JP2023184457A - Magnetic particles and immunoassay particles - Google Patents
Magnetic particles and immunoassay particles Download PDFInfo
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- JP2023184457A JP2023184457A JP2023086755A JP2023086755A JP2023184457A JP 2023184457 A JP2023184457 A JP 2023184457A JP 2023086755 A JP2023086755 A JP 2023086755A JP 2023086755 A JP2023086755 A JP 2023086755A JP 2023184457 A JP2023184457 A JP 2023184457A
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Landscapes
- Soft Magnetic Materials (AREA)
Abstract
Description
本発明は、磁性粒子、及び免疫検査用粒子に関する。 The present invention relates to magnetic particles and particles for immunoassays.
近年、磁性粒子は、多種多様な用途に使用されている。例えば、バイオ用途として、検体物質中の抗原や抗体などを捕捉又は分離するような使用例がある。また、診断に用いるための検体検査用として、磁性粒子が使用されている。具体的には、検体から抗原（抗体）を検出するために、抗原（抗体）と特異的に結合する抗体（抗原）を結合した磁性粒子、及び抗原（抗体）と特異的に結合する抗体（抗原）を固定化したセンサーを用いる方法がある。このような抗体（抗原）を結合した磁性粒子を、光や色等で識別することで陽性、陰性を判定する検査試薬として使用される。このような磁性粒子は、一般的にバイオセパレーション用磁性粒子と呼ばれる。 In recent years, magnetic particles have been used for a wide variety of applications. For example, as a biological application, there is an example of use in which antigens, antibodies, etc. in a specimen substance are captured or separated. Furthermore, magnetic particles are used for specimen testing for diagnosis. Specifically, in order to detect antigens (antibodies) from specimens, we use magnetic particles bound to antibodies (antigens) that specifically bind to antigens (antibodies), and antibodies (antibodies) that specifically bind to antigens (antibodies). There is a method using a sensor immobilized with antigen). Magnetic particles bound to such antibodies (antigens) are used as test reagents to determine positive or negative results by identifying them with light, color, etc. Such magnetic particles are generally called magnetic particles for bioseparation.
これらの磁性粒子は、外部からの磁場で粒子を操作するため、高い磁場応答性を奏するような改良がなされている。例えば、磁性体粒子が内包する磁性体量を高めたり、磁化の高い磁性材料を内包させたりということがなされている。 Since these magnetic particles are manipulated by an external magnetic field, they have been improved to exhibit high magnetic field responsiveness. For example, attempts have been made to increase the amount of magnetic material contained in magnetic particles, or to encapsulate magnetic materials with high magnetization.
このようなバイオセパレーション用磁性粒子として、例えば、樹脂マトリックス中に磁性ナノ粒子を内包させた磁性粒子が提案されている（特許文献１参照）。
また、バイオセパレーション用磁性粒子として、シリカマトリックス中に磁性ナノ粒子を内包させた構造も提案されている（特許文献２参照）。
さらに、単一の磁性粒子表層に樹脂層を形成した粒子構造も提案されている（特許文献３参照）。
As such magnetic particles for bioseparation, for example, magnetic particles in which magnetic nanoparticles are encapsulated in a resin matrix have been proposed (see Patent Document 1).
Furthermore, a structure in which magnetic nanoparticles are encapsulated in a silica matrix has been proposed as magnetic particles for bioseparation (see Patent Document 2).
Furthermore, a particle structure in which a resin layer is formed on the surface layer of a single magnetic particle has also been proposed (see Patent Document 3).
バイオセパレーション用磁性粒子は、溶液に分散させた状態で外部から磁場を印加し粒子を捕捉するが、速やかに粒子を移動させるためには個々の粒子内に高密度に磁性体を内包させる必要がある。本発明者らは、樹脂マトリックス中やシリカマトリックス中に磁性ナノ粒子を内包させた特許文献１及び２に記載の構造に相当する粒子について、磁場を印加し粒子の捕捉速度を調べたが、捕捉速度が遅く十分な性能が得られなかった。また、同様な操作を特許文献３に記載の構造の粒子では、捕捉速度は速くなるものの、いったん捕捉した粒子は印加磁場を停止しても粒子が持つ残留磁化により、容易に再分散させることができずその後の処理が困難であった。上記の検討は、光導波路型システムを備えた検査装置を用いて行われた。
したがって、本発明の目的は、磁場を印加して検体溶液から目的物質を速やかに捕集でき、さらに、磁場印加を停止すると磁性粒子を速やかに再分散させることができる磁性体含有率が高く残留磁化の無い磁性粒子を提供することにある。
Magnetic particles for bioseparation are dispersed in a solution and captured by applying a magnetic field from the outside, but in order to move the particles quickly, it is necessary to encapsulate the magnetic material in each particle at a high density. be. The present inventors applied a magnetic field and investigated the particle capture speed for particles corresponding to the structures described in Patent Documents 1 and 2, in which magnetic nanoparticles are encapsulated in a resin matrix or a silica matrix. The speed was slow and sufficient performance could not be obtained. In addition, when a similar operation is performed on particles having the structure described in Patent Document 3, although the capture speed becomes faster, the captured particles cannot be easily redispersed due to the residual magnetization of the particles even if the applied magnetic field is stopped. This made subsequent processing difficult. The above study was conducted using an inspection device equipped with an optical waveguide type system.
Therefore, an object of the present invention is to provide a material with a high content of magnetic material that can quickly collect a target substance from a sample solution by applying a magnetic field, and furthermore, can quickly redisperse magnetic particles when the magnetic field application is stopped. The object of the present invention is to provide magnetic particles without magnetization.
本発明は、磁性コア粒子と該磁性コア粒子の表面に樹脂層を有する磁性粒子であって、 該磁性コア粒子は、複数の磁性ナノ粒子の会合体を含み、
該樹脂層は、カルボキシル基、アミノ基、チオール基、エポキシ基、マレイミド基、及びスクシンイミジル基からなる群より選択される少なくとも１種の官能基を有する樹脂を有することを特徴とする磁性粒子に関する。
The present invention provides a magnetic particle having a magnetic core particle and a resin layer on the surface of the magnetic core particle, the magnetic core particle including an aggregate of a plurality of magnetic nanoparticles,
The resin layer relates to a magnetic particle characterized in that the resin layer includes a resin having at least one functional group selected from the group consisting of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group.
また、本発明は、前記磁性粒子及びリガンドを有し、前記リガンドと前記官能基とは化学結合を有することを特徴とする免疫検査用粒子に関する。 The present invention also relates to particles for immunoassays, which include the magnetic particles and the ligand, and the ligand and the functional group have a chemical bond.
本発明によれば、検体溶液中の抗原や抗体などの目的物質を捕捉した磁性粒子を磁場で速やかに捕集でき、磁場停止後には速やかに再分散させることができる磁性粒子を提供することができる。 According to the present invention, it is possible to provide magnetic particles that can quickly capture target substances such as antigens and antibodies in a sample solution using a magnetic field, and can be quickly redispersed after the magnetic field has stopped. can.
以下、本発明の実施の形態について、詳細に述べる。各種の物性値は、特に断りのない限り、２５℃における値である。磁性粒子の個数平均粒径は、磁性粒子の表面に樹脂層も含んだ個数平均粒径のことである。以下、検体から検出する測定対象物質として、抗原を例に挙げて説明する。 Embodiments of the present invention will be described in detail below. Various physical property values are values at 25°C unless otherwise specified. The number average particle size of the magnetic particles is the number average particle size including a resin layer on the surface of the magnetic particles. Hereinafter, an example of an antigen will be described as a substance to be measured that is detected from a specimen.
本発明の磁性粒子の一つの形態は、磁性コア粒子と該磁性コア粒子の表面に樹脂層を有する磁性粒子であって、該磁性コア粒子は、複数の磁性ナノ粒子の会合体を含み、該樹脂層は、カルボキシル基、アミノ基、チオール基、エポキシ基、マレイミド基、及びスクシンイミジル基からなる群より選択される少なくとも１種の官能基を有する樹脂を有することを特徴とする。 One form of the magnetic particles of the present invention is a magnetic particle having a magnetic core particle and a resin layer on the surface of the magnetic core particle, the magnetic core particle including an aggregate of a plurality of magnetic nanoparticles, and the magnetic core particle containing an aggregate of a plurality of magnetic nanoparticles. The resin layer is characterized by containing a resin having at least one functional group selected from the group consisting of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group.
検体溶液から目的物質（例えば抗原）を捕集する際には、抗原と特異的に結合する抗体を結合した磁性粒子を用いる。具体的には、抗原を含んだ検体溶液を入れた容器に抗原と特異的に結合する抗体を結合させた磁性粒子を入れ検体溶液中の抗原を磁性粒子に捕捉させる。抗原を捕捉した磁性粒子を容器外から磁石で捕集し上清溶液を除去すれば検体溶液から目的物質を分離することができる。 When collecting a target substance (for example, an antigen) from a sample solution, magnetic particles bound to an antibody that specifically binds to the antigen are used. Specifically, magnetic particles bound to an antibody that specifically binds to the antigen are placed in a container containing a sample solution containing an antigen, and the antigen in the sample solution is captured by the magnetic particles. The target substance can be separated from the sample solution by capturing the antigen-captured magnetic particles with a magnet from outside the container and removing the supernatant solution.
従来は目的物質の捕捉に使用される磁性粒子は、磁性粒子に内包される磁性体成分の含有量が十分ではないため磁性粒子に作用する磁力が小さく、ネオジム磁石等による磁性粒子の捕集に時間がかかり目的物質の分離操作に時間を要していた。 Conventionally, the magnetic particles used to capture the target substance do not have a sufficient content of magnetic components contained in the magnetic particles, so the magnetic force acting on the magnetic particles is small, making it difficult to capture the magnetic particles with neodymium magnets etc. It took a long time to separate the target substance.
そこで本発明は、磁性粒子を磁場で素早く移動させることができるように、磁性粒子中の磁性体の含有率の向上が重要であると考えた。本発明において、磁性体の含有率の向上の手段として、磁性コア粒子は、磁性ナノ粒子の会合体を含むことが必要である。 Therefore, the present invention considered that it is important to improve the content of the magnetic substance in the magnetic particles so that the magnetic particles can be quickly moved by a magnetic field. In the present invention, the magnetic core particles need to contain aggregates of magnetic nanoparticles as a means for increasing the content of magnetic material.
本発明に係る磁性ナノ粒子は、超常磁性であり、個数平均粒径が２０ｎｍ以下であることが好ましい。また、特に、粒径の小さい磁性粒子を用いると粒子の比表面積を大きくすることができ抗原を捕捉する確率が上がるため、従来の磁性粒子構造と比較して目的物質の捕捉能力と磁性粒子の捕集速度の両方を向上することができるため好ましい。 The magnetic nanoparticles according to the present invention are preferably superparamagnetic and have a number average particle diameter of 20 nm or less. In particular, when magnetic particles with a small particle size are used, the specific surface area of the particles can be increased, which increases the probability of capturing antigens. This is preferable because both the collection speed and the collection rate can be improved.
また、本発明は、磁性コア粒子と該磁性コア粒子の表面に樹脂層を有する磁性粒子である。さらに、樹脂層は、カルボキシル基、アミノ基、チオール基、エポキシ基、マレイミド基、及びスクシンイミジル基からなる群より選択される少なくとも１種の官能基を有する樹脂を有することが必要である。これらの官能基を有することで、抗体に結合することが可能となり、抗原を効果的に捕捉することが可能となる。 Further, the present invention is a magnetic particle having a magnetic core particle and a resin layer on the surface of the magnetic core particle. Further, the resin layer needs to contain a resin having at least one functional group selected from the group consisting of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group. Having these functional groups makes it possible to bind to antibodies and effectively capture antigens.
（本実施形態に係る磁性粒子の構成の概要）
本実施形態に係る磁性粒子１００は図１に示すように、磁性ナノ粒子の会合体からなる磁性コア粒子１０１と、磁性コア粒子１０１の表面の無機被覆層１０２とを有し、無機被覆層１０２上には、樹脂層１０３が形成されている。樹脂層１０３は、２種の材料からなることがあり、その場合は、磁性コア粒子１０１に近い方から順に、樹脂層１０３１（第一の樹脂層）と、樹脂層１０３２（第二の樹脂層）と、を有する。樹脂層１０３２はリガンドを結合できる官能基（不図示）を含む。磁性粒子１００の磁性コア粒子１０１は、複数の磁性ナノ粒子が実質的に単独で会合した構造を持つ。この構造であれば、特許文献１、２及び３のような樹脂やセラミックスの非磁性体をマトリックス成分とした媒質中に磁性ナノ粒子を分散させた構造と比較して、磁性成分の比率を向上することができる。その結果、本発明の磁性粒子は、従来構造と比較して磁場応答性を高めることができる。
(Summary of configuration of magnetic particles according to this embodiment)
As shown in FIG. 1, the magnetic particle 100 according to the present embodiment has a magnetic core particle 101 made of an aggregate of magnetic nanoparticles, and an inorganic coating layer 102 on the surface of the magnetic core particle 101. A resin layer 103 is formed on top. The resin layer 103 may be made of two types of materials. In that case, the resin layer 1031 (first resin layer) and the resin layer 1032 (second resin layer) are arranged in order from the one closest to the magnetic core particle 101. ) and has. The resin layer 1032 includes a functional group (not shown) that can bind a ligand. The magnetic core particle 101 of the magnetic particle 100 has a structure in which a plurality of magnetic nanoparticles are substantially individually associated. With this structure, the ratio of magnetic components is improved compared to structures in which magnetic nanoparticles are dispersed in a medium with a non-magnetic material such as resin or ceramic as a matrix component, as in Patent Documents 1, 2, and 3. can do. As a result, the magnetic particles of the present invention can have improved magnetic field responsiveness compared to conventional structures.
＜磁性粒子＞
本発明の磁性粒子は、磁性ナノ粒子の会合体を含む磁性コア粒子と、その表層に非磁性体の無機層が形成され、さらに樹脂層が無機層を介して形成された構造を有する場合が好ましい。本発明において、磁性コア粒子の外観は走査型電子顕微鏡（ＳＥＭ）で観察することができる。また、非磁性層の無機層や樹脂層の厚さは、透過型電子顕微鏡（ＴＥＭ）で観察することができる。個数平均粒径は、乾燥粒子の場合はＳＥＭ観察で１００個の粒子の長径の平均値から求めることができる。
<Magnetic particles>
The magnetic particles of the present invention may have a structure in which a magnetic core particle containing an aggregate of magnetic nanoparticles, a non-magnetic inorganic layer formed on the surface layer, and a resin layer further formed through the inorganic layer. preferable. In the present invention, the appearance of the magnetic core particles can be observed using a scanning electron microscope (SEM). Further, the thickness of the inorganic layer or resin layer of the nonmagnetic layer can be observed using a transmission electron microscope (TEM). In the case of dry particles, the number average particle size can be determined from the average value of the long diameters of 100 particles by SEM observation.
本発明の磁性粒子の個数平均粒径は、特に限定はされないが印加磁場への応答性等を考慮すると、０．１μｍ以上２．０μｍ以下が好ましい。個数平均粒径が０．１μｍ未満になると、磁性粒子の磁石等での捕集作業に時間がかかり過ぎ好ましくない。 The number average particle diameter of the magnetic particles of the present invention is not particularly limited, but in consideration of responsiveness to an applied magnetic field, etc., it is preferably 0.1 μm or more and 2.0 μm or less. If the number average particle diameter is less than 0.1 μm, it is undesirable that it takes too much time to collect the magnetic particles with a magnet or the like.
＜磁性コア粒子＞
本発明の磁性粒子は、磁性コア粒子が磁性を持つ磁性ナノ粒子の会合体を含むことを特徴とする。磁性体は、磁場の印加により磁化される材料のことである。磁性ナノ粒子は、金属、及び金属酸化物からなる群より選択される少なくとも１種を含むことが好ましい。
金属としては、鉄、マンガン、ニッケル、コバルト、クロムなどが挙げられる。金属酸化物としては、マグネタイト（Ｆｅ３Ｏ４）、マグヘマイト（γ－酸化鉄（III））（γ－Ｆｅ２Ｏ３）、フェライトなどの磁性酸化鉄が挙げられる。なかでも、磁化が大きく、かつ、溶液中での安定性等の観点から、マグネタイト（Ｆｅ３Ｏ４）、及びγ－酸化鉄（III）（γ－Ｆｅ２Ｏ３）の少なくともいずれか一方からなる場合が好ましい。また、個数平均粒径が２０ｎｍ以下のマグネタイト微粒子は飽和磁化が大きく、かつ、超常磁性体であるため残留磁化が小さく好ましい。
<Magnetic core particle>
The magnetic particle of the present invention is characterized in that the magnetic core particle contains an aggregate of magnetic nanoparticles having magnetism. A magnetic material is a material that is magnetized by the application of a magnetic field. Preferably, the magnetic nanoparticles include at least one selected from the group consisting of metals and metal oxides.
Examples of metals include iron, manganese, nickel, cobalt, and chromium. Examples of metal oxides include magnetic iron oxides such as magnetite (Fe 3 O 4 ), maghemite (γ-iron (III) oxide) (γ-Fe 2 O 3 ), and ferrite. Among them, from the viewpoint of high magnetization and stability in solution, at least one of magnetite (Fe 3 O 4 ) and γ-iron (III) oxide (γ-Fe 2 O 3 ). It is preferable that In addition, magnetite fine particles having a number average particle size of 20 nm or less have a large saturation magnetization, and since they are superparamagnetic, they have a small residual magnetization and are preferable.
ここで、磁化とは、磁性体に外部磁場をかける際に、その磁性体が分極して磁気モーメントを持つ現象のことであり、飽和磁化とは、磁場の強さとともに増大する磁化が飽和する値のことである。飽和磁化は、磁性ナノ粒子の粒子径等により調整できる。また、残留磁化とは、磁性体に外部磁場をかけた後に磁場ゼロにした場合に、磁性体に残留する磁化のことである。本発明の磁性粒子の残留磁化はゼロとなることがより好ましい。 Here, magnetization is a phenomenon in which a magnetic material is polarized and has a magnetic moment when an external magnetic field is applied to it, and saturation magnetization is a phenomenon in which the magnetization increases with the strength of the magnetic field and saturates. It is about value. Saturation magnetization can be adjusted by adjusting the particle size of the magnetic nanoparticles. Furthermore, residual magnetization refers to magnetization that remains in a magnetic material when the magnetic field is reduced to zero after applying an external magnetic field to the magnetic material. It is more preferable that the magnetic particles of the present invention have zero residual magnetization.
（磁性コア粒子の合成方法）
酸化鉄、特にマグネタイトナノ粒子の会合体からなる磁性コア粒子の形成方法について説明する。マグネタイトナノ粒子の原料は、塩化鉄（ＦｅＣｌ２やＦｅＣｌ３）、硝酸鉄（Ｆｅ（ＮＯ３）２）、硫酸鉄（ＦｅＳＯ４）、及びその水和物（ＦｅＣｌ２・４Ｈ２Ｏ、ＦｅＣｌ３・６Ｈ２Ｏ、Ｆｅ（ＮＯ３）２・６Ｈ２Ｏ、ＦｅＳＯ４・７Ｈ２Ｏ）等から選択することができ、特に好ましくは塩化鉄（ＦｅＣｌ２やＦｅＣｌ３）、及びその水和物（ＦｅＣｌ２・４Ｈ２ＯやＦｅＣｌ３・６Ｈ２Ｏ）から選択されるが、複数の鉄化合物を混合して用いることもできる。
(Method for synthesizing magnetic core particles)
A method for forming magnetic core particles consisting of aggregates of iron oxide, particularly magnetite nanoparticles, will be explained. The raw materials for magnetite nanoparticles are iron chloride (FeCl 2 and FeCl 3 ), iron nitrate (Fe(NO 3 ) 2 ), iron sulfate (FeSO 4 ), and its hydrates (FeCl 2 4H 2 O, FeCl 3・6H 2 O, Fe(NO 3 ) 2.6H 2 O, FeSO 4.7H 2 O), etc., and particularly preferably iron chloride (FeCl 2 or FeCl 3 ) and its hydrate ( The iron compound is selected from FeCl 2 .4H 2 O and FeCl 3 .6H 2 O), but a mixture of a plurality of iron compounds can also be used.
一例として、塩化鉄（III）水和物（ＦｅＣｌ３・６Ｈ２Ｏ）を用いた製法を説明する。塩化鉄（III）水和物を高沸点溶媒のエチレングリコールに十分撹拌し溶解する。この溶解液に、添加剤として酢酸ナトリウムやポリエチレングリコール（ＰＥＧ）等を加えて原料溶液とする。原料溶液をガラス製容器に入れて、ガラス製容器ごとテフロン（登録商標）製内筒を備えた耐圧容器に封入する。 As an example, a manufacturing method using iron (III) chloride hydrate (FeCl 3 .6H 2 O) will be explained. Dissolve iron(III) chloride hydrate in ethylene glycol, a high boiling point solvent, by stirring thoroughly. Additives such as sodium acetate and polyethylene glycol (PEG) are added to this solution to obtain a raw material solution. The raw material solution is placed in a glass container, and the entire glass container is sealed in a pressure-resistant container equipped with an inner tube made of Teflon (registered trademark).
その後、耐圧容器を溶媒の沸点より低い１７０～１９０℃の温度に設定した恒温槽内で加熱すると溶媒中にマグネタイトナノ粒子の会合体が形成される。溶媒を除去し、アルコールと水で十分洗浄した後、６０℃で乾燥させるとマグネタイト会合体が得られる。加熱方式は、恒温槽に限らず、例えばオイルバス等いろいろな方法を採用することができる。 Thereafter, when the pressure container is heated in a constant temperature bath set at a temperature of 170 to 190° C., which is lower than the boiling point of the solvent, aggregates of magnetite nanoparticles are formed in the solvent. After removing the solvent and thoroughly washing with alcohol and water, a magnetite aggregate is obtained by drying at 60°C. The heating method is not limited to a constant temperature oven, and various methods such as an oil bath can be used.
得られたマグネタイト会合体は、ＳＥＭによる観察でナノ粒子が会合した構造であることを確認できる。また、Ｘ線回折測定（ＸＲＤ）により、会合体の結晶構造がマグネタイトであることが確認できる。 It can be confirmed by observation using SEM that the obtained magnetite aggregate has a structure in which nanoparticles are associated. Moreover, it can be confirmed by X-ray diffraction measurement (XRD) that the crystal structure of the aggregate is magnetite.
マグネタイト会合体の好ましい個数平均粒径は、印加磁場への応答性等を考慮すると０．０５μｍ以上１．９５μｍ以下が好ましい。磁性コア粒子の個数平均粒径は、上記磁性粒子と同様の方法で測定することができる。 The preferable number average particle size of the magnetite aggregates is preferably 0.05 μm or more and 1.95 μm or less, taking into consideration the responsiveness to an applied magnetic field and the like. The number average particle diameter of the magnetic core particles can be measured in the same manner as for the magnetic particles described above.
このようにして作製した磁性コア粒子は、従来の樹脂中やシリカ中に磁性ナノ粒子が分散した磁性粒子よりも磁性ナノ粒子の含有量を高くすることができ、印加磁場への応答性が向上する。 The magnetic core particles prepared in this way can have a higher content of magnetic nanoparticles than conventional magnetic particles in which magnetic nanoparticles are dispersed in resin or silica, and have improved responsiveness to an applied magnetic field. do.
＜表面層形成＞
本発明の磁性粒子は、磁性コア粒子の表面に樹脂層を有する。ここで、「磁性コア粒子表面に樹脂層を有する」とは、磁性コア粒子表面に樹脂層が、直接設けられた場合、又は磁性コア粒子に後述のシリカ層などの層を介して樹脂層が設けられた構成である。本発明の磁性粒子は、樹脂で形成された表面層を有する構成が好ましい。
<Surface layer formation>
The magnetic particles of the present invention have a resin layer on the surface of the magnetic core particle. Here, "having a resin layer on the surface of the magnetic core particle" means that the resin layer is directly provided on the surface of the magnetic core particle, or the resin layer is provided on the magnetic core particle through a layer such as a silica layer described below. This is the configuration provided. The magnetic particles of the present invention preferably have a surface layer made of resin.
（無機被覆層の形成）
磁性ナノ粒子の会合体である磁性コア粒子の表面は、ナノ粒子の集合体のため微小な凹凸が生じる。その凹凸を緩和するために、無機のコーティング層を設けることが好ましい。材料は限定されることはないが、安定して形成できる材料としてシリカが好ましい。シリカ層は、例えば、水とエタノール等のアルコールの混合溶媒に磁性コア粒子を分散させて、ＴＥＯＳ（テトラエトキシシラン）と触媒としてアンモニア水を添加することで磁性コア粒子表面にシリカ層を形成することができる。
(Formation of inorganic coating layer)
The surface of the magnetic core particle, which is an aggregate of magnetic nanoparticles, has minute irregularities due to the aggregate of the nanoparticles. In order to alleviate the unevenness, it is preferable to provide an inorganic coating layer. Although the material is not limited, silica is preferred as it can be stably formed. The silica layer is formed by, for example, dispersing magnetic core particles in a mixed solvent of water and alcohol such as ethanol, and adding TEOS (tetraethoxysilane) and ammonia water as a catalyst to form a silica layer on the surface of the magnetic core particles. be able to.
シリカ層の厚さは特に限定はされないが、印加磁場への応答性等を考慮すると、５ｎｍ以上２００ｎｍ以下が好ましく、１０ｎｍ以上１００ｎｍ以下がより好ましい。 The thickness of the silica layer is not particularly limited, but in consideration of responsiveness to an applied magnetic field, etc., it is preferably 5 nm or more and 200 nm or less, more preferably 10 nm or more and 100 nm or less.
（樹脂層の形成）
以下、「（メタ）アクリレート」と記載した場合は、「アクリレート、又はメタクリレート」を表すものとする。
(Formation of resin layer)
Hereinafter, when "(meth)acrylate" is written, it represents "acrylate or methacrylate".
樹脂表層には、抗体を結合することができる官能基を有する。抗体を結合できる官能基は、カルボキシル基、アミノ基、チオール基、エポキシ基、マレイミド基、及びスクシンイミジル基からなる群より選択される少なくとも１種であることが好ましい。 The resin surface layer has a functional group that can bind antibodies. The functional group capable of binding an antibody is preferably at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group.
樹脂層を形成する際は、（メタ）アクリル酸などのカルボキシル基を有するモノマー；（メタ）アクリルアミドなどのアミノ基を有するモノマー；グリシジル（メタ）アクリレートなどのエポキシ基を有するモノマー；Ｎ－スクシンイミジルアクリレートなどのスクシンイミジル基を有するモノマー；を用いることが好ましい。 When forming the resin layer, monomers having a carboxyl group such as (meth)acrylic acid; monomers having an amino group such as (meth)acrylamide; monomers having an epoxy group such as glycidyl (meth)acrylate; N-succine It is preferable to use a monomer having a succinimidyl group such as imidyl acrylate.
また、上記モノマー以外に、グリセロール（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、メトキシエチル（メタ）アクリレート、ポリエチレングリコールモノ（メタ）アクリレートなどの親水性基を有する（メタ）アクリレート類；スチレン、ｐ－クロロスチレン、α－メチルスチレンなどのスチレン類；なども用いることができる。磁性粒子への非特異的な吸着を抑制するために、親水性基を有する（メタ）アクリレート類を用いることが好ましい。 In addition to the above monomers, (meth)acrylates having hydrophilic groups such as glycerol (meth)acrylate, 2-hydroxyethyl (meth)acrylate, methoxyethyl (meth)acrylate, and polyethylene glycol mono(meth)acrylate; styrene Styrenes such as , p-chlorostyrene, and α-methylstyrene can also be used. In order to suppress non-specific adsorption to magnetic particles, it is preferable to use (meth)acrylates having a hydrophilic group.
樹脂層は、必要に応じて架橋することができる。非特異的な吸着の生じるような検体中で磁性粒子を使用する場合は、樹脂層の架橋が有効である。架橋剤としては、一般的に用いられている親水性架橋剤や疎水性架橋剤等を選択することができ、例えば、疎水性であれ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、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が挙げられる。また親水性の架橋剤であれば、例えば、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が挙げられる。 The resin layer can be crosslinked if necessary. When using magnetic particles in a sample where non-specific adsorption occurs, crosslinking of the resin layer is effective. As the crosslinking agent, commonly used hydrophilic crosslinking agents and hydrophobic crosslinking agents can be selected. For example, if the crosslinking agent is hydrophobic, divinylbenzene, ethylene glycol diacrylate, ethylene glycol dimethacrylate, trichloride, etc. Examples include methylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, dipentaerythritol hexamethacrylate, and the like. Examples of hydrophilic crosslinking agents include polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and poly(meth)acrylic ester of polyvinyl alcohol.
これらの樹脂層を形成する際は、下地処理とてシランカップリング剤処理を加えることで、樹脂層を均質に形成することができる。シランカップリング剤の種類は特に限定されないが、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）等が一般的に使用される。 When forming these resin layers, by adding a silane coupling agent treatment as a base treatment, the resin layers can be formed homogeneously. The type of silane coupling agent is not particularly limited, but 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) and the like are generally used.
樹脂層は２種の層で形成することが好ましい。先述の樹脂層の下層に、あらかじめ１層目の樹脂を形成することができる。この場合、１層目の樹脂層を第一の樹脂層とし、２層目の樹脂層を第二の樹脂層とした場合、第二の樹脂層が磁性粒子の最表面層であることが好ましい構成である。第一の樹脂層は、磁性ナノ粒子のコア粒子の保護のために設けることができる。第一の樹脂層としては、疎水性樹脂が好ましくスチレン、ｐ－クロロスチレン、α－メチルスチレンなどのスチレン類などを用いることが好ましい。又、前記第一の樹脂層上に形成された前記磁性粒子の最表面層である第二の樹脂層は、グリシジルメタクリレートに由来するユニットを有する樹脂を含むことが好ましい。第一の樹脂層も第二の樹脂層の最表面樹脂層と同様に架橋することができる。架橋剤種は特に限定はされないが、保護の目的としては疎水性の架橋剤が用いられ、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、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が挙げられる。
生体由来の目的物質（検体）を検出する用途で使用する場合、検体由来の目的物質以外の成分により凝集（非特異凝集）が生じることがあるが、磁性粒子の最表面層の樹脂層を架橋することで非特異凝集を抑制することができ好ましい。
The resin layer is preferably formed of two types of layers. A first layer of resin can be formed in advance under the resin layer described above. In this case, when the first resin layer is the first resin layer and the second resin layer is the second resin layer, it is preferable that the second resin layer is the outermost layer of the magnetic particles. It is the composition. The first resin layer can be provided to protect the core particles of the magnetic nanoparticles. As the first resin layer, a hydrophobic resin is preferably used, and styrenes such as styrene, p-chlorostyrene, and α-methylstyrene are preferably used. Further, it is preferable that the second resin layer, which is the outermost surface layer of the magnetic particles formed on the first resin layer, contains a resin having a unit derived from glycidyl methacrylate. The first resin layer can also be crosslinked in the same way as the outermost resin layer of the second resin layer. The type of crosslinking agent is not particularly limited, but hydrophobic crosslinking agents are used for the purpose of protection, such as divinylbenzene, ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, and trimethylolpropane trimethacrylate. , pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, dipentaerythritol hexamethacrylate, and the like.
When used to detect a target substance (sample) derived from a living body, aggregation (non-specific aggregation) may occur due to components other than the target substance derived from the sample. This is preferable because non-specific aggregation can be suppressed.
樹脂層の厚さは特に限定はされないが、印加磁場への応答性等を考慮すると、第一の樹脂層は５ｎｍ以上１００ｎｍが好ましく、第二の樹脂層は５ｎｍ以上２００ｎｍ以下が好ましい。 The thickness of the resin layer is not particularly limited, but in consideration of responsiveness to an applied magnetic field, etc., the first resin layer preferably has a thickness of 5 nm or more and 100 nm, and the second resin layer preferably has a thickness of 5 nm or more and 200 nm or less.
また、抗体を結合できる官能基は、樹脂の重合の後に付加することも可能である。例えば、グリシジル基を持つ樹脂層にメルカプトプロピオン酸やメルカプトコハク酸等のカルボキシル基を持つ試薬を作用させると、樹脂層にカルボキシル基を導入することができる。また、重合して得られる樹脂に、Ｎ－（２－ヒドロキシエチル）マレイミドを付加することでマレイミド基を導入することができる。 Furthermore, a functional group capable of binding an antibody can also be added after polymerization of the resin. For example, when a reagent having a carboxyl group such as mercaptopropionic acid or mercaptosuccinic acid is applied to a resin layer having a glycidyl group, a carboxyl group can be introduced into the resin layer. Furthermore, a maleimide group can be introduced by adding N-(2-hydroxyethyl)maleimide to the resin obtained by polymerization.
＜リガンド＞
リガンドとは、特定の標的物質が有する受容体に特異的に結合する化合物のことである。リガンドが標的物質と結合する部位は決まっており、選択的又は特異的に高い親和性を有する。例えば、抗原と抗体、酵素タンパク質とその基質、ホルモンや神経伝達物質に代表されるシグナル物質とその受容体、核酸、アビジンとビオチンなどが挙げられるが、上記実施形態の目的を達成可能な範囲においてこれらに限定されない。リガンドとしては、具体的に、抗原、抗体、抗原結合フラグメント（例えば、Ｆａｂ、Ｆ（ａｂ’）２、Ｆ（ａｂ’）、Ｆｖ、ｓｃＦｖなど）、天然由来核酸、人工核酸、アプタマー、ペプチドアプタマー、オリゴペプチド、酵素、補酵素などが挙げられる。
本発明に係る磁性粒子がリガンドを有し、リガンドと樹脂層の官能基とは化学結合を有する免疫検査用粒子が好ましい実施態様である
<ligand>
A ligand is a compound that specifically binds to a receptor possessed by a specific target substance. The site where the ligand binds to the target substance is determined and has high affinity selectively or specifically. Examples include antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, etc., but within the range that can achieve the purpose of the above embodiment. Not limited to these. Specifically, the ligands include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, and peptide aptamers. , oligopeptides, enzymes, coenzymes, etc.
A preferred embodiment is an immunoassay particle in which the magnetic particles according to the present invention have a ligand, and the ligand and the functional group of the resin layer have a chemical bond.
以下、参考例、実施例、及び比較例を挙げて本発明をさらに詳細に説明するが、本発明は、その要旨を超えない限り、下記の実施例によって何ら限定されるものではない。なお、成分量に関して「部」、及び「％」と記載しているものは特に断らない限り質量基準である。 Hereinafter, the present invention will be explained in more detail with reference to Reference Examples, Examples, and Comparative Examples, but the present invention is not limited by the following Examples unless the gist thereof is exceeded. Note that "parts" and "%" regarding component amounts are based on mass unless otherwise specified.
＜参考例１＞
（磁性コア粒子の作製）
マグネタイトナノ粒子が会合した磁性コア粒子を作製した。まず、塩化鉄（III）六水和物（ＦｅＣｌ３・６Ｈ２Ｏ；キシダ化学製）１．２１７ｇ、酢酸ナトリウム（ＣＨ３ＣＯＯＮａ；キシダ化学製）２．７ｇ、ポリエチレングリコール（平均分子量２０００；ＰＥＧ２０００；キシダ化学製）０．７５ｇをそれぞれエチレングリコール（ＥＧ；ＨＯＣＨ２ＣＨ２ＯＨ；キシダ化学製）１０ｍＬに溶解した。ＦｅＣｌ３・６Ｈ２Ｏ／ＥＧにＣＨ３ＣＯＯＮａ／ＥＧ、ＰＥＧ２０００／ＥＧを順番に添加して反応溶液を調製した。この反応溶液の調製は、ガラス製容器を用いた。
次に、この反応溶液をガラス製容器ごと、テフロン（登録商標）製内筒を備えた耐圧容器（耐圧硝子株式会社）中にセットし、１８０℃のオーブンで２４時間加熱した。加熱終了後、室温まで冷却してから、生成物を水とエタノールでそれぞれ二回洗浄し、６０℃に設定した乾燥機で乾燥した。得られた粒子は０．７ｇであった。
<Reference example 1>
(Preparation of magnetic core particles)
We created magnetic core particles in which magnetite nanoparticles were associated. First, 1.217 g of iron chloride hexahydrate (FeCl 3.6H 2 O; manufactured by Kishida Chemical Co., Ltd.), 2.7 g of sodium acetate (CH 3 COONa; manufactured by Kishida Chemical Co., Ltd.), polyethylene glycol (average molecular weight 2000; PEG 2000) ; Kishida Chemical Co., Ltd.) 0.75 g was dissolved in 10 mL of ethylene glycol (EG; HOCH 2 CH 2 OH; Kishida Chemical Co., Ltd.). A reaction solution was prepared by sequentially adding CH 3 COONa/EG and PEG2000/EG to FeCl 3 .6H 2 O/EG. A glass container was used to prepare this reaction solution.
Next, this reaction solution was placed together with the glass container in a pressure container (manufactured by Pressure Glass Co., Ltd.) equipped with an inner tube made of Teflon (registered trademark), and heated in an oven at 180° C. for 24 hours. After heating, the product was cooled to room temperature, washed twice with water and ethanol, and dried in a dryer set at 60°C. The obtained particles weighed 0.7 g.
（磁性コア粒子の分析）
作製した磁性コア粒子のＳＥＭ観察を行った（日立ハイテクノロジー社製、Ｓ－４８００）。５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は４２３ｎｍであった。倍率を５００００倍に上げて粒子表面を観察すると、２０ｎｍ以下の粒径のナノ粒子の会合体であることが確認できた。代表的なＳＥＭ観察像を図２に示す。
(Analysis of magnetic core particles)
The produced magnetic core particles were observed by SEM (manufactured by Hitachi High-Technologies, S-4800). When the long diameter of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 423 nm. When the particle surface was observed at a magnification of 50,000 times, it was confirmed that it was an aggregate of nanoparticles with a particle size of 20 nm or less. A typical SEM observation image is shown in FIG.
次に、ＸＲＤ（Ｐａｎａｌｙｔｉｃａｌ社製、Ｘ’ＰＥＲＴ ＰＲＯ）で得られた磁性コア粒子の結晶構造を分析した。その結果、マグネタイト（Ｆｅ３Ｏ４）の単一層であることを確認した。 Next, the crystal structure of the obtained magnetic core particles was analyzed by XRD (X'PERT PRO, manufactured by Panalytical). As a result, it was confirmed that it was a single layer of magnetite (Fe 3 O 4 ).
＜参考例２＞
参考例１で作製した磁性コア粒子を用いてシリカ層を有する磁性コア粒子（以下、単に「シリカ被覆のコア粒子」とも呼ぶ。）を作製した。
（シリカ層の形成）
参考例１で得られた磁性コア粒子０．５ｇをエタノール７５ｍＬ（キシダ化学製）と純水７５ｍＬの混合溶液に分散させた。次に、ＴＥＯＳ（キシダ化学製）を１．５ｍＬ添加して、触媒として２８％アンモニア水（キシダ化学製）を２２．５ｍＬ追加して撹拌しながら１．５時間反応させた。反応後、粒子をネオジム磁石で捕集して溶媒を除去し、純水で７回洗浄した。粒子の一部を乾燥させてＳＥＭ観察すると、磁性コア粒子表面の観察でみられたナノ粒子の凹凸は無くなっており、磁性コア粒子の表面にシリカ層が形成できていることを確認した。また、５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は４７９ｎｍであった。
<Reference example 2>
Magnetic core particles having a silica layer (hereinafter also simply referred to as "silica-coated core particles") were produced using the magnetic core particles produced in Reference Example 1.
(Formation of silica layer)
0.5 g of the magnetic core particles obtained in Reference Example 1 were dispersed in a mixed solution of 75 mL of ethanol (manufactured by Kishida Chemical) and 75 mL of pure water. Next, 1.5 mL of TEOS (manufactured by Kishida Chemical) was added, and 22.5 mL of 28% ammonia water (manufactured by Kishida Chemical) was added as a catalyst, followed by reaction for 1.5 hours with stirring. After the reaction, the particles were collected with a neodymium magnet to remove the solvent, and washed seven times with pure water. When a part of the particles was dried and observed under SEM, it was confirmed that the unevenness of the nanoparticles observed when observing the surface of the magnetic core particles disappeared, and a silica layer was formed on the surface of the magnetic core particles. Further, when the long diameter of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 479 nm.
＜実施例１＞
（樹脂層の形成）
参考例２で得られたシリカ被覆のコア粒子に樹脂層を形成した。エタノール１０ｍＬと純水１０ｍＬの混合溶液に粒子を分散させて、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を１００μＬ添加して十分に混合した。次に、２８％のアンモニア水２ｍＬを添加し１．５時間撹拌した。次に、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄した後６０ｍＬの窒素バブリングした純水を追加して水分散液を得た。
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングして２００ｒｐｍの撹拌速度で撹拌しながら１５分撹拌した。続いて、窒素バブリングを窒素フローに切り替た後、この分散液にスチレンモノマー（キシダ化学社製）を５０μＬ添加した。
次に、０．０２ｇの過硫酸カリウム（シグマアルドリッチ社製）を予め窒素バブリングで脱気した純水２ｍＬに溶解して１ｍＬをフラスコ内に添加した。次に、オイルバスを用いて、３５℃で３０分加熱した後、６０℃に昇温して１時間保持した。続いて、グリシジルメタクリレート（キシダ化学社製）２００μＬ添加して、さらに１２時間保持して重合を終了した。重合終了後、純水で十分に洗浄して粒子を得た。
<Example 1>
(Formation of resin layer)
A resin layer was formed on the silica-coated core particles obtained in Reference Example 2. Particles were dispersed in a mixed solution of 10 mL of ethanol and 10 mL of pure water, and 100 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added as a silane coupling agent and thoroughly mixed. Next, 2 mL of 28% aqueous ammonia was added and stirred for 1.5 hours. Next, the solvent was removed while collecting the particles with a neodymium magnet, and after thorough washing with pure water, 60 mL of nitrogen-bubbled pure water was added to obtain an aqueous dispersion.
Next, this dispersion was placed in a four-necked flask (200 mL), and stirred for 15 minutes with nitrogen bubbling and stirring at a stirring speed of 200 rpm. Subsequently, after switching the nitrogen bubbling to nitrogen flow, 50 μL of styrene monomer (manufactured by Kishida Chemical Co., Ltd.) was added to this dispersion.
Next, 0.02 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 2 mL of pure water that had been previously degassed by nitrogen bubbling, and 1 mL of the solution was added into the flask. Next, after heating at 35°C for 30 minutes using an oil bath, the temperature was raised to 60°C and held for 1 hour. Subsequently, 200 μL of glycidyl methacrylate (manufactured by Kishida Chemical Co., Ltd.) was added, and the mixture was maintained for an additional 12 hours to complete the polymerization. After the polymerization was completed, the particles were thoroughly washed with pure water to obtain particles.
（官能基の導入）
次に、官能基としてカルボキシル基を導入する処理を行った。得られた粒子１０ｍｇを５ｍＬの純水に分散させた。この分散液とは別に、メルカプトコハク酸（キシダ化学製）３５０ｍｇを純水５ｍＬに溶解して、ｐＨ調整としてトリエチルアミン（東京化成工業製）を０．８ｍＬ添加した。この溶液を、粒子分散液に１ｍＬ添加して十分撹拌して６０℃で３時間加熱処理した。この後、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄し磁性粒子分散液を得た。
(Introduction of functional group)
Next, a process was performed to introduce a carboxyl group as a functional group. 10 mg of the obtained particles were dispersed in 5 mL of pure water. Separately from this dispersion, 350 mg of mercaptosuccinic acid (manufactured by Kishida Chemical) was dissolved in 5 mL of pure water, and 0.8 mL of triethylamine (manufactured by Tokyo Kasei Kogyo) was added to adjust the pH. 1 mL of this solution was added to the particle dispersion, thoroughly stirred, and heat-treated at 60° C. for 3 hours. Thereafter, the solvent was removed while collecting the particles with a neodymium magnet, and the particles were sufficiently washed with pure water to obtain a magnetic particle dispersion.
（磁性粒子の個数平均粒径）
作製した磁性粒子のＳＥＭ観察を行った。磁性粒子分散溶液の一部を乾燥させて５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は６２８ｎｍであった。
得られた磁性粒子を熱分解ガスクロマトグラフィー（フロンティア・ラボ社製ＰＹ－３０３０Ｄ）を用いて、磁性粒子の樹脂層の構成成分を分析した。その結果、樹脂層の構成成分となる、スチレン、グリシジルメタクリレート由来の信号が検出された。
(Number average particle size of magnetic particles)
The produced magnetic particles were observed by SEM. When a portion of the magnetic particle dispersion solution was dried and the length of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 628 nm.
The obtained magnetic particles were analyzed for constituent components of the resin layer of the magnetic particles using pyrolysis gas chromatography (PY-3030D manufactured by Frontier Lab). As a result, signals derived from styrene and glycidyl methacrylate, which are constituent components of the resin layer, were detected.
＜実施例２＞
（樹脂層の形成）
参考例２で得られたシリカ被覆のコア粒子に樹脂層を形成した。エタノール１０ｍＬと純水１０ｍＬの混合溶液に粒子を分散させて、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を１００μＬ添加して十分に混合した。次に、２８％のアンモニア水２ｍＬを添加し１．５時間撹拌した。次に、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄した後６０ｍＬの窒素バブリングした純水を追加して水分散液を得た。
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングして２００ｒｐｍに撹拌速度で撹拌しながら１５分撹拌した。続いて、窒素バブリングを窒素フローに切り替た後、この分散液にスチレンモノマー（キシダ化学社製）を５０μＬ添加した。
次に、０．０２ｇの過硫酸カリウム（シグマアルドリッチ社製）を予め窒素バブリングで脱気した純水２ｍＬに溶解して１ｍＬをフラスコ内に添加した。次に、オイルバスを用いて、３５℃で３０分加熱した後、６０℃に昇温して１時間保持した。続いて、グリシジルメタクリレート（キシダ化学社製）２００μＬとアクリル酸モノマー２００μＬ（東京化成工業製）を添加して、さらに１２時間保持して重合を終了した。重合終了後、純水で十分に洗浄して粒子を得た。
<Example 2>
(Formation of resin layer)
A resin layer was formed on the silica-coated core particles obtained in Reference Example 2. Particles were dispersed in a mixed solution of 10 mL of ethanol and 10 mL of pure water, and 100 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added as a silane coupling agent and thoroughly mixed. Next, 2 mL of 28% aqueous ammonia was added and stirred for 1.5 hours. Next, the solvent was removed while collecting the particles with a neodymium magnet, and after thorough washing with pure water, 60 mL of nitrogen-bubbled pure water was added to obtain an aqueous dispersion.
Next, this dispersion was placed in a four-necked flask (200 mL), and stirred for 15 minutes with nitrogen bubbling and stirring at a stirring speed of 200 rpm. Subsequently, after switching the nitrogen bubbling to nitrogen flow, 50 μL of styrene monomer (manufactured by Kishida Chemical Co., Ltd.) was added to this dispersion.
Next, 0.02 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 2 mL of pure water that had been previously degassed by nitrogen bubbling, and 1 mL of the solution was added into the flask. Next, after heating at 35°C for 30 minutes using an oil bath, the temperature was raised to 60°C and held for 1 hour. Subsequently, 200 μL of glycidyl methacrylate (manufactured by Kishida Chemical Co., Ltd.) and 200 μL of acrylic acid monomer (manufactured by Tokyo Chemical Industry Co., Ltd.) were added, and the mixture was maintained for an additional 12 hours to complete the polymerization. After the polymerization was completed, the particles were thoroughly washed with pure water to obtain particles.
作製した磁性粒子のＳＥＭ観察を行った。磁性粒子分散溶液の一部を乾燥させて５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は５７２ｎｍであった。
得られた磁性粒子を実施例１と同様に熱分解ガスクロマトグラフィーを用いて、磁性粒子の樹脂層の構成成分を分析した。その結果、樹脂層の構成成分となる、スチレン、グリシジルメタクリレート、アクリル酸由来の信号が検出された。
The produced magnetic particles were observed by SEM. When a portion of the magnetic particle dispersion solution was dried and the long diameter of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 572 nm.
The obtained magnetic particles were analyzed for constituent components of the resin layer of the magnetic particles using pyrolysis gas chromatography in the same manner as in Example 1. As a result, signals derived from styrene, glycidyl methacrylate, and acrylic acid, which are constituent components of the resin layer, were detected.
＜実施例３＞
（樹脂層の形成）
参考例２で得られたシリカ被覆のコア粒子に樹脂層を形成した。エタノール１０ｍＬと純水１０ｍＬの混合溶液に粒子を分散させて、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を１００μＬ添加して十分に混合した。次に、２８％のアンモニア水２ｍＬを添加し１．５時間撹拌した。次に、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄した後６０ｍＬの窒素バブリングした純水を追加して水分散液を得た。
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングして２００ｒｐｍに撹拌速度で撹拌しながら１５分撹拌した。続いて、窒素バブリングを窒素フローに切り替た後、この分散液にスチレンモノマー（キシダ化学社製）５０μＬと架橋剤としてジビニルベンゼンモノマー（キシダ化学社製）２．５μＬとを混合して添加した。
次に、０．０２ｇの過硫酸カリウム（シグマアルドリッチ社製）を予め窒素バブリングで脱気した純水２ｍＬに溶解して１ｍＬをフラスコ内に添加した。次に、オイルバスを用いて、３５℃で３０分加熱した後、６０℃に昇温して１時間保持した。続いて、グリシジルメタクリレート（キシダ化学社製）２００μＬと架橋剤としてジビニルベンゼンモノマー（キシダ化学社製）１０μＬとを混合して添加して、さらに１２時間保持して重合を終了した。重合終了後、純水で十分に洗浄して粒子を得た。
<Example 3>
(Formation of resin layer)
A resin layer was formed on the silica-coated core particles obtained in Reference Example 2. Particles were dispersed in a mixed solution of 10 mL of ethanol and 10 mL of pure water, and 100 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added as a silane coupling agent and thoroughly mixed. Next, 2 mL of 28% aqueous ammonia was added and stirred for 1.5 hours. Next, the solvent was removed while collecting the particles with a neodymium magnet, and after thorough washing with pure water, 60 mL of nitrogen-bubbled pure water was added to obtain an aqueous dispersion.
Next, this dispersion was placed in a four-necked flask (200 mL), and stirred for 15 minutes with nitrogen bubbling and stirring at a stirring speed of 200 rpm. Subsequently, after switching the nitrogen bubbling to nitrogen flow, 50 μL of styrene monomer (manufactured by Kishida Chemical Co., Ltd.) and 2.5 μL of divinylbenzene monomer (manufactured by Kishida Chemical Co., Ltd.) as a crosslinking agent were mixed and added to this dispersion.
Next, 0.02 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 2 mL of pure water that had been previously degassed by nitrogen bubbling, and 1 mL of the solution was added into the flask. Next, after heating at 35°C for 30 minutes using an oil bath, the temperature was raised to 60°C and held for 1 hour. Subsequently, 200 μL of glycidyl methacrylate (manufactured by Kishida Chemical Co., Ltd.) and 10 μL of divinylbenzene monomer (manufactured by Kishida Chemical Co., Ltd.) as a crosslinking agent were mixed and added, and the mixture was maintained for an additional 12 hours to complete the polymerization. After the polymerization was completed, the particles were thoroughly washed with pure water to obtain particles.
（官能基の導入）
次に、官能基としてカルボキシル基を導入する処理を行った。得られた粒子１０ｍｇを５ｍＬの純水に分散させた。この分散液とは別に、メルカプトコハク酸（キシダ化学製）３５０ｍｇを純水５ｍＬに溶解して、ｐＨ調整としてトリエチルアミン（東京化成工業製）を０．８ｍＬ添加した。この溶液を、粒子分散液に１ｍＬ添加して十分撹拌して６０℃で３時間加熱処理した。この後、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄し磁性粒子分散液を得た。
作製した磁性粒子のＳＥＭ観察を行った。磁性粒子分散溶液の一部を乾燥させて５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は６１９ｎｍであった。
得られた磁性粒子を実施例１と同様に熱分解ガスクロマトグラフィーを用いて、磁性粒子の樹脂層の構成成分を分析した。その結果、樹脂層の構成成分となる、スチレン、グリシジルメタクリレート、ジビニルベンゼン由来の信号が検出された。
(Introduction of functional group)
Next, a process was performed to introduce a carboxyl group as a functional group. 10 mg of the obtained particles were dispersed in 5 mL of pure water. Separately from this dispersion, 350 mg of mercaptosuccinic acid (manufactured by Kishida Chemical) was dissolved in 5 mL of pure water, and 0.8 mL of triethylamine (manufactured by Tokyo Kasei Kogyo) was added to adjust the pH. 1 mL of this solution was added to the particle dispersion, thoroughly stirred, and heat-treated at 60° C. for 3 hours. Thereafter, the solvent was removed while collecting the particles with a neodymium magnet, and the particles were sufficiently washed with pure water to obtain a magnetic particle dispersion.
The produced magnetic particles were observed by SEM. When a portion of the magnetic particle dispersion solution was dried and the length of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 619 nm.
The obtained magnetic particles were analyzed for constituent components of the resin layer of the magnetic particles using pyrolysis gas chromatography in the same manner as in Example 1. As a result, signals derived from styrene, glycidyl methacrylate, and divinylbenzene, which are constituent components of the resin layer, were detected.
＜実施例４＞
（樹脂層の形成）
参考例２で得られたシリカ被覆のコア粒子に樹脂層を形成した。エタノール１０ｍＬと純水１０ｍＬの混合溶液に粒子を分散させて、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を１００μＬ添加して十分に混合した。次に、２８％のアンモニア水２ｍＬを添加し１．５時間撹拌した。次に、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄した後６０ｍＬの窒素バブリングした純水を追加して水分散液を得た。
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングして２００ｒｐｍに撹拌速度で撹拌しながら１５分撹拌した。続いて、窒素バブリングを窒素フローに切り替た後、この分散液にスチレンモノマー（キシダ化学社製）５０μＬと架橋剤としてジビニルベンゼンモノマー（キシダ化学社製）５μLとを混合して添加した。
次に、０．０２ｇの過硫酸カリウム（シグマアルドリッチ社製）を予め窒素バブリングで脱気した純水２ｍＬに溶解して１ｍＬをフラスコ内に添加した。次に、オイルバスを用いて、３５℃で３０分加熱した後、６０℃に昇温して１時間保持した。続いて、グリシジルメタクリレート（キシダ化学社製）２００μＬと架橋剤としてジビニルベンゼンモノマー（キシダ化学社製）２０μＬとを混合して添加して、さらに１２時間保持して重合を終了した。重合終了後、純水で十分に洗浄して粒子を得た。
<Example 4>
(Formation of resin layer)
A resin layer was formed on the silica-coated core particles obtained in Reference Example 2. Particles were dispersed in a mixed solution of 10 mL of ethanol and 10 mL of pure water, and 100 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added as a silane coupling agent and thoroughly mixed. Next, 2 mL of 28% aqueous ammonia was added and stirred for 1.5 hours. Next, the solvent was removed while collecting the particles with a neodymium magnet, and after thorough washing with pure water, 60 mL of nitrogen-bubbled pure water was added to obtain an aqueous dispersion.
Next, this dispersion was placed in a four-necked flask (200 mL), and stirred for 15 minutes with nitrogen bubbling and stirring at a stirring speed of 200 rpm. Subsequently, after switching the nitrogen bubbling to nitrogen flow, 50 μL of styrene monomer (manufactured by Kishida Chemical Co., Ltd.) and 5 μL of divinylbenzene monomer (manufactured by Kishida Chemical Co., Ltd.) as a crosslinking agent were mixed and added to this dispersion.
Next, 0.02 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 2 mL of pure water that had been previously degassed by nitrogen bubbling, and 1 mL of the solution was added into the flask. Next, after heating at 35°C for 30 minutes using an oil bath, the temperature was raised to 60°C and held for 1 hour. Subsequently, 200 μL of glycidyl methacrylate (manufactured by Kishida Chemical Co., Ltd.) and 20 μL of divinylbenzene monomer (manufactured by Kishida Chemical Co., Ltd.) as a crosslinking agent were mixed and added, and the mixture was maintained for an additional 12 hours to complete the polymerization. After the polymerization was completed, the particles were thoroughly washed with pure water to obtain particles.
（官能基の導入）
次に、官能基としてカルボキシル基を導入する処理を行った。得られた粒子１０ｍｇを５ｍＬの純水に分散させた。この分散液とは別に、メルカプトコハク酸（キシダ化学製）３５０ｍｇを純水５ｍＬに溶解して、ｐＨ調整としてトリエチルアミン（東京化成工業製）を０．８ｍＬ添加した。この溶液を、粒子分散液に１ｍＬ添加して十分撹拌して６０℃で３時間加熱処理した。この後、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄し磁性粒子分散液を得た。
作製した磁性粒子のＳＥＭ観察を行った。磁性粒子分散溶液の一部を乾燥させて５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は６０４ｎｍであった。
得られた磁性粒子を実施例１と同様に熱分解ガスクロマトグラフィーを用いて、磁性粒子の樹脂層の構成成分を分析した。その結果、樹脂層の構成成分となる、スチレン、グリシジルメタクリレート、ジビニルベンゼン由来の信号が検出された。ジビニルベンゼン由来の信号は実施例３の粒子と比較して大きくなっていた。
(Introduction of functional group)
Next, a process was performed to introduce a carboxyl group as a functional group. 10 mg of the obtained particles were dispersed in 5 mL of pure water. Separately from this dispersion, 350 mg of mercaptosuccinic acid (manufactured by Kishida Chemical) was dissolved in 5 mL of pure water, and 0.8 mL of triethylamine (manufactured by Tokyo Kasei Kogyo) was added to adjust the pH. 1 mL of this solution was added to the particle dispersion, thoroughly stirred, and heat-treated at 60° C. for 3 hours. Thereafter, the solvent was removed while collecting the particles with a neodymium magnet, and the particles were sufficiently washed with pure water to obtain a magnetic particle dispersion.
The produced magnetic particles were observed by SEM. When a portion of the magnetic particle dispersion solution was dried and the length of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 604 nm.
The obtained magnetic particles were analyzed for constituent components of the resin layer of the magnetic particles using pyrolysis gas chromatography in the same manner as in Example 1. As a result, signals derived from styrene, glycidyl methacrylate, and divinylbenzene, which are constituent components of the resin layer, were detected. The signal derived from divinylbenzene was increased compared to the particles of Example 3.
＜実施例５＞
（樹脂層の形成）
参考例２で得られたシリカ被覆のコア粒子に樹脂層を形成した。エタノール１０ｍＬと純水１０ｍＬの混合溶液に粒子を分散させて、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を１００μＬ添加して十分に混合した。次に、２８％のアンモニア水２ｍＬを添加し１．５時間撹拌した。次に、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄した後６０ｍＬの窒素バブリングした純水を追加して水分散液を得た。
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングして２００ｒｐｍに撹拌速度で撹拌しながら１５分撹拌した。続いて、窒素バブリングを窒素フローに切り替た後、この分散液にスチレンモノマー（キシダ化学社製）５０μＬと架橋剤としてトリメチロールプロパントリメタクリレート（東京化成工業社製）５μＬとを混合して添加した。
次に、０．０２ｇの過硫酸カリウム（シグマアルドリッチ社製）を予め窒素バブリングで脱気した純水２ｍＬに溶解して１ｍＬをフラスコ内に添加した。次に、オイルバスを用いて、３５℃で３０分加熱した後、６０℃に昇温して１時間保持した。続いて、グリシジルメタクリレート（キシダ化学社製）２００μＬと架橋剤としてトリメチロールプロパントリメタクリレート（東京化成工業社製）２０μＬとを混合して添加して、さらに１２時間保持して重合を終了した。重合終了後、純水で十分に洗浄して粒子を得た。
<Example 5>
(Formation of resin layer)
A resin layer was formed on the silica-coated core particles obtained in Reference Example 2. Particles were dispersed in a mixed solution of 10 mL of ethanol and 10 mL of pure water, and 100 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added as a silane coupling agent and thoroughly mixed. Next, 2 mL of 28% aqueous ammonia was added and stirred for 1.5 hours. Next, the solvent was removed while collecting the particles with a neodymium magnet, and after thorough washing with pure water, 60 mL of nitrogen-bubbled pure water was added to obtain an aqueous dispersion.
Next, this dispersion was placed in a four-necked flask (200 mL), and stirred for 15 minutes with nitrogen bubbling and stirring at a stirring speed of 200 rpm. Subsequently, after switching the nitrogen bubbling to nitrogen flow, a mixture of 50 μL of styrene monomer (manufactured by Kishida Chemical Co., Ltd.) and 5 μL of trimethylolpropane trimethacrylate (manufactured by Tokyo Kasei Kogyo Co., Ltd.) as a crosslinking agent was added to this dispersion. .
Next, 0.02 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 2 mL of pure water that had been previously degassed by nitrogen bubbling, and 1 mL of the solution was added into the flask. Next, after heating at 35°C for 30 minutes using an oil bath, the temperature was raised to 60°C and held for 1 hour. Subsequently, 200 μL of glycidyl methacrylate (manufactured by Kishida Chemical Co., Ltd.) and 20 μL of trimethylolpropane trimethacrylate (manufactured by Tokyo Kasei Kogyo Co., Ltd.) as a crosslinking agent were mixed and added, and the mixture was maintained for an additional 12 hours to complete the polymerization. After the polymerization was completed, the particles were thoroughly washed with pure water to obtain particles.
（官能基の導入）
次に、官能基としてカルボキシル基を導入する処理を行った。得られた粒子１０ｍｇを５ｍＬの純水に分散させた。この分散液とは別に、メルカプトコハク酸（キシダ化学製）３５０ｍｇを純水５ｍＬに溶解して、ｐＨ調整としてトリエチルアミン（東京化成工業製）を０．８ｍＬ添加した。この溶液を、粒子分散液に１ｍＬ添加して十分撹拌して６０℃で３時間加熱処理した。この後、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄し磁性粒子分散液を得た。
作製した磁性粒子のＳＥＭ観察を行った。磁性粒子分散溶液の一部を乾燥させて５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は５９８ｎｍであった。
得られた磁性粒子を実施例１と同様に、熱分解ガスクロマトグラフィーを用いて磁性粒子の構成成分を分析した。その結果、樹脂層構成成分となる、スチレン、グリシジルメタクリレート、トリメチロールプロパントリメタクリレート由来の信号が検出された。
(Introduction of functional group)
Next, a process was performed to introduce a carboxyl group as a functional group. 10 mg of the obtained particles were dispersed in 5 mL of pure water. Separately from this dispersion, 350 mg of mercaptosuccinic acid (manufactured by Kishida Chemical) was dissolved in 5 mL of pure water, and 0.8 mL of triethylamine (manufactured by Tokyo Kasei Kogyo) was added to adjust the pH. 1 mL of this solution was added to the particle dispersion, thoroughly stirred, and heat-treated at 60° C. for 3 hours. Thereafter, the solvent was removed while collecting the particles with a neodymium magnet, and the particles were sufficiently washed with pure water to obtain a magnetic particle dispersion.
The produced magnetic particles were observed by SEM. When a portion of the magnetic particle dispersion solution was dried and the length of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 598 nm.
The constituent components of the obtained magnetic particles were analyzed using pyrolysis gas chromatography in the same manner as in Example 1. As a result, signals derived from styrene, glycidyl methacrylate, and trimethylolpropane trimethacrylate, which are constituent components of the resin layer, were detected.
＜実施例６＞
（樹脂層の形成）
参考例２で得られたシリカ被覆のコア粒子に樹脂層を形成した。エタノール１０ｍＬと純水１０ｍＬの混合溶液に粒子を分散させて、シランカップリング剤として、３－メタクリルオキシプロピルトリメトキシシラン（ＬＳ－３３８０、信越化学工業製）を１００μＬ添加して十分に混合した。次に、２８％のアンモニア水２ｍＬを添加し１．５時間撹拌した。次に、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄した後６０ｍＬの窒素バブリングした純水を追加して水分散液を得た。
次に、この分散液を４つ口のフラスコ（２００ｍＬ）に入れ、窒素バブリングして２００ｒｐｍに撹拌速度で撹拌しながら１５分撹拌した。続いて、窒素バブリングを窒素フローに切り替た後、この分散液にスチレンモノマー（キシダ化学社製）５０μＬと架橋剤としてポリエチレングリコールジアクリレート（東京化成工業社製）５μＬとを混合して添加した。
次に、０．０２ｇの過硫酸カリウム（シグマアルドリッチ社製）を予め窒素バブリングで脱気した純水２ｍＬに溶解して１ｍＬをフラスコ内に添加した。次に、オイルバスを用いて、３５℃で３０分加熱した後、６０℃に昇温して１時間保持した。続いて、グリシジルメタクリレート（キシダ化学社製）２００μＬと架橋剤としてポリエチレングリコールジアクリレート（東京化成工業社製）２０μＬとを混合して添加して、さらに１２時間保持して重合を終了した。重合終了後、純水で十分に洗浄して粒子を得た。
<Example 6>
(Formation of resin layer)
A resin layer was formed on the silica-coated core particles obtained in Reference Example 2. Particles were dispersed in a mixed solution of 10 mL of ethanol and 10 mL of pure water, and 100 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added as a silane coupling agent and thoroughly mixed. Next, 2 mL of 28% aqueous ammonia was added and stirred for 1.5 hours. Next, the solvent was removed while collecting the particles with a neodymium magnet, and after thorough washing with pure water, 60 mL of nitrogen-bubbled pure water was added to obtain an aqueous dispersion.
Next, this dispersion was placed in a four-necked flask (200 mL), and stirred for 15 minutes with nitrogen bubbling and stirring at a stirring speed of 200 rpm. Subsequently, after switching the nitrogen bubbling to nitrogen flow, a mixture of 50 μL of styrene monomer (manufactured by Kishida Chemical Co., Ltd.) and 5 μL of polyethylene glycol diacrylate (manufactured by Tokyo Kasei Kogyo Co., Ltd.) as a crosslinking agent was added to this dispersion.
Next, 0.02 g of potassium persulfate (manufactured by Sigma-Aldrich) was dissolved in 2 mL of pure water that had been previously degassed by nitrogen bubbling, and 1 mL of the solution was added into the flask. Next, after heating at 35°C for 30 minutes using an oil bath, the temperature was raised to 60°C and held for 1 hour. Subsequently, 200 μL of glycidyl methacrylate (manufactured by Kishida Chemical Co., Ltd.) and 20 μL of polyethylene glycol diacrylate (manufactured by Tokyo Kasei Kogyo Co., Ltd.) as a crosslinking agent were mixed and added, and the mixture was maintained for an additional 12 hours to complete the polymerization. After the polymerization was completed, the particles were thoroughly washed with pure water to obtain particles.
（官能基の導入）
次に、官能基としてカルボキシル基を導入する処理を行った。得られた粒子１０ｍｇを５ｍＬの純水に分散させた。この分散液とは別に、メルカプトコハク酸（キシダ化学製）３５０ｍｇを純水５ｍＬに溶解して、ｐＨ調整としてトリエチルアミン（東京化成工業製）を０．８ｍＬ添加した。この溶液を、粒子分散液に１ｍＬ添加して十分撹拌して６０℃で３時間加熱処理した。この後、ネオジム磁石で粒子を捕集しながら溶媒を除去し、純水で十分洗浄し磁性粒子分散液を得た。
作製した磁性粒子のＳＥＭ観察を行った。磁性粒子分散溶液の一部を乾燥させて５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は６２２ｎｍであった。
得られた磁性粒子を実施例１と同様に、熱分解ガスクロマトグラフィーを用いて磁性粒子の構成成分を分析した。その結果、樹脂層の構成成分となる、スチレン、グリシジルメタクリレート、ポリエチレングリコールジアクリレート由来の信号が検出された。
(Introduction of functional group)
Next, a process was performed to introduce a carboxyl group as a functional group. 10 mg of the obtained particles were dispersed in 5 mL of pure water. Separately from this dispersion, 350 mg of mercaptosuccinic acid (manufactured by Kishida Chemical) was dissolved in 5 mL of pure water, and 0.8 mL of triethylamine (manufactured by Tokyo Kasei Kogyo) was added to adjust the pH. 1 mL of this solution was added to the particle dispersion, thoroughly stirred, and heat-treated at 60° C. for 3 hours. Thereafter, the solvent was removed while collecting the particles with a neodymium magnet, and the particles were sufficiently washed with pure water to obtain a magnetic particle dispersion.
The produced magnetic particles were observed by SEM. When a portion of the magnetic particle dispersion solution was dried and the length of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 622 nm.
The constituent components of the obtained magnetic particles were analyzed using pyrolysis gas chromatography in the same manner as in Example 1. As a result, signals derived from styrene, glycidyl methacrylate, and polyethylene glycol diacrylate, which are constituent components of the resin layer, were detected.
＜参考例３＞
マグネタイトナノ粒子が会合した磁性コア粒子を作製した。マグネタイトの原料として、塩化鉄（II）四水和物（ＦｅＣｌ２・４Ｈ２Ｏ、キシダ化学製）０．３０ｇと塩化鉄（III）六水和物（ＦｅＣｌ３・６Ｈ２Ｏ、キシダ化学製）０．８１ｇを用いた以外は、参考例１と同様の手順で作製し、磁性コア粒子を作製した。
<Reference example 3>
We created magnetic core particles in which magnetite nanoparticles were associated. As raw materials for magnetite, 0.30 g of iron (II) chloride tetrahydrate (FeCl 2.4H 2 O, manufactured by Kishida Chemical) and iron (III) chloride hexahydrate (FeCl 3.6H 2 O, manufactured by Kishida Chemical) were used. ) Magnetic core particles were produced in the same manner as in Reference Example 1 except that 0.81 g was used.
作製した磁性コア粒子のＳＥＭ観察を行った。５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は５１９ｎｍであった。 The produced magnetic core particles were observed by SEM. When the long diameter of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 519 nm.
＜比較例１＞
マグネタイトナノ粒子としてＥＭＧ１４００（フェローテック社製）１４ｇに、粒子を形成するためのモノマー、並びに架橋剤としてスチレン１．３５ｇ、ジビニルベンゼン０．１５ｇ及び２，２’－アゾビスイソブチロニトリル０．０６ｇを加え、混合してモノマー混合液を作製した。次に、ドデシル硫酸ナトリウム０．７５ｇを溶解させた水溶液７５ｍＬを、得られたモノマー混合液に加え、超音波ホモジナイザー（ＵＤ－２００、ＴＯＭＹ社製）を用いて、氷冷下で、超音波分散処理（２分間の超音波照射（超音波出力：１５０Ｗ）と、その後の２分間の超音波照射の停止の繰り返しを１０回）を行った。
次に、得られたエマルションを、７０℃で７時間重合反応を行い、ネオジム磁石で捕集し純水で十分洗浄することで、ポリマー中にマグネタイトが分散した磁性粒子を得た。
<Comparative example 1>
To 14 g of EMG1400 (manufactured by Ferrotech) as magnetite nanoparticles, 1.35 g of styrene, 0.15 g of divinylbenzene, and 0.0 g of 2,2'-azobisisobutyronitrile were added as monomers for forming particles, and as crosslinking agents. 06g was added and mixed to prepare a monomer mixture. Next, 75 mL of an aqueous solution in which 0.75 g of sodium dodecyl sulfate was dissolved was added to the obtained monomer mixture, and the mixture was ultrasonically dispersed under ice cooling using an ultrasonic homogenizer (UD-200, manufactured by TOMY). Treatment (repetition of ultrasonic irradiation for 2 minutes (ultrasonic output: 150 W) and then stopping the ultrasonic irradiation for 2 minutes 10 times) was performed.
Next, the obtained emulsion was subjected to a polymerization reaction at 70° C. for 7 hours, collected with a neodymium magnet, and thoroughly washed with pure water to obtain magnetic particles in which magnetite was dispersed in the polymer.
（磁性粒子の分析）
得られた磁性粒子１００個のＳＥＭ観察による粒径サイズは約１００ｎｍであった。また、この磁性粒子を５００℃の加熱による重量減少を熱分析装置（ＴＧＡ日立ハイテクノロジー社製）で分析すると、磁性粒子中に１３質量％の樹脂成分が含有されていた。
作製した磁性粒子のＳＥＭ観察を行った。５０００倍の倍率で粒子１００個の長径を測定すると個数平均粒径は６８７ｎｍであった。
(Analysis of magnetic particles)
The particle size of the 100 magnetic particles obtained was about 100 nm as determined by SEM observation. Further, when this magnetic particle was analyzed for weight loss due to heating at 500° C. using a thermal analyzer (TGA, manufactured by Hitachi High-Technology), it was found that 13% by mass of a resin component was contained in the magnetic particle.
The produced magnetic particles were observed by SEM. When the long diameter of 100 particles was measured at a magnification of 5000 times, the number average particle diameter was 687 nm.
＜比較例２＞
反応容器に塩化鉄（ＩＩＩ）６水和物２．７ｇ、塩化鉄（ＩＩ）４水和物１．０ｇ及び水３７５ｍＬに溶解させて攪拌しながら、２８％アンモニア水４ｍＬと水１００ｍＬを混合した溶液を１時間かけて滴下し、滴下後１時間攪拌し、８０℃に昇温後、オレイン酸１０．５ｇを加え、２時間攪拌を継続した。室温に冷却後、ネオジム磁石で捕集して得られたオレイン酸が吸着したマグネタイト粒子を純水で十分洗浄した。得られたオレイン酸が吸着したマグネタイト粒子を、デカン５．７ｍＬ及びＴＥＯＳ２．２ｍＬを加えて混合し、分散液（Ａ）を調製した。
反応容器に２８％アンモニア水溶液３９．０ｍＬ、イソプロパノール５５．４ｍＬ、ソルビタンモノオレエート２．９ｍＬ（富士フイルム和光純薬製）及びポリオキシエチレンアルキルエーテル（三洋化成工業株式会社製）２．０ｍＬを加えてホモジナイザー（ＵＤ－２００、ＴＯＭＹ社製）を用いて混合し、５０℃に昇温後、上記分散液（Ａ）を１時間かけて滴下後、５０℃で１時間反応させた。反応後、ネオジム磁石で捕集し、捕集できない微粒子が残存する上清を除いた。得られた粒子を純水で十分に洗浄して磁性シリカ粒子を得た。
<Comparative example 2>
In a reaction vessel, 2.7 g of iron (III) chloride hexahydrate, 1.0 g of iron (II) chloride tetrahydrate, and 375 mL of water were dissolved, and while stirring, 4 mL of 28% aqueous ammonia and 100 mL of water were mixed. The solution was added dropwise over 1 hour, stirred for 1 hour after the dropwise addition, heated to 80°C, 10.5 g of oleic acid was added, and stirring was continued for 2 hours. After cooling to room temperature, the oleic acid-adsorbed magnetite particles collected with a neodymium magnet were thoroughly washed with pure water. The obtained magnetite particles adsorbed with oleic acid were mixed with 5.7 mL of decane and 2.2 mL of TEOS to prepare a dispersion liquid (A).
Add 39.0 mL of 28% ammonia aqueous solution, 55.4 mL of isopropanol, 2.9 mL of sorbitan monooleate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 2.0 mL of polyoxyethylene alkyl ether (manufactured by Sanyo Chemical Industries, Ltd.) to a reaction container. The mixture was mixed using a homogenizer (UD-200, manufactured by TOMY), heated to 50°C, the above dispersion (A) was added dropwise over 1 hour, and the mixture was reacted at 50°C for 1 hour. After the reaction, the particles were collected using a neodymium magnet, and the supernatant containing residual fine particles that could not be collected was removed. The obtained particles were thoroughly washed with pure water to obtain magnetic silica particles.
（磁性粒子の分析）
作製した磁性粒子をＸＲＤで結晶構造を分析した。その結果、マグネタイト（Ｆｅ３Ｏ４）のピークと非晶質シリカのブロードなピークの混在した回折像が得られ、マグネタイトとシリカの複合層であることを確認した。
また、磁性粒子１００個のＳＥＭ観察による粒径サイズは約４８８ｎｍであった。
(Analysis of magnetic particles)
The crystal structure of the produced magnetic particles was analyzed by XRD. As a result, a diffraction image containing a mixture of magnetite (Fe 3 O 4 ) peaks and amorphous silica broad peaks was obtained, confirming that it was a composite layer of magnetite and silica.
Furthermore, the particle size of 100 magnetic particles was found to be approximately 488 nm by SEM observation.
（磁場応答性の比較）
参考例２、実施例１、２及び比較例１、２の粒子について、磁場応答性の違いを評価した。各粒子５０ｍｇを４０ｍＬの純水に混合し、５０ｍＬ容量のガラス製スクリュー管内で分散させた。次に、円柱型ネオジム磁石（φ３０ｍｍ×ｔ１８ｍｍ、ネオマグ株式会社製）をスクリュー管底部に配置して磁性粒子の捕捉が完了するまでの時間を比較した（表１）。本発明の磁性粒子は、従来構造の粒子と比較して磁場応答性が高いことを確認した。
(Comparison of magnetic field responsiveness)
The particles of Reference Example 2, Examples 1 and 2, and Comparative Examples 1 and 2 were evaluated for differences in magnetic field responsiveness. 50 mg of each particle was mixed with 40 mL of pure water and dispersed in a 50 mL glass screw tube. Next, a cylindrical neodymium magnet (30 mm in diameter x 18 mm in thickness, manufactured by Neomag Co., Ltd.) was placed at the bottom of the screw tube, and the time required to capture the magnetic particles was compared (Table 1). It was confirmed that the magnetic particles of the present invention have higher magnetic field responsiveness than particles of conventional structure.
本実施形態の開示は、以下の構成を含む。
（構成１）
磁性コア粒子と該磁性コア粒子の表面に樹脂層を有する磁性粒子であって、該磁性コア粒子は、複数の磁性ナノ粒子の会合体を含み、
該樹脂層は、カルボキシル基、アミノ基、チオール基、エポキシ基、マレイミド基、及びスクシンイミジル基からなる群より選択される少なくとも１種の官能基を有する樹脂を有することを特徴とする磁性粒子。
（構成２）
前記磁性ナノ粒子は、個数平均粒径が２０ｎｍ以下であり、磁性酸化鉄を含む構成１に記載の磁性粒子。
（構成３）
前記磁性酸化鉄が、マグネタイト（Ｆｅ３Ｏ４）、及びマグヘマイト（γ－Ｆｅ２Ｏ３）の少なくともいずれか一方からなる構成１又は２に記載の磁性粒子。
（構成４）
前記磁性コア粒子の表面にシリカ層を介して形成された前記樹脂層を有する構成１～３のいずれか１項に記載の磁性粒子。
（構成５）
前記樹脂層は、少なくとも２種の第一の樹脂層及び第二の樹脂層を含み構成される構成１～４のいずれか１項に記載の磁性粒子。
（構成６）
前記第一の樹脂層は疎水性樹脂を含む構成５に記載の磁性粒子。
（構成７）
前記第一の樹脂層上に形成された前記磁性粒子の最表面層である第二の樹脂層は、グリシジルメタクリレートに由来するユニットを有する樹脂を含む構成５に記載の磁性粒子。
（構成８）
前記磁性粒子の個数平均粒径が、０．１μｍ以上２．０μｍ以下である構成１～７のいずれか１項に記載の磁性粒子。
（構成９）
前記官能基が、リガンドと結合できる、構成１～８のいずれか１項に記載の磁性粒子。
（構成１０）
構成１～８のいずれか１項に記載の磁性粒子及びリガンドを有し、該リガンドと前記官能基とは化学結合を有する免疫検査用粒子。
（構成１１）
前記リガンドが抗体、又は抗原である、構成１０に記載の免疫検査用粒子。
The disclosure of this embodiment includes the following configurations.
(Configuration 1)
A magnetic particle having a magnetic core particle and a resin layer on the surface of the magnetic core particle, the magnetic core particle including an aggregate of a plurality of magnetic nanoparticles,
A magnetic particle characterized in that the resin layer contains a resin having at least one functional group selected from the group consisting of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group.
(Configuration 2)
The magnetic particles according to configuration 1, wherein the magnetic nanoparticles have a number average particle size of 20 nm or less and contain magnetic iron oxide.
(Configuration 3)
The magnetic particles according to configuration 1 or 2, wherein the magnetic iron oxide is made of at least one of magnetite (Fe 3 O 4 ) and maghemite (γ-Fe 2 O 3 ).
(Configuration 4)
The magnetic particle according to any one of configurations 1 to 3, wherein the resin layer is formed on the surface of the magnetic core particle via a silica layer.
(Configuration 5)
5. The magnetic particle according to any one of configurations 1 to 4, wherein the resin layer includes at least two types of first resin layer and second resin layer.
(Configuration 6)
The magnetic particles according to configuration 5, wherein the first resin layer includes a hydrophobic resin.
(Configuration 7)
The magnetic particles according to configuration 5, wherein the second resin layer, which is the outermost surface layer of the magnetic particles formed on the first resin layer, includes a resin having a unit derived from glycidyl methacrylate.
(Configuration 8)
Magnetic particles according to any one of configurations 1 to 7, wherein the number average particle diameter of the magnetic particles is 0.1 μm or more and 2.0 μm or less.
(Configuration 9)
9. The magnetic particle according to any one of configurations 1 to 8, wherein the functional group is capable of binding to a ligand.
(Configuration 10)
An immunoassay particle comprising the magnetic particle according to any one of Structures 1 to 8 and a ligand, the ligand and the functional group having a chemical bond.
(Configuration 11)
The particle for immunoassay according to configuration 10, wherein the ligand is an antibody or an antigen.
１００…磁性粒子
１０１…磁性コア粒子
１０２…磁性粒子の無機被覆層
１０３…磁性粒子の樹脂層
１０３１…磁性粒子の第一の樹脂層
１０３２…磁性粒子の第二の樹脂層
100...Magnetic particles 101...Magnetic core particles 102...Inorganic coating layer of magnetic particles 103...Resin layer of magnetic particles 1031...First resin layer of magnetic particles 1032...Second resin layer of magnetic particles
Claims (11)
該磁性コア粒子は、複数の磁性ナノ粒子の会合体を含み、
該樹脂層は、カルボキシル基、アミノ基、チオール基、エポキシ基、マレイミド基、及びスクシンイミジル基からなる群より選択される少なくとも１種の官能基を有する樹脂を有することを特徴とする磁性粒子。 A magnetic particle having a magnetic core particle and a resin layer on the surface of the magnetic core particle,
The magnetic core particle includes an aggregate of a plurality of magnetic nanoparticles,
A magnetic particle characterized in that the resin layer contains a resin having at least one functional group selected from the group consisting of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US18/329,748 US20230408502A1 (en) | 2022-06-17 | 2023-06-06 | Magnetic particle and particle for immunological test |
Applications Claiming Priority (2)
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