり成長していく。成長工程１０では、修飾物質５の成長起点４を金属触媒とし、無電解めっき液を修飾物質５と結合した検体１が固定された支持体２に供給することで、金属の成長物質６を析出させて成長させることができる。無電解めっきの金属触媒としては、Ｐｄが良く知られているが、例えば、Ｆｅ、Ｃｏ、Ｎｉ、Ｃｕ、Ｒｕ、Ｒｈ、Ｏｓ、Ｉｒ、Ｐｔ、Ａｇ、Ａｕ、又はそれらを組み合わせた合金でも良い。また、成長物質６としての金属としては、例えば、Ｃｏ、Ｎｉ、Ｃｕ、Ｒｕ、Ｒｈ、Ｐｄ、Ｐｔ、Ａｇ、Ａｕ又はその合金を用いることができる。無電解めっき液は、上記金属の水溶性ハロゲン化物、硫酸塩及びスルファミン酸塩水溶液とアミンボラン化合物、ヒドラジン化合物、亜リン酸、次亜リン酸又はこれらの塩等の還元剤を組み合わせて作製することができるが、日本カニゼン株式会社、メルテックス株式会社等から市販されている無電解めっき液を使用することもできる。一般的な無電解めっき液としては、Ｎｉ塩と還元剤として次亜リン酸塩が使用されており、成長物質６はリン（Ｐ）を含有する。含有するリン（Ｐ）が多いと成長物質６は非磁性体となるが、ヒドラジン化合物を還元剤とすることで成長物質６を強磁性体とすることができる。無電解めっき法により成長した金属の成長物質６は反射率が高く、検出工程１１において光学顕微鏡でも容易に検出することができ、また、金属である特性を使い、電流センサー等でも検出できる。 In the growth step 10, metal deposition by an electroless plating method can be used. Here, the electroless plating refers to depositing a metal in a solution without using an external power source. In this process, an electroless plating solution containing metal ions and a reducing agent is chemically reduced on the catalyst surface to become metal atoms. Then, the newly deposited metal layer undergoes an autocatalytic reaction that becomes a catalyst for the next deposited metal and grows. In the growth step 10, the growth starting point 4 of the modifying substance 5 is used as a metal catalyst, and the electroless plating solution is supplied to the support 2 to which the specimen 1 combined with the modifying substance 5 is fixed, thereby depositing the growing substance 6 of the metal. Let it grow. Pd is well known as a metal catalyst for electroless plating, but may be, for example, Fe, Co, Ni, Cu, Ru, Rh, Os, Ir, Pt, Ag, Au, or an alloy combining them. . Moreover, as a metal as the growth material 6, for example, Co, Ni, Cu, Ru, Rh, Pd, Pt, Ag, Au, or an alloy thereof can be used. The electroless plating solution is prepared by combining a water-soluble halide, sulfate and sulfamate aqueous solution of the above metal with a reducing agent such as an amine borane compound, a hydrazine compound, phosphorous acid, hypophosphorous acid or a salt thereof. However, an electroless plating solution commercially available from Nippon Kanisen Co., Ltd., Meltex Co., Ltd. or the like can also be used. As a general electroless plating solution, Ni salt and hypophosphite are used as a reducing agent, and the growth material 6 contains phosphorus (P). The growth material 6 becomes a non-magnetic material when a large amount of phosphorus (P) is contained, but the growth material 6 can be made a ferromagnetic material by using a hydrazine compound as a reducing agent. The metal growth material 6 grown by the electroless plating method has high reflectivity, and can be easily detected by an optical microscope in the detection step 11, and can also be detected by a current sensor or the like using the property of being a metal.
また、成長工程１０においては、金属酸化物を成長物質６とすることができる。修飾物質５の成長起点４を水酸基とし、成長工程１０で金属塩水溶液と酸化剤を修飾物質５（修飾物質５と結合した検体１が固定された支持体２）に供給することで、金属酸化物の成長物質６を析出させて成長させることができる。金属塩水溶液と酸化剤は、支持体２への供給前に混合しても良いが、不要な反応を防止するため、別系統で支持体２へ供給することが好ましく、金属塩水溶液と酸化剤を交互に支持体２へ供給することもできる。例えば、金属塩水溶液として塩化第一鉄（ＦｅＣｌ２・４Ｈ２Ｏ）水溶液と酸化剤として亜硝酸ナトリウム（ＮａＮＯ２）水溶液を支持体２へ供給し、修飾物質５の水酸基に２価Ｆｅイオンを吸着させ、吸着した２価Ｆｅイオンの一部を亜硝酸イオンによって酸化するとともにこれを水和させる。この水和された面に再び２価Ｆｅイオンが吸着し、吸着した２価Ｆｅイオンの一部を酸化し水和させる。こうした反応を繰り返すことにより、スピネル構造を有するマグネタイト（Ｆｅ３Ｏ４）を成長物質６として析出させて成長させることができる。また、金属イオンは、特に限定されず、例えば、Ｆｅ、Ｎｉ、Ｃｕ、Ｃｏ、Ｍｎ、Ｖ、Ｌｉ、Ｍｏ、Ｔｉ、Ｍｇ、Ａｌ、Ｓｉ、Ｃｒ、Ｓｎ、Ｃａ、Ｃｄ、ＩｎおよびＧａから１種または２種以上の元素から選択しても良い。酸化剤は、金属イオンに対して適宜選択され、亜硝酸塩、過酸化水素等各種酸化剤が使用できる。本工程は、高価な希少金属触媒を必要とせず、簡便な方法で成長物質６を成長させることができる。 In the growth step 10, a metal oxide can be used as the growth material 6. The growth starting point 4 of the modifying substance 5 is a hydroxyl group, and an aqueous metal salt solution and an oxidizing agent are supplied to the modifying substance 5 (support 2 on which the specimen 1 bound to the modifying substance 5 is fixed) in the growth step 10, thereby oxidizing the metal. The material growth material 6 can be deposited and grown. The aqueous metal salt solution and the oxidizing agent may be mixed before being supplied to the support 2, but in order to prevent unnecessary reaction, it is preferable to supply the aqueous solution and the oxidizing agent to the support 2 separately. Can be alternately supplied to the support 2. For example, a ferrous chloride (FeCl 2 .4H 2 O) aqueous solution as a metal salt aqueous solution and a sodium nitrite (NaNO 2 ) aqueous solution as an oxidizing agent are supplied to the support 2, and divalent Fe ions are added to the hydroxyl group of the modifier 5. It is adsorbed, and a part of the adsorbed divalent Fe ion is oxidized by nitrite ion and hydrated. Divalent Fe ions are adsorbed again on the hydrated surface, and a part of the adsorbed divalent Fe ions is oxidized and hydrated. By repeating such a reaction, magnetite (Fe 3 O 4 ) having a spinel structure can be deposited and grown as the growth material 6. Further, the metal ion is not particularly limited, and for example, Fe, Ni, Cu, Co, Mn, V, Li, Mo, Ti, Mg, Al, Si, Cr, Sn, Ca, Cd, In, and Ga 1 You may select from seeds or two or more elements. The oxidizing agent is appropriately selected for metal ions, and various oxidizing agents such as nitrite and hydrogen peroxide can be used. In this step, an expensive rare metal catalyst is not required, and the growth material 6 can be grown by a simple method.
本発明の成長物質６は、強磁性体とすることができ、その例として、Ｆｅ、Ｍｎ、Ｃｏ、Ｎｉ等の金属、若しくはそれらの少なくとも１つを含む合金または酸化物などが挙げられる。 The growth material 6 of the present invention can be a ferromagnetic material, and examples thereof include metals such as Fe, Mn, Co, and Ni, or alloys or oxides containing at least one of them.
（成長物質の検出）
検出工程では、物理的に成長した成長物質６を検出する。簡便な検出方法としては、例えば光学顕微鏡による検出を用いることができる。光学顕微鏡の分解能は２５０ｎｍ程度であるため、成長物質６を分解能より大きく成長させることで、成長物質６を検出することができる。また、成長物質６を強磁性体とし場合、コイル、電線、磁気抵抗効果センサー、ホール・センサー、平面型ホール・センサー又はフラックス・ゲート・センサー等の磁気センサーを使用して成長物質６を磁気検出により検出することができる。光学顕微鏡による検出は、レンズ構成や照明機器、画像処理装置等のシステムが大きくなり、また、分解能は光の波長の制限を受けるが、磁気検出には、波長制限がなく、高感度であり、システムの小型化が可能である。さらに外部磁界をかけることで、成長物質６をより感度良く検出することができる。 (Growth substance detection)
In the detection step, the physically grown growth material 6 is detected. As a simple detection method, for example, detection by an optical microscope can be used. Since the resolution of the optical microscope is about 250 nm, the growth material 6 can be detected by growing the growth material 6 larger than the resolution. When the growth material 6 is a ferromagnetic material, the growth material 6 is magnetically detected by using a magnetic sensor such as a coil, an electric wire, a magnetoresistive sensor, a Hall sensor, a planar Hall sensor, or a flux gate sensor. Can be detected. Detection by an optical microscope has a large system such as a lens configuration, an illumination device, and an image processing device, and resolution is limited by the wavelength of light, but magnetic detection has no wavelength limitation and is highly sensitive. The system can be miniaturized. Furthermore, the growth substance 6 can be detected with higher sensitivity by applying an external magnetic field.
このように、本実施形態の検出システムによれば、検体１に固定された修飾物質５に成長物質６を成長させ、成長後の成長物質６を検出することで、簡便な方法で高感度の検体検出を行うことができる。 As described above, according to the detection system of the present embodiment, the growth material 6 is grown on the modifying material 5 fixed to the specimen 1, and the growth material 6 after the growth is detected. Specimen detection can be performed.
さらに本実施形態の検出システムによれば、修飾物質５が水溶性であることで、修飾物質５の濃度分布の低減、検体１との結合反応性の向上ができ、安定した検体検出が可能な検出システムを提供できる。 Furthermore, according to the detection system of the present embodiment, since the modifying substance 5 is water-soluble, the concentration distribution of the modifying substance 5 can be reduced, the binding reactivity with the specimen 1 can be improved, and stable specimen detection is possible. A detection system can be provided.
さらに本実施形態の検出システムは、成長起点４が金属触媒であり、成長工程１０において無電解めっき法による金属析出により成長物質６として金属を成長させるので、簡便な方法で、成長物質６を成長させることができる。 Furthermore, in the detection system of this embodiment, the growth starting point 4 is a metal catalyst, and the growth material 6 is grown as a growth material 6 by metal deposition by an electroless plating method in the growth step 10, so that the growth material 6 is grown by a simple method. Can be made.
さらに本実施形態の検出システムは、成長起点４が水酸基であり、成長工程１０において、金属塩水溶液と酸化剤を修飾物質５に供給して、成長物質６として金属酸化物を析出させて成長させるので、希少金属を用いなくても、簡便な方法で成長物質を成長させることができる。 Furthermore, in the detection system of the present embodiment, the growth starting point 4 is a hydroxyl group, and in the growth step 10, a metal salt aqueous solution and an oxidizing agent are supplied to the modifying substance 5, and a metal oxide is deposited as the growing substance 6 to grow. Therefore, the growth material can be grown by a simple method without using a rare metal.
さらに本実施形態の検出システムは、成長物質６が強磁性体であり、磁気検出により成長後の成長物質６を検出するので、検体検出の高感度化、装置の小型化ができる。 Furthermore, in the detection system of the present embodiment, the growth material 6 is a ferromagnetic material, and the growth material 6 after growth is detected by magnetic detection, so that the sensitivity of the specimen detection can be increased and the apparatus can be downsized.
また、本実施形態の修飾物質５は、検体１への選択性結合基３と成長起点４とを有するものであるので、本実施形態の検出システムに用いられることで、簡便な方法で高感度の検体検出を行うことができる。 In addition, since the modifying substance 5 of the present embodiment has the selective binding group 3 and the growth starting point 4 for the specimen 1, it can be used in the detection system of the present embodiment and can be highly sensitive by a simple method. Specimen detection can be performed.
さらに本実施形態の修飾物質５は水溶性であることで、修飾物質５の濃度分布の低減、検体１との結合反応性の向上ができる。 Furthermore, since the modifying substance 5 of this embodiment is water-soluble, the concentration distribution of the modifying substance 5 can be reduced and the binding reactivity with the specimen 1 can be improved.
　なお、本実施形態では、固定工程７、結合工程８、除去工程９、成長工程１０および検出工程１１をこの順に実施しているが、各工程の実施順序はこれに限られない。例えば、結合工程８を固定工程７よりも先に行っても良いし、成長工程１０の後に固定工程７および除去工程９を行っても良い。 In this embodiment, the fixing process 7, the bonding process 8, the removal process 9, the growth process 10 and the detection process 11 are performed in this order, but the execution order of each process is not limited to this. For example, the bonding step 8 may be performed before the fixing step 7, or the fixing step 7 and the removing step 9 may be performed after the growth step 10.
　１　　検体
　２　　支持体
　２ａ　表面修飾
　３　　選択結合基
　４　　成長起点
　５　　修飾物質
　６　　成長物質
　７　　固定工程
　８　　結合工程
　９　　除去工程
１０　　成長工程
１１　　検出工程 DESCRIPTION OF SYMBOLS 1 Specimen 2 Support 2a Surface modification 3 Selective binding group 4 Growth origin 5 Modulating substance 6 Growing substance 7 Fixing process 8 Binding process 9 Removal process 10 Growth process 11 Detection process

Claims (10)

  1. 検体を支持体に固定する固定工程と、前記検体への選択性結合基と成長起点を有する修飾物質を前記検体に結合させる結合工程と、前記検体と結合していない前記修飾物質を除去する除去工程と、前記成長起点に成長物質を成長させる成長工程を有し、成長後の前記成長物質を検出することを特徴とする検出システム。 An immobilization step for immobilizing a specimen on a support, a binding step for binding a modifying substance having a selective binding group and a growth starting point to the specimen to the specimen, and a removal for removing the modifying substance not bound to the specimen. And a growth step of growing a growth material at the growth starting point, and detecting the growth material after growth.
  2. 前記修飾物質が水溶性であることを特徴とする請求項１に記載の検出システム。 The detection system according to claim 1, wherein the modifying substance is water-soluble.
  3. 前記成長起点が金属触媒であり、前記成長工程において無電解めっき法による金属析出により前記成長物質として金属を成長させることを特徴とする請求項１または２に記載の検出システム。 The detection system according to claim 1 or 2, wherein the growth starting point is a metal catalyst, and a metal is grown as the growth material by metal deposition by an electroless plating method in the growth step.
  4. 前記成長起点が水酸基であり、前記成長工程において、金属塩水溶液と酸化剤を前記修飾物質に供給して、前記成長物質として金属酸化物を析出させて成長させることを特徴とする請求項１または２に記載の検出システム。 The growth starting point is a hydroxyl group, and in the growth step, a metal salt aqueous solution and an oxidizing agent are supplied to the modifying substance, and a metal oxide is deposited as the growing substance to grow. 2. The detection system according to 2.
  5. 前記成長物質が強磁性体であり、磁気検出により成長後の前記成長物質を検出することを特徴とする請求項１ないし４のいずれか一項に記載の検出システム。 The detection system according to any one of claims 1 to 4, wherein the growth material is a ferromagnetic substance, and the growth material after growth is detected by magnetic detection.
  6. 検体への選択性結合基と成長起点とを有することを特徴とする修飾物質。 A modifying substance having a selective binding group to a specimen and a growth starting point.
  7. 水溶性であることを特徴とする請求項６に記載の修飾物質。 The modifying substance according to claim 6, wherein the modifying substance is water-soluble.
  8. 前記成長起点が金属触媒である請求項６または７に記載の修飾物質。 The modifying substance according to claim 6 or 7, wherein the growth starting point is a metal catalyst.
  9. 前記成長起点が水酸基である請求項６または７に記載の修飾物質。 The modifying substance according to claim 6 or 7, wherein the growth starting point is a hydroxyl group.
  10. 前記選択性結合基が抗体または核酸である請求項６ないし９のいずれか一項に記載の修飾物質。 The modifying substance according to any one of claims 6 to 9, wherein the selective binding group is an antibody or a nucleic acid.
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