JP4935973B2 - Organic polymer particles and method for producing the same - Google Patents
Organic polymer particles and method for producing the same Download PDFInfo
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- JP4935973B2 JP4935973B2 JP2006030639A JP2006030639A JP4935973B2 JP 4935973 B2 JP4935973 B2 JP 4935973B2 JP 2006030639 A JP2006030639 A JP 2006030639A JP 2006030639 A JP2006030639 A JP 2006030639A JP 4935973 B2 JP4935973 B2 JP 4935973B2
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Landscapes
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Description
本発明は、タンパク質や核酸等の生体関連物質の非特異吸着が少ない有機ポリマー粒子およびその製造方法に関し、特に、生化学・医薬品分野で特出する高感度を発現するプローブ結合粒子に関する。 The present invention relates to organic polymer particles with less non-specific adsorption of biologically related substances such as proteins and nucleic acids, and a method for producing the same, and more particularly to probe-binding particles that exhibit high sensitivity that are outstanding in the fields of biochemistry and medicine.
有機ポリマー粒子および磁性粒子は、例えば、感染症・癌マーカー・ホルモン等の検査対象物質の検出を行なうため、抗原抗体反応を利用した診断薬の反応固相として用いられている。このような診断薬においては、抗体または抗原等の検査用プローブ(一次プローブ)が粒子上に固定化される。サンプル中の検査対象物質は一次プローブを介して粒子上に捕捉された後、第二の検査プローブと反応される。第二の検査プローブ(二次プローブ)は蛍光物質や酵素で標識されており、蛍光や酵素反応によって検出が行われる。近年、疾病の早期発見等の目的のため、検査の高感度化が求められており、診断薬の感度向上は大きな課題となっている。磁性粒子を用いた診断薬においても、感度向上のため、検出法として酵素発色を用いる方式から、より高い感度が得られる蛍光や化学発光を用いる方式へと切り替わりつつある。 Organic polymer particles and magnetic particles are used as a reaction solid phase of a diagnostic agent utilizing antigen-antibody reaction in order to detect test substances such as infectious diseases, cancer markers, and hormones. In such a diagnostic agent, an inspection probe (primary probe) such as an antibody or an antigen is immobilized on the particle. The substance to be inspected in the sample is captured on the particle via the primary probe and then reacted with the second inspection probe. The second inspection probe (secondary probe) is labeled with a fluorescent substance or an enzyme, and is detected by fluorescence or an enzyme reaction. In recent years, for the purpose of early detection of diseases, etc., it has been required to increase the sensitivity of tests, and improving the sensitivity of diagnostic agents has become a major issue. Also in diagnostic agents using magnetic particles, in order to improve sensitivity, a method using enzyme color development as a detection method is being switched to a method using fluorescence or chemiluminescence that can obtain higher sensitivity.
これらの検出技術の発展により、理論上は一分子の検査対象物質の存在まで検出できるレベルに達しているといわれているが、実際には十分な感度が得られていない。その原因としては、粒子表面への二次プローブや夾雑物の非特異的な吸着が挙げられる。例えば、理論上一分子の検査対象物質を検出可能な検査技術であっても、数分子の二次プローブが粒子表面に非特異的に吸着すると、一分子検出は不可能である。このようなことから、粒子表面への検査に使用される物質に対して非特異的な吸着の抑制が強く求められている。 With the development of these detection techniques, it is theoretically said that it has reached a level at which even the presence of a single molecule of a test substance can be detected, but in reality, sufficient sensitivity has not been obtained. The cause is nonspecific adsorption of secondary probes and impurities on the particle surface. For example, even with an inspection technique that can theoretically detect a single molecule of a test target substance, single molecule detection is impossible when several molecules of secondary probes are adsorbed nonspecifically on the particle surface. For these reasons, there is a strong demand for suppression of non-specific adsorption with respect to substances used for inspection on the particle surface.
従来、このような非特異吸着の抑制方法として、ブロッキングと言われる方法が行われてきた。ブロッキングは、一次プローブを粒子上に固定化した後に、二次プローブや夾雑物等の吸着の少ないアルブミンやスキムミルク等のブロッキング剤で粒子表面を被覆する。しかし、ブロッキング剤の被覆効果が十分得られない場合や、生体物質であるブロッキング剤の品質安定性の問題、ブロッキングが十分に行われた場合でもブロッキング剤の変質等によってその作用が経時的に変化し非特異吸着が発生するといった問題点があり、十分な非特異吸着の抑制効果は得られていなかった。 Conventionally, as a method for suppressing such nonspecific adsorption, a method called blocking has been performed. In the blocking, the primary probe is immobilized on the particle, and then the particle surface is coated with a blocking agent such as albumin or skim milk that hardly adsorbs the secondary probe or impurities. However, when the covering effect of the blocking agent cannot be obtained sufficiently, the quality stability of the blocking agent, which is a biological substance, even when blocking is sufficiently performed, the action changes over time due to alteration of the blocking agent, etc. However, there is a problem that non-specific adsorption occurs, and a sufficient effect of suppressing non-specific adsorption has not been obtained.
非特異吸着の問題を解決するための方法として、96ウェルプレートに代表される免疫測定用基材の表面に親水性ポリマーを導入する方法が提案されている(特許文献1〜3)。しかし、このような平面を利用した免疫測定用基材では、一次プローブを固定化する面積が限られること、ならびに、一次プローブと検査対象物質との反応は固液反応であるため、抗原抗体反応の効率が悪く、検査時間が長くなること等の欠点があった。 As a method for solving the problem of non-specific adsorption, a method of introducing a hydrophilic polymer to the surface of an immunoassay substrate typified by a 96-well plate has been proposed (Patent Documents 1 to 3). However, in the immunoassay substrate using such a flat surface, the area for immobilizing the primary probe is limited, and the reaction between the primary probe and the test substance is a solid-liquid reaction. There are disadvantages such as poor efficiency and long inspection time.
さらに、非特異吸着を少なくするための対応策として、スチレン−グリシジルメタクリレート共重合体等からなる有機ポリマー粒子にスペーサを介して生理活性物質を結合したミクロスフィア(特許文献4,5,6)や、粒子表面に親水性のスペーサを導入した有機ポリマー粒子(特許文献7,8)等が提案されている。しかしながら、これらはいずれも、非特異吸着の低減効果が充分ではなく、また、免疫検査用としては感度が不十分であった。 Furthermore, as countermeasures for reducing non-specific adsorption, microspheres (Patent Documents 4, 5, and 6) in which a physiologically active substance is bound to organic polymer particles made of a styrene-glycidyl methacrylate copolymer or the like via a spacer, Organic polymer particles (Patent Documents 7 and 8) in which hydrophilic spacers are introduced on the particle surface have been proposed. However, none of these has a sufficient effect of reducing non-specific adsorption, and the sensitivity is insufficient for immunoassay.
本発明者らは、親水性モノマーとして、ヒドロキシアルキル(メタ)アクリレート、アルコキシアルキル(メタ)アクリレート、ポリオキシアルキレン(C2−C4)基含有(メタ)アクリレート、エポキシ基含有(メタ)アクリレート、ホスホリルコリン類似基含有単量体等を粒子表面に共重合させた、非特異吸着の少ない免疫検査用磁性粒子を提案しているが(特許文献9)、さらなる高感度の発現が望まれる。
本発明の目的は、タンパク質や核酸等の生体関連物質の非特異吸着が少ない有機ポリマー粒子およびその製造方法、ならびにプローブ結合粒子を提供することである。 An object of the present invention is to provide organic polymer particles with little non-specific adsorption of biological substances such as proteins and nucleic acids, a method for producing the same, and probe binding particles.
上記目的を達成するため、本発明者らは鋭意研究を重ね、特定の官能基を有する架橋重合体をシェルとし、かつ、数平均粒径が0.1〜15μmである有機ポリマー粒子が、タンパク質や核酸等の非特異吸着が極めて少ないこと、ならびに、この有機ポリマー粒子を用いることにより、生化学・医薬品分野で特出する高感度を発現するプローブ結合粒子が得られることを見出し、本発明を完成させた。本発明によれば、以下の態様の有機ポリマー粒子およびその製造方法、ならびにプローブ結合粒子を提供することができる。 In order to achieve the above object, the inventors of the present invention have made extensive studies, and organic polymer particles having a cross-linked polymer having a specific functional group as a shell and having a number average particle diameter of 0.1 to 15 μm are protein. It has been found that non-specific adsorption of nucleic acids and nucleic acids is extremely small, and that by using this organic polymer particle, a probe-binding particle that expresses high sensitivity that is special in the biochemistry / pharmaceutical field can be obtained. Completed. According to the present invention, it is possible to provide organic polymer particles of the following aspect, a method for producing the same, and probe binding particles.
本発明の一態様の有機ポリマー粒子は、2,3−ジヒドロキシプロピル基をトシル化して得られた活性基を有する架橋重合体を少なくとも粒子表面に有し、かつ、数平均粒径が0.1〜15μmである。 The organic polymer particle of one embodiment of the present invention has at least a crosslinked polymer having an active group obtained by tosylating 2,3-dihydroxypropyl groups on the particle surface, and has a number average particle size of 0.1. ~ 15 μm.
本発明において、「トシル化する」とは、水酸基を「p−トルエンスルホニル基」へと変換することをいう。 In the present invention, “tosylate” means that a hydroxyl group is converted to a “p-toluenesulfonyl group”.
上記有機ポリマー粒子の水分散液から得られる乾燥塗膜と水との接触角が70°以上であることができる。この場合、上記有機ポリマー粒子は、2,3−ジヒドロキシプロピル基を有する架橋重合体を少なくとも粒子表面に有する有機ポリマー粒子(A)をトシル化処理することにより得られ、前記有機ポリマー粒子(A)の水分散液から得られる乾燥塗膜と水との接触角が40°以下であることができる。 The contact angle between the dried coating film obtained from the aqueous dispersion of organic polymer particles and water can be 70 ° or more. In this case, the organic polymer particle is obtained by tosylating the organic polymer particle (A) having at least a crosslinked polymer having a 2,3-dihydroxypropyl group on the particle surface, and the organic polymer particle (A) The contact angle between the dried coating film obtained from the aqueous dispersion and water can be 40 ° or less.
上記有機ポリマー粒子は、磁性体を含有することができる。この場合、前記架橋重合体は、母粒子を覆うように設けられ、前記母粒子は、核粒子と、該核粒子の表面に設けられた超常磁性微粒子の磁性体層とを含むことができる。 The organic polymer particles can contain a magnetic material. In this case, the cross-linked polymer is provided so as to cover the mother particle, and the mother particle may include a core particle and a magnetic layer of superparamagnetic fine particles provided on the surface of the core particle.
本発明の一態様のプローブ結合粒子は、上記有機ポリマー粒子と、該有機ポリマー粒子に結合するプローブとを含む。 The probe binding particle of one embodiment of the present invention includes the organic polymer particle and a probe that binds to the organic polymer particle.
本発明の一態様の有機ポリマー粒子の製造方法は、
(i)2,3−ジヒドロキシプロピル基を有するモノマー40〜95重量部、(ii)架橋性モノマー5〜30重量部、および(iii)その他のモノマー0〜55重量部からなるモノマー部を重合して架橋重合体を形成することにより、前記架橋重合体を少なくとも粒子表面に有する有機ポリマー粒子(A)を得る工程と、
前記有機ポリマー粒子(A)をトシル化処理する工程と、
を含む。
The method for producing organic polymer particles of one embodiment of the present invention includes:
(I) polymerizing a monomer part comprising 40 to 95 parts by weight of a monomer having a 2,3-dihydroxypropyl group, (ii) 5 to 30 parts by weight of a crosslinkable monomer, and (iii) 0 to 55 parts by weight of other monomers. Forming a crosslinked polymer to obtain organic polymer particles (A) having at least the crosslinked polymer on the particle surface;
A step of tosylating the organic polymer particles (A);
including.
上記有機ポリマー粒子の製造方法において、前記有機ポリマー粒子(A)を得る工程は、母粒子を覆うように前記架橋重合体を形成する工程を含み、前記母粒子は、核粒子と、該核粒子の表面に設けられた超常磁性微粒子の磁性体層とを含むことができる。 In the method for producing organic polymer particles, the step of obtaining the organic polymer particles (A) includes a step of forming the crosslinked polymer so as to cover the mother particles, and the mother particles include the core particles and the core particles. And a magnetic layer of superparamagnetic fine particles provided on the surface.
上記有機ポリマー粒子の製造方法において、前記有機ポリマー粒子(A)の水分散液から得られる乾燥塗膜と水との接触角が40°以下であることができる。 In the method for producing organic polymer particles, the contact angle between the dried coating film obtained from the aqueous dispersion of the organic polymer particles (A) and water may be 40 ° or less.
上記本発明の一態様の有機ポリマー粒子は、タンパク質や核酸等の生体関連物質の非特異吸着量が少ないため、生化学・医薬品分野で特出する高感度を発現する生化学検査用有機ポリマー粒子として好適である。また、上記本発明の一態様のプローブ結合粒子は、タンパク質や核酸等の生体関連物質の非特異吸着量が少ないため、生化学・医薬品分野で特出する高感度を発現し、生化学検査用として高いS/N比を得ることができる。 The organic polymer particle of one embodiment of the present invention has a small amount of non-specific adsorption of biologically related substances such as proteins and nucleic acids, and thus exhibits high sensitivity that is outstanding in the biochemistry / pharmaceutical field. It is suitable as. In addition, since the probe-binding particles of one embodiment of the present invention have a small amount of non-specific adsorption of biologically related substances such as proteins and nucleic acids, they exhibit high sensitivity that is outstanding in the biochemistry / pharmaceutical field, and are suitable for biochemical testing. A high S / N ratio can be obtained.
1.有機ポリマー粒子およびその製造方法
1.1.有機ポリマー粒子の構成
本発明の一態様の有機ポリマー粒子の数平均粒径(以下、単に「粒径」という。)は、通常、0.1〜15μmであり、好ましくは0.3〜10μmであり、より好ましくは1〜10μmである。粒径は、レーザ回折・散乱法により求める。ここで、粒径が0.1μm未満の場合、遠心分離等を用いた分離に長時間を要し、水等の洗浄溶媒と粒子との分離が不十分になるため、目的外の分子(例えば、タンパク質や核酸等の生体関連物質)の除去が不十分になり、充分な精製ができない場合がある。一方、粒径が15μmを超えると、比表面積が小さくなり、生体関連物質の捕捉量が少なくなる結果、感度が低くなる場合がある。
1. Organic polymer particles and production method thereof 1.1. Composition of Organic Polymer Particles The number average particle diameter (hereinafter simply referred to as “particle diameter”) of the organic polymer particles of one embodiment of the present invention is usually 0.1 to 15 μm, preferably 0.3 to 10 μm. Yes, more preferably 1 to 10 μm. The particle size is determined by a laser diffraction / scattering method. Here, when the particle size is less than 0.1 μm, it takes a long time for separation using centrifugation or the like, and the separation between the washing solvent such as water and the particles becomes insufficient. Removal of biologically related substances such as proteins and nucleic acids may be insufficient, and sufficient purification may not be possible. On the other hand, when the particle diameter exceeds 15 μm, the specific surface area becomes small, and the amount of trapped biological substances is reduced, resulting in a decrease in sensitivity.
本発明の一態様の有機ポリマー粒子は、通常、適当な分散媒に分散させて用いられる。使用できる分散媒としては、有機ポリマー粒子を溶解したり、あるいは、有機ポリマー粒子を膨潤させたりしない分散媒が好ましい。好ましい分散媒としては、例えば、水系媒体を用いることができる。ここで、水系媒体とは、水、または水と水に混和する有機溶剤(例えば、アルコール類、アルキレングリコール誘導体等)との混合物をいう。 The organic polymer particles of one embodiment of the present invention are usually used after being dispersed in a suitable dispersion medium. As a dispersion medium that can be used, a dispersion medium that does not dissolve organic polymer particles or swell organic polymer particles is preferable. As a preferable dispersion medium, for example, an aqueous medium can be used. Here, the aqueous medium refers to water or a mixture of water and an organic solvent miscible with water (for example, alcohols, alkylene glycol derivatives, etc.).
本発明の一態様の有機ポリマー粒子は、2,3−ジヒドロキシプロピル基をトシル化して得られた活性基を有する架橋重合体を少なくともその表面に有する。すなわち、本発明の一態様の有機ポリマー粒子は、2,3−ジヒドロキシプロピル基をトシル化した活性基を有する架橋重合体を少なくとも粒子表面に有する。 The organic polymer particle of one embodiment of the present invention has at least a crosslinked polymer having an active group obtained by tosylating 2,3-dihydroxypropyl groups on the surface thereof. That is, the organic polymer particle of one embodiment of the present invention has at least a crosslinked polymer having an active group obtained by tosylating 2,3-dihydroxypropyl group on the particle surface.
2,3−ジヒドロキシプロピル基をトシル化した活性基は、例えば、2,3−ジヒドロキシプロピル基中の水酸基の一方または両方がトシル化された基であり、より具体的には、2−ヒドロキシ−3−(4’−メチルフェニル)スルホニルオキシプロピル基、3−ヒドロキシ−2−(4’−メチルフェニル)スルホニルオキシプロピル基、2,3−ジ(4’−メチルフェニル)スルホニルオキシプロピル基が挙げられる。なお、本発明の一態様の有機ポリマー粒子は、トシル化されていない残余の2,3−ジヒドロキシプロピル基を有していても良い。 The active group obtained by tosylating the 2,3-dihydroxypropyl group is, for example, a group in which one or both of the hydroxyl groups in the 2,3-dihydroxypropyl group are tosylated, and more specifically, 2-hydroxy- Examples include 3- (4′-methylphenyl) sulfonyloxypropyl group, 3-hydroxy-2- (4′-methylphenyl) sulfonyloxypropyl group, and 2,3-di (4′-methylphenyl) sulfonyloxypropyl group. It is done. Note that the organic polymer particles of one embodiment of the present invention may have a residual 2,3-dihydroxypropyl group that is not tosylated.
本発明の一態様の有機ポリマー粒子は、この(4’−メチルフェニル)スルホニル基すなわちトシル基を介して、一次プローブを化学結合させて、免疫検査用のプローブ結合粒子として利用することができる。また、一次プローブの結合後、過剰の一次プローブを洗浄し、未反応のトシル基を不活化した後の残余の2,3−ヒドロキシプロピル基により特出した高感度と低ノイズを発現することができる。このような効果は、例えば、モノヒドロキシプロピル基をトシル化したもの、例えば、3−(4’−メチルフェニル)スルホニルオキシプロピル基のみを有する粒子では発現し得ない。 The organic polymer particle of one embodiment of the present invention can be used as a probe-binding particle for immunoassay by chemically bonding a primary probe via this (4′-methylphenyl) sulfonyl group, that is, a tosyl group. In addition, after binding of the primary probe, the excess primary probe is washed, and the unreacted tosyl group is inactivated and the remaining 2,3-hydroxypropyl group exhibits a high sensitivity and low noise. it can. Such an effect cannot be exhibited in, for example, particles obtained by tosylating a monohydroxypropyl group, for example, particles having only 3- (4'-methylphenyl) sulfonyloxypropyl group.
本発明の一態様の有機ポリマー粒子は、架橋重合体を少なくとも粒子表面に有する。本発明の一態様の有機ポリマー粒子においては、この架橋重合体による架橋構造によって、分散媒による粒子の溶解または粒子表面の膨潤を防止することができる。有機ポリマー粒子において、架橋重合体を少なくともその表面に有さない場合、粒子表面が溶解し、結合した抗体が脱離して感度低下を招いたり、あるいは、粒子表面が膨潤して非特異吸着を増加させたりすることがある。なお、本発明における「少なくとも粒子表面に有する」とは、粒子表面を構成する成分を指し、有機ポリマー粒子が必ずしもコア・シェル構造を必要とするわけではない。よって、本発明においては、有機ポリマー粒子全体が架橋重合体であっても良い。 The organic polymer particle of one embodiment of the present invention has a crosslinked polymer at least on the particle surface. In the organic polymer particle of one embodiment of the present invention, dissolution of the particle by the dispersion medium or swelling of the particle surface can be prevented by the crosslinked structure of the crosslinked polymer. In organic polymer particles, if the crosslinked polymer is not present at least on the surface, the particle surface dissolves and the bound antibody is desorbed, resulting in a decrease in sensitivity, or the particle surface swells to increase nonspecific adsorption. Sometimes In the present invention, “having at least the particle surface” refers to a component constituting the particle surface, and the organic polymer particles do not necessarily require a core-shell structure. Therefore, in the present invention, the whole organic polymer particle may be a crosslinked polymer.
本発明の一態様の有機ポリマー粒子は、2,3−ジヒドロキシプロピル基を有する架橋重合体をシェルとする有機ポリマー粒子(A)をトシル化処理することにより得られる。有機ポリマー粒子(A)の具体的なトシル化処理方法については後述する。また、後述するように、有機ポリマー粒子(A)中の2,3−ジヒドロキシプロピル基の少なくとも一部がトシル化されればよく、また、1つの2,3−ジヒドロキシプロピル基の少なくとも一方の水酸基がトシル化されればよい。 The organic polymer particles of one embodiment of the present invention can be obtained by tosylating organic polymer particles (A) having a crosslinked polymer having 2,3-dihydroxypropyl groups as a shell. A specific tosylation treatment method for the organic polymer particles (A) will be described later. Further, as described later, it is sufficient that at least a part of the 2,3-dihydroxypropyl group in the organic polymer particle (A) is tosylated, and at least one hydroxyl group of one 2,3-dihydroxypropyl group. May be tosylated.
ここで、有機ポリマー粒子(A)の水分散液からの乾燥塗膜と水との接触角は、好ましくは40°以下、さらに好ましくは30°以下、最も好ましくは10°〜25°である。 Here, the contact angle between the dried coating film from the aqueous dispersion of organic polymer particles (A) and water is preferably 40 ° or less, more preferably 30 ° or less, and most preferably 10 ° to 25 °.
また、本発明の一態様の有機ポリマー粒子(以下、有機ポリマー粒子(A)と区別するため有機ポリマー粒子(B)という場合もある)の水分散液からの乾燥塗膜と水との接触角は、好ましくは70°以上、さらに好ましくは90°以上、最も好ましくは105°〜120°である。 Further, the contact angle between water and the dried coating film from the aqueous dispersion of the organic polymer particles of one embodiment of the present invention (hereinafter sometimes referred to as organic polymer particles (B) in order to be distinguished from the organic polymer particles (A)). Is preferably 70 ° or more, more preferably 90 ° or more, and most preferably 105 ° to 120 °.
水分散液からの乾燥塗膜は、50mgの粒子を含む0.2mlの水分散液を、アプリケーター等を用いてスライドガラス等の平滑な基材に塗布し、湿度40%、気温25℃で24時間乾燥することにより得られる。乾燥塗膜と水との接触角は、約1μLの水滴を乾燥塗膜に滴下し、直ちに水平方向からの画像をカメラでデータとして取り込み、水滴の輪郭を円周の一部と仮定して塗膜の水平線との角度から求めることができる。有機ポリマー粒子(A)および(B)の接触角をこれらの範囲とすることにより、低非特異吸着性と高感度とを両立することができる。 The dry coating film from the aqueous dispersion was prepared by applying 0.2 ml of an aqueous dispersion containing 50 mg of particles to a smooth substrate such as a slide glass using an applicator and the like, and having a humidity of 40% and an air temperature of 25 ° C. Obtained by drying for a period of time. The contact angle between the dry coating and water is about 1 μL of water droplets dropped onto the dry coating, and the image from the horizontal direction is immediately captured as data by the camera, and the contour of the water droplet is assumed to be part of the circumference. It can be determined from the angle with the horizontal line of the film. By setting the contact angles of the organic polymer particles (A) and (B) within these ranges, both low non-specific adsorption and high sensitivity can be achieved.
有機ポリマー粒子(A)の水分散液からの乾燥塗膜と水との接触角が40°を超えると、非特異吸着が増加する場合がある。また、有機ポリマー粒子(B)の水分散液からの乾燥塗膜と水との接触角が70°未満であると、感度が低下する場合がある。 If the contact angle between the dried coating film from the aqueous dispersion of organic polymer particles (A) and water exceeds 40 °, non-specific adsorption may increase. Moreover, a sensitivity may fall that the contact angle of the dry coating film from the aqueous dispersion liquid of organic polymer particle (B) and water is less than 70 degrees.
有機ポリマー粒子(A)の水分散液からの乾燥塗膜と水との接触角は、後述のシェルの構成成分である2,3−ジヒドロキシプロピル基を有するモノマーの量およびその他のモノマーの種類および量によって調整することができ、有機ポリマー粒子(B)の水分散液からの乾燥塗膜と水との接触角は、有機ポリマー粒子(A)の接触角の調整因子およびトシル化の度合いによって調整することができる。 The contact angle between the dried coating film from the aqueous dispersion of the organic polymer particles (A) and water is determined based on the amount of the monomer having 2,3-dihydroxypropyl group and the types of other monomers, which are constituent components of the shell described later. The contact angle between the dried coating film from the aqueous dispersion of organic polymer particles (B) and water can be adjusted according to the adjustment factor of the contact angle of organic polymer particles (A) and the degree of tosylation. can do.
1.2.有機ポリマー粒子の製造方法
本発明の一態様の有機ポリマー粒子は、(i)2,3−ジヒドロキシプロピル基を有するモノマー40〜95重量部、(ii)架橋性モノマー5〜30重量部、および(iii)その他のモノマー0〜55重量部からなるモノマー部を重合して架橋重合体(共重合体)を形成することにより、前記架橋重合体をシェルとする有機ポリマー粒子(A)を得る工程と、有機ポリマー粒子(A)をトシル化処理する工程とによって得ることができる。
1.2. Method for Producing Organic Polymer Particle The organic polymer particle of one embodiment of the present invention comprises (i) 40 to 95 parts by weight of a monomer having a 2,3-dihydroxypropyl group, (ii) 5 to 30 parts by weight of a crosslinkable monomer, and ( iii) a step of obtaining organic polymer particles (A) having the crosslinked polymer as a shell by polymerizing a monomer part composed of 0 to 55 parts by weight of other monomers to form a crosslinked polymer (copolymer); And the step of tosylating the organic polymer particles (A).
ここで、有機ポリマー粒子(A)中をトシル化処理する工程において、1つの2,3−ジヒドロキシプロピル基中の2つの水酸基の両方がトシル化されてもよいし、あるいは、1つの2,3−ジヒドロキシプロピル基中の一方の水酸基のみがトシル化されてもよい。また、有機ポリマー粒子(A)中の複数の2,3−ジヒドロキシプロピル基のうち少なくとも一部がトシル化されればよい。さらに、有機ポリマー粒子(A)をトシル化処理する工程において、有機ポリマー粒子(A)中の2,3−ジヒドロキシプロピル基以外の官能基の水酸基がトシル化されてもよい。 Here, in the step of tosylating the organic polymer particles (A), both two hydroxyl groups in one 2,3-dihydroxypropyl group may be tosylated, or one 2,3 -Only one hydroxyl group in the dihydroxypropyl group may be tosylated. Moreover, at least one part should just be tosylated among several 2, 3- dihydroxypropyl groups in an organic polymer particle (A). Furthermore, in the step of tosylating the organic polymer particle (A), the hydroxyl group of a functional group other than the 2,3-dihydroxypropyl group in the organic polymer particle (A) may be tosylated.
1.2.1.モノマー部の組成
(i)2,3−ジヒドロキシプロピル基を有するモノマーとしては、具体的には、グリセロールメタクリレート、グリセロールアクリレート、アリルグリセロールエーテル等を例示することができる。また、(i)2,3−ジヒドロキシプロピル基を有するモノマーの代わりに、グリシジルメタクリレート、グリシジルアクリレート、アリルグリシジルエーテル、2−ヒドロキシ−3−(t−ブトキシ)プロピルメタクリレート、3−ヒドロキシ−2−(t−ブトキシ)プロピルメタクリレート、2,3−ジ(t−ブトキシ)プロピルメタクリレート等を重合中または重合後に加水分解して、(i)2,3−ジヒドロキシプロピル基を有するモノマーに相当する成分を導入しても良い。
1.2.1. Composition of Monomer Part (i) Specific examples of the monomer having a 2,3-dihydroxypropyl group include glycerol methacrylate, glycerol acrylate, and allyl glycerol ether. (I) Instead of the monomer having 2,3-dihydroxypropyl group, glycidyl methacrylate, glycidyl acrylate, allyl glycidyl ether, 2-hydroxy-3- (t-butoxy) propyl methacrylate, 3-hydroxy-2- ( Hydrolysis of t-butoxy) propyl methacrylate, 2,3-di (t-butoxy) propyl methacrylate, etc. during or after polymerization, and (i) introduction of a component corresponding to a monomer having a 2,3-dihydroxypropyl group You may do it.
共重合体形成後の加水分解条件は、例えば、0.05〜0.3mol/Lの硫酸中に、得られた粒子を分散させ、室温〜80℃で1〜24時間反応させることにより行なうことができる。(i)2,3−ジヒドロキシプロピル基を有するモノマーは、粒子表面を構成する共重合体100重量部中に40〜95重量部を占め、好ましくは60〜95重量部を占め、最も好ましくは80〜95重量部を占める。 The hydrolysis conditions after the formation of the copolymer are carried out, for example, by dispersing the obtained particles in 0.05 to 0.3 mol / L sulfuric acid and reacting at room temperature to 80 ° C. for 1 to 24 hours. Can do. (I) The monomer having a 2,3-dihydroxypropyl group occupies 40 to 95 parts by weight, preferably 60 to 95 parts by weight, most preferably 80 parts in 100 parts by weight of the copolymer constituting the particle surface. Occupies ~ 95 parts by weight.
(i)2,3−ジヒドロキシプロピル基を有するモノマーが40重量部未満では、低非特異性および高感度が発現せず、一方、95重量部を超えると、(ii)架橋性モノマーの量が不足し、粒子表面が溶解して結合した抗体が脱離し、感度低下を招いたり、あるいは、粒子表面が膨潤して非特異吸着を増加させたりすることがある。 (I) When the monomer having a 2,3-dihydroxypropyl group is less than 40 parts by weight, low non-specificity and high sensitivity are not expressed. On the other hand, when it exceeds 95 parts by weight, (ii) the amount of the crosslinking monomer is Insufficient, the surface of the particle dissolves and the bound antibody is desorbed, resulting in a decrease in sensitivity, or the surface of the particle may swell and increase nonspecific adsorption.
(ii)架橋性モノマーは、(i)2,3−ジヒドロキシプロピル基を有するモノマー等と共重合可能であり、かつ、1分子中に2個以上のラジカル重合性不飽和結合を有するモノマーである。このような(ii)架橋性モノマーとしては、例えば、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、ペンタエリスリトールトリメタクリレート、ジペンタエリスリトールヘキサアクリレート、ジペンタエリスリトールヘキサメタクリレート等の多官能性(メタ)アクリレート、ブタジエン、イソプレン等の共役ジオレフィン、ジビニルベンゼン、ジアリルフタレート、アリルアクリレート、アリルメタクリレート等を例示することができる。さらに、(ii)架橋性のモノマーとして、ポリエチレングリコールジアクリレート、ポリエチレングリコールジメタクリレート、ポリビニルアルコールのポリ(メタ)アクリルエステル等の親水性のモノマーを例示することができる。 (Ii) The crosslinkable monomer is a monomer that can be copolymerized with (i) a monomer having a 2,3-dihydroxypropyl group or the like and has two or more radically polymerizable unsaturated bonds in one molecule. . Examples of such (ii) crosslinkable monomers include ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexa. Examples thereof include polyfunctional (meth) acrylates such as acrylate and dipentaerythritol hexamethacrylate, conjugated diolefins such as butadiene and isoprene, divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate. Furthermore, (ii) As the crosslinkable monomer, hydrophilic monomers such as polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and poly (meth) acrylic ester of polyvinyl alcohol can be exemplified.
(ii)架橋性モノマーは、架橋重合体を構成する共重合体100重量部中に5〜30重量部を占める。(ii)架橋性モノマーが5重量部未満であると、粒子表面が溶解して結合した抗体が脱離して感度低下を招いたり、あるいは、粒子表面が膨潤して非特異吸着を増加させたりすることがあり、一方、30重量部を超えると、粒子が多孔質化して非特異吸着を増加させることがある。 (Ii) The crosslinking monomer occupies 5 to 30 parts by weight in 100 parts by weight of the copolymer constituting the crosslinked polymer. (Ii) When the crosslinkable monomer is less than 5 parts by weight, the bound antibody dissolves and the bound antibody is desorbed, resulting in a decrease in sensitivity, or the surface of the particle is swollen to increase nonspecific adsorption. On the other hand, if it exceeds 30 parts by weight, the particles may become porous and increase nonspecific adsorption.
(iii)その他のモノマーとしては、例えば、2−ヒドロキシエチルアクリレート、2−ヒドロキシエチルメタクリレート、メトキシエチルアクリレート、メトキシエチルメタクリレート、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等の親水性官能基を有する(メタ)アクリレート、アクリル酸、メタクリル酸、アクリルアミド、メタクリルアミド、N−メチロールアクリルアミド、N−メチロールメタクリルアミド、ダイアセトンアクリルアミド、アリルグリシジルエーテル等の親水性モノマー、ならびに、スチレン、α−メチルスチレン、ハロゲン化スチレン等の芳香族ビニル単量体、酢酸ビニル、プロピオン酸ビニル等のビニルエステル類、アクリロニトリル等の不飽和ニトリル、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、ブチルアクリレート、ブチルメタクリレート、2−エチルヘキシルアクリレート、2−エチルヘキシルメタクリレート、ラウリルアクリレート、ラウリルメタクリレート、ステアリルアクリレート、ステアリルメタクリレート、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、イソボニルアクリレート、イソボニルメタクリレート等のエチレン性不飽和カルボン酸アルキルエステルを例示することができる。 (Iii) Examples of other monomers include hydrophilic functional groups such as 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, methoxyethyl acrylate, methoxyethyl methacrylate, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate (meta ) Hydrophilic monomers such as acrylate, acrylic acid, methacrylic acid, acrylamide, methacrylamide, N-methylol acrylamide, N-methylol methacrylamide, diacetone acrylamide, allyl glycidyl ether, styrene, α-methyl styrene, halogenated styrene Aromatic vinyl monomers such as vinyl acetate, vinyl esters such as vinyl acetate and vinyl propionate, unsaturated nitriles such as acrylonitrile, methyl Acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, butyl acrylate, butyl methacrylate, 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate, lauryl acrylate, lauryl methacrylate, stearyl acrylate, stearyl methacrylate, cyclohexyl acrylate, cyclohexyl methacrylate, isobornyl acrylate, iso Examples thereof include ethylenically unsaturated carboxylic acid alkyl esters such as bonyl methacrylate.
(iii)その他のモノマーの量は、上記(i)2,3−ジヒドロキシプロピル基を有するモノマーおよび(ii)架橋性モノマー以外の残余の量である。 (Iii) The amount of the other monomer is the remaining amount other than (i) the monomer having 2,3-dihydroxypropyl group and (ii) the crosslinkable monomer.
1.2.2.重合方法
有機ポリマー粒子(A)は、例えば、乳化重合、ソープフリー重合、懸濁重合等の定法を用いて製造が可能である。より具体的には、有機ポリマー粒子(A)は、例えば、上記ビニル系モノマーの懸濁重合、あるいはポリマーバルクの粉砕によって得ることができる。例えば、有機ポリマー粒子(A)は、特公昭57−24369号公報記載のシード粒子(母粒子)を用いる二段膨潤重合法、ジャーナル・オブ・ポリマーサイエンス・ポリマーレター・エディション,937頁,第21巻,1963年(J. Polym. Sci., Polymer Letter Ed. 21,937(1963))記載の重合方法、特開昭61−215602号公報、特開昭61−215603号公報、および特開昭61−215604号公報記載の方法によって作製することができる。これらの方法の中では、シード粒子(母粒子)を用いる二段膨潤重合法が、粒径の変動係数を小さくすることができるため好ましい。シード粒子(母粒子)は、ポリスチレンまたはスチレン系共重合体等を用いることができる。そして、二段膨潤重合法により追加されるポリマー部分は、上述の(i)2,3−ジヒドロキシプロピル基を有するモノマーあるいはその前駆体および(ii)架橋性モノマーを必須成分とするモノマー部から形成された共重合体(架橋重合体)からなる。したがって、このようにして得られた有機ポリマー粒子(A)は、2,3−ジヒドロキシプロピル基を少なくともその表面に有する。
1.2.2. Polymerization method The organic polymer particles (A) can be produced using a conventional method such as emulsion polymerization, soap-free polymerization, suspension polymerization or the like. More specifically, the organic polymer particles (A) can be obtained, for example, by suspension polymerization of the vinyl monomer or pulverization of a polymer bulk. For example, the organic polymer particle (A) is a two-stage swelling polymerization method using a seed particle (mother particle) described in JP-B-57-24369, Journal of Polymer Science Polymer Letter Edition, page 937, page 21. Vol., 1963 (J. Polymer. Sci., Polymer Letter Ed. 21, 937 (1963)), JP-A-61-215602, JP-A-61-215603, and JP-A-6-215603. It can be produced by the method described in JP-A-61-215604. Among these methods, the two-stage swelling polymerization method using seed particles (mother particles) is preferable because the coefficient of variation in particle size can be reduced. As the seed particles (mother particles), polystyrene or a styrene copolymer can be used. The polymer portion added by the two-stage swelling polymerization method is formed from the monomer part having (i) the monomer having 2,3-dihydroxypropyl group or the precursor thereof and (ii) the crosslinkable monomer as essential components. Made of a copolymer (crosslinked polymer). Therefore, the organic polymer particles (A) thus obtained have at least a 2,3-dihydroxypropyl group on the surface thereof.
上述したように、有機ポリマー粒子(B)は、有機ポリマー粒子(A)をトシル化処理することにより、有機ポリマー粒子(A)中の2,3−ジヒドロキシプロピル基をトシル化することにより得られる。トシル化は、公知の方法により行なうことができる。例えば、有機ポリマー粒子(A)中の2,3−ジヒドロキシプロピル基とp−トルエンスルホン酸塩とを反応させることにより、2,3−ジヒドロキシプロピル基を、2−ヒドロキシ−3−(4’−メチルフェニル)スルホニルオキシプロピル基に変換することにより、トシル化を達成することができる。 As described above, the organic polymer particle (B) is obtained by tosylating the organic polymer particle (A) to tosylate the 2,3-dihydroxypropyl group in the organic polymer particle (A). . Tosylation can be carried out by a known method. For example, by reacting 2,3-dihydroxypropyl group in organic polymer particles (A) with p-toluenesulfonate, 2,3-dihydroxypropyl group is converted into 2-hydroxy-3- (4′- Tosylation can be achieved by conversion to a methylphenyl) sulfonyloxypropyl group.
p−トルエンスルホン酸塩としては、特に限定されないが、p−トルエンスルホン酸クロライド等を挙げることができる。この工程は、典型的には、有機ポリマー粒子(A)をピリジン等の有機溶剤に分散した後、有機ポリマー粒子(A)100重量部当たり1〜50重量部のp−トルエンスルホン酸クロライドを添加し、室温で1〜6時間反応させることにより行なう。 Although it does not specifically limit as p-toluenesulfonic acid salt, p-toluenesulfonic acid chloride etc. can be mentioned. In this step, typically, after dispersing the organic polymer particles (A) in an organic solvent such as pyridine, 1 to 50 parts by weight of p-toluenesulfonic acid chloride is added per 100 parts by weight of the organic polymer particles (A). And reacting at room temperature for 1 to 6 hours.
あるいは、有機ポリマー粒子(A)中の2,3−ジヒドロキシプロピル基とp−トルエンスルホン酸とを脱水縮合させることにより、2,3−ジヒドロキシプロピル基を2−ヒドロキシ−3−(4’−メチルフェニル)スルホニルオキシプロピル基に変換することにより、前記トシル化を行なってもよい。 Alternatively, the 2,3-dihydroxypropyl group and p-toluenesulfonic acid in the organic polymer particles (A) are subjected to dehydration condensation, whereby the 2,3-dihydroxypropyl group is converted into 2-hydroxy-3- (4′-methyl). The tosylation may be carried out by conversion to a phenyl) sulfonyloxypropyl group.
以上により、本発明の一態様の有機ポリマー粒子(B)を得ることができる。有機ポリマー粒子(B)の分散液は、遠心分離法等により、アセトン洗浄と水洗とを繰り返し、有機ポリマー粒子(B)の水分散体とすることが好ましい。 Through the above steps, the organic polymer particles (B) of one embodiment of the present invention can be obtained. The dispersion of the organic polymer particles (B) is preferably made into an aqueous dispersion of organic polymer particles (B) by repeating acetone washing and water washing by a centrifugal method or the like.
2.磁性体を含有する有機ポリマー粒子およびその製造方法
本発明の一態様の有機ポリマー粒子は、磁性体を含有する有機ポリマー粒子(以下、「磁性体含有有機ポリマー粒子」という。)であってもよい。磁性体含有有機ポリマー粒子は、例えば遠心分離器等を用いずに、磁石を用いて分離することができるため、被検体からの粒子の分離工程を簡素化または自動化することができる点で有用である。
2. Organic polymer particle containing magnetic substance and method for producing the same The organic polymer particle of one embodiment of the present invention may be an organic polymer particle containing a magnetic substance (hereinafter referred to as “magnetic substance-containing organic polymer particle”). . Since the magnetic substance-containing organic polymer particles can be separated using a magnet without using, for example, a centrifuge, it is useful in that the process of separating particles from a specimen can be simplified or automated. is there.
磁性体含有有機ポリマー粒子は、(I)有機ポリマー等の非磁性体の連続相中に磁性体微粒子が分散している粒子、(II)磁性体微粒子の2次凝集体をコアとし、有機ポリマー等の非磁性体をシェルとする粒子、(III)有機ポリマー等の非磁性体からなる核粒子と、該核粒子の表面に設けられた磁性体微粒子の2次凝集体層(磁性体層)とを有する母粒子をコアとし、該母粒子の最外層の有機ポリマー層をシェルとする粒子等が挙げられる。これらの中では、(III)前記磁性体微粒子の2次凝集体層を含む母粒子をコアとし、有機ポリマー層をシェルとする粒子が好ましい。なお、各種構造の磁性体含有有機ポリマー粒子に用いる有機ポリマーは、コア・シェル型粒子のコア部分を除いて、上述の(i)2,3−ジヒドロキシプロピル基を有するモノマーおよび(ii)架橋性モノマーを必須成分とするモノマー部を用いて形成された共重合体であることが必要である。 The magnetic substance-containing organic polymer particles are: (I) particles in which magnetic fine particles are dispersed in a non-magnetic continuous phase such as an organic polymer, and (II) secondary aggregates of magnetic fine particles as a core. (III) Core particles made of non-magnetic material such as organic polymer, and secondary aggregate layer (magnetic material layer) of magnetic fine particles provided on the surface of the core particle And particles having the core as a core and the outermost organic polymer layer of the mother particle as a shell. Among these, (III) particles having a core particle including a secondary aggregate layer of the magnetic fine particles as a core and an organic polymer layer as a shell are preferable. The organic polymer used in the magnetic material-containing organic polymer particles having various structures is the above-mentioned (i) monomer having 2,3-dihydroxypropyl group and (ii) crosslinkability except for the core portion of the core-shell type particle. It is necessary to be a copolymer formed using a monomer part having a monomer as an essential component.
最も好ましい磁性体含有有機ポリマー粒子は、核粒子と、この核粒子の表面に設けられた超常磁性微粒子の磁性体層とを含む母粒子を覆うように、架橋重合体が設けられている。すなわち、この磁性体含有有機ポリマー粒子では、前記母粒子をコアとし、架橋重合体をシェルとする。ここで、架橋重合体は上述の製造方法により得られる。すなわち、架橋重合体は、(i)2,3−ジヒドロキシプロピル基を有するモノマー40〜95重量部、(ii)架橋性モノマー5〜30重量部、および(iii)その他のモノマー0〜55重量部からなるモノマー部を重合することにより得られる。 The most preferable magnetic substance-containing organic polymer particles are provided with a crosslinked polymer so as to cover the mother particles including the core particles and the magnetic layer of superparamagnetic fine particles provided on the surface of the core particles. That is, in the magnetic substance-containing organic polymer particles, the base particles are used as a core, and the crosslinked polymer is used as a shell. Here, the crosslinked polymer is obtained by the above-described production method. That is, the crosslinked polymer comprises (i) 40 to 95 parts by weight of a monomer having a 2,3-dihydroxypropyl group, (ii) 5 to 30 parts by weight of a crosslinkable monomer, and (iii) 0 to 55 parts by weight of other monomers. It is obtained by polymerizing the monomer part consisting of
核粒子の表面に超常磁性微粒子の磁性体層が形成された母粒子の製造方法としては、例えば、非磁性の有機ポリマー粒子と超常磁性微粒子とをドライブレンドして、物理的に強い力を外部から加えることにより双方の粒子を複合化させる方法により作製することができる。物理的に強い力を負荷する方法としては、例えば、乳鉢、自動乳鉢、ボールミル、ブレード加圧式粉体圧縮法、メカノフュージョン法のようなメカノケミカル効果を利用するもの、あるいはジェットミル、ハイブリダイザー等の高速気流中衝撃法を利用するものが挙げられる。効率よくかつ強固に複合化を実施するには、物理的吸着力が強いことが望ましい。その方法としては、攪拌翼付き容器中で攪拌翼の周速度が好ましくは15m/秒以上、より好ましくは30m/秒以上、さらに好ましくは40〜150m/秒で実施することが挙げられる。撹拌翼の周速度が15m/秒より低いと、非磁性の有機ポリマー粒子の表面に超常磁性微粒子を吸着させるのに十分なエネルギーを得ることができないことがある。なお、撹拌翼の周速度の上限については、特に制限はないが、使用する装置、エネルギー効率等の点から自ずと決定される。本発明で使用する超常磁性微粒子は、例えば、粒子径5〜20nm程度のフェライトおよび/またはマグネタイトの微粒子が好適に使用できる。 As a method for producing a mother particle in which a magnetic layer of superparamagnetic fine particles is formed on the surface of a core particle, for example, non-magnetic organic polymer particles and superparamagnetic fine particles are dry blended, and a physically strong force is externally applied. Can be produced by a method of combining both particles. As a method of applying a physically strong force, for example, a mortar, an automatic mortar, a ball mill, a blade pressurizing powder compression method, a method using a mechanochemical effect such as a mechanofusion method, a jet mill, a hybridizer, etc. And those using the high-speed air-flow impact method. It is desirable that the physical adsorption force is strong in order to efficiently and firmly perform the composite. As the method, it is mentioned that the peripheral speed of the stirring blade is preferably 15 m / second or more, more preferably 30 m / second or more, and further preferably 40 to 150 m / second in a vessel with a stirring blade. When the peripheral speed of the stirring blade is lower than 15 m / sec, it may not be possible to obtain sufficient energy to adsorb the superparamagnetic fine particles on the surface of the nonmagnetic organic polymer particles. In addition, although there is no restriction | limiting in particular about the upper limit of the peripheral speed of a stirring blade, It determines automatically from points, such as an apparatus to be used and energy efficiency. As the superparamagnetic fine particles used in the present invention, for example, ferrite and / or magnetite fine particles having a particle diameter of about 5 to 20 nm can be suitably used.
(i)2,3−ジヒドロキシプロピル基を有するモノマー40〜95重量部、(ii)架橋性モノマー5〜30重量部、および(iii)その他のモノマー0〜55重量部からなるモノマー部を重合することにより得られる共重合体(シェル)は、母粒子(コア)の存在下で前記モノマー部を共重合することにより形成することができる。各モノマー成分については上述の通りである。より具体的な重合方法については、特開2004−205481号公報等に開示されている通りである。 (I) A monomer part comprising 40 to 95 parts by weight of a monomer having a 2,3-dihydroxypropyl group, (ii) 5 to 30 parts by weight of a crosslinkable monomer, and (iii) 0 to 55 parts by weight of another monomer is polymerized. The copolymer (shell) obtained by this can be formed by copolymerizing the monomer part in the presence of the base particle (core). Each monomer component is as described above. A more specific polymerization method is as disclosed in JP-A-2004-205481 and the like.
3.用途
本発明の一態様の有機ポリマー粒子は、生化学分野での化合物担体用粒子および診断薬用の化学結合担体用粒子等のアフィニティー担体として利用でき、特に、抗原または抗体等の一次プローブを結合させた免疫検査用のプローブ結合粒子として、特出する高感度および低ノイズを発現することができる。
3. Use The organic polymer particle of one embodiment of the present invention can be used as an affinity carrier such as a particle for a compound carrier in the biochemical field and a particle for a chemical binding carrier for a diagnostic agent. In particular, it binds a primary probe such as an antigen or an antibody. As a probe-binding particle for immunoassay, it is possible to express outstanding high sensitivity and low noise.
本発明の一態様のプローブ結合粒子において、検査対象となる物質は、免疫検査用試薬および被検査試料に含まれる生体関連物質および化学物質である。本発明において、「生体関連物質」とは、生体に関わるすべての物質をいう。生体関連物質としては、例えば、生体に含まれる物質、生体に含まれる物質から誘導された物質、生体内で利用可能な物質が挙げられる。生体関連物質は特に限定されないが、例えば、タンパク質(例えば、酵素、抗体、アプタマー、受容体等)、ペプチド(例えばグルタチオン等)、核酸(例えば、DNAやRNA等)、糖質、脂質、およびその他の細胞または物質(例えば、血小板、赤血球、白血球等の各種血球細胞を含む各種血液由来物質、各種浮遊細胞等)等が挙げられる。 In the probe-binding particles of one embodiment of the present invention, the substances to be tested are biologically relevant substances and chemical substances contained in the immunological test reagent and the sample to be tested. In the present invention, “biologically related substance” refers to all substances related to a living body. Examples of the biological substance include substances contained in the living body, substances derived from the substance contained in the living body, and substances that can be used in the living body. The biological substance is not particularly limited. For example, protein (eg, enzyme, antibody, aptamer, receptor, etc.), peptide (eg, glutathione), nucleic acid (eg, DNA or RNA), carbohydrate, lipid, and others Cells or substances (for example, various blood-derived substances including various blood cells such as platelets, red blood cells, and white blood cells, various floating cells, etc.).
本発明の一態様のプローブ結合粒子によれば、トシル基が粒子の表面に導入されているため、実際に使用するに当たり、一次プローブと粒子とを混合するだけで、一次プローブを粒子の表面に化学的に結合させることができる。 According to the probe-bound particle of one aspect of the present invention, since the tosyl group is introduced on the surface of the particle, the primary probe can be attached to the surface of the particle simply by mixing the primary probe and the particle in actual use. Can be chemically bonded.
一次プローブを粒子の表面に結合させた後、過剰の一次プローブを洗浄し、必要に応じて未反応のトシル基を不活化する。不活化剤として、エタノールアミン、トリス(ヒドロキシアミノ)メタン等の水酸基を含有する不活化剤を使用するのが好ましい。また、一次プローブの活性を阻害しない範囲の酸またはアルカリ条件でトシル基を加水分解してもよい。また、一次プローブを粒子の表面に結合させた後、通常行われるブロッキングの操作は不要であるが、上記不活化工程において、アルブミン等のブロッキング剤を併用してもかまわない。以降は、粒子を用いた通常の分析工程に移行すればよい。 After the primary probe is bound to the surface of the particle, excess primary probe is washed, and unreacted tosyl groups are inactivated as necessary. As the inactivating agent, it is preferable to use an inactivating agent containing a hydroxyl group such as ethanolamine or tris (hydroxyamino) methane. Further, the tosyl group may be hydrolyzed under an acid or alkaline condition in a range that does not inhibit the activity of the primary probe. In addition, after the primary probe is bonded to the surface of the particle, the usual blocking operation is not required, but a blocking agent such as albumin may be used in combination in the inactivation step. Thereafter, a normal analysis process using particles may be performed.
本発明の一態様のプローブ結合粒子に担持することができるプローブは、タンパク質(抗原または抗体)または核酸であり、このうち抗原または抗体が好ましい。この場合、抗原または抗体としては、被検体中に一般に含まれている成分に反応するものであれば特に制限されないが、例えば、アンチプラスミン検査用抗アンチプラスミン抗体、Dダイマー検査用抗Dダイマー抗体、FDP検査用抗FDP抗体、tPA検査用抗tPA抗体、TAT検査用抗トロンビン=アンチトロンビン複合体抗体、FPA検査用抗FPA抗体等の凝固線溶関連検査用抗原または抗体;BFP検査用抗BFP抗体、CEA検査用抗CEA抗体、AFP検査用抗AFP抗体、フェリチン検査用抗フェリチン抗体、CA19−9検査用抗CA19−9抗体等の腫瘍関連検査用抗原または抗体;アポリポタンパク検査用抗アポリポタンパク抗体、β2−ミクロブロブリン検査用抗β2−ミクロブロブリン抗体、α1−ミクログロブリン検査用抗α1―ミクログロブリン抗体、免疫グロブリン検査用抗免疫グロブリン抗体、CRP検査用抗CRP抗体等の血清蛋白関連検査用抗原または抗体;HCG検査用抗HCG抗体等の内分泌機能検査用抗原または抗体;HBs抗原検査用抗HBs抗体、HBs抗体検査用HBs抗原、HCV抗体検査用HCV抗原、HIV−1抗体用HIV−1抗原、HIV−2抗体検査用HIV−2抗原、HTLV−1検査用HTLV−1抗原、マイコプラズマ症検査用マイコプラズマ抗原、トキソプラズマ検査用トキソプラズマ抗原、ASO検査用ストレプトリジンO抗原等の感染症関連検査用抗原または抗体;抗DNA抗体検査用DNA抗原、RF検査用熱変成ヒトIgG等自己免疫関連検査用抗原または抗体;ジゴキシン検査用抗ジゴキシン抗体、リドカイン検査用抗リドカイン抗体等の薬物分析用抗原または抗体等を挙げることができるが、これらに限定されるものではない。抗体としては、ポリクローナル抗体またはモノクローナル抗体のどちらを用いてもかまわない。 The probe that can be carried on the probe-binding particle of one embodiment of the present invention is a protein (antigen or antibody) or nucleic acid, and of these, an antigen or antibody is preferable. In this case, the antigen or antibody is not particularly limited as long as it reacts with a component generally contained in a subject. For example, anti-antiplasmin antibody for antiplasmin test, anti-D dimer antibody for D dimer test Anti-FDP antibody for FDP test, Anti-tPA antibody for tPA test, Anti-thrombin = antithrombin complex antibody for TAT test, Anticoagulation-related test antigen or antibody such as anti-FPA antibody for FPA test; Anti-BFP for BFP test Anti-Apolipoprotein for testing apolipoprotein such as antibodies, anti-CEA antibodies for CEA testing, anti-AFP antibodies for AFP testing, anti-ferritin antibodies for testing ferritin, anti-CA19-9 testing for CA19-9 Antibody, anti-β2-microblob antibody for β2-microblob test, α1-microglob Anti-α1-microglobulin antibody for serum test, anti-immunoglobulin antibody for immunoglobulin test, serum protein-related test antigen or antibody such as anti-CRP antibody for CRP test; endocrine function test antigen such as anti-HCG antibody for HCG test or Antibody: Anti-HBs antibody for HBs antigen test, HBs antigen for HBs antibody test, HCV antigen for HCV antibody test, HIV-1 antigen for HIV-1 antibody, HIV-2 antigen for HIV-2 antibody test, HTLV-1 test HTLV-1 antigen, Mycoplasma antigen for Mycoplasma test, Toxoplasma antigen for Toxoplasma test, Streptridine O antigen for ASO test, etc. Antigen-related test antigen or antibody; Anti-DNA antibody test DNA antigen, RF test heat-modified human Antigens or antibodies for autoimmune related tests such as IgG; antidigoxin anti And antigens for drug analysis such as anti-lidocaine antibodies for lidocaine testing, and the like, but are not limited thereto. As the antibody, either a polyclonal antibody or a monoclonal antibody may be used.
また、本発明の一態様の有機ポリマー粒子は、酵素・ホルモン等のタンパク質、DNA・RNA等の核酸、脂質、あるいは生理活性糖鎖化合物を粒子表面に化学結合法で感作させるアフィニティー担体としても利用できる。さらに、本発明の一態様の有機ポリマー粒子に、解析対象の化学物質(被解析化学物質;リガンド分子に該当する)を化学結合により固定化し、タンパク物質等との特異的相互作用を用いて当該相互作用を解析および/または測定することによって、被解析化学物質と特異的な相互作用を有するタンパク質等(ターゲット分子に該当する)を選別し、精製することが可能である。 In addition, the organic polymer particle of one embodiment of the present invention can be used as an affinity carrier that sensitizes the particle surface with a protein such as an enzyme or hormone, a nucleic acid such as DNA or RNA, a lipid, or a physiologically active sugar chain compound. Available. Furthermore, a chemical substance to be analyzed (analyzed chemical substance; corresponding to a ligand molecule) is immobilized on the organic polymer particle of one embodiment of the present invention by chemical bonding, and the specific chemical substance is used for the interaction. By analyzing and / or measuring the interaction, it is possible to select and purify a protein or the like (corresponding to the target molecule) having a specific interaction with the chemical substance to be analyzed.
具体的には、粒子に結合させるリガンド分子としては、本発明の一態様の有機ポリマー粒子が有するトシル基と反応しうる官能基を有する物質であれば特に限定されないが、例えば、核酸、ペプチド核酸、ホルモン、分子量500〜100万のタンパク質、糖鎖、多糖類、細胞、アプタマー、ウイルス、酵素、各種のアフィニティー用タグ捕捉物質、ビオチン等の補酵素、特定の生理活性作用を有する(あるいは、特定の生理活性作用を有する可能性がある)化学物質等を使用することができる。 Specifically, the ligand molecule to be bonded to the particle is not particularly limited as long as it is a substance having a functional group capable of reacting with the tosyl group included in the organic polymer particle of one embodiment of the present invention. , Hormones, proteins with a molecular weight of 500 to 1,000,000, sugar chains, polysaccharides, cells, aptamers, viruses, enzymes, various affinity tag capture substances, coenzymes such as biotin, and specific physiological activity (or specific A chemical substance or the like that may have a physiologically active action.
4.実施例
以下、実施例を挙げて本発明をさらに詳細に説明するが、本発明はこれらによって制限されるものではない。なお、本実施例において、「%」および「部」は重量基準である。
4). Examples Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. In this example, “%” and “parts” are based on weight.
4.1.評価方法
4.1.1.CLEIA(化学発光酵素免疫測定)
抗AFP(αフェトプロテイン)抗体を感作させた、後述する各実施例・比較例で得られた有機ポリマー粒子の分散液10μl(粒子50μg相当)をテストチューブに取り、ウシ胎児血清(FCS)で1000ng/mLに希釈したAFP抗原(日本バイオテスト社製)の標準検体50μlと混合し、37℃で10分間反応した。遠心または磁気分離して粒子を分離し上清を除いた後、2次抗体としてアルカリフォスファターゼ(以下、「ALP」という。)で標識した抗AFP抗体(富士レビオ株式会社製、ルミパルスAFP−Nに付属の試薬を使用)40μlを添加し、37℃で10分間反応させた。次いで、遠心または磁気分離して粒子を分離し上清を除いた後、PBSで3回遠心洗浄を繰り返して得られた粒子を50μlの0.01%Tween20に分散させ、新しいチューブに移し替えた。ALPの基質液(ルミパルス基質液:富士レビオ株式会社製)100μlを加え、37℃で10分間反応させた後、化学発光量を測定した。化学発光の測定には、ベルトールジャパン株式会社製の化学発光測定装置(商品名:Lumat LB9507)を用いた。
4.1. Evaluation method 4.1.1. CLEIA (chemiluminescence enzyme immunoassay)
10 μl of a dispersion of organic polymer particles (corresponding to 50 μg of particles) obtained in each of the examples and comparative examples described below, sensitized with an anti-AFP (α-fetoprotein) antibody, is placed in a test tube and washed with fetal calf serum (FCS). The mixture was mixed with 50 μl of a standard specimen of AFP antigen (manufactured by Nippon Biotest) diluted to 1000 ng / mL and reacted at 37 ° C. for 10 minutes. After separation of the particles by centrifugation or magnetic separation and removal of the supernatant, an anti-AFP antibody labeled with alkaline phosphatase (hereinafter referred to as “ALP”) as a secondary antibody (Lumipulse AFP-N, manufactured by Fujirebio Inc.) was used. 40 μl was added and reacted at 37 ° C. for 10 minutes. Next, the particles were separated by centrifugation or magnetic separation, and the supernatant was removed. Then, the particles obtained by repeated centrifugal washing with PBS three times were dispersed in 50 μl of 0.01% Tween 20, and transferred to a new tube. . After adding 100 μl of ALP substrate solution (Lumipulse substrate solution: manufactured by Fujirebio Inc.) and reacting at 37 ° C. for 10 minutes, the amount of chemiluminescence was measured. For the measurement of chemiluminescence, a chemiluminescence measuring device (trade name: Lumat LB9507) manufactured by Bertol Japan KK was used.
4.1.2.粒径
レーザ回折式粒度分布測定装置((株)島津製作所製)SALD−200Vにより、粒子の数平均粒径およびその変動係数を測定した。
4.1.2. Particle size The number average particle size of the particles and the coefficient of variation thereof were measured with a laser diffraction particle size distribution analyzer (manufactured by Shimadzu Corporation) SALD-200V.
4.1.3.水分散液から得られる乾燥塗膜と水との接触角
後述する各実施例・比較例で得られた有機ポリマー粒子50mgを1mlの純水で10回洗浄し、最後に0.2mlの純水に分散させた。この粒子を含む水分散液をアプリケーターでスライドガラスに塗布し、湿度40%、気温25℃で24時間乾燥して乾燥塗膜を得た。得られた乾燥塗膜と水との接触角を、協和界面科学製FAMAS接触角測定システム(商品名:DropMaster900)を用いて、以下の手順で測定した。1.0μLの水滴を乾燥塗膜に滴下してから0.15秒後の水平方向からの画像をカメラでデータとして取り込み、水滴の輪郭を円周の一部と仮定して、水滴の輪郭と乾燥塗膜の水平線との角度から、得られた乾燥塗膜と水との接触角を求めた。
4.1.3. Contact angle between water and dry coating film obtained from aqueous dispersion 50 mg of organic polymer particles obtained in each of Examples and Comparative Examples described later were washed 10 times with 1 ml of pure water, and finally 0.2 ml of pure water. Dispersed. The aqueous dispersion containing these particles was applied to a slide glass with an applicator and dried at 40% humidity and 25 ° C. for 24 hours to obtain a dried coating film. The contact angle between the obtained dried coating film and water was measured by the following procedure using a FAMAS contact angle measurement system (trade name: DropMaster 900) manufactured by Kyowa Interface Science. The image taken from the horizontal direction 0.15 seconds after dropping 1.0 μL of water droplets on the dry coating film is captured as data by the camera, and the contour of the water droplet is assumed to be a part of the circumference. From the angle with the horizontal line of the dried coating film, the contact angle between the obtained dried coating film and water was determined.
4.2.合成例1(磁性体を含有しない有機ポリマー粒子の合成)
以下に記載するように、シード粒子(母粒子)を用いる二段膨潤重合法により有機ポリマー粒子(A)を作製した。シード粒子(母粒子)として、ソープフリー重合により得られた粒子径0.98μmのポリスチレン粒子を用い、このポリスチレン粒子を窒素雰囲気下で水500gに分散させて、水分散体(固形分量5.0g)を調製した。この水分散体に、一段目として有機溶剤(シェルゾールTK0.1g)、二段目としてメタクリル酸メチル(以下、「MMA」という。)40g、エチレングリコールジメタクリレート10g、およびグリセロールメタクリレート(以下、「GLM」という。)50gを加えてそれぞれ吸収させた後、AIBN(アゾビスイソブチロニトリル)を2g添加して75℃で24時間ゆっくり撹拌した。次に、この反応液を冷却した後、500メッシュ金網でろ過したところ、99%が通過し、良好な重合安定性であった。重合収率は99%であった。上記工程により得られた有機ポリマー粒子(A)を以下「A−1」とする。
4.2. Synthesis Example 1 (Synthesis of organic polymer particles containing no magnetic substance)
As described below, organic polymer particles (A) were prepared by a two-stage swelling polymerization method using seed particles (mother particles). As seed particles (mother particles), polystyrene particles having a particle diameter of 0.98 μm obtained by soap-free polymerization were used. The polystyrene particles were dispersed in 500 g of water in a nitrogen atmosphere, and an aqueous dispersion (solid content: 5.0 g) was obtained. ) Was prepared. In this aqueous dispersion, an organic solvent (shell sol 0.1 g) is used as the first stage, methyl methacrylate (hereinafter referred to as “MMA”) 40 g, ethylene glycol dimethacrylate 10 g, and glycerol methacrylate (hereinafter “ It was referred to as “GLM”.) 50 g was added for absorption, and 2 g of AIBN (azobisisobutyronitrile) was added thereto, followed by slow stirring at 75 ° C. for 24 hours. Next, the reaction solution was cooled and then filtered through a 500 mesh wire net. As a result, 99% passed and the polymerization stability was good. The polymerization yield was 99%. The organic polymer particles (A) obtained by the above step are hereinafter referred to as “A-1”.
このA−1粒子を、遠心分離を用いて蒸留水で洗浄した後、上述の方法にて、水分散液からの乾燥塗膜と水との接触角を測定した結果、該接触角は34°であった。 After the A-1 particles were washed with distilled water using centrifugation, the contact angle between the dried coating film from the aqueous dispersion and water was measured by the method described above. As a result, the contact angle was 34 °. Met.
次に、このA−1粒子5.0gを、遠心分離を用いて蒸留水で洗浄した後、凍結乾燥して得られた乾燥粒子1.0gを8mlのピリジンに分散させ、次いで、p−トルエンスルホン酸クロライド(和光純薬工業製)0.2gを加えて室温で2時間撹拌した。反応後、遠心分離機を用いて分離した粒子を採取した後、この粒子をアセトンで4回、続いて蒸留水で4回洗浄して、有機ポリマー粒子(B)(以下、「B−1」とする。)を得た。B−1粒子の粒子径は2.6μmであり、粒子径の変動係数は4%であった。B−1粒子の水分散液からの乾燥塗膜と水との接触角は80°であった。 Next, after 5.0 g of this A-1 particle was washed with distilled water using centrifugation, 1.0 g of dry particles obtained by lyophilization was dispersed in 8 ml of pyridine, and then p-toluene. 0.2 g of sulfonic acid chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred at room temperature for 2 hours. After the reaction, the separated particles were collected using a centrifuge, and the particles were washed 4 times with acetone and then 4 times with distilled water to obtain organic polymer particles (B) (hereinafter referred to as “B-1”). And obtained. The particle diameter of the B-1 particles was 2.6 μm, and the coefficient of variation of the particle diameter was 4%. The contact angle between the dried coating film and water from the aqueous dispersion of B-1 particles was 80 °.
得られたB−1粒子のトシル基含量を、エタノールアミンとの反応量から求めた。B−1粒子5mgに0.2mol/Lのエタノールアミンを含有するホウ酸緩衝液(0.1mol/L、pH9.5)溶液500μlを加え、37℃で16時間撹拌して反応した。反応後、反応によって生じたp−トルエンスルホン酸の生成量を反応液の上澄みの吸光度から測定した結果から(ε262=440)、B−1粒子の粒子のトシル基量は、0.17μモル/mgであった。 The tosyl group content of the obtained B-1 particles was determined from the amount of reaction with ethanolamine. 500 μl of a borate buffer solution (0.1 mol / L, pH 9.5) containing 0.2 mol / L ethanolamine was added to 5 mg of B-1 particles, and the mixture was reacted at 37 ° C. for 16 hours with stirring. From the result of measuring the production amount of p-toluenesulfonic acid generated by the reaction from the absorbance of the supernatant of the reaction solution (ε 262 = 440), the amount of tosyl groups of the particles of the B-1 particle was 0.17 μmol. / Mg.
4.3.合成例2(磁性体を含有する有機ポリマー粒子の合成)
75%ジ(3,5,5−トリメチルヘキサノイル)パーオキサイド溶液(日本油脂製「パーロイル355−75(S)」2質量部を1%ドデシル硫酸ナトリウム水溶液20質量部に混合し、超音波分散機にて微細乳化した。これを粒径0.77μmのポリスチレン粒子13質量部および水41質量部の入ったリアクターに入れ、25℃で12時間攪拌した。別の容器にて、スチレン96質量部およびジビニルベンゼン4質量部を0.1%ドデシル硫酸ナトリウム水溶液400質量部で乳化させた液を前記リアクターに入れ、40℃で2時間攪拌した後、75℃に昇温して8時間重合した。室温まで冷却した後、遠心分離により粒子のみ取り出したものをさらに水洗し、乾燥および粉砕した。これを核粒子とする(核粒子の作製)。数平均粒径は1.5μmであった。
4.3. Synthesis Example 2 (Synthesis of organic polymer particles containing magnetic substance)
2 parts by mass of 75% di (3,5,5-trimethylhexanoyl) peroxide solution (“Perroyl 355-75 (S)” manufactured by NOF Corporation) is mixed with 20 parts by mass of 1% aqueous sodium dodecyl sulfate solution, and ultrasonically dispersed. The mixture was finely emulsified with a machine and placed in a reactor containing 13 parts by mass of polystyrene particles having a particle size of 0.77 μm and 41 parts by mass of water and stirred for 12 hours at 25 ° C. In another container, 96 parts by mass of styrene A solution prepared by emulsifying 4 parts by mass of divinylbenzene with 400 parts by mass of a 0.1% aqueous sodium dodecyl sulfate solution was placed in the reactor, stirred at 40 ° C. for 2 hours, then heated to 75 ° C. and polymerized for 8 hours. After cooling to room temperature, the particles taken out by centrifugation were further washed with water, dried and pulverized, and used as core particles (preparation of core particles). It was 1.5μm.
次に、油性磁性流体(商品名:「EXPシリーズ」,(株)フェローテック製)にアセトンを加えて粒子を析出沈殿させた後、これを乾燥することにより、疎水化処理された表面を有するフェライト系の磁性体微粒子(平均一次粒子径:0.01μm)を得た。 Next, acetone is added to an oil-based magnetic fluid (trade name: “EXP series”, manufactured by Ferrotec Co., Ltd.) to precipitate and precipitate particles, which are then dried to have a surface that has been hydrophobized. Ferrite-based magnetic fine particles (average primary particle size: 0.01 μm) were obtained.
次いで、上記核粒子15gおよび上記疎水化された磁性体微粒子15gをミキサーでよく混合し、この混合物をハイブリダイゼーションシステムNHS−0型(奈良機械製作所(株)製)を使用して、羽根(撹拌翼)の周速度100m/秒(16200rpm)で5分間処理し、平均数粒子径が2.0μmの磁性体微粒子からなる磁性体層を表面に有する母粒子を得た。 Next, 15 g of the core particles and 15 g of the hydrophobized magnetic fine particles are mixed well with a mixer, and this mixture is mixed with a blade (stirring) using a hybridization system NHS-0 type (manufactured by Nara Machinery Co., Ltd.). The peripheral particles were treated at a peripheral speed of 100 m / sec (16200 rpm) for 5 minutes to obtain mother particles having a magnetic layer composed of magnetic fine particles having an average number particle diameter of 2.0 μm on the surface.
次に、ドデシルベンゼンスルホン酸ナトリウム0.25重量%およびノニオン性乳化剤(商品名:「エマルゲン150」,花王(株)製)0.25重量%を含む水溶液375gを1Lセパラブルフラスコに投入し、次いで、前記磁性体層を有する母粒子15gを投入し、ホモジナイザーで分散した後、60℃に加熱した。ドデシルベンゼンスルホン酸ナトリウム0.25重量%およびノニオン性乳化剤(商品名:「エマルゲン150」,花王(株)製)0.25重量%を含む水溶液150gに、MMA27g、トリメチロールプロパントリメタクリレート(以下、「TMP」という。)3g、およびジ(3,5,5−トリメチルヘキサノイル)パーオキサイド(日本油脂社製;パーロイル355)0.6gを入れて分散させたプレエマルジョンを、60℃にコントロールした前記1Lセパラブルフラスコに1時間30分かけて滴下した。滴下終了後、60℃に保持し1時間攪拌した後、ドデシルベンゼンスルホン酸ナトリウム0.25重量%およびノニオン性乳化剤(商品名:「エマルゲン150」,花王(株)製)0.25重量%を含む水溶液75gに、グリシジルメタクリレート(以下、「GMA」という。)13.5g、TMP1.5g、およびジ(3,5,5−トリメチルヘキサノイル)パーオキサイド(日本油脂社製;パーロイル355)0.3gを入れて分散させたプレエマルジョンを、60℃にコントロールした上記1Lセパラブルフラスコに1時間30分かけて滴下した。その後75℃に昇温した後さらに2時間重合を続けて、反応を完了させた。続けて、この1Lセパラブルフラスコに1mol/L 硫酸60mlを入れ、60℃で6時間撹拌した。次いで、前記セパラブルフラスコ中の粒子を、磁気を用いて分離した後、蒸留水を用いて繰り返し洗浄した。以上により、磁性体を含有する有機ポリマー粒子(A)の分散液を得た(以下、「A−2」とする。)。A−2粒子の水分散液からの乾燥塗膜と水との接触角は20°であった。 Next, 375 g of an aqueous solution containing 0.25 wt% of sodium dodecylbenzenesulfonate and 0.25 wt% of a nonionic emulsifier (trade name: “Emulgen 150”, manufactured by Kao Corporation) was charged into a 1 L separable flask, Next, 15 g of mother particles having the magnetic layer were added, dispersed with a homogenizer, and heated to 60 ° C. To 150 g of an aqueous solution containing 0.25 wt% of sodium dodecylbenzenesulfonate and 0.25 wt% of a nonionic emulsifier (trade name: “Emulgen 150”, manufactured by Kao Corporation), 27 g of MMA, trimethylolpropane trimethacrylate (hereinafter, The pre-emulsion in which 3 g of “TMP”) and 0.6 g of di (3,5,5-trimethylhexanoyl) peroxide (manufactured by NOF Corporation; Parroyl 355) were dispersed was controlled at 60 ° C. The solution was added dropwise to the 1 L separable flask over 1 hour 30 minutes. After completion of dropping, the mixture was kept at 60 ° C. and stirred for 1 hour, and then 0.25% by weight of sodium dodecylbenzenesulfonate and 0.25% by weight of a nonionic emulsifier (trade name: “Emulgen 150”, manufactured by Kao Corporation) 75 g of an aqueous solution containing 13.5 g of glycidyl methacrylate (hereinafter referred to as “GMA”), 1.5 g of TMP, and di (3,5,5-trimethylhexanoyl) peroxide (manufactured by NOF Corporation; Parroyl 355) The pre-emulsion in which 3 g was dispersed was dropped into the 1 L separable flask controlled at 60 ° C. over 1 hour and 30 minutes. Thereafter, the temperature was raised to 75 ° C., and then polymerization was continued for another 2 hours to complete the reaction. Subsequently, 60 ml of 1 mol / L sulfuric acid was placed in this 1 L separable flask and stirred at 60 ° C. for 6 hours. Next, the particles in the separable flask were separated using magnetism, and then washed repeatedly using distilled water. Thus, a dispersion of organic polymer particles (A) containing a magnetic material was obtained (hereinafter referred to as “A-2”). The contact angle between the dried coating film from the aqueous dispersion of A-2 particles and water was 20 °.
次に、A−2粒子を凍結乾燥して得られた乾燥粒子1.0gを8mlのピリジンに分散させた後、p−トシルクロライド0.2gを加えて室温で2時間撹拌した。反応後、磁気を用いて粒子を分離し、アセトンで4回、続いて蒸留水で4回洗浄して、有機ポリマー粒子(B)(以下、「B−2」とする。)を得た。この磁性粒子(B−2粒子)の平均数粒子径は2.9μmであった。また、B−2粒子の水分散液からの乾燥塗膜と水との接触角は112°であった。 Next, 1.0 g of dry particles obtained by freeze-drying the A-2 particles was dispersed in 8 ml of pyridine, 0.2 g of p-tosyl chloride was added, and the mixture was stirred at room temperature for 2 hours. After the reaction, the particles were separated using magnetism and washed 4 times with acetone and then 4 times with distilled water to obtain organic polymer particles (B) (hereinafter referred to as “B-2”). The average number particle diameter of the magnetic particles (B-2 particles) was 2.9 μm. Moreover, the contact angle of the dry coating film and water from the aqueous dispersion of B-2 particles was 112 °.
得られたB−2粒子のトシル基量を、エタノールアミンとの反応から求めた結果、0.24μmol/mgであった。 As a result of obtaining the tosyl group amount of the obtained B-2 particles from the reaction with ethanolamine, it was 0.24 μmol / mg.
4.4.合成例3:(抗体を感作させた、磁性体を含有しない有機ポリマー粒子の合成)
合成例1で得られた有機ポリマー粒子(B−1粒子)10mgをホウ酸緩衝液(0.1mol/L、pH9.5)溶液1.0mlに分散させ、腫瘍マーカーであるヒトαフェトプロテイン(AFP)に対する抗体(以下、「抗AFP抗体」という。コスモ・バイオ株式会社製)100μgを加えて室温で18時間反応させた。反応後、粒子を遠心分離し、洗浄液(25mmol/L Tris−HCl,pH7.4、0.01%Tween20含有)で繰り返し洗浄した後、粒子濃度が0.5%になるように洗浄液で希釈して、抗AFP抗体を感作させた有機ポリマー粒子(以下、「C−1」とする。)の分散液を得た。
4.4. Synthesis Example 3: (Synthesis of antibody-sensitized organic polymer particles not containing a magnetic substance)
10 mg of the organic polymer particles (B-1 particles) obtained in Synthesis Example 1 were dispersed in 1.0 ml of a borate buffer solution (0.1 mol / L, pH 9.5), and human alpha-fetoprotein (AFP) as a tumor marker was dispersed. ) (Hereinafter referred to as “anti-AFP antibody”, manufactured by Cosmo Bio Inc.) 100 μg was added and allowed to react at room temperature for 18 hours. After the reaction, the particles are centrifuged, washed repeatedly with a washing solution (containing 25 mmol / L Tris-HCl, pH 7.4, 0.01% Tween 20), and then diluted with the washing solution so that the particle concentration becomes 0.5%. Thus, a dispersion of organic polymer particles (hereinafter referred to as “C-1”) sensitized with an anti-AFP antibody was obtained.
4.5.合成例4(抗体を感作させた、磁性体を含有する有機ポリマー粒子の合成)
有機ポリマー粒子(B−1粒子)に替えて、合成例2で得られた、磁性体を含有する有機ポリマー粒子(B−2粒子)を用い、遠心分離に替えて磁気分離を用いた他は、合成例3と同様の方法にて、抗AFP抗体を感作させた有機ポリマー粒子(以下、「C−2」とする。)の分散液を得た。
4.5. Synthesis Example 4 (Synthesis of organic polymer particles containing a magnetic material sensitized with an antibody)
Other than using organic polymer particles (B-2 particles) containing a magnetic substance obtained in Synthesis Example 2 instead of organic polymer particles (B-1 particles) and using magnetic separation instead of centrifugation. In the same manner as in Synthesis Example 3, an organic polymer particle sensitized anti-AFP antibody (hereinafter referred to as “C-2”) dispersion was obtained.
4.6.比較合成例1(抗体を感作させた、磁性体を含有しない有機ポリマー粒子の合成)
合成例1でGLMの代わりにGMAを用いて得られたグリシジル基含有ポリマー粒子10mgを1mol/L硫酸アンモニウム含有ホウ酸緩衝液(0.1mol/L、pH9.5)溶液1.0mlに分散させ、抗AFP抗体100μgを加えて室温で18時間反応させた。反応後、粒子を遠心分離し、洗浄液(25mmol/L Tris−HCl,pH7.4、0.01%Tween20含有)で繰り返し洗浄した後、粒子濃度0.5%になるように洗浄液で希釈して、抗AFP抗体を感作させた有機ポリマー粒子(以下、「C−3」とする。)の分散液を得た。
4.6. Comparative synthesis example 1 (synthesis of organic polymer particles sensitized with an antibody and containing no magnetic substance)
Disperse 10 mg of glycidyl group-containing polymer particles obtained by using GMA instead of GLM in Synthesis Example 1 in 1.0 ml of a 1 mol / L ammonium sulfate-containing borate buffer solution (0.1 mol / L, pH 9.5), 100 μg of anti-AFP antibody was added and allowed to react at room temperature for 18 hours. After the reaction, the particles are centrifuged, washed repeatedly with a washing solution (containing 25 mmol / L Tris-HCl, pH 7.4, 0.01% Tween 20), and then diluted with a washing solution to a particle concentration of 0.5%. A dispersion of organic polymer particles (hereinafter referred to as “C-3”) sensitized with an anti-AFP antibody was obtained.
4.7.比較合成例2(抗体分子を感作した、磁性体を含有する粒子の合成)
有機ポリマー粒子(B−2粒子)に替えて、Dynabeads M280 Tosylactivated(粒子径2.8μm;DYNAL BIOTECH社、3−ヒドロキシプロピル基をトシル化した活性基を有する)を用いた他は、合成例4と同様に、抗AFP抗体を感作させた有機ポリマー粒子(以下、「C−4」とする。)を得た。
4.7. Comparative Synthesis Example 2 (Synthesis of particles containing a magnetic material sensitized with antibody molecules)
Synthetic Example 4 except that Dynabeads M280 Tosylactivated (particle size: 2.8 μm; DYNAL BIOTECH, having an active group tosylated 3-hydroxypropyl group) was used instead of organic polymer particles (B-2 particles) In the same manner, organic polymer particles (hereinafter referred to as “C-4”) sensitized with an anti-AFP antibody were obtained.
4.8.実施例1
合成例3で得られた、抗体を感作させた、磁性体を含有しない有機ポリマー粒子(C−1)の分散液を用いて、化学発光酵素免疫測定(CLEIA)を実施した。AFPを含まない検体のノイズ強度は155RIU(Relative intensity units)であった。AFP濃度1000ng/mLの時のシグナル強度は445849(RIU)であった。
4.8. Example 1
The chemiluminescent enzyme immunoassay (CLEIA) was performed using the dispersion liquid of the organic polymer particle (C-1) obtained by the synthesis example 3 and sensitized with the antibody and containing no magnetic substance. The noise intensity of the specimen not containing AFP was 155 RIU (Relativistic intensity units). The signal intensity at an AFP concentration of 1000 ng / mL was 445849 (RIU).
4.9.実施例2
有機ポリマー粒子(C−1)の分散液に替えて、合成例4で得られた、抗体を感作させた、磁性体を含有する有機ポリマー粒子(C−2)の分散液を用いた他は実施例1と同様にして、CLEIAを実施した。AFPを含まない検体のノイズ強度は52(RIU)であった。AFP濃度1000ng/mLの時のシグナル強度は584221(RIU)であった。
4.9. Example 2
Other than using the dispersion liquid of organic polymer particles (C-2) containing a magnetic substance sensitized with the antibody obtained in Synthesis Example 4, instead of the dispersion liquid of organic polymer particles (C-1) CLEIA was carried out in the same manner as in Example 1. The noise intensity of the specimen not containing AFP was 52 (RIU). The signal intensity at an AFP concentration of 1000 ng / mL was 58221 (RIU).
4.10.比較例1
有機ポリマー粒子(C−1)の分散液に替えて、比較合成例1で得られた、抗体分子を感作させた、磁性体を含有しない有機ポリマー粒子(C−3)の分散液を用いた他は、実施例1と同様にしてCLEIAを実施した。AFPを含まない検体のノイズ強度は250(RIU)であった。AFP濃度1000ng/mLの時のシグナル強度は42045(RIU)であった。
4.10. Comparative Example 1
Instead of the dispersion of the organic polymer particles (C-1), the dispersion of the organic polymer particles (C-3) containing no magnetic substance, sensitized with the antibody molecules obtained in Comparative Synthesis Example 1, was used. CLEIA was carried out in the same manner as in Example 1 except that. The noise intensity of the specimen not containing AFP was 250 (RIU). The signal intensity at an AFP concentration of 1000 ng / mL was 42045 (RIU).
4.11.比較例2
有機ポリマー粒子(C−1)の分散液に替えて、比較合成例2で得られた、抗体分子を感作させた、磁性体を含有する粒子(C−4)の分散液を用いた他は、実施例1と同様にしてCLEIAを実施した。AFPを含まない検体のノイズ強度は580(RIU)であった。AFP濃度1000ng/mLの時のシグナル強度は157898(RIU)であった。
4.11. Comparative Example 2
Other than using the dispersion of organic polymer particles (C-1), the dispersion of particles (C-4) containing a magnetic material sensitized with antibody molecules obtained in Comparative Synthesis Example 2 was used. Performed CLEIA in the same manner as in Example 1. The noise intensity of the specimen not containing AFP was 580 (RIU). The signal intensity at an AFP concentration of 1000 ng / mL was 157898 (RIU).
Claims (6)
前記架橋重合体は、母粒子を覆うように設けられ、
前記母粒子は、核粒子と、該核粒子の表面に設けられた超常磁性微粒子の磁性体層とを含む、有機ポリマー粒子。 Having a 2,3-dihydroxypropyl group at least at the particle surface cross-linked polymer having an active group obtained by tosylation and, Ri number average particle diameter of 0.1~15μm der,
The crosslinked polymer is provided so as to cover the mother particles,
The mother particle is an organic polymer particle including a core particle and a magnetic layer of superparamagnetic fine particles provided on the surface of the core particle.
前記有機ポリマー粒子の水分散液から得られる乾燥塗膜と水との接触角が70°以上である、有機ポリマー粒子。 In claim 1,
Organic polymer particles, wherein the contact angle between the dried coating film obtained from the aqueous dispersion of organic polymer particles and water is 70 ° or more.
2,3−ジヒドロキシプロピル基を有する架橋重合体を少なくとも粒子表面に有する有機ポリマー粒子(A)をトシル化処理することにより得られ、
前記有機ポリマー粒子(A)の水分散液から得られる乾燥塗膜と水との接触角が40°以下である、有機ポリマー粒子。 In claim 2,
It is obtained by tosylating organic polymer particles (A) having a crosslinked polymer having a 2,3-dihydroxypropyl group at least on the particle surface,
Organic polymer particles, wherein the contact angle between the dried coating film obtained from the aqueous dispersion of the organic polymer particles (A) and water is 40 ° or less.
前記有機ポリマー粒子(A)をトシル化処理する工程と、
を含む、有機ポリマー粒子の製造方法。 (I) polymerizing a monomer part comprising 40 to 95 parts by weight of a monomer having a 2,3-dihydroxypropyl group, (ii) 5 to 30 parts by weight of a crosslinkable monomer, and (iii) 0 to 55 parts by weight of other monomers. Forming a crosslinked polymer to obtain organic polymer particles (A) having at least the crosslinked polymer on the particle surface;
A step of tosylating the organic polymer particles (A);
A method for producing organic polymer particles, comprising:
前記有機ポリマー粒子(A)を得る工程は、
母粒子を覆うように前記架橋重合体を形成する工程を含み、
前記母粒子は、核粒子と、該核粒子の表面に設けられた超常磁性微粒子の磁性体層とを含む、有機ポリマー粒子の製造方法。 In claim 4 ,
The step of obtaining the organic polymer particles (A)
Forming the crosslinked polymer so as to cover the mother particles,
The method for producing organic polymer particles, wherein the mother particle includes a core particle and a magnetic layer of superparamagnetic fine particles provided on a surface of the core particle.
前記有機ポリマー粒子(A)の水分散液から得られる乾燥塗膜と水との接触角が40°以下である、有機ポリマー粒子の製造方法。 In claim 4 or 5 ,
The manufacturing method of the organic polymer particle whose contact angle of the dry coating film obtained from the aqueous dispersion liquid of the said organic polymer particle (A) and water is 40 degrees or less.
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