JP4660431B2 - Biological material detection / recovery method and apparatus, and biological material analysis method - Google Patents
Biological material detection / recovery method and apparatus, and biological material analysis method Download PDFInfo
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Description
本発明は、試料溶液中に含まれる生体分子を検出し、生体物質検出・回収方法およびその装置及び生体物質解析方法に関する。 The present invention relates to a biological material detection / recovery method, a device for detecting a biomolecule contained in a sample solution, and a biological material analysis method.
生体物質は、核酸、ｍＲＮＡなどのＲＮＡ、アミノ酸、ジペプチド、トリペプチドなどのオリゴペプチド、タンパク質などのポリペプチド、単糖、２単糖やオリゴ糖、多糖類などの糖類、ステロイドなどのホルモン類、ノルアドレナリン、ドーパミン、セロトニンなどの神経伝達物質、そのほか内分泌攪乱剤、各種薬剤、カリウム、ナトリウム、塩化物イオン、水素イオン、また、近年様々な用途に応用される高分子ポリマーなどが挙げられる。このように生命現象に関わる物質は多種多様で、様々な化学的特性を有している。従来、物質ごとの特性を利用した多種多様な検出、分離方法が考案され利用されてきた。 Biological substances include nucleic acids, RNA such as mRNA, amino acids, dipeptides, oligopeptides such as tripeptides, polypeptides such as proteins, monosaccharides, saccharides such as disaccharides and oligosaccharides, polysaccharides, hormones such as steroids, Examples include neurotransmitters such as noradrenaline, dopamine, and serotonin, other endocrine disruptors, various drugs, potassium, sodium, chloride ions, hydrogen ions, and high-molecular polymers that are applied to various uses in recent years. In this way, there are a wide variety of substances related to life phenomena and various chemical properties. Conventionally, a wide variety of detection and separation methods using the characteristics of each substance have been devised and used.
アミノ酸や糖の分離に最も用いられている方法として、液体クロマトグラフィーが挙げられる。この方法では、形状のバリエーションだけではなく、分離単体の種類や分離溶媒の種類が多様であるので、最適な条件下で実行することで多くの生化学物質や化学物質を分離することが可能である。あるいはこれに類似の技術で、溶液の搬送を電気浸透流で行う方法も利用されている。また、タンパク質やポリヌクレオチド（ＤＮＡやＲＮＡ）などの、電荷を持った高分子の分離には一般的に電気泳動が用いられる。電気泳動においても、分離単体の選択と、溶媒の選択（多くの場合、ｐＨと静電力のコントロールを行う）により、一般的には、サイズの２％くらいまでの違いを識別し分離できる。あるいはチャージの違いで分ける等電点電気泳動では０．０２ｐＨの違いに対応する等電点の違いでタンパク質を分離できる。ポリヌクレオチドの分野で、ＤＮＡシーケンサーに用いられている技術では、類似配列のＤＮＡに限れば、７００ｂｐと７０１ｂｐのＤＮＡを長さの差で分離することも可能である。 The most used method for separating amino acids and sugars is liquid chromatography. In this method, not only variations in shape but also various types of separation units and types of separation solvents are available, so it is possible to separate many biochemical substances and chemical substances by executing them under the optimum conditions. is there. Alternatively, a method in which the solution is transported by electroosmotic flow using a similar technique is also used. Electrophoresis is generally used to separate charged polymers such as proteins and polynucleotides (DNA and RNA). Also in electrophoresis, the difference of up to about 2% in size can be generally identified and separated by selecting a single unit of separation and selecting a solvent (in many cases, controlling pH and electrostatic force). Alternatively, in isoelectric focusing separated by charge difference, proteins can be separated by isoelectric point differences corresponding to 0.02 pH differences. In the field of polynucleotides, in the technique used for DNA sequencers, it is possible to separate 700 bp and 701 bp DNAs by the difference in length, as long as the DNAs have similar sequences.
タンパク質を高感度に検出する方法として、イムノアッセイが挙げられる。この方法は臨床検査にも応用されており、タンパク質だけではなく抗体が認識することができる化合物の検出も可能である。細胞中で発現しているｍＲＮＡを解析する方法としては網羅的に解析するＤＮＡチップやｍＲＮＡがＰＣＲにて増幅する速度から被検出対象ｍＲＮＡの濃度を検出するリアルタイムＰＣＲ法が有効である。 An immunoassay is mentioned as a method for detecting a protein with high sensitivity. This method is also applied to clinical tests, and it is possible to detect not only proteins but also compounds that can be recognized by antibodies. As a method for analyzing mRNA expressed in cells, a real-time PCR method for detecting the concentration of mRNA to be detected from the speed of amplification of the DNA chip or mRNA to be comprehensively analyzed by PCR is effective.
上記したように、生化学研究の分野は様々な分離法や検出法に支えられて発展してきた。生命の構成要素を成分ごとに分離し、それらの特性を明らかにすることで、生命現象全体が再構築できると考えられていたからである。
一方で、近年のゲノム研究をはじめとするオーミクス研究では、生体の構成要因は遺伝子だけでも数万に及び、それ以外に、ゲノム情報によらずに関係し合う化学物質や物質間の相互作用は膨大な数にのぼることが明らかになりつつある。このため、生命現象は物質の複雑な相互作用の結果であるという古典的な解釈が再浮上している。
As described above, the field of biochemical research has been developed supported by various separation methods and detection methods. This is because it was thought that the whole life phenomenon could be reconstructed by separating the components of life into components and clarifying their characteristics.
On the other hand, in recent omics research such as genome research, there are tens of thousands of components of living organisms alone, and in addition to this, there are no relations between chemical substances and interrelated substances regardless of genomic information. It is becoming clear that the number is enormous. For this reason, the classical interpretation that life phenomena are the result of complex interactions of matter has resurfaced.
前記生体物質の相互作用を検出する方法としては、溶液系の反応場で検出する方法と固相表面を反応場とし測定する方法がある。
前者の方法では、物質間が相互作用した際に生じる熱量を測定する等温滴定カロリメトリー法、核磁気共鳴法（ＮＭＲ）によって分子の構造変化をモニターする方法、蛍光共鳴エネルギー転移法が挙げられる。
後者の方法では、表面プラズモン共鳴法や水晶振動子を利用した方法が挙げられる。表面プラズモン共鳴センサーは、金属薄膜に全反射する光を入射した際に生じる微弱なエネルギー波（エバネッセント波）が誘電体と接触している金属表面における粗密波（表面プラズモン）と共鳴する事で全反射光が減衰する現象（ＳＰＲ現象）を応用する。生体物質間の相互作用によって生じた金属薄膜表面の誘電率変化をＳＰＲ現象の減衰ピークの生じる角度変化によって検出する水晶振動子は、水晶板の圧電効果を利用し、水晶板に一定の電圧を印加することで一定の周波数で発振する素子を用いる。水晶板表面に負荷される質量や粘性および弾性の変化によって周波数が変化し、生体物質が相互作用した際に生じる質量負荷の変化を周波数として検出することができる。この他に、表面の屈折率を計測するエリプソメーター、二面編波式干渉法、表面弾性波を利用した方法がある。
As a method for detecting the interaction of the biological substance, there are a method for detecting in a solution reaction field and a method for measuring using a solid phase surface as a reaction field.
Examples of the former method include an isothermal titration calorimetry method for measuring the amount of heat generated when substances interact with each other, a method for monitoring molecular structural changes by nuclear magnetic resonance (NMR), and a fluorescence resonance energy transfer method.
Examples of the latter method include a surface plasmon resonance method and a method using a crystal resonator. A surface plasmon resonance sensor resonates with a minute energy wave (evanescent wave) generated when light that is totally reflected on a metal thin film is incident and resonates with a rough wave (surface plasmon) on a metal surface in contact with a dielectric. A phenomenon (SPR phenomenon) in which reflected light attenuates is applied. A quartz crystal resonator that detects changes in the dielectric constant on the surface of a metal thin film caused by interaction between biological materials by the angle change that causes the decay peak of the SPR phenomenon, uses the piezoelectric effect of the quartz plate to apply a constant voltage to the quartz plate. An element that oscillates at a constant frequency when applied is used. The frequency changes due to changes in mass, viscosity, and elasticity loaded on the quartz plate surface, and a change in mass load that occurs when a biological substance interacts can be detected as a frequency. In addition, there are an ellipsometer for measuring the refractive index of the surface, a two-sided knitting wave interferometry, and a method using surface acoustic waves.
また、近年急速に発展しているDrug Delivery System（以下「DDS」とする。）と呼ばれる技術では、薬剤を患部組織へ直接作用させると同時に、正常細胞への影響を抑えることで薬剤の副作用を軽減を図る技術である。DDS技術にはactive targetingとpassive targetingという二つの概念が存在し、前者はモノクローナル抗体のように物質間の特異的結合能を利用し標的組織への輸送を図る。後者は、腫瘍血管の増生やリンパ系の未熟、また著しい腫瘍血管透過性の亢進などの特徴を利用するものであり、生体親和性に富む高子ポリマー修飾薬剤またはミセル、リポソームなどのナノ粒子に含有される薬剤は正常血管からは漏出せず、腫瘍血管からは漏れやすく結果として、passive targeting が可能となる。この他にも、人工臓器および医療用デバイスの開発によって様々な脂質分子やそのアナログ分子が開発され利用されている。 In addition, a technology called Drug Delivery System (hereinafter referred to as “DDS”), which has been rapidly developing in recent years, allows the drug to act directly on the affected tissue and at the same time reduce the side effects of the drug by suppressing the effect on normal cells. This is a technology for mitigation. The DDS technology has two concepts, active targeting and passive targeting. The former uses specific binding ability between substances like a monoclonal antibody to transport to the target tissue. The latter uses features such as tumor vascular proliferation, lymphatic immaturity, and markedly increased tumor vascular permeability, and is highly biocompatible with high-molecular weight polymer-modified drugs or micelles and liposomes. The contained drug does not leak from normal blood vessels, but easily leaks from tumor blood vessels. As a result, passive targeting is possible. In addition to this, various lipid molecules and analog molecules thereof have been developed and used by the development of artificial organs and medical devices.
上記生体物質の相互作用の検出、さらに、先に述べた物質の検出、同定及び分離方法には各種のものがあるが、次のような問題点がある。 There are various methods for detecting the interaction of the biological substances, and the methods for detecting, identifying and separating the substances described above, but have the following problems.
従来から所定量の試料溶液から生体物質を分離する方法としては、分離部（カラム）においてその担体と溶媒界面における媒質の分配に依存し分離されるクロマトグラフィー技術や担体と相互作用をする物質を分離する電気泳動が多く用いられてきた。分離された生体物質は質量分析法やＤＮＡシーケンサーによって同定され、機能解析が行われる。生体物質を機能も様々であるため多種多様な方法で物質間相互作用や分光学的手法による構造解析などが行われる。
その一例として、表面プラズモン共鳴センサーを用いて、認識物質に対し相互作用した物質の活性を測定した後、センサー上で捕捉した物質を回収して質量分析法にて同定する報告がされている（非特許文献１）。
しかし、固相表面と液相間との反応はLangmuirの法則に従う平衡反応であると同時に、ごく限られた領域の検出部で物質を捕捉、回収することになるので、元の試料溶液中の被検出対象物質濃度がとても濃くなければ、後の分析に耐えうるサンプル量を得る事は困難であった。また、抗原抗体反応、ＤＮＡの相補鎖同士のハイブリダイゼーションおよび認識物質が一般的にアプタマーと呼ばれているある特定物質に対し特異的に結合するポリヌクレオチドであれば結合定数も強く、非常に安定した結合体を形成するが、結合能が低い物質間相互作用においてはほとんどが試料溶液中に未反応で残ってしまい、回収が困難であった。
Conventionally, as a method of separating a biological substance from a predetermined amount of a sample solution, a separation technique (column) is a chromatography technique that is separated depending on the distribution of the medium at the carrier-solvent interface, or a substance that interacts with the carrier. Separation electrophoresis has often been used. The separated biological material is identified by mass spectrometry or a DNA sequencer and subjected to functional analysis. Since biological materials have various functions, the interaction between substances and structural analysis by spectroscopic techniques are performed by various methods.
As an example of this, after measuring the activity of a substance that interacted with a recognition substance using a surface plasmon resonance sensor, there has been a report of collecting the substance captured on the sensor and identifying it by mass spectrometry ( Non-patent document 1).
However, the reaction between the solid phase surface and the liquid phase is an equilibrium reaction according to Langmuir's law, and at the same time, the substance is captured and recovered by the detection part in a very limited area. Unless the concentration of the target substance to be detected was very high, it was difficult to obtain a sample amount that could withstand subsequent analysis. In addition, antigen-antibody reaction, hybridization between complementary strands of DNA, and a recognition substance that binds specifically to a specific substance, generally called an aptamer, has a strong binding constant and is very stable. However, most of the interactions between substances having low binding ability remain unreacted in the sample solution, making it difficult to recover.
また、極性脂質を基本構成成分とする生体物質、たとえば脂質膜やそれに含まれる糖鎖、脂質膜中に存在することで正常に機能する膜タンパク質などは成分ごとに単離してしまうことによって、本来の機能を示さない。また脂質膜にはそれ自体を反応場とすることが知られており、脂質膜と相互作用している状態で初めて機能を発現する生体物質がある。このように極性脂質を基本構成成分とする認識物質により特定の機能を有する生体物質を解析する場合には、相互作用解析、具体的には結合の有無やその親和性を解析し、それと同時に構造解析などの機能解析を行う必要がある。
また、DDS技術や医療用デバイス、人工臓器といった技術の発展によって、細胞膜を構成するphosphatidylcholineの極性基と同一の構造をもつ2-メタクリロイルオキシエチルホスホリルコリンの重合体（以下「MPCポリマ」とする。）のような様々なポリマーやリポソーム試薬が開発されており、その生体物質の非特異的吸着や適合性を評価する方法が求められている。
In addition, biological substances that have polar lipids as basic constituents, such as lipid membranes, sugar chains contained in them, and membrane proteins that function normally when present in lipid membranes are isolated for each component. Does not show the function. Lipid membranes are known to use the reaction field as a reaction field, and there are biological substances that express their functions only when they interact with the lipid membrane. In this way, when analyzing a biological substance having a specific function with a recognition substance having a polar lipid as a basic component, an analysis of the interaction, specifically the presence or absence of binding and its affinity, is performed simultaneously with the structure. It is necessary to perform functional analysis such as analysis.
With the development of technologies such as DDS technology, medical devices, and artificial organs, a polymer of 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as “MPC polymer”) having the same structure as the polar group of phosphatidylcholine constituting the cell membrane. As described above, various polymers and liposome reagents have been developed, and a method for evaluating non-specific adsorption and compatibility of the biological material is required.
そこで、本発明は、極性脂質を基本成分とする生体物質を含む試料溶液を複数の試料溶液の中から確実に選択できるようにし、さらに、その選択された試料溶液の中から生体物質の本来の機能を阻害することなく回収して、他の解析に使用することが可能な生体物質検出・回収方法及びその装置を提供する。また、得られた生体物質を使用して該物質の解析を行う方法を提供することを目的とする。 Therefore, the present invention makes it possible to reliably select a sample solution containing a biological material having a polar lipid as a basic component from among a plurality of sample solutions, and further, from the selected sample solution, the original biological material. Provided are a biological material detection / recovery method and apparatus which can be collected without impairing the function and used for other analyses . Another object of the present invention is to provide a method for analyzing the obtained biological material.
本発明の生体物質検出・回収方法は、請求項１に記載の通り、試料溶液中の標的物質の検出を、固相表面に極性脂質を基本成分とする認識物質を固定化して前記標的物質との生体物質間の相互作用により生じる前記固相表面の物理的特性の変化に基づいて行う工程、前記極性脂質のリポソーム構造を認識素子として用い前記標的物質が検出された前記試料溶液中の前記標的物質を捕捉する工程及び前記標的物質を捕捉した前記認識素子を回収する工程を有することを特徴とする。
また、請求項２に記載の本発明は、請求項１に記載の生体物質検出・回収方法において、前記生体物質間の相互作用を水晶振動子の周波数変化によって検出することを特徴とする。
また、請求項３に記載の本発明は、請求項１に記載の生体物質検出・回収方法において、前記生体物質間相互作用を表面プラズモン共鳴法センサ表面の誘電率変化により検出することを特徴とする。
また、請求項４に記載の本発明は、請求項１に記載の生体物質検出・回収方法において、前記標的物質がペプチド分子であることを特徴とする。
また、本発明の生体物質解析方法は、請求項５に記載の通り、請求項１に記載の生体物質検出・回収方法により回収された前記標的物質が捕捉された前記認識素子を分光学的手法によって構造解析を行う工程を有することを特徴とする。
また、本発明の生体物質解析方法は、請求項６に記載の通り、請求項１に記載の生体物質検出・回収方法により回収された前記標的物質が捕捉された前記認識素子を固体核磁気共鳴法によって構造解析を行うことを特徴とする。
また、本発明の生体物質検出・回収装置は、請求項７に記載の通り、試料溶液中の標的物質と相互作用する極性脂質を基本成分とする認識物質を固定化するための固相表面に備え、前記固相表面の物理的特性の変化に基づき前記標的物質を検出するための検出手段、前記標的物質が検出された前記試料溶液と前記認識物質のリポソーム構造である認識素子とを混合するための混合手段及び前記試料溶液から前記標的物質が捕捉された認識素子を回収するための遠心分離手段を備えたことを特徴とする。
また、請求項８に記載の本発明は、請求項７に記載の生体物質検出・回収装置において、前記検出手段は、水晶振動子のセンサーであることを特徴とする。
また、請求項９に記載の本発明は、請求項７に記載の生体物質検出・回収装置において、前記検出手段は、表面プラズモン共鳴センサーであることを特徴とする。
また、請求項１０に記載の本発明は、請求項７に記載の生体物質検出・回収装置において、前記検出手段の前記検出部と前記混合手段とは、前記試料溶液を搬送するための流路により接続されていることを特徴とする。
また、請求項１１に記載の本発明は、請求項７に記載の生体物質検出・回収装置において、前記検出手段の前記検出部と前記混合手段との間に、前記試料溶液を排出するための他の流路を備えたことを特徴とする。
According to the biological substance detection / recovery method of the present invention, as described in claim 1, the target substance in the sample solution is detected by immobilizing a recognition substance having a polar lipid as a basic component on the solid phase surface and the target substance. A step performed based on a change in physical properties of the solid phase surface caused by an interaction between the biological substances, and the target in the sample solution in which the target substance is detected using the liposome structure of the polar lipid as a recognition element The method includes a step of capturing a substance and a step of recovering the recognition element that has captured the target substance.
The present invention according to claim 2 is characterized in that in the biological material detection / recovery method according to claim 1, the interaction between the biological materials is detected by a change in the frequency of a crystal resonator.
The present invention according to claim 3 is characterized in that in the biological material detection / recovery method according to claim 1, the interaction between the biological materials is detected by a change in dielectric constant of a surface plasmon resonance sensor surface. To do.
According to a fourth aspect of the present invention, in the biological substance detection / recovery method according to the first aspect, the target substance is a peptide molecule.
In addition, the biological material analysis method of the present invention, as described in claim 5, is a spectroscopic technique in which the recognition element in which the target substance recovered by the biological material detection / recovery method according to claim 1 is captured is used. And a step of performing a structural analysis.
Further, according to the biological material analysis method of the present invention, as described in claim 6, the recognition element in which the target substance recovered by the biological material detection / recovery method according to claim 1 is captured is solid-state nuclear magnetic resonance. you and performing a structural analysis by law.
Also, the biological material detection and recovery device of the present invention, as described in claim 7, solid surface for immobilizing the recognition substance to a polar lipid that interacts with a target substance in a sample solution as basic components A detection means for detecting the target substance based on a change in physical properties of the solid phase surface, and mixing the sample solution in which the target substance is detected and a recognition element having a liposome structure of the recognition substance And a centrifuge for collecting the recognition element in which the target substance is captured from the sample solution.
The invention according to claim 8 is the biological material detection / collection device according to claim 7 , wherein the detection means is a sensor of a crystal resonator.
The present invention according to claim 9 is the biological substance detection / recovery device according to claim 7 , wherein the detection means is a surface plasmon resonance sensor.
The present invention according to claim 10 is the biological material detection / recovery device according to claim 7 , wherein the detection unit of the detection unit and the mixing unit are flow paths for transporting the sample solution. It is connected by this.
Further, the invention of claim 11 is the biological material detection and recovery device according to claim 7, between said detecting unit and said mixing means of said detecting means, for discharging said sample solution Another flow path is provided.
本発明により、生命機能の一端を担う重要な成分である脂質膜やそれに結合することで機能する物質に対する生化学物質の相互作用が検出できると同時に、回収することができる。これによって、より詳細な機能解析が行える。
また、極性脂質により構成されたリポソームを認識素子を使用することにより、固相表面と液相の平衡状態において捕捉できる標的物質を使用する場合に比べ、結合積が増えより効率的に試料溶液中の標的物質を回収することができる。この結果、相互作用の測定と標的物質の回収を効率的に行うことができ、回収された標的試料は他の分析手段に使用することができる。また、極性脂質を基本構成とする認識物質により特定の機能を有する生体物質を解析する場合に、相互作用解析、具体的には結合の有無やその親和性を解析し、それと同時に構造解析などの機能解析を行うことができる。
また、本発明における認識素子は、緩衝液の比重よりも重い糖類を主成分とした溶液等を含むために、沈殿しやすく遠心分離により容易に回収することができる。
認識物質と相互作用する標的物質を検出し効率的に標的物質を回収できるので、認識物質と相互作用する標的物質を検出し、効率的に標的物質を回収することが可能となるため、標的物質を他の手法、例えば固体核磁気共鳴法などによる解析が可能となる（Biophys. J 78, 2405-2417(2000)）。
固相表面での生体物質間相互作用は、水晶振動子センサーや表面プラズモンセンサーを利用できるため、容易にシステムを構築することが可能となる。
According to the present invention, the interaction of a biochemical substance with a lipid membrane, which is an important component that plays a role in vital functions, and a substance that functions by binding to the lipid film can be detected and simultaneously recovered. As a result, more detailed functional analysis can be performed.
In addition, by using a recognition element for liposomes composed of polar lipids, the binding product is increased more efficiently in the sample solution than when using a target substance that can be captured in an equilibrium state between the solid surface and the liquid phase. Target substance can be recovered. As a result, interaction measurement and target substance recovery can be performed efficiently, and the recovered target sample can be used for other analysis means. In addition, when analyzing biological substances with specific functions using recognition substances based on polar lipids, analysis of interaction, specifically the presence or absence of binding and its affinity, and at the same time, structural analysis, etc. Functional analysis can be performed.
In addition, since the recognition element in the present invention includes a solution mainly composed of a saccharide heavier than the specific gravity of the buffer solution, it easily precipitates and can be easily recovered by centrifugation.
Since the target substance that interacts with the recognition substance can be detected and the target substance can be efficiently recovered, it becomes possible to detect the target substance that interacts with the recognition substance and efficiently recover the target substance. Can be analyzed by other methods such as solid nuclear magnetic resonance (Biophys. J 78, 2405-2417 (2000)).
Since the interaction between biological materials on the solid phase surface can use a quartz crystal sensor or a surface plasmon sensor, a system can be easily constructed.
上述の通り、本発明は、標的物質を含む試料溶液の検出を、固相表面（本明細書において、固相表面あるいは固相表面に設けられた電極等を含む。）に極性脂質を基本成分とする認識物質を固定化して前記標的物質との生体物質間の相互作用により生じる前記固相表面の物理的特性の変化に基づいて行う工程、前記極性脂質により構成されたリポソームを認識素子として用い前記標的物質が検出された前記試料溶液中の前記標的物質を捕捉する工程及び前記標的物質を捕捉した前記認識素子を回収する工程を有するものである。
本明細書において、生体物質というのは生命現象に関わる物質一般を指し、本明細書における認識物質は極性脂質膜および極性脂質と複合体を形成しているものとする。そして、この認識物質と相互作用し得る物質を標的物質とする。
As described above, the present invention detects a sample solution containing a target substance by using a polar lipid as a basic component on a solid phase surface (in this specification, including a solid phase surface or an electrode provided on the solid phase surface). A step of immobilizing the recognition substance to be performed based on a change in physical properties of the solid phase surface caused by the interaction between the target substance and the biological substance, using a liposome composed of the polar lipid as a recognition element The method includes a step of capturing the target material in the sample solution in which the target material is detected and a step of recovering the recognition element that has captured the target material.
In this specification, a biological substance generally refers to a substance related to a life phenomenon, and the recognition substance in this specification is assumed to form a complex with a polar lipid membrane and a polar lipid. And let the substance which can interact with this recognition substance be a target substance.
以下に、各工程について説明する。
（標的物質を含む試料溶液の検出工程）
本発明における検出手段は、固相表面を反応検出場とし、標的物質と認識物質との生体物質間の相互作用により生じる固相表面の物理的特性を測定することができるセンサーであればよく、水晶振動子、表面プラズモン、エリプソメトリー、二面編波式干渉法および表面弾性波を利用したセンサーを使用することができる。
検出手段として水晶振動子を使用する場合には、水晶板の両面に設けられた電極のうちの一方の電極に、認識物質を固定化し、認識物質が固定化されている水晶板の片側を試料溶液に浸し、前記両電極に電圧を所定周波数で印加するようにし、その際に、電極上の認識物質と標的物質との相互作用による水晶板の周波数変動を両電極を介して測定すればよい。
また、検出手段として、表面プラズモンセンサーを使用する場合には、プリズム底面に金属層を設け、認識物質を積層し、プリズム底面を試料溶液を浸漬させ誘電率の変化を測定する。具体的には、表面プラズモン共鳴現象の減衰ピークの生じる角度変化を測定すればよい。
Below, each process is demonstrated.
(Detection process of sample solution containing target substance)
The detection means in the present invention may be any sensor that can measure the physical characteristics of the solid phase surface caused by the interaction between the biological substance of the target substance and the recognition substance, using the solid phase surface as a reaction detection field, It is possible to use a sensor utilizing a quartz oscillator, surface plasmon, ellipsometry, two-surface knitting wave interferometry, and surface acoustic wave.
When using a crystal resonator as the detection means, the recognition substance is fixed to one of the electrodes provided on both sides of the crystal plate, and one side of the crystal plate on which the recognition substance is fixed is sampled. Immerse it in a solution and apply a voltage to both electrodes at a predetermined frequency. At that time, the frequency fluctuation of the quartz plate due to the interaction between the recognition substance on the electrode and the target substance may be measured via both electrodes. .
When a surface plasmon sensor is used as the detecting means, a metal layer is provided on the bottom surface of the prism, a recognition substance is laminated, and a sample solution is immersed in the bottom surface of the prism to measure a change in dielectric constant. Specifically, it is only necessary to measure an angle change in which an attenuation peak of the surface plasmon resonance phenomenon occurs.
前記検出手段を構成する固相表面に、被検出対象である標的物質を特異的に認識する認識物質を固定化しておき、標的物質を含む試料溶液が認識物質と相互作用することにより、固相表面の物理的特性が変化し、認識物質と標的物質の相互作用、即ち、試料溶液中に標的物質を含むことが検出される。この際、試料溶液中の標的物質と認識物質との反応は平衡であるために、標的物質のほとんどが未反応で試料中に残るために以後の工程において捕捉と回収を行って、他の解析等に使用できる。
認識物質と相互作用が確認して標的物質を含むことが確認できた試料溶液は、そのまま次の工程に送られる。尚、標的物質を含むか否かの判断については、ゼロかそうでないか以外にも、所定の濃度をボーダーとしてその判断を行うようにしてもよい。また、標的物質を含まない試料溶液についてはドレイン等を介して排出する工程を設けることが好ましい。複数の試料溶液を連続して検出する場合に、標的物質を含まない溶液を排出すれば作業効率が高めることができるからである。
A recognition substance that specifically recognizes a target substance to be detected is immobilized on the solid phase surface that constitutes the detection means, and a sample solution containing the target substance interacts with the recognition substance, whereby a solid phase is obtained. The physical characteristics of the surface change, and the interaction between the recognition substance and the target substance, that is, the inclusion of the target substance in the sample solution is detected. At this time, since the reaction between the target substance and the recognition substance in the sample solution is in equilibrium, most of the target substance remains unreacted in the sample. Can be used for etc.
The sample solution whose interaction with the recognition substance has been confirmed and the target substance has been confirmed is sent directly to the next step. It should be noted that the determination as to whether or not the target substance is contained may be performed using a predetermined concentration as a border, in addition to whether it is zero or not. Further, it is preferable to provide a step of discharging the sample solution not containing the target substance through a drain or the like. This is because when a plurality of sample solutions are continuously detected, the working efficiency can be improved by discharging a solution that does not contain the target substance.
（標的物質を捕捉する工程）
標的物質を回収するために前記極性脂質をリポソーム構造とした認識素子を前記試料溶液中に添加することにより、標的物質を捕捉する。認識素子は微粒子としてみなすことができ、標的物質との衝突確率が高く、試料中の標的物質のほとんどが認識素子により捕捉することができる。
また、上記試料溶液中の標的物質の検出時においては、標的物質とセンサー上の認識素子との反応は平衡であるために、標的物質のほとんどが未反応で試料溶液中に存在している。認識素子は、この未反応の標的物質を捕捉することもできる。
(Step of capturing the target substance)
In order to collect the target substance, a recognition element having a liposome structure of the polar lipid is added to the sample solution to capture the target substance. The recognition element can be regarded as a fine particle, has a high collision probability with the target substance, and most of the target substance in the sample can be captured by the recognition element.
Further, at the time of detecting the target substance in the sample solution, since the reaction between the target substance and the recognition element on the sensor is in equilibrium, most of the target substance is unreacted and exists in the sample solution. The recognition element can also capture this unreacted target substance.
（認識素子を回収する工程）
標的物質を捕捉した認識素子は、遠心分離や電気泳動法等の公知の分離方法により容易に回収することができる。
(Step of collecting the recognition element)
The recognition element that has captured the target substance can be easily recovered by a known separation method such as centrifugation or electrophoresis.
また、前記試料溶液には通常緩衝液が使用されるが、この緩衝液の比重よりも、前記リポソームに含まれる溶液の比重を重くすることが好ましく、より好ましくは緩衝液の比重に対してリポソームの比重は１．０２から１．０８とすることが好ましい。サイズの小さい認識素子を遠心分離によって回収しやすくなるからである。具体的な例を挙げると、グリセロール等の糖類を主成分とする溶液を前記比重の範囲とすればよい。更に好ましくは３００ｍＭのグルコース水溶液とする。
尚、緩衝液とはpHを保つための溶液で、中性付近で用いられる緩衝液としてはリン酸、Tris、HEPES等が挙げられる。生体内での反応を再現するためにはこれにNaCl等の塩やタンパク質の変性防止目的としてグリセロール等の糖が加えられ用いる。尚、本明細書においては、これら生理緩衝液一般を指す。
In addition, a buffer solution is usually used as the sample solution, but it is preferable to make the specific gravity of the solution contained in the liposome heavier than the specific gravity of the buffer solution, and more preferably the liposome with respect to the specific gravity of the buffer solution. The specific gravity is preferably 1.02 to 1.08. This is because a small-size recognition element can be easily collected by centrifugation. As a specific example, a solution containing saccharides such as glycerol as a main component may be set in the specific gravity range. More preferably, a 300 mM glucose aqueous solution is used.
The buffer solution is a solution for maintaining pH, and examples of the buffer solution used in the vicinity of neutrality include phosphoric acid, Tris, and HEPES. In order to reproduce the reaction in the living body, a salt such as NaCl or a sugar such as glycerol is added for the purpose of preventing protein denaturation. In addition, in this specification, the physiological buffer is generally indicated.
また、認識素子を回収する工程の後に、回収された認識素子から、認識物質と標的物質の結合を解離させ、認識素子を回収することで残った標的物質を得るようにしてもよい。これにより、質量分析、電気泳動、ＤＮＡシーケンサーまたはアミノ酸配列分析等を使用して測定するために十分なサンプル量を確保することができる。 Further, after the step of recovering the recognition element, the binding between the recognition substance and the target substance may be dissociated from the recovered recognition element, and the remaining target substance may be obtained by recovering the recognition element. Thereby, it is possible to ensure a sufficient sample amount for measurement using mass spectrometry, electrophoresis, DNA sequencer, amino acid sequence analysis, or the like.
次に、本発明における極性脂質を基本成分とする認識物質をリポソーム構造にし、認識素子として調整する方法、および生体物質間の相互作用を検出する固相表面に認識物質を固定化する方法について説明する。 Next, a description will be given of a method for preparing a recognition substance having a polar lipid as a basic component in the present invention into a liposome structure and preparing it as a recognition element, and a method for immobilizing a recognition substance on a solid surface for detecting an interaction between biological substances. To do.
前記認識素子を調整する方法の一例として、Bioscience, Biotechnology, and Biochemistry 65,2638-2643 (2001)の論文に記載されている方法が挙げられる。上記論文では卵黄由来のphosphatidyl choline（以下「egg-PC」とする。）を用いて、300 mMのグルコース水溶液が含有されている単層ベシクル小胞（以下「SUV」とする。）を調整している。egg-PCをクロロホルムに溶解し、egg-PC溶液をガラス上にまき真空乾燥させることでegg-PCフィルムを作成する。egg-PCフィルムを300 mMのグリセロール水溶液で懸濁すると、egg-PCの多重ベシクル小胞（以下「MLV」とする。）を得ることができる。egg-PCのSUVはMLVを超音波処理することで調整される。 As an example of a method for adjusting the recognition element, there is a method described in a paper of Bioscience, Biotechnology, and Biochemistry 65, 2638-2643 (2001). In the above paper, phosphatidyl choline derived from egg yolk (hereinafter referred to as “egg-PC”) was used to prepare unilamellar vesicle vesicles (hereinafter referred to as “SUV”) containing 300 mM glucose aqueous solution. ing. Egg-PC is dissolved in chloroform, egg-PC solution is sprinkled on glass and vacuum-dried to create egg-PC film. When an egg-PC film is suspended in a 300 mM aqueous glycerol solution, egg-PC multi-vesicle vesicles (hereinafter referred to as “MLV”) can be obtained . The egg-PC SUV is adjusted by sonicating the MLV.
上記論文ではSUVを用いているが、本発明ではリポソームを認識素子として用いるため、MLVを利用しても構わないが、サイズや内包する溶液の比重が大きいと容易に沈殿して認識素子としての捕捉効率にも悪影響を与え、サイズや含有させる溶液組成を考慮する必要がある。サイズが大きすぎるとリポソームは使い勝手が悪くなるため粒径20〜500 nmとすることが好ましい。
また、SUVの調整やサイズをコントロールする方法として市販されているフィルターを用いることができる。この他の脂質組成においても上記論文記載の方法で十分対応ができるが、Dimyristoyl phosphatidylglycerol（以下「DMPG」とする。）のような極性の偏った極性脂質においてはクロロホルムだけでは溶解できないのでエタノールなどの極性溶媒を用いればよい。また、膜タンパク質を有する認識素子を作成する場合は、上記方法で得られるリポソームに膜タンパク質を再構成する方法で可能となるし、膜表在性ペプチドなどは上記リポソームと混合することで修飾することができる。
Although SUV is used in the above paper, MLV may be used in the present invention because liposome is used as a recognition element. However, if the size or the specific gravity of the encapsulated solution is large, it is easily precipitated and used as a recognition element. The trapping efficiency is also adversely affected, and it is necessary to consider the size and composition of the solution to be included. If the size is too large, the liposomes are unusable, so the particle size is preferably 20 to 500 nm.
Moreover, a commercially available filter can be used as a method for adjusting the SUV and controlling the size. Other lipid compositions can be adequately handled by the method described in the above paper, but polar lipids such as Dimyristoyl phosphatidylglycerol (hereinafter referred to as “DMPG”) cannot be dissolved with chloroform alone. A polar solvent may be used. In addition, when creating a recognition element having a membrane protein, it is possible to reconstitute the membrane protein into the liposome obtained by the above method, and membrane superficial peptides and the like are modified by mixing with the liposome. be able to.
続いて固相表面を検出器とするセンサー上に基本成分が極性脂質である認識物質を固定化する方法について説明する。脂質膜や膜タンパク質のセンサーへの固定化方法は従来技術を応用すればよい。例えば、上記と同様にリポソームに調整した認識物質を固定化する方法（非特許文献２：Biochemistry 38,15659-15665(1999)、非特許文献３：Journal of Medical Chemistry 43,2083-2086(2000)、）、Langmuir-Blodgett膜を作成し固定化する方法や疎水性表面にSUVに調整したリポソームを接触させることによる単層膜での固定化方法（非特許文献４：Biochimica et Biophysica Acta 1462,89-108(1999)、非特許文献５：Analytical Biochemistry 226,342-348(1995)）、SiO2といった親水性表面やBiacore社の表面プラズモンチップでSensor Chip L1（登録商標）を用いて二重膜として固定化する方法（非特許文献６：Langmuir 20,7526-7513(2004)、非特許文献７：FEBS Letter 559,96-98(2004)）、脂質膜をフィルムで固定化する方法（非特許文献８：Analytical Chemistry 62,1431-1438(1990)）が挙げられる。また、膜タンパク質を固定化する方法として、界面活性剤で可溶化した膜タンパク質をセンサー上に固定化し、センサー表面上で質膜に再構成する方法（非特許文献９：Analytical Biochemistry 316,243-250(2003)）や膜タンパク質をリポソームに再構成後センサー表面に固定化する方法（非特許文献１０：Biophysical Chemistry 85,141-152(2000)）が考案されている。 Next, a method for immobilizing a recognition substance whose basic component is a polar lipid on a sensor having a solid surface as a detector will be described. Conventional methods may be applied to the method for immobilizing lipid membranes and membrane proteins on the sensor. For example, a method for immobilizing a recognition substance prepared on a liposome as described above (Non-patent Document 2: Biochemistry 38, 15659-15665 (1999), Non-patent Document 3: Journal of Medical Chemistry 43, 2083-2086 (2000) )), A method for preparing and immobilizing a Langmuir-Blodgett membrane, and a method for immobilizing a monolayer membrane by contacting liposomes adjusted to SUV on a hydrophobic surface (Non-patent Document 4: Biochimica et Biophysica Acta 1462,89) -108 (1999), Non-Patent Document 5: Analytical Biochemistry 226,342-348 (1995)), fixed as a double membrane using Sensor Chip L1 (registered trademark) with hydrophilic surface such as SiO 2 or Biacore surface plasmon chip (Non-patent document 6: Langmuir 20,7526-7513 (2004), Non-patent document 7: FEBS Letter 559, 96-98 (2004)), a method of immobilizing a lipid membrane with a film (non-patent document 8) : Analytical Chemistry 62,1431-1438 (1990)). As a method for immobilizing membrane proteins, a membrane protein solubilized with a surfactant is immobilized on a sensor and reconstituted into a membrane on the sensor surface (Non-patent Document 9: Analytical Biochemistry 316,243-250 ( 2003)) and a method of reconstituting membrane proteins into liposomes and immobilizing them on the sensor surface (Non-patent Document 10: Biophysical Chemistry 85, 141-152 (2000)).
また、本発明の生体物質解析方法は、上述した生体物質検出・回収方法の後に、生体物質検出・回収方法により回収された前記標的物質が捕捉された前記認識素子を分光学的手法によって構造解析を行うものである。これにより、少量の試料を有効に活用して生体物質の解析を行うことができる。 In addition, the biological material analysis method of the present invention is configured to analyze the structure of the recognition element in which the target substance recovered by the biological material detection / recovery method is captured by a spectroscopic technique after the biological material detection / recovery method described above. Is to do. Thereby, a biological material can be analyzed by effectively using a small amount of sample.
また、本発明の生体物質解析方法は、標的物質を含む試料溶液の検出を、固相表面に固定化された極性脂質を基本成分とする認識物質と前記標的物質との生体物質間の相互作用により生じる前記固相表面の物理的特性の変化に基づいて行う工程、前記極性脂質のリポソーム構造体である認識素子により前記標的物質を含むことが検出された前記試料溶液中の前記標的物質を捕捉する工程、前記標的物質を捕捉した前記認識素子を回収するとともに固体核磁気共鳴法によって構造解析を行うものである。 In addition, the biological material analysis method of the present invention can detect a sample solution containing a target substance by interacting the recognition substance with a polar lipid immobilized on a solid surface as a basic component and the target substance. A step performed based on a change in physical properties of the solid phase surface caused by the step, capturing the target substance in the sample solution detected to contain the target substance by a recognition element that is a liposome structure of the polar lipid And a step of recovering the recognition element that has captured the target substance and performing structural analysis by a solid nuclear magnetic resonance method.
次に、本発明の生体物質検出・回収装置について説明する。
本発明は、試料溶液中の標的物質と相互作用する極性脂質を基本成分とする認識物質を固定化するための固相表面に備え、前記固相表面の物理的特性の変化に基づき前記標的物質を検出するための検出手段、前記標的物質が検出された前記試料溶液と前記極性脂質のリポソーム構造を認識素子と混合するための混合手段及び前記試料溶液から前記標的物質が捕捉された認識素子を回収するための遠心分離手段を備えたものである。
前記検出手段は、生体物質検出・回収方法のところで説明した検出手段と同じである。前記混合手段は、認識素子を試料溶液に加え混合できるものであればよく、また、遠心分離手段についても、遠心分離により認識素子を回収できるものであればよく、いずれも市販されている。
上記検出手段の検出部から混合手段への試料溶液の移動は、手作業によっても行うことができるが、好ましくは検出手段を容器内に設け、この容器と混合手段とを流路により接続してポンプ等を利用して移動できるように構成することが好ましい。検出を行った試料溶液をそのまま混合手段に移動することができ、作業効率がよいからである。また、前記容器又は前記流路をドレインに接続するようにしてもよい。検出により、不要と判断した試料溶液を排出し、更に回収することができるからである。更に、電磁弁等により、前記ドレインへの切換ができるようにすれば、操作がより簡便となるため好ましい。また、試料溶液の量については特に制限はないが、通常10μL〜5 mL程度である。
Next, the biological material detection / recovery device of the present invention will be described.
The present invention comprises a solid phase surface for immobilizing a recognition substance having a polar lipid that interacts with a target substance in a sample solution as a basic component, and the target substance is based on a change in physical properties of the solid phase surface. Detection means for detecting the target substance, mixing means for mixing the sample solution in which the target substance is detected and the liposome structure of the polar lipid with a recognition element, and a recognition element in which the target substance is captured from the sample solution A centrifuge for collecting is provided.
The detection means is the same as the detection means described in the biological material detection / recovery method. The mixing means may be any means that can add and recognize the recognition element to the sample solution, and any centrifugal means may be used as long as it can collect the recognition element by centrifugation, and all are commercially available.
The sample solution can be moved manually from the detection section of the detection means to the mixing means. Preferably, the detection means is provided in a container, and the container and the mixing means are connected by a flow path. It is preferable to configure so that it can be moved using a pump or the like. This is because the detected sample solution can be directly moved to the mixing means, and the working efficiency is good. Moreover, you may make it connect the said container or the said flow path to a drain. This is because the sample solution determined to be unnecessary can be discharged and further recovered by detection. Furthermore, it is preferable that switching to the drain can be performed by an electromagnetic valve or the like because the operation becomes simpler. The amount of the sample solution is not particularly limited, but is usually about 10 μL to 5 mL.
次に、本発明の実施例を図面を参照して説明する。 Next, embodiments of the present invention will be described with reference to the drawings.
（実施例１）
抗菌性ペプチドであるMagainin2（以下「MG2」とする。）を標的物質のモデルとし、Dimyristoyl phosphatidylcholine(以下「DMPC」とする。)膜とDMPG膜を認識物質として採用し、試料溶液中の標的物質と認識物質との相互作用を検出する手段として表面プラズモン共鳴（以下「SPR」とする。）センサーを使用して行う。その後、標的物質を含む試料溶液中の標的物質を各極性脂質膜から構成されるリポソーム（認識素子）により回収するとともに遠心分離器により、リポソームを分離するとともにこれに対して円偏光二色性法および固体高分解能核磁気共鳴法（以下「NMR」とする。）によってMG2および各脂質膜の構造解析を行う例を示す。
Example 1
Magainin2 (hereinafter referred to as “MG2”), an antibacterial peptide, is used as a target substance model, Dimyristoyl phosphatidylcholine (hereinafter referred to as “DMPC”) membrane and DMPG membrane are used as recognition substances, and target substances in the sample solution. As a means for detecting the interaction between the substance and the recognition substance, a surface plasmon resonance (hereinafter referred to as “SPR”) sensor is used. Thereafter, the target substance in the sample solution containing the target substance is collected by liposomes (recognition elements) composed of polar lipid membranes, and the liposomes are separated by a centrifuge and the circular dichroism method is used. In addition, an example of structural analysis of MG2 and each lipid membrane by solid high-resolution nuclear magnetic resonance (hereinafter referred to as “NMR”) is shown.
図１は、実施例１の方法の説明及び使用する装置の概念図である。
図１の最上段部に符号１０１で示す容器には、試料溶液と測定緩衝液である１％ＢＳＡ, 10 mM Tris(pH 7.4), 150 mM NaCl溶液と試料が導入される。この容器（１０１）内には、検出手段としてSPRセンサー（１２７）が設けられており、SPRセンサー（１２７）表面上に固定化されている認識物質（１２５）と標的物質（１２４）の相互作用を検出できるように構成される。
FIG. 1 is a conceptual diagram of an apparatus used and description of the method of the first embodiment.
A sample solution, 1% BSA, 10 mM Tris (pH 7.4), 150 mM NaCl solution and sample are introduced into a container denoted by reference numeral 101 in the uppermost part of FIG. In this container (101), an SPR sensor (127) is provided as a detection means, and the interaction between the recognition substance (125) immobilized on the surface of the SPR sensor (127) and the target substance (124). Is configured to be detected.
DMPCもしくはDMPGとする認識物質（１２５）を、SPRセンサー（１２７）に固定化する方法は以下の通りである。
金表面であるSPRセンサー（１２７）をオゾンプラズマで１０秒間処理し、さらにピランハ溶液（30%過酸化水素水と濃硫酸の混合溶液）で１０分間金表面を洗浄する。続いて、トルエンに溶解した5 mM n-Octadecanethiol溶液をセンサー表面に置き、１時間、室温で放置することで、n-Octadecanethiolの自己組織化膜が形成される。n-Octadecanethiolの自己組織化膜ではメチル基が表面にくるため、SPRセンサー（１２７）表面が疎水性に改質される。次に上記している方法にて50 nmのサイズに調整された、DMPCまたはDMPGのSUVを0.5 mMの濃度で疎水性センサー表面に反応させることで、認識物質（１２５）である各極性脂質を単相膜としてセンサー（１２７）表面に固定化することができる。
The method of immobilizing the recognition substance (125) as DMPC or DMPG on the SPR sensor (127) is as follows.
The SPR sensor (127), which is the gold surface, is treated with ozone plasma for 10 seconds, and the gold surface is washed with a piranha solution (mixed solution of 30% hydrogen peroxide and concentrated sulfuric acid) for 10 minutes. Subsequently, a 5 mM n-Octadecanethiol solution dissolved in toluene is placed on the sensor surface and allowed to stand at room temperature for 1 hour, whereby a self-assembled film of n-Octadecanethiol is formed. In the n-Octadecanethiol self-assembled film, the methyl group comes to the surface, so the surface of the SPR sensor (127) is modified to be hydrophobic. Next, by reacting DMPC or DMPG SUV adjusted to the size of 50 nm by the method described above to the surface of the hydrophobic sensor at a concentration of 0.5 mM, each polar lipid as the recognition substance (125) is reacted. It can be immobilized on the surface of the sensor (127) as a single phase film.
標的物質であるMG2（１２４）を含まない試料Ａ（１２１）として、VECTOR社から購入した正常マウス血清IgG分画（商品コードＪ９２０）を用いることとする。正常マウス血清IgG分画（１２２）は１％ ＢＳＡ, 10 mM Tris(pH 7.4), 150 mM NaCl溶液とし、DMPC（１２５）の固定化されたSPRセンサー（１２７）に導入すると、SPRシグナルに変化はなく相互作用が検出されない。これに対し、試料Ａ（１２１）と1 μMのMG2（１２４）の混合試料Ｂ（１２３）をSPRセンサー（１２７）に導入するとSPRシグナルに変化が生じ、DMPC（１２５）と相互作用する物質が検出される。 As a sample A (121) that does not contain the target substance MG2 (124), a normal mouse serum IgG fraction (product code J920) purchased from VECTOR is used. Normal mouse serum IgG fraction (122) is 1% BSA, 10 mM Tris (pH 7.4), 150 mM NaCl solution, and changes to SPR signal when introduced into SPR sensor (127) immobilized with DMPC (125) There is no interaction detected. In contrast, when the mixed sample B (123) of sample A (121) and 1 μM MG2 (124) is introduced into the SPR sensor (127), the SPR signal changes, and the substance that interacts with DMPC (125) Detected.
図２に、試料溶液Ａ（１２１）および試料溶液Ｂ（１２３）をSPRセンサー（１２７）にて測定した結果の模式図を示す。符号２０２で示す結果は試料溶液Ａ（１２１）を測定した結果でSPRシグナルに変化は見られないことがわかる。これに対し、試料溶液Ｂ（１２３）を測定すると符号２０１のようにＳＰＲシグナルに変化が検出される。
DMPGを認識物質とした場合においても、DMPCの時と同様に試料溶液Ｂ（１２３）でのみシグナルが変化し標的物質（１２４）が検出されることが確認される。
FIG. 2 shows a schematic diagram of the results of measuring the sample solution A (121) and the sample solution B (123) with the SPR sensor (127). The result indicated by reference numeral 202 is a result of measuring the sample solution A (121), and it can be seen that no change is observed in the SPR signal. On the other hand, when the sample solution B (123) is measured, a change in the SPR signal is detected as indicated by reference numeral 201.
Even when DMPG is used as the recognition substance, it is confirmed that the signal changes only in the sample solution B (123) and the target substance (124) is detected as in the case of DMPC.
認識物質（１２５）と相互作用する標的物質が検出されなかった試料溶液Ａ（１２１）は、流路（１１１）を介してポンプ（１０２）によってドレインへ回収するようにする。標的物質MG2（１２４）が検出された試料溶液Ｂ（１２３）は流路（１１２）を通って混合手段である混合槽（１０３）に移され、認識物質（１２５）のリポソームである認識素子（１２８）と混合される。認識素子（１２８）であるリポソームはサイズが100 nmのSUVで、200 mMのグルコース溶液を内包するように調整しておく。
その際、上述したSPRセンサー（１２７）における標的物質（１２４）と固定化された認識物質（１２５）との相互作用は、Langmuirの固相表面−液相間の法則に従うため、図１の中段右側に示すように試料溶液Ｂ（１２３'）中の標的物質（１２４）のほとんどが未反応な状態で残る。このため、粒子状の認識素子（１２８）は試料溶液Ｂ（１２３’）中の標的物質MG2（１２４）のほとんどと結合し複合体（１２９）の状態で捕捉される。
十分な時間、試料溶液Ｂ（１２３’）と認識素子（１２８）を混合した後、この混合液を遠心分離手段である遠心分離槽（１０４）に流路（１１３）を介して移動し、130,000gで５分間遠心することで沈殿物として複合体（１２９）を回収する。
遠心後の上清は流路（１１４）、ポンプ（１０５）及び流路（１１５）を介してドレインへと排出する。
上記の方法により、複合体（１２９）は測定緩衝液に再懸濁された状態で回収することができる。
The sample solution A (121) in which the target substance that interacts with the recognition substance (125) is not detected is recovered to the drain by the pump (102) through the flow path (111). The sample solution B (123) in which the target substance MG2 (124) is detected is transferred to the mixing tank (103) as the mixing means through the flow path (112), and the recognition element (the liposome of the recognition substance (125) ( 128). The liposome which is the recognition element (128) is an SUV having a size of 100 nm and is adjusted so as to enclose a 200 mM glucose solution.
In this case, the interaction between the target substance (124) and the immobilized recognition substance (125) in the SPR sensor (127) described above follows the law between the solid surface and the liquid phase of Langmuir. As shown on the right side, most of the target substance (124) in the sample solution B (123 ′) remains unreacted. Therefore, the particulate recognition element (128) binds to most of the target substance MG2 (124) in the sample solution B (123 ′) and is captured in the state of the complex (129).
After mixing the sample solution B (123 ′) and the recognition element (128) for a sufficient time, the mixture is moved to the centrifuge tank (104), which is a centrifuge, through the flow path (113), and 130,000 The complex (129) is recovered as a precipitate by centrifugation at g for 5 minutes.
The supernatant after centrifugation is discharged to the drain through the channel (114), the pump (105) and the channel (115).
By the above method, the complex (129) can be recovered in a state resuspended in the measurement buffer.
上記方法によって回収されたDMPCおよびDMPGのリポソームである認識素子（１２８）とMG2（１２４）との結合複合体（１２９）を円偏光二色性法および固体NMR法によって構造解析を行う。円偏光二色性法によって、MG2（１２４）の二次構造解析を行った結果、222 nmにおけるellipticityから、DMPC膜に結合したMG2（１２４）に比べDMPG膜に結合したMG2（１２４）ではαへリックス構造をとっていることが分かる。固体NMR法は、Biochimica et Biophysica Acta 1558,34-44(2002)（非特許文献１１）記載の方法によって31Pを観測核として測定した。結果、DMPCおよびDMPGの相転移点温度(Tm)がMG2（１２４）が結合した場合に変化していることが分かる。また、マジック角回転（MAS）法を用いて等方化学シフト値を比較すると、DMPCとDMPGともにMG2（１２４）が結合することで変化し、また、その変化量はDMPG膜のほうが大きいことが分かる。このことから、MG2（１２４）は酸性脂質であるDMPGに対して２次構造変化を伴うより安定した結合を行い、より大きな構造変化を誘起していることが示唆される。 The structure of the binding complex (129) of the recognition element (128), which is a liposome of DMPC and DMPG collected by the above method, and MG2 (124) is analyzed by a circular dichroism method and a solid state NMR method. As a result of the secondary structure analysis of MG2 (124) by the circular dichroism method, it was found from the ellipticity at 222 nm that MG2 (124) bonded to the DMPG film was less α than MG2 (124) bonded to the DMPC film. It can be seen that it has a helix structure. In the solid-state NMR method, 31 P was measured as an observation nucleus by the method described in Biochimica et Biophysica Acta 1558, 34-44 (2002) (Non-patent Document 11). As a result, it can be seen that the phase transition temperature (T m ) of DMPC and DMPG changes when MG2 (124) is bonded. In addition, when the isotropic chemical shift values are compared using the magic angle rotation (MAS) method, both DMPC and DMPG change when MG2 (124) binds, and the amount of change is larger in the DMPG film. I understand. This suggests that MG2 (124) binds more stably with secondary structure change to DMPG, which is an acidic lipid, and induces a larger structural change.
このように、本実施例によれば極性脂質膜を認識物質とした物質間相互作用をモニターすると同時に被検出対象標的物質を認識物質のリポソームである認識素子に捕捉したサンプルによって円偏光二色性法および固体NMR法によって詳細な構造解析に使用することができる。また、動的光散乱やゼータ電位によって認識素子であるリポソームのサイズを計測し、MG2の極性脂質膜に対する機能を知ることも可能である。
また、本実施例では相互作用解析は単純化するために検出のみとしたが、様々な濃度の試料をセンサー上にアプライすることによって結合（解離）定数を求めるアフィニティー解析やその結合および解離速度から速度論解析を行う事もできる。
As described above, according to the present example, the circular dichroism is detected by the sample in which the target substance to be detected is captured by the recognition element, which is a liposome of the recognition substance, while monitoring the interaction between substances using the polar lipid membrane as the recognition substance. And can be used for detailed structural analysis by solid state NMR. It is also possible to know the function of MG2 on the polar lipid membrane by measuring the size of the liposome, which is a recognition element, by dynamic light scattering and zeta potential.
In this example, the interaction analysis is performed only for the sake of simplification. However, from the affinity analysis to obtain the binding (dissociation) constant by applying samples of various concentrations on the sensor and the binding and dissociation rates. Kinetic analysis can also be performed.
（実施例２）
次に薬物送達系（Drug Delivery System,DDS）として応用するためのリポソームの評価として、脂質とDNAの複合体の評価を行った例を示す。固相表面で生体物質間相互作用を検出手段として27 MHzを基本周波数とする水晶振動子センサーを採用し、中性脂質である1,2-dioleoyl-sn-glycero-3- phosphatidylcholine（以下「DOPC」とする。）とDOPCとカチオン脂質である1,2-dioleoyl-3-3trimethyl-ammoniumpropane（以下「DOTAP」とする。）の混合膜を認識物質として採用する。DOPCとDOTAPの混合比は５：１のものを調整する。各脂質はクロロホルムとメタノールの混合溶媒に溶解し、フィルムを作成後、0.2 wt %のAnthraceneを含む10 mM PBS(pH 7.5), 150 mM NaClの緩衝液で懸濁し、0.2 μmのフィルターを通したMLVを調整し、遠心、回収することで認識素子とする。ここで調整されたリポソームのアルキル鎖疎水部にはAnthraceneが修飾され、蛍光寿命測定法による解析が可能となる。認識物質であるDNAはClontech社から購入したPlasmid DNA pCMVSport-β-Gal DNA（以下「DNA β-Gal」とする。）を使用する。
(Example 2)
Next, as an evaluation of liposomes for application as a drug delivery system (Drug Delivery System, DDS), an example of evaluating a complex of lipid and DNA is shown. Using a quartz crystal sensor with a fundamental frequency of 27 MHz as a means of detecting interactions between biological materials on the solid surface, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (hereinafter referred to as “DOPC”) is a neutral lipid. )) And DOPC and 1,2-dioleoyl-3-3trimethyl-ammoniumpropane (hereinafter referred to as “DOTAP”), which is a cationic lipid, are used as a recognition substance. Adjust the mixing ratio of DOPC and DOTAP to 5: 1. Each lipid was dissolved in a mixed solvent of chloroform and methanol, and after creating a film, it was suspended in a buffer of 10 mM PBS (pH 7.5) and 150 mM NaCl containing 0.2 wt% Anthracene and passed through a 0.2 μm filter. The MLV is adjusted, centrifuged, and collected to make a recognition element. Anthracene is modified in the hydrophobic part of the alkyl chain of the liposome prepared here, and analysis by a fluorescence lifetime measurement method becomes possible. As the recognition substance DNA, Plasmid DNA pCMVSport-β-Gal DNA (hereinafter referred to as “DNA β-Gal”) purchased from Clontech is used.
図３に、本実施例を行うに当たって使用する装置の概略図を示す。図３の上部から測定緩衝液（10 mM PBS(pH 7.5), 150 mM NaCl）と標的物質DNA β-Galの試料溶液が導入される。溶液が導入される流路（３０５）は、流路（３０６）と流路（３０７）に分岐され、一方は検出手段（３０１）に接続され、他方は混合手段（３０３）に接続される。尚、流路の分岐箇所には図示しないが電磁切換弁が設けられ、流路（３０６）又は流路（３０７）への試料溶液の移動を任意に切り換えることができるように構成される。
検出手段（３０１）においては、水晶振動子センサーが設けられている。水晶振動子センサーの電極上には認識物質であるDOPCもしくはDOPC/DOTAP混合膜が二重膜で固定化されている。DOPCもしくはDOPC/DOTAP混合膜の固定化方法は、水晶振動子表面にSiO2膜を0.1 μmの膜厚で蒸着し、オゾンプラズマアッシングを10秒間行った表面に、SUVのDOPCもしくはDOPC/DOTAPを結合させることにより行う。
FIG. 3 shows a schematic diagram of an apparatus used in carrying out this embodiment. A sample buffer solution (10 mM PBS (pH 7.5), 150 mM NaCl) and a target substance DNA β-Gal are introduced from the upper part of FIG. The flow path (305) into which the solution is introduced is branched into a flow path (306) and a flow path (307), one connected to the detection means (301) and the other connected to the mixing means (303). Although not shown, an electromagnetic switching valve is provided at a branch point of the flow path so that the movement of the sample solution to the flow path (306) or the flow path (307) can be arbitrarily switched.
In the detection means (301), a quartz vibrator sensor is provided. A DOPC or DOPC / DOTAP mixed film, which is a recognition substance, is fixed on the electrode of the quartz crystal sensor with a double film. The DOPC or DOPC / DOTAP mixed film is immobilized by depositing a SiO 2 film with a thickness of 0.1 μm on the surface of the quartz crystal and performing ozone plasma ashing for 10 seconds. This is done by combining them.
検出手段（３０１）の水晶振動子センサーにより相互作用が検出された試料溶液は、混合手段である混合槽（３０３）に移動する。ここでは、試料溶液に前記した認識素子を導入し混合を行う。ここで認識素子と標的物質が結合し、認識素子−標的物質複合体が形成される。十分な時間混合した後、流路（３０８）を介して分離手段（３０４）である遠心分離装置により認識素子および認識素子−標的物質複合体を分離し、流路（３０９）を介して上清をポンプ（３０２）によりドレインから排出し、沈殿物を試料として回収する。 The sample solution whose interaction is detected by the quartz vibrator sensor of the detection means (301) moves to the mixing tank (303) which is a mixing means. Here, the above-described recognition element is introduced into the sample solution and mixed. Here, the recognition element and the target substance are combined to form a recognition element-target substance complex. After mixing for a sufficient time, the recognition element and the recognition element-target substance complex are separated through the flow path (308) by the centrifugal separator as the separation means (304), and the supernatant is obtained through the flow path (309). Is discharged from the drain by the pump (302), and the precipitate is collected as a sample.
図４に水晶振動子センサーにて、DOPCに対する標的物質DNA β-Galの相互作用を解析した結果の概要図を示す。
水晶振動子センサー表面に3μM, 6μM, 9μM, 12μMのDNA β-Galを導入しその周波数の経時変化の模式図が図４（ａ）である。
導入するDNA β-Galの濃度が高くなるにつれ、水晶振動子センサーの周波数減少の減衰時定数が短く、DOPCとDNA β-Galとの相互作用が平衡に達するまでの時間が早いことが分かる。
FIG. 4 shows a schematic diagram of the results of analyzing the interaction of the target substance DNA β-Gal with DOPC using a quartz crystal sensor.
FIG. 4 (a) is a schematic diagram of the frequency change with time of introducing 3 μM, 6 μM, 9 μM, and 12 μM DNA β-Gal on the surface of the quartz oscillator sensor.
It can be seen that as the concentration of DNA β-Gal to be introduced increases, the decay time constant of the frequency decrease of the quartz crystal sensor is shorter, and the time until the interaction between DOPC and DNA β-Gal reaches equilibrium is faster.
この周波数変化を用いて動力学解析を行ったものを同図（ｂ）に概念図として示す。
動力学解析方法は１対１の結合モデルで行う。水晶振動子センサー上に固定化されている認識物質（Ａ）とそれと相互作用する標的物質（Ｂ）は結合し複合体（Ｃ）を形成する過程はＡ＋Ｂ⇔Ｃに従う平衡であり、その親和性は下記数１で定義される結合（解離）定数Ka(Kd)で定量化することができる（ここで、［Ａ］、［Ｂ］、［Ｃ］はそれぞれのモル濃度を示す）。
The dynamic analysis method is performed by a one-to-one coupled model. The process of forming the complex (C) by binding the recognition substance (A) immobilized on the quartz crystal sensor and the target substance (B) interacting with it is an equilibrium according to A + B⇔C, and its affinity binding is defined by the following Expression 1 can be quantified by (dissociation) constant K a (K d) (here, the [a], [B], [C] each molar).
図４（ｂ）は図４（ａ）の周波数変化を数（３）でフィットし、各DNA β-Gal濃度における1/τを求め、DNA β-Gal濃度に対してプロット（４０５）した図である。このプロットの回帰直線（４０６）の切片および傾きから、DOPCとDNA β-Galの相互作用における結合速度定数k+、解離速度定数k-および結合（解離）定数Ka (Kd)が求まる。
図４で示した方法と同様、DOPC/DOTAPとDNA β-Galの相互作用における結合速度定数k+、解離速度定数k-を求めた結果、DNA β-Galの親和性はカチオン脂質が含まれるDOPC/DOTAPの方が中性脂質であるDOPCに比べ高いことがわかる。
FIG. 4B is a diagram in which the frequency change of FIG. 4A is fitted by the number (3), 1 / τ at each DNA β-Gal concentration is obtained and plotted (405) against the DNA β-Gal concentration. It is. From the intercept and the slope of the regression line of the plot (406), association rate constant in the interaction of DOPC and DNA β-Gal k +, the dissociation rate constant k - and binding (dissociation) constant K a (K d) is obtained.
Similar to the method shown in FIG. 4, the binding rate constant k + and the dissociation rate constant k − in the interaction between DOPC / DOTAP and DNA β-Gal were determined. As a result, the affinity of DNA β-Gal includes cationic lipids. It can be seen that DOPC / DOTAP is higher than DOPC, which is a neutral lipid.
3μMのDNA β-Galを混合槽（３０３）に導入し、各脂質から構成される前記認識素子に結合させ、DNA β-Galと認識素子の複合体を回収した試料を、蛍光寿命測定法を用いて解析を行う。これにより、DOPCおよびDOPC/DOTAPの流動性に関する知見を得ることができ、DNA β-GalがMLVの脂質二重膜間に捕捉されていることが分かる。 3 μM DNA β-Gal was introduced into the mixing vessel (303), bound to the recognition element composed of each lipid, and the sample from which the complex of DNA β-Gal and recognition element was recovered was subjected to a fluorescence lifetime measurement method. To analyze. Thereby, knowledge about the fluidity of DOPC and DOPC / DOTAP can be obtained, and it can be seen that DNA β-Gal is trapped between the lipid bilayers of MLV.
このように、本発明によって回収された脂質膜を基本構成とする認識物質とそれに機能を示す生体物質に関する解析を様々な手法で行うことができる。
上記した実施例１および実施例２では、水晶振動子の計測では共振点周波数のみを計測例を示しているが、特願2005-192612に示されるような水晶振動子の基本波の共振点付近のコンダクタンスの最大値の1/2になる周波数（Ｆ１、Ｆ２）およびこのオーバートーンのＦ１、Ｆ２を用いる方法や、Q-sence AB社が提案しているQCM-Dと呼ばれる測定方法により、質量付加の正確な測定、固定化されたフィルム膜の粘弾性変化および溶液の粘性変化を測定することにより、より正確な相互作用解析が行えることは言うまでもない。また、その他の手法、例えばエリプソメーターや二面編波式干渉法といった方法でも計測は可能である。
As described above, various methods can be used to analyze a recognition substance based on a lipid membrane collected according to the present invention and a biological substance having a function thereof.
In the first and second embodiments described above, only the resonance point frequency is shown in the measurement of the crystal resonator. However, in the vicinity of the resonance point of the fundamental wave of the crystal resonator as shown in Japanese Patent Application No. 2005-192612. By using the frequency (F1, F2) that is half the maximum conductance of F and F1 and F2 of this overtone and the measurement method called QCM-D proposed by Q-sence AB, It goes without saying that a more accurate interaction analysis can be performed by measuring the precise measurement of the addition, the change in the viscoelasticity of the fixed film membrane and the change in the viscosity of the solution. Measurement can also be performed by other methods such as an ellipsometer or a two-sided knitting wave interferometry.
本発明は、生化学研究等の研究・開発分野やこれに関する製品の分野において広く利用することができる。 The present invention can be widely used in research / development fields such as biochemical research and related product fields.
１０１ 容器
１０２ ポンプ
１０３ 混合槽
１２１ 試料溶液Ａ
１２３ 試料溶液Ｂ
１２４ ＭＧ２
１２４ 標的物質
１２５ ＤＭＰＣ
１２５ 認識物質
１２７ ＳＰＲセンサー
１２９ 複合体
１１１，１１２，１１３，１１４，１１５ 流路
３０１ 検出手段
３０３ 混合手段
３０４ 分離手段
３０５，３０６，３０７，３０８，３０９ 流路
101 Container 102 Pump 103 Mixing tank 121 Sample solution A
123 Sample solution B
124 MG2
124 Target substance 125 DMPC
125 Recognizing substance 127 SPR sensor 129 Complex 111, 112, 113, 114, 115 Flow path 301 Detection means 303 Mixing means 304 Separation means 305, 306, 307, 308, 309 Flow path
Claims (11)
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| JP7239403B2 (en) * | 2019-06-27 | 2023-03-14 | 日本無線株式会社 | CONCENTRATION DETECTION DEVICE AND CONCENTRATION DETECTION PROGRAM FOR SAW SENSOR |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003116515A (en) * | 2001-10-18 | 2003-04-22 | Hitachi Ltd | Method for recovering biomolecule and apparatus therefor |
| JP2004028832A (en) * | 2002-06-26 | 2004-01-29 | Fuji Photo Film Co Ltd | Sensor and measurement chip using total reflection attenuation |
| JP2005098733A (en) * | 2003-09-22 | 2005-04-14 | Ulvac Japan Ltd | Biosensor, measuring method by biosensor, and biosensor device |
| JP2005512019A (en) * | 2001-05-11 | 2005-04-28 | イェール ユニバーシティ | Comprehensive analysis of protein activity using proteome chips |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2005512019A (en) * | 2001-05-11 | 2005-04-28 | イェール ユニバーシティ | Comprehensive analysis of protein activity using proteome chips |
| JP2003116515A (en) * | 2001-10-18 | 2003-04-22 | Hitachi Ltd | Method for recovering biomolecule and apparatus therefor |
| JP2004028832A (en) * | 2002-06-26 | 2004-01-29 | Fuji Photo Film Co Ltd | Sensor and measurement chip using total reflection attenuation |
| JP2005098733A (en) * | 2003-09-22 | 2005-04-14 | Ulvac Japan Ltd | Biosensor, measuring method by biosensor, and biosensor device |
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