JP7363327B2 - Magnetic particles for antibody binding and their manufacturing method - Google Patents
Magnetic particles for antibody binding and their manufacturing method Download PDFInfo
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- JP7363327B2 JP7363327B2 JP2019185800A JP2019185800A JP7363327B2 JP 7363327 B2 JP7363327 B2 JP 7363327B2 JP 2019185800 A JP2019185800 A JP 2019185800A JP 2019185800 A JP2019185800 A JP 2019185800A JP 7363327 B2 JP7363327 B2 JP 7363327B2
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- Polymerisation Methods In General (AREA)
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Description
本発明は、免疫診断に用いられる抗体結合用磁性粒子およびその製造方法に関する。 The present invention relates to antibody-binding magnetic particles used in immunodiagnosis and a method for producing the same.
磁性粒子は、その磁気的特性を利用し、免疫診断用の生理活性物質担持用担体として使用されている。しかし、抗体等の生理活性物質を磁性粒子の表面に結合する際、酸性水溶液中で反応させるため、磁性体の脱離や鉄イオン溶出など、磁性体成分に由来する物質が流出するという問題があった。鉄は免疫反応の干渉物質として働くため、免疫診断の感度の低下や感作可能な生理活性物質は限定されるという問題があり、このような磁性体成分の流出が少ない磁性粒子が望まれている。また、免疫診断用担体としての磁性粒子には、免疫反応に無関係な成分が非特異吸着することを抑制した低ノイズ化や、多くの生理活性物質を担持可能とする高シグナル化等、免疫診断の高感度化が可能な磁性粒子が求められている。さらに、免疫診断では磁性粒子を水溶液に分散させて使用することから、水への分散性が良好な磁性粒子が求められている。 Magnetic particles are used as carriers for supporting physiologically active substances for immunodiagnosis by utilizing their magnetic properties. However, when binding physiologically active substances such as antibodies to the surface of magnetic particles, the reaction is carried out in an acidic aqueous solution, which causes problems such as detachment of the magnetic substance and elution of iron ions, resulting in the outflow of substances derived from the magnetic substance components. there were. Since iron acts as an interfering substance in immune reactions, there is a problem that the sensitivity of immunodiagnosis decreases and the number of physiologically active substances that can be sensitized is limited.Therefore, magnetic particles with less leakage of magnetic components are desired. There is. In addition, magnetic particles used as carriers for immunodiagnosis have features such as low noise that suppresses nonspecific adsorption of components unrelated to immune reactions, and high signal that can support many physiologically active substances. There is a need for magnetic particles that can achieve high sensitivity. Furthermore, since magnetic particles are used in immunodiagnosis by being dispersed in an aqueous solution, there is a need for magnetic particles that have good dispersibility in water.
これらの課題を解決するため、コア粒子表面にフェライト被覆層を有する磁性粒子の表面をアミノ基等の官能基を有する有機シランカップリング剤で被覆し、さらにカルボキシル基を導入した免疫診断用粒子が提案されている(例えば、特許文献1参照。)。また、疎水性の有機シランカップリング剤で被覆したナノ磁性体をコア粒子に吸着させた後、ナノ磁性体が埋没するように疎水性重合体で被覆し、さらにその表面に官能基を導入した診断薬用粒子が提案されている(例えば、特許文献2参照。)。 In order to solve these problems, we have developed immunodiagnostic particles in which the surface of magnetic particles having a ferrite coating layer on the core particle surface is coated with an organic silane coupling agent having a functional group such as an amino group, and further a carboxyl group is introduced. It has been proposed (for example, see Patent Document 1). In addition, after adsorbing nanomagnetic material coated with a hydrophobic organosilane coupling agent onto core particles, the nanomagnetic material was coated with a hydrophobic polymer so that it was buried, and functional groups were further introduced onto the surface. Particles for diagnostic reagents have been proposed (see, for example, Patent Document 2).
特許文献1では、有機シランカップリング剤で表面を処理することによって鉄の溶出を抑制できるものの、抑制が不十分であるという問題があった。 In Patent Document 1, the elution of iron can be suppressed by treating the surface with an organic silane coupling agent, but there is a problem that the suppression is insufficient.
特許文献2では、疎水性の有機シランカップリング剤で処理した磁性体をコア粒子表面に被覆せしめた後、疎水性重合体層を形成することで鉄の溶出を抑制しているものの、疎水性重合体層は磁性粒子の水への分散性を悪化させるという問題があった。また、疎水性重合体層で被覆することでナノ磁性体が重合体で埋没するため、磁性粒子の比表面積が小さく、抗体等の生理活性物質を目的量担持させるためには多量の磁性粒子が必要であるという問題があった。 In Patent Document 2, the surface of the core particle is coated with a magnetic material treated with a hydrophobic organosilane coupling agent, and then a hydrophobic polymer layer is formed to suppress the elution of iron. The polymer layer has the problem of worsening the dispersibility of magnetic particles in water. Furthermore, since the nanomagnetic material is buried in the polymer by being coated with a hydrophobic polymer layer, the specific surface area of the magnetic particles is small, and a large amount of magnetic particles is required to support the desired amount of physiologically active substances such as antibodies. The problem was that it was necessary.
本発明は上記課題に鑑みてなされたものであり、耐酸性に優れ酸性水溶液で処理しても鉄の溶出が少なく、さらに水への分散性が良好であり、また、非特異吸着が少なく、並びに、粒子の比表面積が大きく生理活性物質の担持量が多い、免疫診断の高感度化が可能な磁性粒子を提供することにある。 The present invention was made in view of the above problems, and has excellent acid resistance, little elution of iron even when treated with an acidic aqueous solution, good dispersibility in water, and low nonspecific adsorption. Another object of the present invention is to provide magnetic particles that have a large specific surface area, support a large amount of physiologically active substances, and are capable of increasing the sensitivity of immunodiagnosis.
本発明者らは、以上の点を鑑み鋭意研究を重ねた結果、磁性粒子によって上記課題を解決できることを見出し、本発明を完成するに至った。 In view of the above points, the present inventors have conducted extensive research, and as a result have found that the above-mentioned problems can be solved by using magnetic particles, and have completed the present invention.
本発明の各態様は以下に示す[1]~[4]である。
[1] コア粒子と、前記コア粒子表面に形成された第1の被覆層と、前記第1の被覆層の表面に形成された第2の被覆層を有する磁性粒子であって、
前記第1の被覆層は、ナノ磁性体を含有する架橋無機物層であり、
前記第2の被覆層は、ヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有する重合体層であり、第1の被覆層と共有結合を形成し、前記共有結合とは反対側の高分子鎖末端にカルボキシル基を有することを特徴とする、抗体結合用磁性粒子。
[2] 前記第1の被覆層の架橋無機物層がシリカを含有することを特徴とする、[1]に記載の抗体結合用磁性粒子。
[3] 前記第2の被覆層である重合体層が、架橋構造であることを特徴とする、[1]または[2]に記載の抗体結合用磁性粒子。
[4] 下記(i)~(v)の工程を含むことを特徴とする、[1]~[3]のいずれかに記載の抗体結合用磁性粒子の製造方法。
(i)コア粒子の表面にナノ磁性体を物理吸着させる工程
(ii)前記ナノ磁性体を吸着させた粒子の表面に架橋無機物層を形成する工程
(iii)前記架橋無機物層の表面に、リビングラジカル重合の開始剤又は連鎖移動剤を有する、シランカップリング剤を結合させる工程
(iv)前記シランカップリング剤を結合させた粒子を溶媒に分散させ、溶媒中でヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有するモノマーをリビングラジカル重合することにより粒子の表面に重合体層を形成する工程
(v)共重合体層を形成した粒子にメルカプトカルボン酸を反応させる工程
Each aspect of the present invention is [1] to [4] shown below.
[1] A magnetic particle having a core particle, a first coating layer formed on the surface of the core particle, and a second coating layer formed on the surface of the first coating layer,
The first coating layer is a crosslinked inorganic layer containing nanomagnetic material,
The second coating layer is a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group, and forms a covalent bond with the first coating layer, and forms a covalent bond with the first coating layer. Magnetic particles for antibody binding characterized by having a carboxyl group at the end of the polymer chain on the opposite side from the binding.
[2] The magnetic particle for antibody binding according to [1], wherein the crosslinked inorganic layer of the first coating layer contains silica.
[3] The antibody-binding magnetic particle according to [1] or [2], wherein the polymer layer that is the second coating layer has a crosslinked structure.
[4] The method for producing antibody-binding magnetic particles according to any one of [1] to [3], which comprises the following steps (i) to (v).
(i) A step of physically adsorbing the nanomagnetic material on the surface of the core particle (ii) A step of forming a crosslinked inorganic layer on the surface of the particle to which the nanomagnetic material is adsorbed (iii) A step of physically adsorbing the nanomagnetic material on the surface of the core particle Step (iv) of bonding a silane coupling agent having a radical polymerization initiator or chain transfer agent: dispersing the particles bound with the silane coupling agent in a solvent, and dispersing the particles selected from hydroxyl groups and polyethylene glycol groups in the solvent. step (v) of forming a polymer layer on the surface of the particles by living radical polymerization of a monomer having at least one hydrophilic group-containing structural unit, (v) reacting the particles with the copolymer layer formed with mercaptocarboxylic acid; process of letting
本発明の一態様にかかる抗体結合用磁性粒子は、鉄の溶出抑制と粒子の水への分散性を有するものであり、また、抗体等の生理活性物質の担持量が多く、免疫診断の高感度化が可能となる。 The magnetic particles for antibody binding according to one embodiment of the present invention have iron elution suppression and particle dispersibility in water, and also have a large amount of physiologically active substances such as antibodies supported, making them highly suitable for immunodiagnosis. Sensitization becomes possible.
以下、本発明を実施するための形態(以下、単に「本実施の形態」という。)について詳細に説明する。以下の本実施の形態は、本発明を説明するための例示であり、本発明を以下の内容に限定する趣旨ではない。本発明は、その趣旨の範囲内で適宜に変形して実施できる。 Hereinafter, a mode for carrying out the present invention (hereinafter simply referred to as "this embodiment") will be described in detail. The present embodiment below is an illustration for explaining the present invention, and is not intended to limit the present invention to the following content. The present invention can be implemented with appropriate modifications within the scope of its spirit.
本発明の一態様にかかる抗体結合用磁性粒子は、
コア粒子と、前記コア粒子表面に形成された第1の被覆層と、前記第1の被覆層の表面に形成された第2の被覆層を有する磁性粒子であって、
前記第1の被覆層は、ナノ磁性体を含有する架橋無機物層であり、
前記第2の被覆層は、ヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有する重合体層であり、第1の被覆層と共有結合を形成し、前記共有結合とは反対側の高分子鎖末端にカルボキシル基を有する。
Magnetic particles for antibody binding according to one aspect of the present invention include:
A magnetic particle having a core particle, a first coating layer formed on the surface of the core particle, and a second coating layer formed on the surface of the first coating layer,
The first coating layer is a crosslinked inorganic layer containing nanomagnetic material,
The second coating layer is a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group, and forms a covalent bond with the first coating layer, and forms a covalent bond with the first coating layer. It has a carboxyl group at the end of the polymer chain on the opposite side from the bond.
抗体結合用磁性粒子に用いられるコア粒子は、磁性粒子の最も中心に位置する粒子であり、コア粒子の形状や材質に特に限定はない。コア粒子の形状は適宜選択可能であり、真球に近い球状、楕円体や立方体、円柱、多角柱等の非球状、多孔質状や突起を有するもの等の比表面積の大きな凹凸を有する粒子等を挙げることができる。コア粒子の表面に多くのナノ磁性体を被覆させるのに好適のため、多孔質状や突起を有するもの等の比表面積の大きな凹凸を有する粒子が好ましい。 The core particle used in the antibody-binding magnetic particle is a particle located at the center of the magnetic particle, and there are no particular limitations on the shape or material of the core particle. The shape of the core particles can be selected as appropriate, including spherical shapes that are close to true spheres, non-spherical shapes such as ellipsoids, cubes, cylinders, and polygonal columns, and particles that have irregularities with a large specific surface area such as porous shapes and those with protrusions. can be mentioned. Particles having irregularities with a large specific surface area, such as porous particles or those having protrusions, are preferable because they are suitable for coating the surface of the core particle with a large amount of nanomagnetic material.
磁気応答性が良好な磁性粒子とするため、コア粒子の粒径としては0.1μm以上が好ましく、0.5μm以上がさらに好ましく、1μm以上が特に好ましく、2μm以上が最も好ましい。また、水への再分散性が良好な磁性粒子とするために、コア粒子の粒径は100μm以下が好ましく、50μm以下がさらに好ましく、10μm以下が特に好ましく、5μm以下が最も好ましい。 In order to obtain magnetic particles with good magnetic responsiveness, the particle size of the core particles is preferably 0.1 μm or more, more preferably 0.5 μm or more, particularly preferably 1 μm or more, and most preferably 2 μm or more. Further, in order to obtain magnetic particles with good redispersibility in water, the particle size of the core particles is preferably 100 μm or less, more preferably 50 μm or less, particularly preferably 10 μm or less, and most preferably 5 μm or less.
コア粒子の材質としては、例えば重合体等の有機化合物、無機化合物等を挙げることができる。これらから適宜選択できるが、例えばスチレン系単量体単位、アクリレート系単量体単位若しくはメタクリレート系単量体単位を含む重合体粒子、又はシリカ粒子を挙げることができる。 Examples of the material of the core particles include organic compounds such as polymers, inorganic compounds, and the like. Although it can be appropriately selected from these, examples include polymer particles containing a styrene monomer unit, an acrylate monomer unit, or a methacrylate monomer unit, or silica particles.
前記のスチレン系単量体としては、スチレン、α-メチルスチレン、ビニルトルエン、p-メチルスチレン、2-メチルスチレン、3-メチルスチレン、4-メチルスチレン、4-エチルスチレン、4-tert-ブチルスチレン、3,4-ジメチルスチレン、4-メトキシスチレン、4-エトキシスチレン、2-クロロスチレン、3-クロロスチレン、4-クロロスチレン、2,4-ジクロロスチレン、2,6-ジクロロスチレン、4-クロロ-3-メチルスチレン、ジビニルベンゼン、p-スチレンスルホン酸ナトリウム等を挙げることができる。 Examples of the styrene monomers include styrene, α-methylstyrene, vinyltoluene, p-methylstyrene, 2-methylstyrene, 3-methylstyrene, 4-methylstyrene, 4-ethylstyrene, and 4-tert-butyl. Styrene, 3,4-dimethylstyrene, 4-methoxystyrene, 4-ethoxystyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 2,4-dichlorostyrene, 2,6-dichlorostyrene, 4- Examples include chloro-3-methylstyrene, divinylbenzene, and sodium p-styrenesulfonate.
前記のアクリレート系単量体若しくはメタアクリレート系単量体としては、(メタ)アクリレート、メチル(メタ)アクリレート、エチル(メタ)アクリレート、プロピル(メタ)アクリレート、n-へキシル(メタ)アクリレート、2-エチルヘキシル(メタ)アクリレート、シクロヘキシル(メタ)アクリレート等の(シクロ)アルキル(メタ)アクリレート類; 2-メトキシエチル(メタ)アクリレート、p-メトキシシクロヘキシル(メタ)アクリレート等のアルコキシ(シクロ)アルキル(メタ)アクリレート類;トリメチロールプロパントリ(メタ)アクリレート等の多価(メタ)アクレート類;酢酸ビニル、プロピオン酸ビニル、バーサチック酸ビニル等のビニルエステル類;2-シアノエチル(メタ)アクリレート、2-シアノプロピル(メタ)アクリレート、3-シアノプロピル(メタ)アクリレート等のシアノアクリレート類;ヒドロキシメチル(メタ)アクリレート、2-ヒドロキシエチル(メタ)アクリレート、6-ヒドロキシヘキシル(メタ)アクリレート、4-ヒドロキシシクロヘキシル(メタ)アクリレート、ネオペンチルグリコールモノ(メタ)アクリレート、 3-クロロ-2-ヒドロキシプロピル(メタ)アクリレート、3-アミノ-2-ヒドロキシプロピル(メタ)アクリレート等の置換ヒドロキシ(メタ)アクリレート類;グリシジル(メタ)アクリレート、メチルグリシジルメチルアクリレート、エポキシ化シクロヘキシル(メタ)アクリレート等のグリシジル基含有アクリレート類;トリメチロールプロパンエトキシトリアクリレート、ペンタエリスリトールエトキシテトラアクリレート、トリメチロールプロパンプロポキシトリアクリレート、ペンタエリスリトールトリアクリレート、ペンタエリスリトールテトラアクリレート、ジペンタエリスリトールヘキサアクリレート、ポリエチレングリコールジアクリレート、ポリプロピレングリコールジアクリレート、トリシクロデカンジメタノールジアクリレート、エトキシ化フェニルアクリレート等の多官能(メタ)アクリレート類等の(メタ)アクリレートを単量体単位とする重合体粒子が挙げられる。また、前記アクリレートは架橋したものであってもよい。 Examples of the acrylate monomer or methacrylate monomer include (meth)acrylate, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-hexyl (meth)acrylate, 2 - (Cyclo)alkyl (meth)acrylates such as ethylhexyl (meth)acrylate and cyclohexyl (meth)acrylate; Alkoxy(cyclo)alkyl (meth)acrylates such as 2-methoxyethyl (meth)acrylate and p-methoxycyclohexyl (meth)acrylate; ) Acrylates; Polyvalent (meth)acrylates such as trimethylolpropane tri(meth)acrylate; Vinyl esters such as vinyl acetate, vinyl propionate, vinyl versatate; 2-cyanoethyl (meth)acrylate, 2-cyanopropyl Cyanoacrylates such as (meth)acrylate, 3-cyanopropyl (meth)acrylate; hydroxymethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 6-hydroxyhexyl (meth)acrylate, 4-hydroxycyclohexyl (meth)acrylate; ) acrylate, neopentyl glycol mono(meth)acrylate, 3-chloro-2-hydroxypropyl(meth)acrylate, 3-amino-2-hydroxypropyl(meth)acrylate; substituted hydroxy(meth)acrylates; glycidyl(meth)acrylate; ) acrylate, acrylates containing glycidyl groups such as methylglycidyl methyl acrylate, epoxidized cyclohexyl (meth)acrylate; trimethylolpropane ethoxy triacrylate, pentaerythritol ethoxytetraacrylate, trimethylolpropane propoxy triacrylate, pentaerythritol triacrylate, pentaerythritol Monomers containing (meth)acrylates such as polyfunctional (meth)acrylates such as tetraacrylate, dipentaerythritol hexaacrylate, polyethylene glycol diacrylate, polypropylene glycol diacrylate, tricyclodecane dimethanol diacrylate, and ethoxylated phenyl acrylate. Examples include polymer particles used as units. Furthermore, the acrylate may be crosslinked.
必要に応じて、コア粒子はナノ磁性体を含有してもよい。また、第1の被覆層を設けるための前処理として、コア粒子の表面に正電荷又は負電荷を有する物質を被覆してもよい。また、コア粒子の表面に常磁性の薄膜の被覆層を設けてもよい。常磁性の被覆層を設ける場合、粒子への残留磁化を低減し、粒子同士の凝集を抑制するのに好適のため、厚み0.001~1μmが好ましく、0.001~0.1μmがさらに好ましく、0.001~0.01μmが特に好ましく、0.001~0.005μmが最も好ましい。コア粒子の表面に正電荷又は負電荷を有する物質を被覆する方法としては特に限定はないが、側鎖に電荷を有する単量体を用いてコア粒子の表面に重合体層を形成する方法、電荷を有する重合体を交互積層法(Layer-by-Layer法)によりコア粒子表面に被覆する方法等を挙げることができる。 If necessary, the core particle may contain nanomagnetic material. Further, as a pretreatment for providing the first coating layer, the surface of the core particle may be coated with a substance having a positive charge or a negative charge. Further, a paramagnetic thin film coating layer may be provided on the surface of the core particle. When providing a paramagnetic coating layer, the thickness is preferably 0.001 to 1 μm, more preferably 0.001 to 0.1 μm, since it is suitable for reducing residual magnetization of particles and suppressing agglomeration of particles. , 0.001 to 0.01 μm is particularly preferred, and 0.001 to 0.005 μm is most preferred. There are no particular limitations on the method of coating the surface of the core particle with a substance having a positive or negative charge, but methods include forming a polymer layer on the surface of the core particle using a monomer having a charge on the side chain; Examples include a method in which the surface of the core particle is coated with a charged polymer by a layer-by-layer method.
コア粒子表面に形成された第1の被覆層は、ナノ磁性体を含有する架橋無機物層である。ナノ磁性体は、前記コア粒子よりも粒径の小さな粒子であり、さらに磁気応答性を有するものである。超常磁性を有するものであることが好ましいことから、ナノ磁性体の粒径は0.001~1μmが好ましく、0.001~0.5μmがさらに好ましく、0.001~0.1μmが特に好ましく、0.001~0.05μmが最も好ましい。材質としては例えば、酸化鉄を挙げることができる。ナノ磁性体は表面に脂質やシランカップリング剤等の無機表面改質剤を含んでいてもよい。前記シランカップリング剤としては、例えば、ビニルトリメトキシシラン、ビニルトリエトキシシラン、2-(3,4-エポキシシクロヘキシル)エチルトリメトキシシラン、3-グリシドキシプロピルトリメトキシシラン、p-スチリルトリメトキシシラン、3-メタクリロキシプロピルメチルジメトキシシラン、3-メタクリロキシプロピルトリメトキシシラン、3-メタクリロキシプロピルメチルジエトキシシラン、3-メタクリロキシプロピルトリエトキシシラン、3-アクリロキシプロピルトリメトキシシラン、N-2-(アミノエチル)-3-アミノプロピルメチルジメトキシシラン、3-アミノプロピルトリメトキシシラン、3-アミノプロピルトリエトキシシラン、トリス-(トリメトキシシリルプロピル)イソシアヌレート、3-ウレイドプロピルトリアルコキシシラン、3-メルカプトプロピルメチルジメトキシシラン、ビス(トリエトキシシリルプロピル)テトラスルフィド、3-イソシアネートプロピルトリエトキシシラン等が挙げられる。 The first coating layer formed on the surface of the core particle is a crosslinked inorganic layer containing nanomagnetic material. The nanomagnetic material is a particle having a smaller particle size than the core particle, and further has magnetic responsiveness. Since it is preferable to have superparamagnetism, the particle size of the nanomagnetic material is preferably 0.001 to 1 μm, more preferably 0.001 to 0.5 μm, particularly preferably 0.001 to 0.1 μm, Most preferably 0.001 to 0.05 μm. Examples of the material include iron oxide. The nanomagnetic material may contain an inorganic surface modifier such as a lipid or a silane coupling agent on the surface. Examples of the silane coupling agent include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropyltrimethoxysilane, and p-styryltrimethoxysilane. Silane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N- 2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyltrialkoxysilane, Examples include 3-mercaptopropylmethyldimethoxysilane, bis(triethoxysilylpropyl)tetrasulfide, and 3-isocyanatepropyltriethoxysilane.
ナノ磁性体は架橋無機物層中に分散した状態にあることが好ましい。ナノ磁性体が架橋無機物層中に分散した状態にあることで、全てのナノ磁性体の表面を架橋無機物層で均一に被覆することができ、磁性粒子の耐酸性を高めることができる。 The nanomagnetic material is preferably dispersed in the crosslinked inorganic layer. By dispersing the nanomagnetic material in the crosslinked inorganic layer, the surfaces of all the nanomagnetic materials can be uniformly coated with the crosslinked inorganic layer, and the acid resistance of the magnetic particles can be improved.
ナノ磁性体の含有量としては、磁気応答性の良好な磁性粒子とするのに好適のため、磁性粒子1g当たりのナノ磁性体の含有量として、0.1g/g以上が好ましく、0.2g/g以上がさらに好ましく、0.5g/g以上が特に好ましく、0.7g/g以上が最も好ましい。また、磁性粒子の水への分散性を高めるのに好適であることから、磁性粒子1g当たりのナノ磁性体の含有量として、0.9g/g以下が好ましく、0.8g/g以下がさらに好ましく、0.7g/g以下が特に好ましく、0.6g/g以下が最も好ましい。 The content of nanomagnetic material is preferably 0.1 g/g or more, and 0.2 g as the content of nanomagnetic material per 1 g of magnetic particles, since it is suitable for forming magnetic particles with good magnetic response. /g or more is more preferable, 0.5 g/g or more is particularly preferable, and 0.7 g/g or more is most preferable. In addition, since it is suitable for increasing the dispersibility of magnetic particles in water, the content of nanomagnetic material per gram of magnetic particles is preferably 0.9 g/g or less, and more preferably 0.8 g/g or less. It is preferably 0.7 g/g or less, particularly preferably 0.6 g/g or less, and most preferably 0.6 g/g or less.
架橋無機物層はコア粒子に架橋無機物層を形成することにより形成される。架橋無機物としては、シリカやガラス、酸化マグネシウム、酸化アルミナ等の酸化物、ジルコニアやヒドロキシアパタイト等のセラミックスを挙げることができる。これらの中で耐酸性を高めるのに特に好適のため、シリカが好ましい。架橋無機物層を設けることで、親水性でありながら耐酸性を高めることが可能であることから、鉄の溶出抑制と粒子の水への分散性という、相反する特性を付与することができる。また、架橋無機物層は粒子表面の磁性体の形状に沿って被覆され、磁性体を完全に埋没させないため、比表面積の大きな磁性粒子を得ることができ、抗体等の生理活性物質の担持量を増やすことができる。架橋無機物層の厚みとしては、耐酸性を高めるのに好適であることから、0.001μm以上が好ましく、0.005μm以上がさらに好ましく、0.01μm以上が特に好ましく、0.05μm以上が最も好ましい。また、比表面積の大きな磁性粒子とするのに好適のため、架橋無機物層の厚みは1μm以下が好ましく、0.5μm以下がさらに好ましく、0.1μm以下が特に好ましく、0.01以下が最も好ましい。 The crosslinked inorganic layer is formed by forming a crosslinked inorganic layer on the core particle. Examples of crosslinked inorganic substances include silica, glass, oxides such as magnesium oxide and alumina oxide, and ceramics such as zirconia and hydroxyapatite. Among these, silica is preferred because it is particularly suitable for increasing acid resistance. By providing a crosslinked inorganic layer, it is possible to increase acid resistance while being hydrophilic, and therefore it is possible to impart contradictory properties of suppressing elution of iron and dispersibility of particles in water. In addition, the crosslinked inorganic layer is coated along the shape of the magnetic material on the particle surface and does not completely bury the magnetic material, making it possible to obtain magnetic particles with a large specific surface area and reducing the amount of physiologically active substances such as antibodies carried. can be increased. The thickness of the crosslinked inorganic layer is preferably 0.001 μm or more, more preferably 0.005 μm or more, particularly preferably 0.01 μm or more, and most preferably 0.05 μm or more, since it is suitable for increasing acid resistance. . Further, since it is suitable for forming magnetic particles with a large specific surface area, the thickness of the crosslinked inorganic layer is preferably 1 μm or less, more preferably 0.5 μm or less, particularly preferably 0.1 μm or less, and most preferably 0.01 μm or less. .
第1の被覆層の表面に形成された第2の被覆層は、ヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有する重合体層であり、第1の被覆層と共有結合を形成し、前記共有結合とは反対側の高分子鎖末端にカルボキシル基を有する。 The second coating layer formed on the surface of the first coating layer is a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group; It forms a covalent bond with the layer and has a carboxyl group at the end of the polymer chain on the opposite side from the covalent bond.
コア粒子表面に第1の被覆層を形成した粒子は、抗体等の生理活性物質を粒子に結合するための官能基を有していないことから、生理活性物質を担持することができないため、架橋無機物層を設けた粒子の表面に第2の被覆層を設けることにより、非特異吸着を抑制しつつ抗体を担持させることができるようになり、免疫診断の高感度化が可能となる。カルボキシル基含有構造単位だけでは非特異吸着が高く、免疫診断の高感度化はできない。カルボキシル基含有構造単位及び親水性基含有構造単位の2つを有することにより、免疫診断の高感度化が可能となる。 Particles with a first coating layer formed on the surface of the core particles do not have functional groups for binding physiologically active substances such as antibodies to the particles, and therefore cannot support physiologically active substances, so crosslinking is not possible. By providing the second coating layer on the surface of the particles provided with the inorganic layer, it becomes possible to carry antibodies while suppressing non-specific adsorption, and it becomes possible to increase the sensitivity of immunodiagnosis. Carboxyl group-containing structural units alone result in high nonspecific adsorption, making it impossible to increase the sensitivity of immunodiagnosis. By having two structural units, a carboxyl group-containing structural unit and a hydrophilic group-containing structural unit, it becomes possible to increase the sensitivity of immunodiagnosis.
第2の被覆層は、ヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有する単量体を重合することにより形成される。前記単量体としては、重合速度が速く、磁性粒子の量産性を高めるのに好適のため、ヒドロキシル基含有アクリレート若しくはヒドロキシル基含有メタクリレート、又は、ポリエチレングリコール基含有アクリレート若しくはポリエチレングリコール基含有メタクリレートが好ましい。また、免疫診断の高感度化に特に好適のため、ポリエチレングリコール基含有アクリレート又はポリエチレングリコール基含有メタクリレートがさらに好ましい。ヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有する単量体としては、ヒドロキシエチルアクリレート、ヒドロキシエチルメタクリレート、N-(2-ヒドロキシエチル)アクリルアミド、2-ヒドロキシフェニルアクリレート、2-ヒドロキシフェニルメタクリレート、3-ヒドロキシフェニルアクリレート、3-ヒドロキシフェニルメタクリレート、4-ヒドロキシフェニルアクリレート、4-ヒドロキシフェニルメタクリレート、ポリエチレングリコールモノアクリレート、ポリエチレングリコールモノメタクリレート、ポリプロピレングリコールモノアクリレート、メトキシポリエチレングリコールモノアクリレート、メトキシポリエチレングリコールモノメタクリレート、ジエチレングリコールモノメチルエーテルアクリレート、ジエチレングリコールモノメチルエーテルメタクリレート、ジエチレングリコールモノエチルエーテルアクリレート、ジエチレングリコールモノエチルエーテルメタクリレート、ポリエチレングリコールメチルエーテルアクリレート、ポリエチレングリコールメチルエーテルメタクリレート等を挙げることができる。 The second coating layer is formed by polymerizing a monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group. The monomer is preferably a hydroxyl group-containing acrylate or a hydroxyl group-containing methacrylate, or a polyethylene glycol group-containing acrylate or a polyethylene glycol group-containing methacrylate because the polymerization rate is fast and it is suitable for increasing the mass productivity of magnetic particles. . Moreover, polyethylene glycol group-containing acrylate or polyethylene glycol group-containing methacrylate is more preferable because it is particularly suitable for increasing the sensitivity of immunodiagnosis. Examples of the monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group include hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, and 2-hydroxyphenyl. Acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, 4-hydroxyphenyl acrylate, 4-hydroxyphenyl methacrylate, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, methoxypolyethylene glycol Examples include monoacrylate, methoxypolyethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, polyethylene glycol methyl ether acrylate, polyethylene glycol methyl ether methacrylate, and the like.
また、第2の被覆層は、親水性基含有構造単位を有する単量体以外の単量体を共重合してもよい。例えば、抗体を粒子表面に吸着又は結合させるための疎水性を付与するために好適のため、炭素数1~6の長鎖及び環状アルキル基から選択される少なくとも1種の疎水性基含有構造単位を有することが好ましい。この疎水性基含有構造単位は、ヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有する単量体に炭素数1~6の長鎖及び環状アルキル基から選択される少なくとも1種の疎水性基含有構造単位を有する単量体を共重合することにより導入できる。疎水性基含有構造単位を有する単量体としては、疎水性基含有アクリレート又は疎水性基含有メタクリレートが好ましく、例えば、メチルアクリレート、メチルメタクリレート、エチルアクリレート、エチルメタクリレート、プロピルアクリレート、プロピルメタクリレート、n-ブチルアクリレート、n-ブチルメタクリレート、t-ブチルアクリレート、t-ブチルメタクリレート、n-ペンタノイルアクリレート、n-ペンタノイルメタクリレート、n-ヘキシルチルアクリレート、n-ヘキシルメタクリレート、シクロプロピルアクリレート、シクロプロピルメタクリレート、シクロヘキシルアクリレート、シクロヘキシルメタクリレート、フェニルアクリレート、フェニルメタクリレート等を挙げることができる。また、前記疎水性基含有単量体以外の単量体としては、スチレンを挙げることができる。 Further, the second coating layer may be formed by copolymerizing a monomer other than the monomer having a hydrophilic group-containing structural unit. For example, at least one hydrophobic group-containing structural unit selected from long chain and cyclic alkyl groups having 1 to 6 carbon atoms is suitable for imparting hydrophobicity for adsorbing or bonding antibodies to particle surfaces. It is preferable to have. This hydrophobic group-containing structural unit is a monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group, and a long chain and cyclic alkyl group having 1 to 6 carbon atoms. It can be introduced by copolymerizing a monomer having at least one hydrophobic group-containing structural unit. The monomer having a hydrophobic group-containing structural unit is preferably a hydrophobic group-containing acrylate or a hydrophobic group-containing methacrylate, such as methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, propyl acrylate, propyl methacrylate, n- Butyl acrylate, n-butyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-pentanoyl acrylate, n-pentanoyl methacrylate, n-hexyltyl acrylate, n-hexyl methacrylate, cyclopropyl acrylate, cyclopropyl methacrylate, cyclohexyl Acrylate, cyclohexyl methacrylate, phenyl acrylate, phenyl methacrylate, etc. can be mentioned. Furthermore, examples of monomers other than the hydrophobic group-containing monomer include styrene.
また、その他の単量体としては、重合性官能基を2以上有する単量体を架橋剤として併用してもよい。前記架橋剤としては、例えば、ジビニルベンゼン、エチレングリコールジアクリレート、エチレングリコールジメタクリレート、トリメチロールプロパントリアクリレート、トリメチロールプロパントリメタクリレート、ペンタエリスリトールトリアクリレート、ジトリメチロールプロパンテトラアクリレート、ペンタエリスリトールテトラアクリレート等を挙げることができる。架橋剤を併用することで第2の被覆層に高分子鎖が複数の結合点で互いにつながった架橋構造を形成することができ、粒子の耐溶剤性及び耐熱性、力学的強度を高めることができる。 Further, as other monomers, monomers having two or more polymerizable functional groups may be used in combination as a crosslinking agent. Examples of the crosslinking agent include divinylbenzene, ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, pentaerythritol tetraacrylate, and the like. can be mentioned. By using a crosslinking agent in combination, it is possible to form a crosslinked structure in which polymer chains are connected to each other at multiple bonding points in the second coating layer, thereby increasing the solvent resistance, heat resistance, and mechanical strength of the particles. can.
第2の被覆層は、第1の被覆層と共有結合を形成し、前記共有結合とは反対側の高分子鎖末端にカルボキシル基を有する。カルボキシル基含有構造単位は、カルボキシル基を有するものであれば特に限定はない。第2の被覆層に第1の被覆層との共有結合を形成させる方法としては、第1の被覆層に、リビングラジカル重合のための開始剤又は連鎖移動剤を有するシランカップリング剤を共有結合させた後、リビングラジカル重合により第2の被覆層を形成することで可能である。また、前記共有結合とは反対側の高分子鎖末端にカルボキシル基を設ける方法としては、前記リビングラジカル重合のための開始剤又は連鎖移動剤と反応する有機反応試薬を用いて、前記開始剤又は連鎖移動剤にカルボキシル基を導入することで可能である。 The second coating layer forms a covalent bond with the first coating layer and has a carboxyl group at the end of the polymer chain opposite to the covalent bond. The carboxyl group-containing structural unit is not particularly limited as long as it has a carboxyl group. As a method for forming a covalent bond with the first coating layer in the second coating layer, a silane coupling agent having an initiator or chain transfer agent for living radical polymerization is covalently bonded to the first coating layer. This can be done by forming the second coating layer by living radical polymerization after the reaction. Further, as a method for providing a carboxyl group at the end of the polymer chain opposite to the covalent bond, an organic reaction reagent that reacts with the initiator or chain transfer agent for living radical polymerization is used. This is possible by introducing a carboxyl group into the chain transfer agent.
カルボキシル基含有構造単位は、ヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有する重合体層を形成した粒子にメルカプトカルボン酸を反応させて、重合体層に導入することができる。架橋無機物層の表面にカルボキシル基が導入されていても良い。メルカプトカルボン酸としては、メルカプト酢酸、メルカプトプロピオン酸、メルカプトシュウ酸、メルカプトコハク酸、メルカプトマレイン酸、メルカプトフタル酸、メルカプト安息香酸等を挙げることができる。 The carboxyl group-containing structural unit is introduced into the polymer layer by reacting mercaptocarboxylic acid with particles forming a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group. can do. A carboxyl group may be introduced onto the surface of the crosslinked inorganic layer. Examples of the mercaptocarboxylic acid include mercaptoacetic acid, mercaptopropionic acid, mercaptooxalic acid, mercaptosuccinic acid, mercaptomaleic acid, mercaptophthalic acid, mercaptobenzoic acid, and the like.
第2の被覆層である共重合体層は、第1の被覆層と共有結合している。共有結合していることで、耐酸性及び耐溶剤性を高めることができる。共有結合の存在は、重合体が溶解する溶剤で洗浄しても重合体が消失していないことにより確認できる。共有結合していない場合は、被覆した重合体がほぼ全て溶けてなくなる。 The second coating layer, a copolymer layer, is covalently bonded to the first coating layer. By being covalently bonded, acid resistance and solvent resistance can be improved. The presence of covalent bonds can be confirmed by the fact that the polymer does not disappear even after washing with a solvent that dissolves the polymer. If it is not covalently bonded, almost all of the coated polymer will dissolve.
第2の被覆層を第1の被覆層と共有結合で固定化させることで、磁性粒子の耐酸性及び耐溶剤性を高めるのに好適であることから、架橋無機物層の表面に重合性官能基(及び残基)を有するシランカップリング剤を有していることが好ましい。前記シランカップリング剤としては、例えば、ビニルトリメトキシシラン、ビニルトリエトキシシラン、2-(3,4-エポキシシクロヘキシル)エチルトリメトキシシラン、3-グリシドキシプロピルトリメトキシシラン、p-スチリルトリメトキシシラン、3-メタクリロキシプロピルメチルジメトキシシラン、3-メタクリロキシプロピルトリメトキシシラン、3-メタクリロキシプロピルメチルジエトキシシラン、3-メタクリロキシプロピルトリエトキシシラン、3-アクリロキシプロピルトリメトキシシラン等を挙げることができる。 By covalently bonding the second coating layer to the first coating layer, it is suitable for increasing the acid resistance and solvent resistance of the magnetic particles. It is preferable to have a silane coupling agent having (and residues). Examples of the silane coupling agent include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropyltrimethoxysilane, and p-styryltrimethoxysilane. Silane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, etc. be able to.
第2の被覆層における重合体の被覆量としては特に限定はないが、比表面積の大きな磁性粒子を作製するのに好適のため、磁性粒子1g当たりの重合体の被覆量として、1g/g以下が好ましく、0.5g/g以下がさらに好ましく、0.1g/g以下が特に好ましく、0.05g/g以下が最も好ましい。また、耐酸性を高めるのに好適のため、磁性粒子1g当たりの重合体の被覆量として、0.001g/g以上が好ましく、0.01g/g以上がさらに好ましく、0.1g/g以上が特に好ましく、0.5g/g以上が最も好ましい。重合体の被覆量は、重合体を被覆する前後の粒子のTG-DTAを測定し、重合体被覆前後の熱重量減少の差分を求めることにより測定可能である。また、粒子断面の走査型電子顕微鏡像、又は粒子の透過型電子顕微鏡像により、重合体の厚みを求めることでも算出可能である。 There is no particular limitation on the amount of polymer coated in the second coating layer, but since it is suitable for producing magnetic particles with a large specific surface area, the amount of polymer coated per 1 g of magnetic particles is 1 g/g or less. is preferable, 0.5 g/g or less is more preferable, 0.1 g/g or less is particularly preferable, and 0.05 g/g or less is most preferable. In addition, since it is suitable for increasing acid resistance, the amount of polymer coating per gram of magnetic particles is preferably 0.001 g/g or more, more preferably 0.01 g/g or more, and 0.1 g/g or more. Particularly preferred, and most preferably 0.5 g/g or more. The amount of polymer coated can be measured by measuring the TG-DTA of the particles before and after coating with the polymer, and determining the difference in thermogravimetric loss before and after coating with the polymer. It can also be calculated by determining the thickness of the polymer from a scanning electron microscope image of a particle cross section or a transmission electron microscope image of the particles.
磁性粒子のカルボキシル基量としては、多くの抗体を結合可能な磁性粒子とするのに好適のため、磁性粒子1g当たりのカルボキシル基量として、5μmol/g以上が好ましく、10μmol/g以上がさらに好ましく、20μmol/g以上が特に好ましく、50μmol/g以上が最も好ましい。また、磁性粒子への抗体以外の物質が非特異吸着し、免疫診断における測定ノイズが発生することを抑制するのに好適のため、磁性粒子1g当たりのカルボキシル基量として、200μmol/g以下が好ましく、100μmol/g以下がさらに好ましく、50μmol/g以下が特に好ましく、20μmol/g以下が最も好ましい。カルボキシル基量の測定方法としては、ピレニルジアゾメタンと磁性粒子を混合し、反応により消失したピレニルジアゾメタンを定量する方法、又は電位差滴定により磁性粒子表面のカルボキシル基量を直接定量する方法を挙げることができる。 The amount of carboxyl groups in the magnetic particles is preferably 5 μmol/g or more, more preferably 10 μmol/g or more, since the magnetic particles are suitable for binding many antibodies. , 20 μmol/g or more is particularly preferable, and 50 μmol/g or more is most preferable. In addition, the amount of carboxyl groups per 1 g of magnetic particles is preferably 200 μmol/g or less because it is suitable for suppressing non-specific adsorption of substances other than antibodies to magnetic particles and measurement noise in immunodiagnosis. , more preferably 100 μmol/g or less, particularly preferably 50 μmol/g or less, and most preferably 20 μmol/g or less. Examples of methods for measuring the amount of carboxyl groups include a method of mixing pyrenyldiazomethane and magnetic particles and quantifying the amount of pyrenyldiazomethane that has disappeared through the reaction, or a method of directly quantifying the amount of carboxyl groups on the surface of the magnetic particles by potentiometric titration. I can do it.
本発明の一態様にかかる抗体結合用磁性粒子は、下記(i)~(v)の工程を含む製造方法により得ることができる。
(i)コア粒子の表面にナノ磁性体を物理吸着させる工程
(ii)前記ナノ磁性体を吸着させた粒子の表面に架橋無機物層を形成する工程
(iii)前記架橋無機物層の表面に、リビングラジカル重合の開始剤又は連鎖移動剤を有する、シランカップリング剤を結合させる工程
(iv)前記シランカップリング剤を結合させた粒子を溶媒に分散させ、溶媒中でヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有するモノマーをリビングラジカル重合することにより粒子の表面に重合体層を形成する工程
(v)共重合体層を形成した粒子にメルカプトカルボン酸を反応させる工程
工程(i)において、コア粒子の表面電荷と反対の電荷を有するナノ磁性体を接触させる方法、疎水性のコア粒子に疎水性のナノ磁性体を接触させる方法、または常磁性を有するコア粒子にナノ磁性体を接触させる方法により、コア粒子の表面にナノ磁性体を物理吸着させる。コア粒子とナノ磁性体を接触させる方法としては特に限定はなく、液相中、気相中で攪拌することで可能である。
The antibody-binding magnetic particles according to one aspect of the present invention can be obtained by a manufacturing method including the following steps (i) to (v).
(i) A step of physically adsorbing the nanomagnetic material on the surface of the core particle (ii) A step of forming a crosslinked inorganic layer on the surface of the particle to which the nanomagnetic material is adsorbed (iii) A step of physically adsorbing the nanomagnetic material on the surface of the core particle Step (iv) of bonding a silane coupling agent having a radical polymerization initiator or chain transfer agent: dispersing the particles bound with the silane coupling agent in a solvent, and dispersing the particles selected from hydroxyl groups and polyethylene glycol groups in the solvent. step (v) of forming a polymer layer on the surface of the particles by living radical polymerization of a monomer having at least one hydrophilic group-containing structural unit, (v) reacting the particles with the copolymer layer formed with mercaptocarboxylic acid; In the step (i), a method of contacting a nanomagnetic material having a charge opposite to the surface charge of the core particle, a method of contacting a hydrophobic nanomagnetic material with a hydrophobic core particle, or a method of contacting a hydrophobic nanomagnetic material with a core particle having paramagnetism. By bringing the nanomagnetic material into contact with the particles, the nanomagnetic material is physically adsorbed onto the surface of the core particle. There is no particular limitation on the method of bringing the core particles into contact with the nanomagnetic material, and it is possible to do so by stirring in a liquid phase or a gas phase.
工程(ii)において、工程(i)でナノ磁性体を吸着させた粒子表面に架橋無機物層を形成する。架橋無機物層を形成する方法に特に限定はないが、架橋無機物層の原料となる試薬と反応触媒を有する溶液中に粒子を分散させ、反応を進行させることで粒子表面に架橋無機物層を形成する方法、粒子表面に前記架橋無機物層又はその原料を被覆する方法等を挙げることができる。 In step (ii), a crosslinked inorganic layer is formed on the surface of the particles to which the nanomagnetic material was adsorbed in step (i). There is no particular limitation on the method of forming the crosslinked inorganic layer, but particles are dispersed in a solution containing a reagent and a reaction catalyst that are raw materials for the crosslinked inorganic layer, and the reaction is allowed to proceed to form a crosslinked inorganic layer on the surface of the particles. Examples include a method of coating the particle surface with the crosslinked inorganic layer or its raw material.
工程(iii)において、工程(ii)で形成した架橋無機物層の表面にリビングラジカル重合の開始剤又は連鎖移動剤を有するシランカップリング剤を結合させる。架橋無機物層の表面にシランカップリング剤を結合させる方法としては特に限定はなく、溶媒中に粒子を分散させ、シランカップリング剤を加える方法、粒子表面にシランカップリング剤を直接被覆する方法から選択可能である。また、必要に応じて反応触媒となる酸性又は塩基性水溶液を併用してもよい。 In step (iii), a silane coupling agent having a living radical polymerization initiator or chain transfer agent is bonded to the surface of the crosslinked inorganic layer formed in step (ii). There are no particular limitations on the method of bonding the silane coupling agent to the surface of the crosslinked inorganic layer, and methods include methods of dispersing particles in a solvent and adding the silane coupling agent, and methods of directly coating the particle surface with the silane coupling agent. Selectable. Furthermore, an acidic or basic aqueous solution serving as a reaction catalyst may be used in combination, if necessary.
工程(iv)において、工程(ii)で架橋無機物層を形成した粒子を溶媒に分散させ、溶媒中でヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有するモノマーをリビングラジカル重合することにより粒子の表面に重合体層を形成する。重合方法としては、リビングラジカル重合であること以外に特に限定はなく、粒子以外の成分が全て溶媒に溶解した状態で重合を行う分散重合法、溶媒に相溶しない単量体を使用し、粒子に単量体を吸着させた状態で重合を行うシード重合法等を挙げることができる。また、シード重合法としては、乳化剤を使用しないソープフリー重合、乳化剤を使用する乳化重合から適宜選択できる。リビングラジカル重合の方法としては、原子移動ラジカル重合(ATRP)又は可逆的付加開裂連鎖移動(RAFT)重合が好ましい。原子移動ラジカル重合の場合は、ブロモ基等のハロゲン基を有するシランカップリング剤を粒子に結合させ、触媒として遷移金属錯体の存在下で重合を行う。可逆的付加開裂連鎖移動重合の場合は、ジチオエステル等の連鎖移動剤を有するシランカップリング剤を粒子に結合させ、アズ化合物等のラジカル発生剤の存在下で重合を行う。 In step (iv), the particles forming the crosslinked inorganic layer in step (ii) are dispersed in a solvent, and a monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group is added in the solvent. A polymer layer is formed on the surface of the particles by living radical polymerization. There are no particular limitations on the polymerization method other than living radical polymerization, and there are dispersion polymerization methods in which polymerization is performed with all components other than particles dissolved in a solvent, and monomers that are incompatible with the solvent are used to form particles. Examples include seed polymerization methods in which polymerization is carried out with monomers adsorbed on the polymer. Further, the seed polymerization method can be appropriately selected from soap-free polymerization that does not use an emulsifier and emulsion polymerization that uses an emulsifier. As the method of living radical polymerization, atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT) polymerization is preferred. In the case of atom transfer radical polymerization, a silane coupling agent having a halogen group such as a bromo group is bonded to particles, and polymerization is performed in the presence of a transition metal complex as a catalyst. In the case of reversible addition-fragmentation chain transfer polymerization, a silane coupling agent having a chain transfer agent such as a dithioester is bonded to particles, and polymerization is performed in the presence of a radical generator such as an az compound.
また、工程(iv)において、ヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有する単量体をリビングラジカル重合し、重合体層を形成した後、工程(v)でメルカプトカルボン酸を反応させ、粒子表面にカルボキシル基を導入しても良い。粒子表面とカルボン酸無水物の反応方法は特に限定はなく、水を含まない溶媒中に粒子を分散させ、過剰量のメルカプトカルボン酸を加えることで可能である。 In step (iv), a monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group is subjected to living radical polymerization to form a polymer layer, and then step (v) ) may be reacted with mercaptocarboxylic acid to introduce a carboxyl group onto the particle surface. The reaction method between the particle surface and the carboxylic acid anhydride is not particularly limited, and can be achieved by dispersing the particles in a water-free solvent and adding an excess amount of mercaptocarboxylic acid.
本発明の一態様にかかる抗体結合用磁性粒子は、粒子の表面に抗体を固定化し、免疫診断用装置で使用することができる。抗体の結合方法としては、粒子表面のカルボキシル基を介して結合させることが好ましく、例えば、カルボキシル基にカルボジイミド等の縮合剤を反応させることで活性化させ、活性化させたカルボキシル基を抗体が有するアミノ基と反応させることで結合できる。 The antibody-binding magnetic particles according to one embodiment of the present invention have antibodies immobilized on the surface of the particles, and can be used in immunodiagnosis devices. As for the binding method of the antibody, it is preferable to bind through the carboxyl group on the particle surface. For example, the carboxyl group is activated by reacting a condensing agent such as carbodiimide, and the antibody has the activated carboxyl group. Can be bonded by reacting with an amino group.
以下、本発明を実施するための形態を挙げて本発明について詳細に説明するが、本発明を説明するための例示であり、本発明を以下の内容に限定する趣旨ではない。また本発明の要旨の範囲内で適宜に変更して実施することができる。なお、断りのない限り、試薬は市販品を用いた。
[重合体被覆量の測定]
重合体被覆前後の粒子についてそれぞれTG-DTA測定を行い、100℃から560℃までの熱重量減少を求めた。重合体被覆前の粒子における熱重量減少をΔTG1、重合体被覆後の粒子における熱重量減少をΔTG2としたとき、ΔTG2-ΔTG1を重合体被覆量とした。
[カルボキシル基量の測定]
磁性粒子10mgに酢酸エチル2mLを加え、室温で20分間攪拌。この粒子分散液にピレニルジアゾメタン0.12mgを溶解させた酢酸エチル2mLを加え、室温で50分間攪拌。粒子を除去した後、上澄み液をメタノールで10倍希釈し、392nmの吸光度を測定し、この吸光度をA1とし、粒子を使用せずに同様の操作を行った場合の溶液の吸光度をA0とした時、下記式からカルボキシル基量を求めた。
138×(A0-A1)μmol/g
[水分散性の評価]
乾燥させた粉末状の磁性粒子0.1gを10gのイオン交換水に加え、手で振って攪拌して評価した。
○:水に均一分散可能
×:水に分散しない
[鉄イオン溶出の評価]
pH3.5のクエン酸緩衝液1mLに磁性粒子10mgを加え、37℃で17時間攪拌。溶液から粒子を除いた溶液を測定試料とし、光の波長460nmにおける溶液の吸光度を測定することで鉄イオンの溶出量を評価した。
[希釈固相懸濁液と検出用標識抗体溶液の調製]
緩衝液(pH6.0)中に分散させた磁性粒子に1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩を過剰量加え、37℃で1時間インキュベートした。緩衝液(pH3.5)中で抗甲状腺刺激ホルモン(TSH)抗体を加え、37℃で3時間インキュベートして抗TSH抗体を磁性粒子に結合させた。次にブロッキング剤溶液で17時間インキュベートして磁性粒子にブロッキング処理を行い、抗TSH抗体固定化磁性粒子とした(A)。さらに(A)を8%コラーゲンペプチドを含む緩衝液(pH6.0)で希釈し、希釈固相懸濁液(B)を調製した。また抗TSH抗体とアルカリ性ホスファターゼの結合物を、5%ウシ血清アルブミンを含む緩衝液(pH6.2)で希釈し、検出用標識抗体溶液(C)を調製した。
[NSB(非特異吸着)及びポジカウント評価用サンプルの調製]
5%コラーゲンペプチドを含む0.03mol/L トリス塩酸緩衝液(pH8.0)を調製し、NSB(非特異吸着)評価用サンプル(D)とした。また5%コラーゲンペプチドを含む0.03mol/L トリス塩酸緩衝液(pH8.0)に甲状腺刺激ホルモン(TSH)を添加して、ポジカウント評価用サンプル(E)とした。
[NSB(非特異吸着)及びポジカウントの測定、感度の評価]
測定試薬(希釈固相懸濁液(B)と検出用標識抗体溶液(C)を含む凍結乾燥品)でNSB及びポジカウントの測定、感度の評価を行った
NSBの測定は以下の手順で行った。
Hereinafter, the present invention will be described in detail by citing embodiments for carrying out the present invention, but these are merely examples for explaining the present invention, and are not intended to limit the present invention to the following contents. Further, the present invention can be modified and implemented as appropriate within the scope of the gist of the present invention. In addition, unless otherwise specified, commercially available reagents were used.
[Measurement of polymer coverage]
TG-DTA measurements were performed on the particles before and after coating with the polymer, and the thermogravimetric loss from 100°C to 560°C was determined. When the thermal weight loss in the particles before polymer coating is ΔTG1, and the thermal weight loss in the particles after polymer coating is ΔTG2, ΔTG2−ΔTG1 is defined as the polymer coating amount.
[Measurement of carboxyl group amount]
Add 2 mL of ethyl acetate to 10 mg of magnetic particles, and stir at room temperature for 20 minutes. 2 mL of ethyl acetate in which 0.12 mg of pyrenyldiazomethane was dissolved was added to this particle dispersion, and the mixture was stirred at room temperature for 50 minutes. After removing the particles, the supernatant liquid was diluted 10 times with methanol, the absorbance at 392 nm was measured, and this absorbance was designated as A1, and the absorbance of the solution when the same operation was performed without using particles was designated as A0. At that time, the amount of carboxyl groups was determined from the following formula.
138×(A0-A1)μmol/g
[Evaluation of water dispersibility]
0.1 g of dried powdered magnetic particles was added to 10 g of ion-exchanged water, and the mixture was shaken and stirred by hand for evaluation.
○: Uniformly dispersible in water ×: Not dispersible in water [Evaluation of iron ion elution]
Add 10 mg of magnetic particles to 1 mL of pH 3.5 citrate buffer and stir at 37°C for 17 hours. A solution obtained by removing particles from the solution was used as a measurement sample, and the amount of iron ions eluted was evaluated by measuring the absorbance of the solution at a wavelength of 460 nm.
[Preparation of diluted solid phase suspension and labeled antibody solution for detection]
An excess amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the magnetic particles dispersed in a buffer solution (pH 6.0), and the mixture was incubated at 37°C for 1 hour. Anti-thyroid stimulating hormone (TSH) antibodies were added in a buffer solution (pH 3.5) and incubated at 37°C for 3 hours to bind the anti-TSH antibodies to the magnetic particles. Next, the magnetic particles were subjected to blocking treatment by incubation with a blocking agent solution for 17 hours to obtain anti-TSH antibody-immobilized magnetic particles (A). Furthermore, (A) was diluted with a buffer solution (pH 6.0) containing 8% collagen peptide to prepare a diluted solid phase suspension (B). Further, a conjugate of anti-TSH antibody and alkaline phosphatase was diluted with a buffer solution (pH 6.2) containing 5% bovine serum albumin to prepare a labeled antibody solution for detection (C).
[Preparation of samples for NSB (non-specific adsorption) and positive count evaluation]
A 0.03 mol/L Tris-HCl buffer (pH 8.0) containing 5% collagen peptide was prepared and used as a sample (D) for NSB (non-specific adsorption) evaluation. In addition, thyroid stimulating hormone (TSH) was added to 0.03 mol/L Tris-HCl buffer (pH 8.0) containing 5% collagen peptide to prepare a positive count evaluation sample (E).
[Measurement of NSB (non-specific adsorption) and positive count, evaluation of sensitivity]
Measurement of NSB and positive counts and evaluation of sensitivity were performed using measurement reagents (lyophilized product containing diluted solid phase suspension (B) and labeled antibody solution for detection (C)) NSB measurement was performed using the following procedure. Ta.
凍結乾燥した希釈固相懸濁液(B)を、界面活性剤を含む純水及びNSB評価用サンプル(D)で溶解した後、37℃にて5分間免疫反応を行い、1回目のB/F分離を行った。次に界面活性剤を含む純水で溶解した検出用標識抗体溶液(C)を加えて37℃にて3分間免疫反応を行い、2回目のB/F分離を行った。その後、アルカリ性ホスファターゼに対する化学発光基質(DIFURAT、3-(5-tert-ブチル-4,4-ジメチル-2,6,7-トリオキサビシクロ[3,2,0]ヘプト-1-イル)フェニルリン酸エステル ジナトリウム塩)及び化学発光補助試薬を加え、発光強度(count/sec(cps))を測定しNSBとした。 After dissolving the lyophilized diluted solid phase suspension (B) in pure water containing a surfactant and the NSB evaluation sample (D), immunoreaction was performed at 37°C for 5 minutes, and the first B/ F separation was performed. Next, a labeled antibody solution for detection (C) dissolved in pure water containing a surfactant was added, and an immunoreaction was performed at 37° C. for 3 minutes, followed by a second B/F separation. Thereafter, a chemiluminescent substrate for alkaline phosphatase (DIFURAT, 3-(5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3,2,0]hept-1-yl)phenylphosphorus Acid ester disodium salt) and chemiluminescence auxiliary reagent were added, and the luminescence intensity (count/sec (cps)) was measured and determined as NSB.
ポジカウントの測定は以下の手順で行った。 The positive count was measured using the following procedure.
凍結乾燥した希釈固相懸濁液(B)を、界面活性剤を含む純水及びポジカウント評価用サンプル(E)で溶解した後、37℃にて5分間免疫反応を行い、1回目のB/F分離を行った。次に界面活性剤を含む純水で溶解した検出用標識抗体溶液(C)を加えて37℃にて3分間免疫反応を行い、2回目のB/F分離を行った。その後、アルカリ性ホスファターゼに対する化学発光基質(DIFURAT、3-(5-tert-ブチル-4,4-ジメチル-2,6,7-トリオキサビシクロ[3,2,0]ヘプト-1-イル)フェニルリン酸エステル ジナトリウム塩)及び化学発光補助試薬を加え、発光強度(count/sec(cps))を測定しポジカウントとした。 After dissolving the lyophilized diluted solid phase suspension (B) in pure water containing a surfactant and a positive count evaluation sample (E), an immunoreaction was performed at 37°C for 5 minutes, and the first B /F separation was performed. Next, a labeled antibody solution for detection (C) dissolved in pure water containing a surfactant was added, and an immunoreaction was performed at 37° C. for 3 minutes, followed by a second B/F separation. Thereafter, a chemiluminescent substrate for alkaline phosphatase (DIFURAT, 3-(5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3,2,0]hept-1-yl)phenylphosphorus Acid ester disodium salt) and a chemiluminescence auxiliary reagent were added, and the luminescence intensity (count/sec (cps)) was measured as a positive count.
感度はポジカウント/NSBの比を求めることによって評価した。
[実施例1]
(磁性化微粒子の作製)
ポリジビニルベンゼン粒子5.0g(粒子径2.5μm)、ポリビニルピロリドン1.2g、1N-塩酸水溶液6mLを純水160mLに回転数180rpmで分散させた。窒素雰囲気下、80℃に昇温した後に、尿素6g、塩化鉄(II)四水和物2g、塩化鉄(III)六水和物3gを添加し5時間反応させた。この粒子分散液をろ過にて粒子と反応液を分離した後に、再度粒子を純水200mLに回転数180rpmで分散させた。窒素雰囲気下、80℃に昇温した後に、0.2N-水酸化ナトリウム水溶液を滴下し15時間反応させた。純水にて洗浄し、酸化鉄被覆された粒子5.75gを得た。
Sensitivity was evaluated by determining the ratio of positive counts/NSB.
[Example 1]
(Preparation of magnetized fine particles)
5.0 g of polydivinylbenzene particles (particle size 2.5 μm), 1.2 g of polyvinylpyrrolidone, and 6 mL of a 1N hydrochloric acid aqueous solution were dispersed in 160 mL of pure water at a rotation speed of 180 rpm. After raising the temperature to 80° C. in a nitrogen atmosphere, 6 g of urea, 2 g of iron (II) chloride tetrahydrate, and 3 g of iron (III) chloride hexahydrate were added and reacted for 5 hours. After this particle dispersion liquid was filtered to separate the particles and the reaction liquid, the particles were again dispersed in 200 mL of pure water at a rotation speed of 180 rpm. After raising the temperature to 80° C. under a nitrogen atmosphere, a 0.2N aqueous sodium hydroxide solution was added dropwise and the mixture was allowed to react for 15 hours. After washing with pure water, 5.75 g of particles coated with iron oxide were obtained.
得られた酸化鉄被覆された粒子1gを純水50mLに室温にて回転数100rpmで分散させた。この粒子分散液に、表面にカチオン系分散剤を有する磁性体(材質:マグネタイト、粒子径:10nm、商品名:EMG607、フェローテック社製)1.0gを添加し、室温にて回転数100rpmで2時間反応させた。純水にて洗浄し、さらに50℃で15時間乾燥させることにより、磁性化された粒子を1.93g得た。磁性粒子1g当たりの磁性体の含有量は0.32g/gであった。
(磁性化粒子へのシリカ被覆)
前記磁性化された微粒子1gをエタノール400mLに分散させ、オルトけい酸テトラエチル3gを加え、室温にて回転数300rpmで攪拌した。25%アンモニア水21gを加え、35℃にて回転数300rpmで4時間反応させた。純水及びエタノールにて洗浄し、減圧乾燥させた。透過型電子顕微鏡により確認したシリカ層の厚みは約20nmであった。
(シリカ被覆粒子へのATRP開始剤導入)
シリカ被覆された粒子1gをメタノール75mLに分散させ、3-(トリメトキシシリルプロピル)-2-ブロモ-2-メチルプロピオン酸0.5g、25%アンモニア水15gを加え、60℃にて回転数300rpmで3時間反応させた。メタノール及び純水にて洗浄し、減圧乾燥させた。
(ATRP開始剤導入粒子への重合体被覆)
前記ATRP開始剤導入された粒子0.1gをメタノール18mLに分散させ、2-ヒドロキシエチルメタクリレート:トリメチロールプロパントリアクリレート=60:40の混合物0.4g、臭化銅(II)0.0022g、アスコルビン酸ナトリウム0.02g、2,2’-ビピリジル0.015gを加え、脱気を行った後、窒素雰囲気下にて40℃で3時間反応させた。純水及びメタノールにて洗浄し、減圧乾燥させた。
(カルボキシル基導入)
前記重合体被覆された粒子0.1gを、純水:メタノール=50:50の混合溶媒20mLに分散させ、炭酸リチウム0.074g、チオグリコール酸0.092gを加え、37℃で12時間反応させた。純水及びメタノールにて洗浄し、減圧乾燥させた。
(評価結果)
重合体被覆量は0.090g/gであった。カルボキシル基量は32μmol/gであった。
1 g of the obtained iron oxide-coated particles was dispersed in 50 mL of pure water at room temperature and at a rotation speed of 100 rpm. To this particle dispersion, 1.0 g of a magnetic material having a cationic dispersant on the surface (material: magnetite, particle size: 10 nm, product name: EMG607, manufactured by Ferrotech) was added, and the mixture was heated at room temperature and at a rotation speed of 100 rpm. The reaction was allowed to proceed for 2 hours. By washing with pure water and further drying at 50° C. for 15 hours, 1.93 g of magnetized particles were obtained. The content of magnetic material per gram of magnetic particles was 0.32 g/g.
(Silica coating on magnetized particles)
1 g of the magnetized fine particles were dispersed in 400 mL of ethanol, 3 g of tetraethyl orthosilicate was added, and the mixture was stirred at room temperature at 300 rpm. 21 g of 25% ammonia water was added, and the mixture was reacted for 4 hours at 35° C. and a rotational speed of 300 rpm. It was washed with pure water and ethanol, and dried under reduced pressure. The thickness of the silica layer confirmed by a transmission electron microscope was about 20 nm.
(Introduction of ATRP initiator into silica-coated particles)
Disperse 1 g of silica-coated particles in 75 mL of methanol, add 0.5 g of 3-(trimethoxysilylpropyl)-2-bromo-2-methylpropionic acid and 15 g of 25% ammonia water, and rotate at 300 rpm at 60°C. The mixture was allowed to react for 3 hours. It was washed with methanol and pure water, and dried under reduced pressure.
(Polymer coating on ATRP initiator-introduced particles)
Disperse 0.1 g of the ATRP initiator-introduced particles in 18 mL of methanol, add 0.4 g of a mixture of 2-hydroxyethyl methacrylate:trimethylolpropane triacrylate = 60:40, 0.0022 g of copper(II) bromide, and ascorbin. After adding 0.02 g of sodium chloride and 0.015 g of 2,2'-bipyridyl and degassing, the mixture was reacted at 40° C. for 3 hours under a nitrogen atmosphere. It was washed with pure water and methanol and dried under reduced pressure.
(Introduction of carboxyl group)
0.1 g of the polymer-coated particles were dispersed in 20 mL of a mixed solvent of pure water: methanol = 50:50, 0.074 g of lithium carbonate and 0.092 g of thioglycolic acid were added, and the mixture was reacted at 37° C. for 12 hours. Ta. It was washed with pure water and methanol and dried under reduced pressure.
(Evaluation results)
Polymer coverage was 0.090 g/g. The amount of carboxyl groups was 32 μmol/g.
作製された磁性粒子は水分散性に優れ、鉄イオンの溶出が少ないものであった。また、NSBは67、ポジカウントは953269、感度は14228で、感度に優れるものであった。
[実施例2]
実施例1における重合体被覆の単量体としてトリメチロールプロパントリアクリレートの代わりに、トリメチロールプロパントリメタクリレートを用い、その他は実施例1と同様にして粒子を作製した。
(評価結果)
重合体被覆量は0.106g/gであった。カルボキシル基量は30μmol/gであった。
The prepared magnetic particles had excellent water dispersibility and little elution of iron ions. Further, the NSB was 67, the positive count was 953,269, and the sensitivity was 14,228, indicating excellent sensitivity.
[Example 2]
Particles were produced in the same manner as in Example 1 except that trimethylolpropane trimethacrylate was used instead of trimethylolpropane triacrylate as the monomer for the polymer coating in Example 1.
(Evaluation results)
Polymer coverage was 0.106 g/g. The amount of carboxyl groups was 30 μmol/g.
作製された磁性粒子は水分散性に優れ、鉄イオンの溶出が少ないものであった。また、NSBは59、ポジカウントは1018909、感度は17270で、感度に優れるものであった。
[実施例3]
実施例1における重合体被覆の単量体として2-ヒドロキシエチルメタクリレートの代わりに、ポリエチレングリコールメチルエーテルアクリレート(Mn=300)を用い、その他は実施例1と同様にして粒子を作製した。
(評価結果)
重合体被覆量は0.096g/gであった。カルボキシル基量は29μmol/gであった。
The prepared magnetic particles had excellent water dispersibility and little elution of iron ions. Further, the NSB was 59, the positive count was 1018909, and the sensitivity was 17270, indicating excellent sensitivity.
[Example 3]
Particles were produced in the same manner as in Example 1 except that polyethylene glycol methyl ether acrylate (Mn=300) was used instead of 2-hydroxyethyl methacrylate as the monomer for the polymer coating in Example 1.
(Evaluation results)
Polymer coverage was 0.096 g/g. The amount of carboxyl groups was 29 μmol/g.
作製された磁性粒子は水分散性に優れ、鉄イオンの溶出が少ないものであった。また、NSBは48、ポジカウントは959202、感度は19983で、感度に優れるものであった。
[実施例4]
実施例1における重合体被覆の単量体として2-ヒドロキシエチルメタクリレートの代わりに、ポリエチレングリコールメチルエーテルアクリレート(Mn=300)を用い、また、トリメチロールプロパントリアクリレートの代わりに、トリメチロールプロパントリメタクリレートを用い、その他は実施例1と同様にして粒子を作製した。
(評価結果)
重合体被覆量は0.102g/gであった。カルボキシル基量は34μmol/gであった。
The prepared magnetic particles had excellent water dispersibility and little elution of iron ions. Further, the NSB was 48, the positive count was 959,202, and the sensitivity was 19,983, indicating excellent sensitivity.
[Example 4]
As the monomer for the polymer coating in Example 1, polyethylene glycol methyl ether acrylate (Mn=300) was used instead of 2-hydroxyethyl methacrylate, and trimethylolpropane trimethacrylate was used instead of trimethylolpropane triacrylate. Particles were produced in the same manner as in Example 1 except for the following.
(Evaluation results)
Polymer coverage was 0.102 g/g. The amount of carboxyl groups was 34 μmol/g.
作製された磁性粒子は水分散性に優れ、鉄イオンの溶出が少ないものであった。また、NSBは66、ポジカウントは1273013、感度は19288で、感度に優れるものであった。
[比較例1]
実施例1におけるシリカ被覆工程、ATRP開始剤導入工程、重合体被覆工程、カルボン酸導入工程の全てを実施せず、その他は実施例1と同様にして粒子を作製した。
(評価結果)
作製された磁性粒子は水分散性が乏しく、鉄イオンの溶出が多いものであった。
[比較例2]
実施例1におけるシリカ被覆工程、ATRP開始剤導入工程、重合体被覆工程、カルボン酸導入工程の全てを実施せず、代わりに以下の方法で磁性化粒子への有機シランカップリング剤修飾を行った。その他は実施例1と同様にして粒子を作製した。
(磁性化粒子への有機シランカップリング剤修飾)
前記磁性化された微粒子1gをメタノール75mLに分散させた。3-メタクリロキシプロピルトリメトキシシラン0.5g、25%アンモニア水15gを加え、60℃にて回転数300rpmで3時間反応させた。メタノール及び純水にて洗浄し、減圧乾燥させた。
(評価結果)
作製された磁性粒子は水への分散性が乏しく、鉄イオンの溶出が多いものであった。
[比較例3]
実施例1におけるシリカ被覆工程、ATRP開始剤導入工程、カルボン酸導入工程の全てを実施せず、代わりに以下の方法で磁性化粒子への重合体被覆を行った。その他は実施例1と同様にして粒子を作製した。
(磁性化粒子への重合体被覆)
前記磁性化された粒子0.1gを純水18mLに分散させ、メタクリル酸メチル4g、過硫酸カリウム0.02gを加え、脱気を行った後、窒素雰囲気下にて65℃で3時間反応させた。純水及びメタノールにて洗浄し、減圧乾燥させた。
(評価結果)
作製された粒子は水への分散性に乏しいものであった。比表面積は1.8m2/gで、比表面積が小さなものであった。
[比較例4]
実施例1におけるシリカ被覆工程、ATRP開始剤導入工程、カルボン酸導入工程の全てを実施せず、代わりに以下の方法で磁性化粒子への重合体被覆を行った。その他は実施例1と同様にして粒子を作製した。
(磁性化粒子への重合体被覆)
前記磁性化された粒子0.1gを純水18mLに分散させ、2-ヒドロキシエチルメタクリレート4g、過硫酸カリウム0.02gを加え、脱気を行った後、窒素雰囲気下にて65℃で3時間反応させた。純水及びメタノールにて洗浄し、減圧乾燥させた。
(評価結果)
作製された粒子は鉄イオンの溶出が多いものであった。
[比較例5]
実施例1におけるカルボン酸導入工程を実施せず、その他は実施例1と同様にして粒子を作製した。
(評価結果)
作製された磁性粒子のNSBは66、ポジカウントは477882、感度は7241で、感度に劣るものであった。
The prepared magnetic particles had excellent water dispersibility and little elution of iron ions. Further, the NSB was 66, the positive count was 1273013, and the sensitivity was 19288, indicating excellent sensitivity.
[Comparative example 1]
Particles were produced in the same manner as in Example 1 except that all of the silica coating step, ATRP initiator introduction step, polymer coating step, and carboxylic acid introduction step in Example 1 were omitted.
(Evaluation results)
The produced magnetic particles had poor water dispersibility and a large amount of iron ions were eluted.
[Comparative example 2]
All of the silica coating step, ATRP initiator introduction step, polymer coating step, and carboxylic acid introduction step in Example 1 were not performed, and instead, the magnetized particles were modified with an organic silane coupling agent by the following method. . Particles were produced in the same manner as in Example 1 in other respects.
(Modification of magnetized particles with organosilane coupling agent)
1 g of the magnetized fine particles was dispersed in 75 mL of methanol. 0.5 g of 3-methacryloxypropyltrimethoxysilane and 15 g of 25% aqueous ammonia were added, and the mixture was reacted at 60° C. and a rotational speed of 300 rpm for 3 hours. It was washed with methanol and pure water, and dried under reduced pressure.
(Evaluation results)
The produced magnetic particles had poor dispersibility in water, and a large amount of iron ions were eluted.
[Comparative example 3]
The silica coating step, the ATRP initiator introduction step, and the carboxylic acid introduction step in Example 1 were all omitted, and instead, the magnetized particles were coated with the polymer in the following manner. Particles were produced in the same manner as in Example 1 in other respects.
(Polymer coating on magnetized particles)
0.1 g of the magnetized particles were dispersed in 18 mL of pure water, 4 g of methyl methacrylate and 0.02 g of potassium persulfate were added, and after degassing, the mixture was reacted at 65° C. for 3 hours under a nitrogen atmosphere. Ta. It was washed with pure water and methanol and dried under reduced pressure.
(Evaluation results)
The prepared particles had poor dispersibility in water. The specific surface area was 1.8 m 2 /g, which was small.
[Comparative example 4]
The silica coating step, the ATRP initiator introduction step, and the carboxylic acid introduction step in Example 1 were all omitted, and instead, the magnetized particles were coated with the polymer in the following manner. Particles were produced in the same manner as in Example 1 in other respects.
(Polymer coating on magnetized particles)
0.1 g of the magnetized particles were dispersed in 18 mL of pure water, 4 g of 2-hydroxyethyl methacrylate and 0.02 g of potassium persulfate were added, and after degassing, the mixture was heated at 65° C. for 3 hours under a nitrogen atmosphere. Made it react. It was washed with pure water and methanol and dried under reduced pressure.
(Evaluation results)
The prepared particles had a large amount of iron ions eluted.
[Comparative example 5]
Particles were produced in the same manner as in Example 1 except that the carboxylic acid introduction step in Example 1 was not performed.
(Evaluation results)
The produced magnetic particles had an NSB of 66, a positive count of 477,882, and a sensitivity of 7241, which were poor in sensitivity.
1 コア粒子
2 ナノ磁性体
3 架橋無機物層
4 親水性基含有構造単位を有する重合体層
1 Core particle 2 Nanomagnetic material 3 Crosslinked inorganic layer 4 Polymer layer having a hydrophilic group-containing structural unit
Claims (4)
前記第1の被覆層は、ナノ磁性体を含有する架橋無機物層であり、
前記第2の被覆層は、ヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有する重合体層であり、第1の被覆層と共有結合を形成し、前記共有結合とは反対側の高分子鎖末端にカルボキシル基を有することを特徴とする、抗体結合用磁性粒子。 A magnetic particle having a core particle, a first coating layer formed on the surface of the core particle, and a second coating layer formed on the surface of the first coating layer,
The first coating layer is a crosslinked inorganic layer containing nanomagnetic material,
The second coating layer is a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group, and forms a covalent bond with the first coating layer, and forms a covalent bond with the first coating layer. Magnetic particles for antibody binding characterized by having a carboxyl group at the end of the polymer chain on the opposite side from the binding.
(i)コア粒子の表面にナノ磁性体を物理吸着させる工程
(ii)前記ナノ磁性体を吸着させた粒子の表面に架橋無機物層を形成する工程
(iii)前記架橋無機物層の表面に、リビングラジカル重合の開始剤又は連鎖移動剤を有する、シランカップリング剤を結合させる工程
(iv)前記シランカップリング剤を結合させた粒子を溶媒に分散させ、溶媒中でヒドロキシル基及びポリエチレングリコール基から選択される少なくとも1種の親水性基含有構造単位を有するモノマーをリビングラジカル重合することにより粒子の表面に重合体層を形成する工程
(v)共重合体層を形成した粒子にメルカプトカルボン酸を反応させる工程 The method for producing antibody-binding magnetic particles according to any one of claims 1 to 3, which comprises the following steps (i) to (v).
(i) A step of physically adsorbing the nanomagnetic material on the surface of the core particle (ii) A step of forming a crosslinked inorganic layer on the surface of the particle to which the nanomagnetic material is adsorbed (iii) A step of physically adsorbing the nanomagnetic material on the surface of the core particle Step (iv) of bonding a silane coupling agent having a radical polymerization initiator or chain transfer agent: dispersing the particles bound with the silane coupling agent in a solvent, and dispersing the particles selected from hydroxyl groups and polyethylene glycol groups in the solvent. step (v) of forming a polymer layer on the surface of the particles by living radical polymerization of a monomer having at least one hydrophilic group-containing structural unit, (v) reacting the particles with the copolymer layer formed with mercaptocarboxylic acid; process of letting
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JP2008081574A (en) | 2006-09-27 | 2008-04-10 | Jsr Corp | Magnetic particle and method for producing the same, and probe-bound particle |
US20090017518A1 (en) | 2007-07-11 | 2009-01-15 | Industrial Technology Research Institute | Magnetic particles and fabrication method thereof |
JP2016501286A (en) | 2012-11-16 | 2016-01-18 | エスエヌユー アールアンドディービー ファウンデーショ | Coded polymer particles |
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