JP2009186356A - Analytical method using magnetic particle - Google Patents

Analytical method using magnetic particle Download PDF

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JP2009186356A
JP2009186356A JP2008027551A JP2008027551A JP2009186356A JP 2009186356 A JP2009186356 A JP 2009186356A JP 2008027551 A JP2008027551 A JP 2008027551A JP 2008027551 A JP2008027551 A JP 2008027551A JP 2009186356 A JP2009186356 A JP 2009186356A
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magnetic particles
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JP4964161B2 (en
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Takayuki Kanda
隆之 神田
Kyoko Imai
恭子 今井
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Hitachi High Tech Corp
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  • Investigating Or Analyzing Materials By The Use Of Magnetic Means (AREA)

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analytical method using magnetic particles, which has a high performance of magnetic separation and has improved reactivity. <P>SOLUTION: The method which analyzes a test substance by simultaneously using two particles dispersed in a solution is characterized in that a substance interacting with the test substance is fixed on a first magnetic particle, and the particle size of a second magnetic particle is large in comparison with the first magnetic particle. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

本発明は、固相として磁性粒子を用いた分析方法、例えば血清、尿等の体液成分の免疫学的な特異的結合や核酸の相補鎖結合を利用した微量成分の検出に用いる磁性粒子を用いた分析方法に関する。   The present invention uses an analysis method using magnetic particles as a solid phase, for example, magnetic particles used for detection of trace components using the specific immunological binding of body fluid components such as serum and urine and the complementary strand binding of nucleic acids. Related to the analysis method.

磁性粒子は、磁力を利用することにより容易に分離回収することができる特性(磁気分離性)を有する。当該特性に起因して、主に医学や生化学分野において、磁性粒子は、診断薬、細菌又は細胞の分離、核酸、タンパク質や糖類の分離又は精製、ドラックデリバリー、酵素反応、細胞培養等のための担体として優れた取り扱い性が得られるものと期待されている。   Magnetic particles have a characteristic (magnetic separation property) that can be easily separated and recovered by using magnetic force. Due to these characteristics, mainly in the medical and biochemical fields, magnetic particles are used for diagnostic agents, separation of bacteria or cells, separation or purification of nucleic acids, proteins and sugars, drug delivery, enzymatic reactions, cell culture, etc. It is expected that excellent handling properties can be obtained as a carrier of the above.

一般に、磁性粒子について高い磁気分離性を得る手段としては、(1)粒子中に含有される磁性体の割合を高くすること、（2）磁化(飽和磁化)が高い磁性体を粒子中に含有させることが考えられる。   In general, the means to obtain high magnetic separation for magnetic particles are: (1) Increasing the proportion of magnetic material contained in the particle; (2) Containing magnetic material with high magnetization (saturation magnetization) in the particle It is possible to make it.

しかし、上記(1)の方法は、磁気分離性の向上に有効な手段であるものの、粒子中に含有される磁性体の割合が高くなることにより、当該粒子の表面に露出する磁性体の量が増加し、磁性体の構成成分(例えば鉄分等)が溶出しやすくなる。このような磁性粒子を医学や生化学分野において使用する場合には、分析等において重大な支障が生じる。従って、粒子中に含有される磁性体の割合を高くすることには制約があるため、上記(1)の方法によって磁気分離性の向上を図るには限界がある。   However, although the method (1) is an effective means for improving the magnetic separation property, the amount of the magnetic substance exposed on the surface of the particle is increased by increasing the ratio of the magnetic substance contained in the particle. Increases, and the constituent components of the magnetic substance (for example, iron) are easily eluted. When such magnetic particles are used in the medical and biochemical fields, a serious trouble occurs in analysis and the like. Therefore, since there is a restriction on increasing the ratio of the magnetic substance contained in the particles, there is a limit to improving the magnetic separation property by the method (1).

上記(2)の方法においては、通常、磁化が高い磁性体は強磁性体であり、高い残留磁化を示す。このような磁性体を含有する磁性粒子に磁場を作用させた場合、磁場の作用を停止しても当該粒子自体が磁石となり凝集状態が維持され、磁気履歴後の磁性粒子を再分散させることが困難となる。   In the above method (2), normally, a magnetic material having high magnetization is a ferromagnetic material and exhibits high residual magnetization. When a magnetic field is applied to magnetic particles containing such a magnetic substance, the particles themselves become magnets even if the magnetic field action is stopped, and the aggregated state is maintained, and the magnetic particles after the magnetic history can be redispersed. It becomes difficult.

一方、現在の医学や生化学分野において、担体として磁性粒子に求められる条件は、(i)標的物質と特異的に反応する物質をできる限り多く粒子表面に付加させるため、できるだけ粒径が小さく、比表面積が大きいこと、(ii)その表面に、標的物質以外の物質の吸着ができるだけ少ないこと(すなわち、非特異的吸着が起こりにくいこと)、(iii)磁性粒子自体が反応液中に悪影響を与えないこと、(iv)反応液中で凝集することなく分散し、磁力を与えた場合に迅速に分離でき、且つ無磁力化した際に容易に再分散できることである。   On the other hand, in the current medical and biochemical fields, the conditions required for magnetic particles as a carrier are (i) adding as much as possible to the particle surface a substance that specifically reacts with the target substance, so that the particle size is as small as possible, (Ii) Adsorption of substances other than the target substance on the surface is as small as possible (that is, nonspecific adsorption is unlikely to occur), (iii) Magnetic particles themselves adversely affect the reaction solution. (Iv) It disperses without agglomerating in the reaction solution, can be rapidly separated when a magnetic force is applied, and can be easily redispersed when it is demagnetized.

上述した条件から、粒径が非常に小さく、単一の磁区から成る超常磁性磁性体を含有する磁性ポリマー粒子が実用化されている。しかしながら、当該磁性ポリマー粒子はμmサイズの比較的大きな粒径を有する磁性粒子であり、比表面積も比較的小さい。そのため、当該磁性ポリマー粒子を担体として用いた場合、反応に必要な化学物質や生体物質を表面に固定できる量が少なく、反応効率が低い。この点では、比表面積の大きいnmサイズの粒径を有する磁性粒子を用いることが望ましいものの、これらは磁化が弱く、磁気分離が困難である。   From the above-mentioned conditions, magnetic polymer particles having a very small particle diameter and containing a superparamagnetic material composed of a single magnetic domain have been put into practical use. However, the magnetic polymer particles are magnetic particles having a relatively large particle size of μm size and a relatively small specific surface area. Therefore, when the magnetic polymer particles are used as a carrier, the amount of chemical substances and biological substances necessary for the reaction can be fixed on the surface, and the reaction efficiency is low. In this respect, although it is desirable to use magnetic particles having a particle size of nm size with a large specific surface area, they are weakly magnetized and difficult to separate.

また、磁性体の含有量が高く、且つnmサイズの粒径を有する磁性粒子が開発されている。当該磁性粒子を同じ粒径の磁性粒子と比較した場合には、その磁気分離時間は飽和磁化が高いほど短くなる。一方、飽和磁化が同じで、粒径の異なる磁性粒子と比較すると、粒径の大きな磁性粒子ほど、磁気分離時間が短い。従って、nmサイズの磁性粒子を用いた場合には、磁気分離における飽和磁化と粒径のバランスが、反応液中で磁性粒子に影響を及ぼすミクロブラウン運動より大きいか否かが重要となってくる。   Further, magnetic particles having a high content of magnetic substance and having a particle size of nm size have been developed. When the magnetic particles are compared with magnetic particles having the same particle size, the magnetic separation time becomes shorter as the saturation magnetization becomes higher. On the other hand, as compared with magnetic particles having the same saturation magnetization and different particle sizes, the magnetic particles having a larger particle size have a shorter magnetic separation time. Therefore, when using nano-sized magnetic particles, it is important whether the saturation magnetization and particle size balance in magnetic separation is greater than the micro-Brownian motion that affects magnetic particles in the reaction solution. .

さらに、磁性粒子に含まれる磁性体の含有量が大きいほど、磁性粒子の粒子密度が上がる。このため、反応液中での磁性粒子の分散性は低くなる。分散性を上げるために、反応液の密度を上げる等の工夫が必要となるが、当該工夫には、利用する化学物質や生体物質等への影響を避けるために限界がある。   Furthermore, the larger the content of the magnetic substance contained in the magnetic particles, the higher the particle density of the magnetic particles. For this reason, the dispersibility of the magnetic particles in the reaction solution is lowered. In order to increase the dispersibility, it is necessary to devise such as increasing the density of the reaction solution. However, this contrivance has a limit in order to avoid an influence on a chemical substance or a biological substance to be used.

また、粒径の大きな磁性粒子では、磁気分離性を大きく阻害するミクロブラウン運動の影響が少なくなる。さらに、粒径の大きな磁性粒子では、粒子の運動抵抗と比較して、粒子が受ける磁力の相対比率が大きくなるので、磁気分離性の向上に大いに有効である。しかしながら、上述したように、粒径の大きな磁性粒子は表面積が小さいため、実用上の性能が低い。   In addition, in the case of magnetic particles having a large particle size, the influence of the micro Brownian motion that greatly impairs the magnetic separation property is reduced. Furthermore, a magnetic particle having a large particle size has a large relative ratio of magnetic force applied to the particle as compared with the movement resistance of the particle, and is thus very effective in improving magnetic separation. However, as described above, a magnetic particle having a large particle size has a small surface area and thus has low practical performance.

従来行われている磁性粒子を用いた分析は、反応を迅速に、且つ均一に進めるため、粒径が小さく且つ粒径にばらつきの無い磁性粒子が用いられてきた。粒径が小さい磁性粒子は分析上の反応場となる表面の有効面積の増加のためには非常に有効である。しかしながら、上述したように、ミクロブラウン運動の影響に起因して、粒径が小さい磁性粒子の磁気分離性は非常に低くなる。   In the conventional analysis using magnetic particles, magnetic particles having a small particle size and no variation in particle size have been used in order to advance the reaction quickly and uniformly. Magnetic particles having a small particle size are very effective for increasing the effective area of the surface that serves as an analytical reaction field. However, as described above, due to the influence of the micro Brownian motion, the magnetic separation property of the magnetic particles having a small particle size becomes very low.

そこで、本発明は、上述した実状に鑑み、磁気分離性が高く且つ反応性が向上した、磁性粒子を用いた分析方法を提供することを目的とする。   In view of the above, the present invention has an object to provide an analysis method using magnetic particles having high magnetic separation and improved reactivity.

上記のように、粒径の大きな磁性粒子では、粒径が小さい場合に磁気分離性を大きく阻害するミクロブラウン運動の影響が少なくなり、且つ、粒子の運動抵抗と比較して、粒子が受ける磁力の相対比率が大きくなり、磁気分離性は大きい。さらに、反応液中での移動速度が速い。しかしながら、粒子表面の有効面積は小さく、反応性の向上には不利に働く。   As described above, in the case of a magnetic particle having a large particle size, when the particle size is small, the influence of the micro-Brownian motion that greatly impairs the magnetic separation property is reduced, and the magnetic force that the particle receives compared to the motion resistance of the particle The relative ratio of is increased, and the magnetic separation property is large. Furthermore, the moving speed in the reaction solution is fast. However, the effective area of the particle surface is small, which adversely affects the reactivity.

一方、粒径の小さな磁性粒子では、磁気分離性は小さくなるが、粒子表面の有効面積が大きく、反応性の向上には有利に働く。   On the other hand, a magnetic particle having a small particle size has a small magnetic separation property, but has a large effective area on the particle surface, which is advantageous for improving the reactivity.

そこで、上述した目的を達成するため鋭意検討した結果、粒径の小さい磁性粒子と大きい磁性粒子の双方を同時に用いることで、分析方法において磁気分離性が高く、且つ反応性が向上することを見出し、本発明を完成するに至った。   Therefore, as a result of intensive studies to achieve the above-mentioned object, it was found that by using both a small magnetic particle and a large magnetic particle at the same time, the magnetic separation property is high and the reactivity is improved in the analysis method. The present invention has been completed.

すなわち、本発明は、溶液中に分散した2種類の磁性粒子を同時に用いて、被験物質を分析する方法であって、第１磁性粒子上に前記被験物質と相互作用する物質が固定されており、且つ第２磁性粒子の粒径が第１磁性粒子と比較して大きいことを特徴とする方法である。また、本発明は、当該第１磁性粒子と第２磁性粒子とを含む被験物質分析用キットである。   That is, the present invention is a method for analyzing a test substance using two kinds of magnetic particles dispersed in a solution at the same time, and a substance that interacts with the test substance is fixed on the first magnetic particles. In addition, the second magnetic particles have a larger particle size than the first magnetic particles. Further, the present invention is a test substance analysis kit including the first magnetic particles and the second magnetic particles.

本発明は、分析方法において、反応物質間の反応性が向上し、且つ当該反応物質の磁気分離性が高いという効果を奏する。   The present invention has the effects that the reactivity between the reactants is improved and the magnetic separation of the reactants is high in the analysis method.

以下、本発明を詳細に説明する。   Hereinafter, the present invention will be described in detail.

本発明に係る方法は、溶液中に分散した2種類の磁性粒子を同時に用いて、被験物質を分析する方法である。当該2種類の磁性粒子のうち、第１磁性粒子上には前記被験物質と相互作用する物質(以下、「相互作用物質」という)が固定されている。また、第２磁性粒子の粒径は第１磁性粒子と比較して大きい。第１磁性粒子と第２磁性粒子とを同時に用いると、磁力の相対比率が大きい第２磁性粒子は、第１磁性粒子に比べて早く反応液中を移動し、第１磁性粒子に接近して移動する。この際、第１磁性粒子は第２磁性粒子に結合し、第１磁性粒子単独に比べて、移動時間が短くなる。また、粒子表面の有効面積が大きい第１磁性粒子のみに相互作用物質が固定されているので、反応自体の時間も短くすることができる。   The method according to the present invention is a method for analyzing a test substance by simultaneously using two types of magnetic particles dispersed in a solution. Of the two types of magnetic particles, a substance that interacts with the test substance (hereinafter referred to as “interactive substance”) is fixed on the first magnetic particle. The particle size of the second magnetic particles is larger than that of the first magnetic particles. When the first magnetic particles and the second magnetic particles are used at the same time, the second magnetic particles having a large relative ratio of magnetic force move in the reaction liquid faster than the first magnetic particles and approach the first magnetic particles. Moving. At this time, the first magnetic particles are bonded to the second magnetic particles, and the moving time is shorter than that of the first magnetic particles alone. Moreover, since the interaction substance is fixed only to the first magnetic particles having a large effective area on the particle surface, the reaction time can be shortened.

ここで、「分析」とは、被験物質と相互作用物質間の相互作用の分析、並びに被験物質と相互作用物質間の相互作用による被験物質の検出、定量及び分離を意味する。当該相互作用には、共有結合及び非共有結合が含まれる。具体的な相互作用としては、例えば、水素結合、抗原抗体反応による結合、ビオチン-アビジン相互作用、ビオチン-ストレプトアビジン相互作用、エポキシ基-アミノ基相互作用、トシル基-スルフヒドリル基相互作用、カルボン酸基-アミノ基相互作用、核酸共有結合が挙げられる。さらに、具体的な相互作用の例示としては、被験物質が抗原であり、相互作用物質が抗体である場合(又はその逆である場合)の抗原抗体反応、被験物質と相互作用物質とが共にDNA、RNA等の核酸である場合の相補鎖結合等が挙げられる。   Here, “analysis” means analysis of the interaction between the test substance and the interacting substance, and detection, quantification and separation of the test substance by the interaction between the test substance and the interacting substance. Such interactions include covalent and non-covalent bonds. Specific interactions include, for example, hydrogen bonding, binding by antigen-antibody reaction, biotin-avidin interaction, biotin-streptavidin interaction, epoxy group-amino group interaction, tosyl group-sulfhydryl group interaction, carboxylic acid Examples include group-amino group interaction and nucleic acid covalent bond. Furthermore, specific examples of interaction include antigen-antibody reaction when the test substance is an antigen and the interacting substance is an antibody (or vice versa), and the test substance and the interacting substance are both DNA. And complementary strand binding in the case of nucleic acids such as RNA.

また、「被験物質」としては、例えば、タンパク質、核酸、糖類、細菌、細胞等が挙げられる。例えば、被験物質を含有する体液成分(血清や尿)等のサンプルを被験物質として使用することができる。なお、分析に応じて、被験物質又は相互作用物質は、例えば蛍光標識、化学発光標識、放射性標識、酵素標識等の標識がなされていてもよい。例えば、被験物質の検出又は定量を行う際には、当該標識を指標として被験物質の検出又は定量を行うことができる。   Examples of the “test substance” include proteins, nucleic acids, saccharides, bacteria, cells and the like. For example, a sample such as a body fluid component (serum or urine) containing the test substance can be used as the test substance. Depending on the analysis, the test substance or interaction substance may be labeled with, for example, a fluorescent label, a chemiluminescent label, a radioactive label, or an enzyme label. For example, when detecting or quantifying a test substance, the test substance can be detected or quantified using the label as an index.

本発明に係る方法では、先ず上述した2種類の磁性粒子を準備する。   In the method according to the present invention, first, the two types of magnetic particles described above are prepared.

第１磁性粒子には、相互作用物質が固定される。固定方法としては、ポリマーやオリゴマーを用いた方法が挙げられる。例えば、第１磁性粒子表面を1種又は複数のエポキシド化合物及びエポキシド類の混合物でコーティングした後、物理吸着や化学結合により相互作用物質を固定化する。   An interactive substance is fixed to the first magnetic particles. Examples of the fixing method include a method using a polymer or an oligomer. For example, after the surface of the first magnetic particle is coated with a mixture of one or more epoxide compounds and epoxides, the interaction substance is immobilized by physical adsorption or chemical bonding.

一方、第２磁性粒子は、非特異的吸着を避けるため、分析反応に関与する物質や官能基等が付加されていない。分析反応の溶液に応じて親水性又は疎水性の表面処理を行い、分析反応に対する影響を最小限にすることが好ましい。   On the other hand, in order to avoid non-specific adsorption, the second magnetic particles are not added with substances or functional groups involved in the analytical reaction. It is preferable to perform a hydrophilic or hydrophobic surface treatment according to the solution of the analytical reaction to minimize the influence on the analytical reaction.

第１及び第２磁性粒子のコア部分は、無機質のみで形成されていてもよいが、低比重にすることにより溶液中での沈降を遅らせ、分散を容易にするため、有機物が含まれていることが望ましい。あるいは、低比重となるように有機物又は無機物から成る多孔質粒子を用いても良い。コア部分としては、例えばポリマー粒子中に磁性体を分散させたもの、ポリマー粒子表面に磁性体を物理的に付着させたもの、ポリマー粒子や多孔質粒子の表面に磁性体を析出させたもの等が挙げられる。   The core portions of the first and second magnetic particles may be formed only of an inorganic substance, but contain an organic substance in order to delay the sedimentation in the solution and facilitate the dispersion by making the specific gravity low. It is desirable. Or you may use the porous particle which consists of an organic substance or an inorganic substance so that it may become low specific gravity. Examples of the core portion include those in which a magnetic substance is dispersed in polymer particles, those in which a magnetic substance is physically attached to the surface of polymer particles, and those in which a magnetic substance is deposited on the surface of polymer particles or porous particles. Is mentioned.

第１及び第２磁性粒子のコア部分を形成する際に使用する磁性体の材質としては特に限定されないが、酸化鉄系の物質が代表的であり、例えば、XFe2O4(式中、X=Mn、Co、Ni、Mg、Cu、Li0.5、Fe0.5等)として示されるフェライト、Fe3O4として示されるマグネタイト、あるいはγFe2O3が挙げられる。また、上記磁性体を複合して用いても良い。特に、飽和磁化が高く且つ残留磁化が低い磁性体である、γFe2O3及び/又はFe3O4から成る磁性体が好ましい。 The material of the magnetic material used when forming the core portions of the first and second magnetic particles is not particularly limited, but iron oxide-based materials are typical, for example, XFe 2 O 4 (wherein X = Mn, Co, Ni, Mg, Cu, Li0.5, Fe0.5, etc.), magnetite shown as Fe 3 O 4 , or γFe 2 O 3 . Moreover, you may use combining the said magnetic body. In particular, a magnetic body made of γFe 2 O 3 and / or Fe 3 O 4 which is a magnetic body having a high saturation magnetization and a low residual magnetization is preferable.

第１磁性粒子の粒径(直径)は、例えば10nm〜1μm、好ましくは50nm〜500nmである。第２磁性粒子の粒径(直径)は、第１磁性粒子と比較して大きければよく、例えば1μm〜10μm、好ましくは1.5μm〜3.5μmである。このような粒径の違いにより、両者を同時に用いると、磁力の相対比率が大きい第２磁性粒子は、小さい第１磁性粒子に比べて早く反応液中を移動するが、この際、小さい第１磁性粒子に接近して移動することとなる。   The particle diameter (diameter) of the first magnetic particles is, for example, 10 nm to 1 μm, preferably 50 nm to 500 nm. The particle size (diameter) of the second magnetic particles may be larger than that of the first magnetic particles, and is, for example, 1 μm to 10 μm, preferably 1.5 μm to 3.5 μm. Due to such a difference in particle size, when both are used simultaneously, the second magnetic particles having a large relative ratio of magnetic force move in the reaction solution faster than the small first magnetic particles. It will move closer to the magnetic particles.

また、反応に用いる第２磁性粒子の使用総重量は、第１磁性粒子の使用総重量よりも低いことが望ましい。例えば、第１磁性粒子と第２磁性粒子との使用総重量比が第１磁性粒子：第２磁性粒子＝9：1〜99：1であることが好ましい。これにより、反応性を損なうことなく、第１磁性粒子の移動時間を短くすることが可能となる。   Further, it is desirable that the total use weight of the second magnetic particles used in the reaction is lower than the total use weight of the first magnetic particles. For example, the total weight ratio of the first magnetic particles to the second magnetic particles is preferably 1st magnetic particles: 2nd magnetic particles = 9: 1 to 99: 1. Thereby, it becomes possible to shorten the moving time of the first magnetic particles without impairing the reactivity.

さらに、磁性粒子の飽和磁化に関しては、一方の磁性粒子が他方の磁性粒子と比較して大きくても小さくてもよいが、第２磁性粒子の飽和磁化が、第１磁性粒子と比較して大きいことが好ましい。   Further, regarding the saturation magnetization of the magnetic particles, one magnetic particle may be larger or smaller than the other magnetic particle, but the saturation magnetization of the second magnetic particle is larger than that of the first magnetic particle. It is preferable.

以上に説明した磁性粒子は、例えばDynal社、micromod社等により市販されており、当該市販品を使用してもよい。   The magnetic particles described above are commercially available from, for example, Dynal and micromod, and the commercially available products may be used.

次いで、本発明に係る方法では、第１磁性粒子と第２磁性粒子とを含有する溶液を準備する。なお、ここで「溶液」は、懸濁液、混合液等と互換的に用いられる。当該溶液では、第１磁性粒子と第２磁性粒子とを均一に分散させる。例えば、当該溶液をピペッティングに供し、十分に撹拌することで、均一に分散させることができる。溶液中の第１磁性粒子と第２磁性粒子との割合は、上述の総重量比に準じた割合とすることができる。   Next, in the method according to the present invention, a solution containing the first magnetic particles and the second magnetic particles is prepared. Here, the “solution” is used interchangeably with a suspension, a mixed solution or the like. In the solution, the first magnetic particles and the second magnetic particles are uniformly dispersed. For example, the solution can be uniformly dispersed by subjecting the solution to pipetting and stirring sufficiently. The ratio of the first magnetic particles and the second magnetic particles in the solution can be set to a ratio according to the above total weight ratio.

このようにして用意した第１磁性粒子と第２磁性粒子を含有する溶液及び被験物質を含有するサンプルを分析装置に供することで、目的とする分析を行うことができる。具体的には、第１磁性粒子と第２磁性粒子を含有する溶液と被験物質とを接触させるようにサンプルを調製する。次いで、第１磁性粒子上の相互作用物質と被験物質との結合体を磁石により集磁する。集磁後、集磁した結合体を、所望の分析に供することとなる。ここで、「接触」とは、相互作用物質と被験物質とが結合又は会合するように、相互作用物質と被験物質とを近接した状態に配置することを意味する。   By providing the solution containing the first magnetic particles and the second magnetic particles thus prepared and the sample containing the test substance to the analyzer, the target analysis can be performed. Specifically, a sample is prepared so that a solution containing the first magnetic particles and the second magnetic particles is brought into contact with the test substance. Next, a combined body of the interaction substance and the test substance on the first magnetic particles is collected by a magnet. After collecting the magnetic flux, the collected magnetic flux is subjected to a desired analysis. Here, the “contact” means that the interacting substance and the test substance are arranged in close proximity so that the interacting substance and the test substance are bound or associated with each other.

本発明に係る方法における分析の例示を、図１〜３を用いて説明する。なお、図１〜３において、構成要素は、各図において符号が異なっていても名称が同一である場合には、同一のものを指す。図１は、化学分析装置の構成を示す傾視図である。   An example of analysis in the method according to the present invention will be described with reference to FIGS. 1 to 3, the constituent elements are the same when the names are the same even if the reference numerals are different in each figure. FIG. 1 is a perspective view showing the configuration of the chemical analyzer.

先ず、図１を用いて化学分析装置全体の主な構成を説明する。当該化学分析装置は、サンプル(被験物質を含有するサンプル)を含むサンプルカップ１０１と、その複数のサンプルカップを格納するサンプルディスク１０２と、試薬ボトル１０４を格納する試薬保冷庫(試薬ディスク)１０３と、サンプルを分注するサンプル分注ノズル１０６と、試薬(第１磁性粒子と第２磁性粒子とを含有する溶液を含む)を分注する試薬分注ユニット１０５と、サンプル及び試薬を分注して反応を行わせる分離容器１０８を格納し、且つ分離容器内の温度を管理するインキュベーター１０７とを備えている。また、図中にはその一部しか示されていないが、撹拌と磁性粒子の吸着を行う撹拌/吸着ユニット１１１と、被験物質と第１磁性粒子上の相互作用物質との結合体の洗浄を行う際に希釈液を分注・吸引する吐出/吸引ユニット１１２と、標識が修飾された結合体の標識を計数するユニット１０９と、ユーザとのインターフェースや各ユニットの制御を行うコンソール１１０より構成される。   First, the main structure of the whole chemical analyzer will be described with reference to FIG. The chemical analyzer includes a sample cup 101 containing a sample (sample containing a test substance), a sample disk 102 storing a plurality of sample cups, a reagent cold storage (reagent disk) 103 storing a reagent bottle 104, and the like. A sample dispensing nozzle 106 for dispensing a sample, a reagent dispensing unit 105 for dispensing a reagent (including a solution containing first magnetic particles and second magnetic particles), and dispensing the sample and the reagent And an incubator 107 for storing the separation container 108 for performing the reaction and managing the temperature in the separation container. Although only a part of the drawing is shown in the figure, the stirring / adsorption unit 111 for stirring and adsorbing the magnetic particles, and washing of the conjugate of the test substance and the interaction substance on the first magnetic particles are washed. It comprises a discharge / aspiration unit 112 that dispenses and aspirates the diluent when it is performed, a unit 109 that counts the label of the conjugate whose label is modified, and a console 110 that controls the user interface and each unit. The

次に、図２を用いて撹拌/吸着ユニット１１１及びその周りの構成について詳しく説明する。撹拌/吸着ユニット１１１は、インキュベーター１０７に埋め込まれるように設置されている。   Next, the agitation / adsorption unit 111 and the configuration around it will be described in detail with reference to FIG. The agitation / adsorption unit 111 is installed so as to be embedded in the incubator 107.

図２において、分離容器２０１は円筒形で底部が丸く、上部には開口部が設けられている。この分離容器２０１は、容器ホルダ２０２の上面に設けられた挿入口に挿入される。挿入口の底部は、分離容器２０１の底部と同様に丸く形成されている。容器ホルダ２０２には、その上部に挿入口を挟んで磁石２０３が埋め込まれている。当該磁石２０３は永久磁石又は電磁石である。   In FIG. 2, the separation container 201 has a cylindrical shape with a round bottom and an opening at the top. The separation container 201 is inserted into an insertion port provided on the upper surface of the container holder 202. The bottom of the insertion port is formed round like the bottom of the separation container 201. A magnet 203 is embedded in the container holder 202 with an insertion opening interposed therebetween. The magnet 203 is a permanent magnet or an electromagnet.

容器ホルダ２０２の上方には、吐出/吸引ノズル２０４を保持する吐出/吸引ユニット２０５と再懸濁液ノズル２０６を保持する吐出/吸引ユニット２０７とが設置されている。各ノズル２０４及び２０６は、分離容器２０１の開口部に挿入可能である。また、吐出/吸引ノズル２０４はディスポーザブルであり、吐出/吸引ユニット２０５に自動的に脱着される。このように、吐出/吸引ノズル２０４がディスポーザブルである場合には、以前の測定でノズルに付着した物質で汚されることがなく、複数の種類の分離精製を連続して汚れなく行うことが可能である。なお、図２において、「２０８」は懸濁液であり、「２０９」は磁性粒子であり、「２１０」は被験物質と第１磁性粒子上の相互作用物質との結合体の集磁物である。   Disposed above the container holder 202 is a discharge / suction unit 205 that holds a discharge / suction nozzle 204 and a discharge / suction unit 207 that holds a resuspension nozzle 206. Each nozzle 204 and 206 can be inserted into the opening of the separation container 201. The discharge / suction nozzle 204 is disposable and is automatically attached to and detached from the discharge / suction unit 205. As described above, when the discharge / suction nozzle 204 is disposable, it is not contaminated by the substance adhering to the nozzle in the previous measurement, and a plurality of types of separation and purification can be continuously performed without contamination. is there. In FIG. 2, “208” is a suspension, “209” is a magnetic particle, and “210” is a magnetic collector of a combined body of a test substance and an interaction substance on the first magnetic particle. is there.

次に、磁性粒子の撹拌及び分離動作を、図３を用いて説明する。まず、分離容器３０１にサンプル液を分注する。サンプル液は、被験物質(例えば、血清)と、磁性粒子(第１磁性粒子及び第２磁性粒子)との混合物である。第１磁性粒子上の相互作用物質としては、抗原、抗体、核酸等が利用可能である。血清中の特定生体分子の存在により、当該特定生体分子が第１磁性粒子上の相互作用物質に特異的に結合することとなる。結合した第１磁性粒子上の相互作用物質と、結合せずに浮遊した第１磁性粒子上の相互作用物質の比率は血清中の特定生体分子の濃度に依存する。なお、分注したサンプル液は分離容器３０１内では懸濁液３０４を形成する。   Next, the stirring and separating operation of the magnetic particles will be described with reference to FIG. First, a sample liquid is dispensed into the separation container 301. The sample liquid is a mixture of a test substance (for example, serum) and magnetic particles (first magnetic particles and second magnetic particles). As the interaction substance on the first magnetic particle, an antigen, an antibody, a nucleic acid, or the like can be used. Due to the presence of the specific biomolecule in the serum, the specific biomolecule is specifically bound to the interacting substance on the first magnetic particle. The ratio of the interacting substance on the bound first magnetic particle to the interacting substance on the first magnetic particle that has floated without being bound depends on the concentration of the specific biomolecule in the serum. Note that the dispensed sample liquid forms a suspension 304 in the separation container 301.

懸濁液３０４を形成した後、分離容器３０１を容器ホルダの挿入口に挿入して、磁石３０２によって分離容器３０１内に磁界を作用させると、懸濁液３０４中の磁性粒子３０５(第１磁性粒子と第２磁性粒子)は分離容器３０１の壁面に集磁され、集磁物３０６を形成する。この際、磁石３０２は容器ホルダの上部に配置されているので、集磁物３０６は分離容器３０１の底部よりも上方位置に形成される。また、分離容器３０１の下部には磁場があまり作用しないので、懸濁液３０４の下部には集磁されていない磁性粒子３０５が存在することとなる。   After the suspension 304 is formed, when the separation container 301 is inserted into the insertion port of the container holder and a magnetic field is applied to the separation container 301 by the magnet 302, the magnetic particles 305 (first magnetic particles) in the suspension 304 are formed. Particles and second magnetic particles) are collected on the wall surface of the separation container 301 to form a magnetized material 306. At this time, since the magnet 302 is disposed on the upper part of the container holder, the magnetic collector 306 is formed at a position above the bottom of the separation container 301. Further, since the magnetic field does not act much at the lower part of the separation container 301, magnetic particles 305 that are not magnetized exist at the lower part of the suspension 304.

次に、吐出/吸引ノズル３０３を懸濁液３０４の液面位置まで上昇させる。そして、懸濁液３０４中の液成分を吸引しながら、液面の下降速度に合わせて吐出/吸引ノズル３０３を下降させる。このようにして、分離容器３０１の壁面には集磁物３０６が残されたままとなる。   Next, the discharge / suction nozzle 303 is raised to the liquid level position of the suspension 304. Then, while sucking the liquid component in the suspension 304, the discharge / suction nozzle 303 is lowered in accordance with the lowering speed of the liquid level. In this way, the magnetic current collector 306 remains on the wall surface of the separation container 301.

さらに、分離容器３０１内の開口部に再懸濁液ノズル３０７を挿入し、不要物質を含まない再懸濁液３０８を吐出する。吐出終了後、容器ホルダから分離容器３０１を取り出して攪拌し、再懸濁する。このように集磁した結合体(再懸濁液)を、所望の分析に供することができる。なお、分析は、集磁と同時に、又は集磁後に行うことができる。例えば、図１に示すユニット１０９によれば、標識が修飾された結合体の標識を集磁と同時に、又は集磁後に計数することで、被験物質を定量することができる。   Further, the resuspension nozzle 307 is inserted into the opening in the separation container 301, and the resuspension 308 containing no unnecessary substances is discharged. After completion of the discharge, the separation container 301 is taken out from the container holder, stirred and resuspended. The combined body (resuspension) collected in this way can be subjected to a desired analysis. The analysis can be performed at the same time as the magnetic collection or after the magnetic collection. For example, according to the unit 109 shown in FIG. 1, the test substance can be quantified by counting the label of the conjugate whose label is modified at the same time as or after the magnet collection.

以上に説明したように、本発明に係る方法によれば、担体として粒径の小さい磁性粒子をより粒径の大きい磁性粒子と共に用いることで、分析対象の反応の迅速性を損なわず、集磁時間を短縮することができ、分析の時間を短縮することができる。   As described above, according to the method of the present invention, by using magnetic particles having a small particle size together with magnetic particles having a larger particle size as a carrier, the magnetic collection is performed without impairing the speed of the reaction to be analyzed. Time can be shortened and analysis time can be shortened.

また、本発明に係るキットは、上記に説明した第１磁性粒子と第２磁性粒子とを含む被験物質分析用キットである。当該キットにおいて、第１磁性粒子と第２磁性粒子とは、別々の容器又は混合して一緒に容器中に用意することができる。さらに、当該キットは、第１磁性粒子と第２磁性粒子以外に、所望の分析に必要な試薬や使用説明書等を含んでいてもよい。なお、当該キットの使用の際には、対象となる被験物質や使用する装置等に応じて、第１磁性粒子及び第２磁性粒子のそれぞれの粒径や混合比等を上記の範囲内で最適化することが好ましい。   The kit according to the present invention is a test substance analysis kit including the first magnetic particles and the second magnetic particles described above. In the kit, the first magnetic particles and the second magnetic particles can be prepared in separate containers or mixed together and prepared in the containers. Further, the kit may contain reagents necessary for the desired analysis, instructions for use, etc. in addition to the first magnetic particles and the second magnetic particles. When using the kit, depending on the test substance to be used, the device to be used, etc., the respective particle sizes and mixing ratios of the first magnetic particles and the second magnetic particles are optimal within the above range. Is preferable.

以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲はこれら実施例に限定されるものではない。   EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

本実施例では、粒径の異なる市販の2つの磁性粒子を同時に用いて磁性粒子が磁石に集まるまでの時間が変化するか否かを検討した。   In this example, two commercially available magnetic particles having different particle diameters were used at the same time to examine whether or not the time until the magnetic particles gathered in the magnet changed.

1．磁性粒子懸濁液の調製
磁性粒子は、Dynal社Dynabeads MyOne Streptavidin T1(粒径1.05μm、以下「MyOne」という；本発明における「第１の磁性粒子」に相当する)とDynal社Dynabeads M-280 Streptavidin(粒径2.8μm、以下「M-280」という；本発明における「第２の磁性粒子」に相当する）の2種類を用いた。
これら2種類の磁性粒子をそれぞれ1mg/mLになるように、PBS溶液に懸濁した。これら懸濁液を、一定の割合で混合して使用した。 1． Preparation of Magnetic Particle Suspension Magnetic particles include Dynalads MyOne Streptavidin T1 from Dynal (particle size 1.05 μm, hereinafter referred to as “MyOne”; corresponding to “first magnetic particle” in the present invention) and Dynabeads M-280 from Dynal. Two types of Streptavidin (particle size: 2.8 μm, hereinafter referred to as “M-280”; corresponding to “second magnetic particle” in the present invention) were used.
These two kinds of magnetic particles were suspended in a PBS solution so as to be 1 mg / mL each. These suspensions were mixed and used at a certain ratio.

2．集磁時間の測定
集磁時間の測定は、分光光度計を用いた濁度測定により行った。懸濁液中の磁性粒子は、時間の変化とともに磁石に集められる。従って、集磁に従い、懸濁液中の磁性粒子濃度が低下する。 2． Measurement of magnetic collection time Measurement of magnetic collection time was performed by turbidity measurement using a spectrophotometer. The magnetic particles in the suspension are collected in a magnet over time. Therefore, the concentration of magnetic particles in the suspension decreases as the magnetic flux is collected.

溶液の物質濃度と吸光度とはランバート・ベールの法則により比例関係にあるので、測定開始直後の懸濁液の吸光度を集磁率0％とし、測定を続け、最も吸光度が低くなり、且つ変化がなくなったときの吸光度を集磁率100％とした。このようにして、集磁率50％、90％、95％に達するまでの時間を求めた。   Since the substance concentration and absorbance of the solution are proportional to each other according to Lambert-Beer's law, the absorbance of the suspension immediately after the start of measurement is assumed to be 0% and the measurement is continued, the absorbance becomes the lowest and there is no change. The absorbance at that time was taken as 100% magnetic collection. In this way, the time required to reach 50%, 90%, and 95% of the magnetic flux collection factor was obtained.

懸濁液の吸光度測定では、先ず2種類の磁性粒子懸濁液を一定の割合で混合し、当該混合液2mlを10mm角型セルに分注した。分光光度計の測定室内に磁石を設置し、当該磁石上に混合液を含有する10mm角型セルを置き、685nmでの吸光度を5分間測定した。なお、角型セルに分注した混合液は、測定室内に置く前にピペッティングにより十分に撹拌した。   In measuring the absorbance of the suspension, first, two types of magnetic particle suspensions were mixed at a constant ratio, and 2 ml of the mixed solution was dispensed into a 10 mm square cell. A magnet was placed in the measurement chamber of the spectrophotometer, a 10 mm square cell containing the mixed solution was placed on the magnet, and the absorbance at 685 nm was measured for 5 minutes. The mixed solution dispensed into the square cell was sufficiently agitated by pipetting before being placed in the measurement chamber.

3．2種類の磁性粒子を用いた場合の濁度による集磁時間の計測
MyOne：M-280＝8：2又は9：1の割合で混合した混合液の吸光度を測定した。結果を図４及び表１に示す。 3. Measurement of magnetic collection time by turbidity when two kinds of magnetic particles are used
The absorbance of the mixed solution mixed at a ratio of MyOne: M-280 = 8: 2 or 9: 1 was measured. The results are shown in FIG.

図４は、MyOne懸濁液(MyOneのみ)、MyOne：M-280＝8：2の混合液、MyOne：M-280＝9：1の混合液の吸光度の変化を測定した結果である。図４において、縦軸が吸光度であり、横軸が測定時間である。図４において、略号は以下を示す。
「MyOne」：MyOne懸濁液
「8：2」：MyOne：M-280＝8：2の混合液
「9：1」：MyOne：M-280＝9：1の混合液
図４に示すように、MyOneのみの懸濁液と比較して、M-280と混合した混合液の集磁時間が短くなっていることが分かる。 FIG. 4 shows the results of measuring changes in absorbance of MyOne suspension (only MyOne), MyOne: M-280 = 8: 2 mixture, and MyOne: M-280 = 9: 1 mixture. In FIG. 4, the vertical axis represents absorbance and the horizontal axis represents measurement time. In FIG. 4, abbreviations indicate the following.
“MyOne”: MyOne suspension “8: 2”: MyOne: M-280 = 8: 2 mixture “9: 1”: MyOne: M-280 = 9: 1 mixture As shown in FIG. It can be seen that the magnetic collection time of the mixed solution mixed with M-280 is shorter than the suspension of MyOne alone.

一方、以下の表１は、懸濁液又は混合液の集磁率50％、90％、95％に達するまでの時間を示したものである。   On the other hand, Table 1 below shows the time required to reach 50%, 90%, and 95% of the magnetic flux collection ratio of the suspension or mixed solution.

Figure 2009186356
Figure 2009186356

表１に示すように、MyOne：M-280＝8：2又は9：1の割合で混合した混合液は、MyOneのみの懸濁液と比較して、約15〜20％集磁時間を短縮できたことが分かる。   As shown in Table 1, MyOne: M-280 = 8: 2 or 9: 1 mixed solution shortens the magnetic collection time by about 15-20% compared to MyOne-only suspension. You can see that it was made.

化学分析装置の構成を示す傾視図である。It is a perspective view which shows the structure of a chemical analyzer. 図１に示す化学分析装置に装備されている撹拌/吸着ユニットを示す図である。It is a figure which shows the stirring / adsorption unit with which the chemical analyzer shown in FIG. 1 is equipped. 図１に示す化学分析装置における磁性粒子の撹拌及び分離動作を示す図である。It is a figure which shows the stirring and separation operation | movement of a magnetic particle in the chemical analyzer shown in FIG. 集磁において磁性粒子の懸濁液又は混合液の吸光度の変化を示す特性図である。It is a characteristic figure which shows the change of the light absorbency of the suspension or mixed liquid of a magnetic particle in magnetic collection.

Claims (17)

溶液中に分散した2種類の磁性粒子を同時に用いて、被験物質を分析する方法であって、第１磁性粒子上に前記被験物質と相互作用する物質が固定されており、且つ第２磁性粒子の粒径が第１磁性粒子と比較して大きいことを特徴とする、前記方法。   A method for analyzing a test substance by simultaneously using two types of magnetic particles dispersed in a solution, wherein a substance interacting with the test substance is fixed on the first magnetic particle, and the second magnetic particle The method described above is characterized in that the particle size of is larger than that of the first magnetic particles. 被験物質と相互作用する物質が固定された第１磁性粒子及び第２磁性粒子を含有する溶液と被験物質とを接触させる工程と、
前記第１磁性粒子上の被験物質と相互作用する物質と前記被験物質との結合体を集磁する工程と、
集磁した前記結合体を分析に供する工程と、
を含み、前記第２磁性粒子の粒径が前記第１磁性粒子と比較して大きいことを特徴とする、被験物質を分析する方法。 Contacting the test substance with a solution containing the first magnetic particles and the second magnetic particles to which a substance that interacts with the test substance is fixed;
Collecting a substance that interacts with the test substance on the first magnetic particle and the test substance;
A step of subjecting the combined magnetic flux to analysis;
A method for analyzing a test substance, wherein the second magnetic particles have a larger particle size than the first magnetic particles. 前記第１磁性粒子の粒径が10nm〜1μmであることを特徴とする、請求項１又は２記載の方法。   The method according to claim 1, wherein the first magnetic particles have a particle size of 10 nm to 1 μm. 前記第２磁性粒子の粒径が1μm〜10μmであることを特徴とする、請求項１又は２記載の方法。   The method according to claim 1 or 2, wherein the second magnetic particles have a particle size of 1 µm to 10 µm. 前記第２磁性粒子の使用総重量が前記第１磁性粒子の使用総重量と比較して低いことを特徴とする、請求項１又は２記載の方法。   The method according to claim 1 or 2, wherein the total used weight of the second magnetic particles is lower than the total used weight of the first magnetic particles. 前記第１磁性粒子と前記第２磁性粒子との使用総重量比が9：1〜99：1であることを特徴とする、請求項１又は２記載の方法。   The method according to claim 1 or 2, wherein the total weight ratio of the first magnetic particles and the second magnetic particles is 9: 1 to 99: 1. 前記第２磁性粒子の飽和磁化が前記第１磁性粒子と比較して大きいことを特徴とする、請求項１又は２記載の方法。   The method according to claim 1, wherein the saturation magnetization of the second magnetic particle is larger than that of the first magnetic particle. 前記被験物質がタンパク質、核酸、糖類、細菌、細胞及び体液成分から成る群より選択される、請求項１又は２記載の方法。   The method according to claim 1 or 2, wherein the test substance is selected from the group consisting of proteins, nucleic acids, saccharides, bacteria, cells and body fluid components. 前記分析が、前記被験物質と前記被験物質と相互作用する物質との間の相互作用並びに前記被験物質の検出、定量及び分離から成る群より選択される、請求項１又は２記載の方法。   The method according to claim 1 or 2, wherein the analysis is selected from the group consisting of an interaction between the test substance and a substance that interacts with the test substance, and detection, quantification, and separation of the test substance. 被験物質と相互作用する物質が固定された第１磁性粒子と第２磁性粒子とを含み、第２磁性粒子の粒径が第１磁性粒子と比較して大きいことを特徴とする、被験物質分析用キット。   Test substance analysis comprising first magnetic particles and second magnetic particles to which a substance interacting with a test substance is fixed, wherein the second magnetic particles have a larger particle size than the first magnetic particles For kit. 前記第１磁性粒子の粒径が10nm〜1μmであることを特徴とする、請求項１０記載のキット。   The kit according to claim 10, wherein the first magnetic particles have a particle size of 10 nm to 1 μm. 前記第２磁性粒子の粒径が1μm〜10μmであることを特徴とする、請求項１０記載のキット。   The kit according to claim 10, wherein the second magnetic particles have a particle size of 1 μm to 10 μm. 使用において、前記第２磁性粒子の使用総重量が前記第１磁性粒子の使用総重量と比較して低いことを特徴とする、請求項１０記載のキット。   11. The kit according to claim 10, wherein, in use, the total used weight of the second magnetic particles is lower than the total used weight of the first magnetic particles. 使用において、前記第１磁性粒子と前記第２磁性粒子との使用総重量比が9：1〜99：1であることを特徴とする、請求項１０記載のキット。   The kit according to claim 10, wherein the total weight ratio of the first magnetic particles and the second magnetic particles in use is 9: 1 to 99: 1. 前記第２磁性粒子の飽和磁化が前記第１磁性粒子と比較して大きいことを特徴とする、請求項１０記載のキット。   The kit according to claim 10, wherein the saturation magnetization of the second magnetic particles is larger than that of the first magnetic particles. 前記被験物質がタンパク質、核酸、糖類、細菌、細胞及び体液成分から成る群より選択される、請求項１０記載のキット。   The kit according to claim 10, wherein the test substance is selected from the group consisting of proteins, nucleic acids, saccharides, bacteria, cells, and body fluid components. 前記分析が、前記被験物質と前記被験物質と相互作用する物質との間の相互作用並びに前記被験物質の検出、定量及び分離から成る群より選択される、請求項１０記載のキット。   The kit according to claim 10, wherein the analysis is selected from the group consisting of an interaction between the test substance and a substance that interacts with the test substance, and detection, quantification and separation of the test substance.
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