JP6998004B2 - Magnetic particles for supporting physiologically active substances and their manufacturing methods - Google Patents

Description

The present invention relates to magnetic particles for supporting a physiologically active substance and a method for producing the same.
The "analysis" in the present specification includes both "measurement" for quantitatively or semi-quantitatively determining the amount of the substance to be analyzed and "detection" for determining the presence or absence of the substance to be analyzed. Is done.

Currently, in the field of clinical diagnostic tests, it is required to measure a wide variety of substances that are indicators of disease diagnosis in a short time and with high accuracy for a large number of samples, and to feed back the results to the treatment site promptly and accurately. There is. For example, an analysis system in which a physiologically active substance (protein such as antibody, enzyme, receptor, antigen, nucleic acid substance such as DNA or RNA, sugar chain, etc.) is bound to the particle surface, or immunology using an antigen-antibody reaction. Many accurate quantifications of minute amounts of substances to be analyzed have been carried out by targeted measurements. In particular, as a method for improving detection sensitivity and accuracy, particles in which an antigen or antibody against a substance to be analyzed is supported on the surface of fine particles (so-called magnetic particles) made of a polymer such as polystyrene containing a magnetic substance are used. The method used is known.

Specifically, the substance to be analyzed is trapped by the first antibody bound to the magnetic particles, accumulated by a magnet, and the unreacted substance or the like is washed (so-called B / F separation), and then a signal such as an enzyme or a fluorescent agent is generated. A sandwich method or the like in which a second antibody labeled with a substance is added and analyzed is used. B / F separation can also be used in biochemical applications such as quantification, separation, purification or analysis of target substances such as cells, proteins, nucleic acids or chemicals. Compared with methods such as centrifugation, column separation, and electrophoresis, this analysis method and separation method can be performed on a small amount of sample, and can be performed in a short time without denaturing the target substance. Therefore, magnetic particles are extremely useful functional materials.

When analyzing a biological sample using magnetic particles, it is necessary to immobilize a physiologically active substance such as an antigen or an antibody on the surface of the magnetic particles. As the method, a method of physically or chemically supporting the carrier is used. For example, a physiologically active substance such as an antigen or an antibody is directly immobilized on the magnetic particle by physical adsorption, or a functional group on the surface of the magnetic particle, for example, a carboxyl group, a maleimide group, an amino group, a mercapto group, or a hydroxyl group. A method of binding a physiologically active substance such as an antigen or an antibody to a magnetic particle by a covalent bond via a group, an aldehyde group, an epoxy group or the like is used.
The advantage of the chemical bond method over the physical bond method is that the antibody is bound to the surface of the magnetic particles via a covalent bond, so that the physiologically active substance can be stably supported on the surface.
In particular, in the analysis using magnetic particles, it is desirable to immobilize the primary antibody by a chemical bond because the primary antibody immobilized by a physical bond may be peeled off from the magnetic particles during the B / F separation operation.

The performance of in vitro diagnostic agents used in clinical diagnostic tests largely depends on the performance of the main ingredient. There are two main types of main ingredients. One is a physiologically active substance typified by an antibody that recognizes a target substance, and the other is particles carrying the physiologically active substance. The physical characteristics of particles used as diagnostic agents are mainly evaluated from the viewpoints of particle size, dispersibility, magnetic substance content, and non-specific reaction. It can be said that each of these items has a trade-off relationship, and it is not easy to stably produce and supply particles having an excellent balance of performance.

It can be said that the amount of the bioactive substance fixed on the particle surface depends on the specific surface area, that is, the particle size of the particles. That is, when the specific surface area of a particle is small, the number of physiologically active substances bound to one particle is reduced, and the amount of target substance that can be bound to one particle is also reduced, resulting in a decrease in detection sensitivity. Therefore, it is preferable that the specific surface area of the particles is large to some extent.
However, if the specific surface area of the particles becomes too large more than necessary, many non-specific bonds to which substances other than the target substance bind to the particles occur, which is not preferable. Non-specific adsorption in which magnetic particles capture a substance (non-target substance) that is not the target is caused by the physical and chemical interaction between the non-target substance and the magnetic particles.
Increased non-specific adsorption can lead to inaccurate measurement results and inadequate purification and separation. Therefore, in the past, non-specific adsorption has been reduced or avoided by a process called blocking. Blocking is a method of immobilizing a physiologically active substance on particles and then coating the surface of the particles with a biological material such as albumin or casein. However, there are cases where the covering effect of the blocking agent is not sufficiently obtained, or the action of the blocking agent changes with time due to alteration of the blocking agent, and non-specific adsorption occurs. Therefore, it cannot be said that the effect of the blocking agent on reducing non-specific adsorption is sufficient.

On the other hand, as a problem at the time of producing particles, there is a problem of dispersibility of the particles and, in the case of magnetic particles, the content of magnetic substances contained in the particles.
Water accounts for a very high proportion of the constituent elements of living organisms, and most molecular reactions in living organisms are carried out in an environment involving water. Therefore, in the above-mentioned quantification, separation, purification, analysis, or the like of a target substance in a biological sample, fine particles having a hydrophilic particle surface are often useful.
However, the fine particles are generally hydrophobic, such as metals, inorganic substances, and polymers constituting the particles such as polystyrene and polymethacrylate, and the particles are aggregated and settled, so that the stability cannot be said to be high. Stabilizers such as surfactants may be used to improve dispersibility and storage stability, but the stabilizer itself may cause non-specific adsorption or magnetic particles used in the manufacturing stage. There is a secondary problem that the raw material remains and causes non-specific adsorption.
In particular, when magnetic particles are produced, a magnetic material is used, but the magnetic material itself has the property of easily aggregating.
Further, magnetic particles are required to have magnetic responsiveness such that they can quickly collect magnetism when magnetic is applied. In order to obtain such magnetic responsiveness, it is natural to increase the content of magnetic material in the magnetic particles. There is a need. However, if the content is increased, there arises a problem that the stability of the magnetic particles is lowered. In addition, the difficulty of synthesizing magnetic particles also increases.

Various methods for introducing a functional group onto the surface of magnetic particles have been reported, but most of them are methods in which a monomer having a functional group is added at the time of particle synthesis. For example, by copolymerizing styrene with acrylic acid having a carboxyl group and a magnetic substance, magnetic particles having a carboxyl group introduced on the particle surface can be obtained. For example, there is a technique of copolymerizing a compound having a double bond and further having a functional group via a hydrophilic polymer such as polyethyleglycol as a macromonomer with a monomer such as styrene and bonding the compound to the latex surface. In such a method, how to control the amount of functional groups and the distance between the functional groups becomes a problem, and this causes a difference in production lots, and stably and accurately supports the bioactive substance. The problem is that it may not be possible to produce the particles. This makes it difficult to estimate the functionality of the magnetic particles as a diagnostic agent, and makes it impossible to stably supply the magnetic particles to the production of clinical diagnostic agents.

As a method for quantifying the amount of functional groups on the surface of the introduced magnetic particles as described above, for example, there is a method of performing acid-base titration used for quantifying a carboxyl group and quantifying from the change in electrical conductivity. However, accurate quantification is difficult with this method. The reason is that magnetic particles used for diagnostic agents have a small particle size and a small amount of functional groups, the metal oxides constituting the magnetic material are ionic, and the particles are hydrophobic and have poor dispersibility. This is because it also affects the quantification of the amount of functional groups.
In addition, controlling the distance between the surface of magnetic particles and the functional group for carrying a physiologically active substance for binding a biological sample is important when considering application to biomaterials such as clinical diagnostic agents. This is because the distance from the particle surface is expected to greatly affect the activity of bioactive substances such as antibodies. When a monomer having a carboxyl group is used in the particle synthesis stage, it is considered difficult to precisely control the characteristics of the bioactive substance because the distance between the functional group as the bioactive site and the particles is unknown. ..

In addition to the difficulty in quantifying the amount of functional groups on the surface of the particles, it is difficult to analyze under what conditions the polyethylene glycol derived from the macromonomer is present on the surface of the synthesized particles. Further, in the case of emulsion polymerization or mini-emulsion polymerization, the copolymerization of styrene and macromonomer is difficult to analyze because the polymerization system is more complicated than that of ordinary radical polymerization.

Therefore, it has an excellent performance balance of being able to carry a physiologically active substance and having excellent magnetic responsiveness, and it is difficult to manufacture magnetic particles that can be stably supplied as diagnostic agents without the need for a complicated evaluation system after manufacturing. It cannot be said that it has been fully achieved because there are some problems and the difficulty is increasing because each condition is complicatedly related to each other.

Japanese Unexamined Patent Publication No. 2004-99844 Japanese Unexamined Patent Publication No. 2007-127426 Special Table 2007-527454 Japanese Unexamined Patent Publication No. 2008-191128 Japanese Unexamined Patent Publication No. 2016-207879 Japanese Unexamined Patent Publication No. 2010-123984

As a result of diligent research, the present inventors have succeeded in overcoming the above-mentioned problem regarding the synthesis of magnetic particles. That is, the subject of the present invention is that the particle size distribution is narrow, uniform, excellent dispersibility, and the magnetic substance content is not small, which can provide an analytical reagent having high analysis accuracy and sensitivity and stable production. New magnetic particles for carrying a physiologically active substance, which are magnetic particles, can control the amount of functional groups that carry a physiologically active substance, and can introduce a hydrophilic compound that suppresses a non-specific reaction onto the surface of the magnetic particles. The purpose is to provide the manufacturing method.

In the past, only one problem could be solved, and it could not be solved sufficiently, and only a very complicated and difficult method was known. Surprisingly, the above-mentioned problem is a monomer and a radical polymerization initiator. When performing mini-emulsifier polymerization using an emulsifier or a magnetic material, the emulsifier has a functional group for carrying a physiologically active substance in one molecule as represented by the following general formula (1), and is a hydrophilic segment. And by using a polymer emulsifier which is an amphipathic block copolymer composed of hydrophobic segments and in which the hydrophilic segments suppress non-specific reactions, all the problems can be solved at the same time by a simpler method. be able to.

〔式中、ｎは、２以上の整数であり、ｍは、２以上の整数であり、
Ｒ１及びＲ４は、それぞれ独立して、少なくともいずれか一方が生理活性物質担持用官能基であり、
Ｒ２_－１及びＲ２_－２は、それぞれ独立して、水素原子、ハロゲン原子、アルキル基であり、
Ｒ３は、親水性化合物に由来する官能基であり、
Ｒ５は、疎水性を付与する官能基であり、
Ｒ６は、ハロゲン原子、アルキル基、不飽和結合を含む炭化水素基、又は、乳化剤合成時の開始剤由来の官能基である〕
で表される両親媒性ブロックコポリマーである、前記生理活性物質担持用磁性粒子。
［２］前記Ｒ１又はＲ４が、カルボキシル基、マレイミド基、アミノ基、メルカプト基、ヒドロキシル基、アルデヒド基、及びエポキシ基からなる群から選択される基である、［１］の生理活性物質担持用磁性粒子。
［３］前記Ｒ３における前記親水性化合物が、オリゴエチレングリコール、ポリエチレングリコール、２－メタクリロイルオキシエチルホスホリルコリン（ＭＰＣ）ポリマー、ポリビニルアルコール、ポリビニルピロリドン、ポリアミノ酸、ポリペプチド、単糖類、及び多糖類からなる群から選択される化合物である、［１］又は［２］の生理活性物質担持用磁性粒子。
［４］前記Ｒ５が、水素原子、ハロゲン原子、アルキル基、置換または非置換の芳香族化合物基、カルボニル基、アミド基、アミノ基、アルデヒド基、及びケト基、並びに、アミン、アルデヒド、ケトン、及びエーテルの各化合物に由来する官能基からなる群から選択される１又はそれ以上の官能基である、［１］～［３］のいずれかの生理活性物質担持用磁性粒子。
［５］前記乳化剤の分子量が、４００～１００万である、［１］～［４］のいずれかの生理活性物質担持用磁性粒子。
［６］前記親水性セグメントの分子量が、２００～５０万である、［１］～［５］のいずれかの生理活性物質担持用磁性粒子。
［７］前記疎水性セグメントの分子量が、２００～５０万である、［１］～［６］のいずれかの生理活性物質担持用磁性粒子。
［８］前記Ｒ１又はＲ４のカルボキシル基を有する官能基が、ポリアクリル酸、ポリメタクリル酸、ポリイタコン酸、ポリマレイン酸、ポリフマル酸からなる群から選択されるポリカルボン酸に由来する、請求項１～８のいずれか１項に記載の生理活性物質担持用磁性粒子。
［９］前記Ｒ１又はＲ４のカルボキシル基が１個以上であることを特徴とする、［１］～［８］のいずれか１項に記載の生理活性物質担持用磁性粒子。
［１０］前記Ｒ６が、疎水性側に二重結合が導入された官能基である、［１］～［９］のいずれか一項に記載の生理活性物質担持用磁性粒子。
［１１］モノマー、ラジカル重合開始剤、乳化剤を用いてミニエマルション重合を行う際に、該乳化剤が下記一般式（１）：

〔式中、ｎは、２以上の整数であり、ｍは、２以上の整数であり、
Ｒ１及びＲ４は、それぞれ独立して、少なくともいずれか一方が生理活性物質担持用官能基であり、
Ｒ２_－１及びＲ２_－２は、それぞれ独立して、水素原子、ハロゲン原子、アルキル基であり、
Ｒ３は、親水性化合物に由来する官能基であり、
Ｒ５は、疎水性を付与する官能基であり、
Ｒ６は、ハロゲン原子、アルキル基、不飽和結合を含む炭化水素基、又は、乳化剤合成時の開始剤由来の官能基である〕
で表される化合物であることを特徴とする、生理活性物質担持用磁性粒子の製造方法。
［１２］前記乳化剤の合成が、制御／リビングラジカル重合又はイオン重合である、［１１］の製造方法。 The present invention relates to:
[1] Magnetic particles for carrying a physiologically active substance obtained by polymerizing a monomer, a radical polymerization initiator, an emulsifier, and a magnetic substance, wherein the emulsifier has the following general formula (1):

[In the equation, n is an integer of 2 or more, m is an integer of 2 or more, and
Each of R1 and R4 is independent, and at least one of them is a functional group for carrying a physiologically active substance.
_R2-1 and _R2-2 are independent hydrogen atoms, halogen atoms, and alkyl groups, respectively.
R3 is a functional group derived from a hydrophilic compound.
R5 is a functional group that imparts hydrophobicity and is
R6 is a halogen atom, an alkyl group, a hydrocarbon group containing an unsaturated bond, or a functional group derived from an initiator at the time of emulsifier synthesis]
The magnetic particles for supporting a physiologically active substance, which are amphipathic block copolymers represented by.
[2] For carrying a physiologically active substance according to [1], wherein R1 or R4 is a group selected from the group consisting of a carboxyl group, a maleimide group, an amino group, a mercapto group, a hydroxyl group, an aldehyde group, and an epoxy group. Magnetic particles.
[3] The hydrophilic compound in the R3 comprises oligoethylene glycol, polyethylene glycol, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, polyvinyl alcohol, polyvinylpyrrolidone, polyamino acids, polypeptides, monosaccharides, and polysaccharides. The magnetic particles for carrying a physiologically active substance according to [1] or [2], which are compounds selected from the group.
[4] The R5 contains a hydrogen atom, a halogen atom, an alkyl group, a substituted or unsubstituted aromatic compound group, a carbonyl group, an amide group, an amino group, an aldehyde group, and a keto group, as well as an amine, an aldehyde, a ketone, and the like. The magnetic particle for carrying a physiologically active substance according to any one of [1] to [3], which is one or more functional groups selected from the group consisting of functional groups derived from each compound of ether and ether.
[5] The magnetic particles for supporting a physiologically active substance according to any one of [1] to [4], wherein the emulsifier has a molecular weight of 4 to 1,000,000.
[6] The magnetic particles for supporting a physiologically active substance according to any one of [1] to [5], wherein the hydrophilic segment has a molecular weight of 2 to 500,000.
[7] The magnetic particles for supporting a physiologically active substance according to any one of [1] to [6], wherein the hydrophobic segment has a molecular weight of 2 to 500,000.
[8] Claims 1 to 1, wherein the functional group having a carboxyl group of R1 or R4 is derived from a polycarboxylic acid selected from the group consisting of polyacrylic acid, polymethacrylic acid, polyitaconic acid, polymaleic acid, and polyfumalic acid. Item 8. The magnetic particle for carrying a physiologically active substance according to any one of 8.
[9] The magnetic particle for supporting a physiologically active substance according to any one of [1] to [8], which comprises one or more carboxyl groups of R1 or R4.
[10] The magnetic particle for supporting a physiologically active substance according to any one of [1] to [9], wherein the R6 is a functional group having a double bond introduced on the hydrophobic side.
[11] When performing mini-emulsion polymerization using a monomer, a radical polymerization initiator, and an emulsifier, the emulsifier is used in the following general formula (1):

[In the equation, n is an integer of 2 or more, m is an integer of 2 or more, and
Each of R1 and R4 is independent, and at least one of them is a functional group for carrying a physiologically active substance.
_R2-1 and _R2-2 are independent hydrogen atoms, halogen atoms, and alkyl groups, respectively.
R3 is a functional group derived from a hydrophilic compound.
R5 is a functional group that imparts hydrophobicity and is
R6 is a halogen atom, an alkyl group, a hydrocarbon group containing an unsaturated bond, or a functional group derived from an initiator at the time of emulsifier synthesis]
A method for producing magnetic particles for supporting a physiologically active substance, which is characterized by being a compound represented by.
[12] The production method according to [11], wherein the synthesis of the emulsifier is controlled / living radical polymerization or ionic polymerization.

According to the present invention, magnetic particles having a narrow particle size distribution, uniform distribution, excellent dispersibility, and a magnetic substance content not small, which can provide an analytical reagent having high analysis accuracy and sensitivity and stable production. A novel magnetic particle for carrying a physiologically active substance and its production, which can control the amount of functional groups carrying a physiologically active substance and can introduce a hydrophilic compound that suppresses a non-specific reaction onto the surface of the magnetic particle. A method can be provided.

Evaluation of in vitro diagnostic agents for magnetic particles for carrying physiologically active substances modified with antibodies

As used herein, the term "alkyl group" means a saturated linear or branched chain monovalent hydrocarbon. The alkyl group may be, for example, 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 1 to. Contains 3 carbon atoms.

As used herein, the term "hydrocarbon containing an unsaturated bond" means a linear or branched monovalent hydrocarbon having at least one unsaturated bond, eg, a double bond or a triple bond. For example, an alkenyl group having a double bond and an alkynyl group having a triple bond can be mentioned. The hydrocarbon containing the unsaturated bond is, for example, 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, and 2 to 4 carbon atoms. Contains carbon atoms, or 2-3 carbon atoms.

As used herein, the term "polyamino acid" is not particularly limited as long as the basic skeleton is a dehydrated and condensed amino acid and is a polyamino acid exhibiting hydrophilicity as a whole. For example, glutamate and asparagine. 20 kinds of essential amino acids such as acid, arginine and lysine, L-ornithin, a series of α-amino acids, β-alanine, γ-aminobutyric acid, neutral amino acids, acidic amino acids, ω-esters of acidic amino acids, basic amino acids, Examples thereof include N-substituted basic amino acids, amino acids such as aspartic acid-L-phenylalanine dimer (aspartame) and amino acid derivatives, and aminosulfonic acids such as L-cysteine acid. The α-amino acid may be an optically active substance (L form, D form) or a racemate form. These amino acids may be homopolyamino acids consisting of only one type of amino acid, such as polyglutamic acid, polyaspartic acid, polyarginine, polylysine, etc., or polyamino acids consisting of two or more types of amino acids. You can also. Examples of the copolymer component containing two or more kinds of amino acids include aminocarboxylic acid, aminosulfonic acid, aminophosphonic acid, hydroxycarboxylic acid, mercaptocarboxylic acid, mercaptosulfonic acid, mercaptophosphonic acid and the like.

In the present specification, the "monosaccharide" or "polysaccharide" used as the hydrophilic compound is not particularly limited as long as it is a monosaccharide or a polysaccharide exhibiting hydrophilicity as a whole, and the monosaccharide may be used. For example, glucose, galactose, xylose, fructose and the like can be mentioned, and examples of the polysaccharide include trehalose, sucrose, lactose, maltose, raffinose, gluconic acid, dextran, dextrin, agarose and carboxymethyl (CM)-. Examples thereof include cellulose, heparin, and soluble starch. The polysaccharide may be a straight chain or a polysaccharide having a fractionated chain.

In the present specification, the "aromatic compound group" in the "substituted or unsubstituted aromatic compound group" is preferably toluene, styrene, benzene or the like, and styrene is more preferable in terms of cost and ease of polymerization.
Examples of the substituent include an α-, o-, m- or p-form alkyl group (for example, an alkyl group having 1 to 20 carbon atoms), a hydroxyl group, and an alkoxyl group (for example, an alkoxyl having 1 to 20 carbon atoms). Groups), halogen atoms (eg, fluorine atoms, chlorine atoms, bromine atoms), cyano groups, amino groups, nitro groups, acyl groups, carboxylic acid (salt) groups, haloalkyls, ester substituents and the like. Examples of those having these substituents include α-methylstyrene, halogenated styrene, divinylbenzene, styrenesulfonic acid, 2,4-dimethylstyrene, paradimethylaminostyrene, vinylbenzyl chloride, vinylbenzaldehyde, inden, and 1, -A polymerizable unsaturated aromatic compound such as methylinden, acenaphthalene, vinylnaphthalene, vinylanthracene, vinylcarbazole, 2-vinylpyridine, 4-vinylpyridine, and 2-vinylstyrene can be mentioned.

The magnetic particles for carrying a physiologically active substance of the present invention include a monomer, a radical polymerization initiator, an emulsifier, and a magnetic substance, and the emulsifier has the characteristics described below.

で表され、少なくとも以下の特徴：
[１]疎水性セグメント及び親水性セグメントを有する両親媒性ブロックコポリマーであって、
[２]前記親水セグメント（Ｒ１又はＲ４のいずれか一カ所以上）に生理活性物質担持用官能基を有する。
本願発明における、ブロックコポリマーとは、構成単位となる化合物が２種類以上である場合とする。 The emulsifier that can be used in the present invention has the following general formula (1):

Represented by, at least the following features:
[1] An amphipathic block copolymer having a hydrophobic segment and a hydrophilic segment.
[2] The hydrophilic segment (either one or more of R1 or R4) has a functional group for supporting a physiologically active substance.
In the present invention, the block copolymer is a case where two or more kinds of compounds as constituent units are used.

The emulsifier and the monomer may or may not be covalently bonded. For example, if the emulsifier has a polymerizable double bond within or at the end of the hydrophobic segment, it can be covalently bonded to the magnetic particles. On the other hand, since the hydrophobic segment of the emulsifier strongly interacts with the magnetic particles, the emulsifier added during the polymerization reaction is strongly bonded to the magnetic particles and can maintain a stable state without covalent bonding. The polymerization reaction is easy and preferable.

The main chain of the emulsifier that can be used in the present invention [-(CH ₂ -C) _n- (CH ₂ -C) _m- ] consists of an alkyl chain.
The molecular weight (number average molecular weight) of the emulsifier that can be used in the present invention is 400 or more and 1 million or less, preferably 500 or more and 500,000 or less, more preferably 600 or more and 300,000 or less, and particularly preferably 1000 or more and 200,000 or less. Is. The lower limit and the upper limit may be arbitrarily combined as desired.
Further, as the molecular weight distribution of the emulsifier that can be used in the present invention, Mw (weight average molecular weight) / Mn (number average molecular weight) having a polydispersity is 1 or more and 2 or less, preferably 1.8 or less, more preferably. It should be 1.3 or less. It can be easily synthesized by controlled / living radical polymerization or ionic polymerization. The lower limit and the upper limit may be arbitrarily combined as desired.

The hydrophilic segment of the emulsifier that can be used in the present invention may be hydrophilic as a whole. Further, the lengths of the main chain [-( _CH2 -C) _m- ] and the graft chain [-R3-R4] of the hydrophilic segment should be appropriately selected from known ones or synthesized arbitrarily. Can be done.
The hydrophilic segment has a molecular weight of 200 or more and 500,000 or less, preferably 250 or more and 400,000 or less, more preferably 300 or more and 300,000 or less, and particularly preferably 500 or more and 250,000 or less. m is an integer of 2 or more, preferably an integer of 2 or more and 2000 or less, and more preferably an integer of 3 or more and 1000 or less. The lower limit and the upper limit may be arbitrarily combined as desired.

The graft chain of the hydrophilic segment contains a hydrophilic compound moiety and has a functional group derived from the hydrophilic compound as the group R3.
Examples of the hydrophilic compound that can be used in the present invention include oligoethylene glycol, polyethylene glycol, oligoethylene glycol alkyl ether (meth) acrylate, polyethylene glycol alkyl ether (meth) acrylate, and 2-methacryloyloxyethyl phosphorylcholine (MPC). Examples include polymers, polyvinyl alcohols, polyvinylpyrrolidones, polycarboxylic acids (eg, polyacrylic acid, polymethacrylic acid, polyitaconic acid, polymaleic acid, polyfumaric acid), polyamino acids, polypeptides, monosaccharides, polysaccharides and the like. Any hydrophilic compound that can be synthesized as an emulsifier that can be used in the present invention can be appropriately selected and used from known ones.
The molecular weight of the group R3 is preferably 40 to 10,000, more preferably 80 to 5000. The lower limit and the upper limit may be arbitrarily combined as desired.

At least one of the groups R1 and R4 in the hydrophilic segment is a functional group for carrying a bioactive substance. The bioactive substance is supported by chemically bonding with the functional group for supporting the bioactive substance in the hydrophilic segment. As the functional group for carrying a physiologically active substance that can be used in the present invention, a known functional group for carrying a physiologically active substance can be appropriately selected and used. For example, a carboxyl group, a maleimide group, an amino group, or a mercapto group can be used. , A hydroxyl group, an aldehyde group, an epoxy group and the like. The type and number can be appropriately selected according to the purpose. By using an emulsifier into which a functional group for supporting a physiologically active substance is introduced, the amount of the functional group for supporting a physiologically active substance to be introduced into one fine particle can be easily controlled.

For example, the group R3 is oligoethylene glycol, polyethylene glycol, oligoethylene glycol alkyl ether (meth) acrylate, polyethylene glycol alkyl ether (meth) acrylate, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, polyvinyl alcohol, polyvinylpyrrolidone, poly. In the case of a functional group derived from an amino acid, a polypeptide, a monosaccharide or a polysaccharide, the hydroxyl group of these hydrophilic compounds may be the above-mentioned carboxyl group, maleimide group, amino group, mercapto group, hydroxyl group, aldehyde group or epoxy. By substituting with a group or the like, the group R4 can be used as a functional group for carrying a physiologically active substance.
Alternatively, when the group R3 is a functional group derived from a polycarboxylic acid (for example, polyacrylic acid, polymethacrylic acid, polyitaconic acid, polymaleic acid, polyfumaric acid), the carboxyl group of the polycarboxylic acid is the group R4 (that is, that is). , Functional group for carrying physiologically active substances).

When the group R1 or R4 is not a functional group for carrying a physiologically active substance, it can be appropriately selected from known atoms and functional groups and used. For example, a hydrogen atom, a halogen atom and an alkyl group (preferably having 1 carbon number) can be used. 20 to 20 lower alkyl groups, such as methyl group, ethyl group).

The groups _R2-1 and _R2-2 are not particularly limited as long as they do not interfere with the amphipathic nature of the emulsifier and the function of the functional group for carrying a physiologically active substance, but are not particularly limited, for example, hydrogen atom, halogen atom and alkyl group. Etc. are preferable. _R2-1 and _R2-2 may be the same or different. It can be appropriately selected and used according to each compound and synthesis method.

で表されるように、２種類以上のモノマーユニットを含む構造であってもよい。この場合、１分子内に多数の生理活性物質を担持できることから、生理活性物質を効率よく磁性粒子に修飾することが期待できる。 Further, the hydrophilic segment is described by the following general formula (2) :.

As shown by, the structure may include two or more types of monomer units. In this case, since a large number of physiologically active substances can be supported in one molecule, it can be expected that the physiologically active substances can be efficiently modified into magnetic particles.

The monomer unit can exist as a block copolymer or a random copolymer in the hydrophilic segment. Further, it may be located at the end of the hydrophilic segment, or may have a structure in which the hydrophilic segment is mixed, for example, as a repeating sequence. The hydrophilic segment located at the end (for example, the hydrophilic segment 2 in the general formula (2)) can function as a segment for carrying a physiologically active substance, and the hydrophilic segment located inside (for example, the general formula (2)). The hydrophilic segment 1) in) can function as a non-specific reaction suppressing segment and / or a physiologically active substance carrying segment.

In the emulsifier of the present invention (hereinafter referred to as a polyhydrophilic segment-containing emulsifier) containing two or more types of hydrophilic segments as in the general formula (2), the present invention is similarly used in the case of the general formula (1). The main chain of the emulsifiers that can be used [eg-( _CH2 -C) _n- ( _CH2 -C) _m- ( _CH2 -C) _O ] consists of an alkyl chain.
The molecular weight (number average molecular weight) of the emulsifier that can be used in the present invention is 400 or more and 1 million or less, preferably 500 or more and 500,000 or less, more preferably 600 or more and 300,000 or less, and particularly preferably 1000 or more and 200,000 or less. Is. The lower limit and the upper limit may be arbitrarily combined as desired.
Further, as the molecular weight distribution of the emulsifier that can be used in the present invention, Mw (weight average molecular weight) / Mn (number average molecular weight) having a polydispersity is 1 or more and 2 or less, preferably 1.8 or less, more preferably. It should be 1.3 or less. It can be easily synthesized by controlled / living radical polymerization or ionic polymerization. The lower limit and the upper limit may be arbitrarily combined as desired.

In the emulsifier containing a polyhydrophilic segment, the hydrophilic segment of the emulsifier that can be used in the present invention may be hydrophilic as a whole. Further, the lengths of the main chain [-(CH ₂ -C) _m- ] and the graft chain [-R3-1 _{- R4-1} ] of the hydrophilic segment can be appropriately selected from known ones or synthesized arbitrarily. Can be done.
The hydrophilic segment has a molecular weight of 200 or more and 500,000 or less, preferably 250 or more and 400,000 or less, more preferably 300 or more and 300,000 or less, and particularly preferably 500 or more and 250,000 or less. m is an integer of 2 or more, preferably an integer of 2 or more and 2000 or less, and more preferably an integer of 3 or more and 1000 or less. The lower limit and the upper limit may be arbitrarily combined as desired.

In the polyhydrophilic segment-containing emulsifier, the graft chain of the hydrophilic segment contains a hydrophilic compound moiety, and is functionally derived from the hydrophilic compound, for example, as the group _R3-1 or _R3-2 in the general formula (2). Has a group.
Examples of the hydrophilic compound that can be used in the present invention include oligoethylene glycol, polyethylene glycol, oligoethylene glycol alkyl ether (meth) acrylate, polyethylene glycol alkyl ether (meth) acrylate, and 2-methacryloyloxyethyl phosphorylcholine (MPC). Examples include polymers, polyvinyl alcohols, polyvinylpyrrolidones, polycarboxylic acids (eg, polyacrylic acid, polymethacrylic acid, polyitaconic acid, polymaleic acid, polyfumaric acid), polyamino acids, polypeptides, monosaccharides, polysaccharides and the like. Any hydrophilic compound that can be synthesized as an emulsifier that can be used in the present invention can be appropriately selected and used from known ones.

In the polyhydrophilic segment-containing emulsifier (for example, the general formula (2)), at least one of the groups R1, _R4-1 , or _R4-2 in the hydrophilic segment is a functional group for supporting a physiologically active substance. The bioactive substance is supported by chemically bonding with the functional group for supporting the bioactive substance in the hydrophilic segment. As the functional group for carrying a physiologically active substance that can be used in the present invention, a known functional group for carrying a physiologically active substance can be appropriately selected and used. For example, a carboxyl group, a maleimide group, an amino group, or a mercapto group can be used. , A hydroxyl group, an aldehyde group, an epoxy group and the like. The type and number can be appropriately selected according to the purpose. By using an emulsifier into which a functional group for supporting a physiologically active substance is introduced, the amount of the functional group for supporting a physiologically active substance to be introduced into one fine particle can be easily controlled.

For example, the groups _R3-1 or _R3-2 are oligoethylene glycol, polyethylene glycol, oligoethylene glycol alkyl ether (meth) acrylate, polyethylene glycol alkyl ether (meth) acrylate, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, polyvinyl. In the case of a functional group derived from alcohol, polyvinylpyrrolidone, polyamino acid, polypeptide, monosaccharide or polysaccharide, the hydroxyl group of these hydrophilic compounds may be the above-mentioned carboxyl group, maleimide group, amino group, mercapto group or hydroxyl group. , The group _R4-1 and _R4-2 can be used as a functional group for carrying a physiologically active substance by substituting with an aldehyde group, an epoxy group or the like.
Alternatively, when the group _R3-1 or _R3-2 is a functional group derived from a polycarboxylic acid (for example, polyacrylic acid, polymethacrylic acid, polyitaconic acid, polymaleic acid, polyfumaric acid), the carboxyl of the polycarboxylic acid. The group functions as a group _R4-1 or _R4-2 (that is, a functional group for carrying a physiologically active substance).

When the group R1, _R4-1 or _R4-2 is not a functional group for carrying a physiologically active substance, it can be appropriately selected from known atoms and functional groups and used. For example, a hydrogen atom, a halogen atom and an alkyl group can be used. (Preferably, a lower alkyl group having 1 to 20 carbon atoms, for example, a methyl group or an ethyl group) can be mentioned.

The groups _R2-1 , _R2-2 , and _R2-3 are not particularly limited as long as they do not interfere with the amphipathic nature of the emulsifier and the function of the functional group for carrying a physiologically active substance, but are not particularly limited, and are, for example, hydrogen atoms. Halogen atoms, alkyl groups and the like are preferred. _R2-1 and _R2-2 may be the same or different. It can be appropriately selected and used according to each compound and synthesis method.

The hydrophobic segment of the emulsifier that can be used in the present invention may be hydrophobic as a whole. Further, the lengths of the main chain [-(CH ₂ -C) _n- ] and the graft chain [-R5] of the hydrophobic segment can be appropriately selected from known ones or synthesized arbitrarily. ..
The hydrophobic segment has a molecular weight of 200 or more and 500,000 or less, preferably 300 or more and 400,000 or less, more preferably 500 or more and 300,000 or less, and particularly preferably 1,000 or more and 250,000 or less. n is an integer of 2 or more, preferably an integer of 2 or more and 5000 or less, and more preferably an integer of 3 or more and 2000 or less. The lower limit and the upper limit may be arbitrarily combined as desired.

The group R5 is not particularly limited as long as it is hydrophobic as a whole of the hydrophobic segment, but for example, a hydrogen atom, a halogen atom, an alkyl group, a substituted or unsubstituted aromatic compound group, a carbonyl group, an amide group, and the like. Examples thereof include an amino group, an aldehyde group, a keto group, or a functional group derived from each compound of amine, aldehyde, ketone, or ether (for example, a group obtained by removing one or more atoms from each of the above compounds). .. R5 can all be the same group, or can be two or more different groups.

The main chain and _R2-1 and R5 of such a hydrophobic segment can be obtained, for example, by polymerizing a monomer having a carbon double bond. Examples of the monomer include ethylene, propylene, styrene sulfonic acid, styrene, methacrylate, acrylate, acrylamide, methacrylamide, and the like, and one of these can be used alone or in combination of two or more.

The terminal R6 of the hydrophobic segment of the emulsifier that can be used in the present invention can be appropriately selected from known ones and used according to the polymerization method of the emulsifier. For example, a functional group derived from the starting group used for the polymerization of the emulsifier, a halogen atom, an alkyl group, and a hydrocarbon group containing an unsaturated bond can be mentioned. Further, according to the polymerization method for synthesizing the magnetic particles, it is also possible to appropriately select and use from known ones. For example, when covalently bonding with a monomer when synthesizing magnetic particles, a double bond can be introduced.

For the synthesis of emulsifiers that can be used in the present invention, for example, controlled / living radical polymerization, ionic polymerization can be used. Examples of the controlled / living radical polymerization include RAFT (Reversible Addition Fragmentation chain Transfer Polymerization), NMP (Nitroxide Distributed Polymerization), ATRP (Atom Transfer Radicalization) and the like. By using an emulsifier synthesized by controlled / living radical polymerization and ionic polymerization, the difference between production lots of the magnetic particles for carrying a physiologically active substance of the present invention can be reduced.
As these synthetic methods, a suitable method can be selected depending on the functional group for supporting the physiologically active substance to be introduced into the hydrophilic segment.

The synthesis of the emulsifier that can be used in the present invention can be appropriately selected from known methods and used. For example, when the synthesis is started from the hydrophobic segment and then the hydrophilic segment is synthesized, or the hydrophilic segment is synthesized. There is a method of synthesizing from, and then synthesizing a hydrophobic segment. A suitable method can be selected according to the purpose.

As the radical polymerization initiator that can be used in the present invention, a radical polymerization initiator that can be used in ordinary mini-emulsion polymerization can be used, and for example, a peroxidation initiator, a persulfate initiator, an azo-based initiator, and the like. Examples thereof include an azo-based low-temperature initiator or a redox initiator. In the present invention, it is preferable to use a redox initiator or an azo-based low-temperature initiator in that the polymerization can be carried out at a temperature lower than the phase inversion emulsification temperature, but there are no particular restrictions.

Examples of the peroxide initiator include benzoyl peroxide (BPO), di-t-butyl peroxide (DBPO), and ammonium peroxide. Examples of the persulfate initiator include potassium persulfate (KPS), ammonium persulfate (APS), and sodium persulfate (NPS).

Examples of the azo-based initiator include azobisisobutyronitrile (AIBN), dimethyl 2,2'-azobisisobutyrate (MAIB), 4,4'-azobis (4-cyanovaleric acid), and 2 , 2'-azobis (2,4-dimethylvaleronitrile) can be mentioned. Among the azo-based initiators, the azo-based low-temperature initiator that can be carried out at a low temperature includes, for example, VA-044 (Wako Pure Chemical Industries, Ltd.), which is a water-soluble azo polymerization initiator, or V, which is an oil-soluble azo polymerization initiator. -70 (Wako Pure Chemical Industries, Ltd.) and the like can be mentioned.

Examples of the redox initiator include N, N, N', N'-tetramethylethylenediamine [N, N, N', N'-tetramethylethylenediamine (TMEDA)] / potassium persulfate [potassium persulfate (KPS)], and the like. Examples thereof include FeSO ₄ / KPS, FeSO ₄ / H ₂ O ₂ , ascorbic acid (vitamin C) / H ₂ O ₂ .

The magnetic substance contained in the magnetic substance-containing fine particles of the present invention is not particularly limited as long as it is a magnetic substance, but iron oxide-based substances are typical, and ferrite and magnetite represented by Fe ₃ O ₄ are mentioned. Be done. In particular, γFe ₂ O ₃ and Fe ₃ O ₄ are preferable as magnetic materials having high saturation magnetization and low residual magnetization. The magnetic fine particles used in the present invention are required to have low residual magnetization, and for example, ferrite and / or magnetite fine particles having a particle diameter of about 5 to 20 nm can be preferably used.

The content of the magnetic particles in the fine particles is 10% by weight or more with respect to the weight of the polymer. In order to exhibit sufficient magnetic responsiveness, a high magnetic substance content is preferable, the content is preferably 20% by weight or more, more preferably 25% by weight or more, still more preferably 30% by weight or more. Is. On the other hand, in order to prevent the magnetic material from being exposed on the surface of the resin fine particles, the magnetic material content is usually 70% by weight or less, preferably 60% by weight or less, and particularly preferably 50% by weight or less. be.

As the monomer that can be used in the present invention, a monomer that can be used in ordinary miniemulsion polymerization can be used. For example, styrene, styrene derivatives (eg, chloromethylstyrene, sodium styrene sulfonate), divinylbenzene, acrylic acid or methacrylic acid, itaconic acid, maleic anhydride, maleic acid, phthalic acid, acrylic acid ester or methacrylic acid ester [eg , Methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, hexadecyl (meth) acrylate], vinyl acetate. Further, these monomers may be used as a mixture of two or more kinds.

The method for producing the magnetic particles for carrying a physiologically active substance of the present invention is not particularly limited as long as the magnetic particles for carrying a physiologically active substance of the present invention can be produced. Other than the use of available emulsifiers, conventionally known mini-emulsion polymerizations (eg, M. Antonietti, K. Landfester, Prog. Polym. Sci., 2002, 27, 689-757, JM Asua, Prog. Polym. It can be carried out in the same manner as Sci., 2002, 27, 1283-1346).

Normal mini-emulsion polymerization is not limited to this, and for example, a step of mixing a monomer, a radical polymerization initiator, an emulsifier, and a magnetic substance, a step of shearing the mixture, and a step of shearing the mixture up to the polymerization start temperature. It can include a step of heating and polymerizing. In miniemulsion polymerization, after mixing the monomer for polymerization and the emulsifier, for example, by performing a shearing step by ultrasonic irradiation, the monomer is torn off by the shearing force, and monomer micro oil droplets covered with the emulsifier are formed. Will be done. Then, by heating to the polymerization start temperature of the radical polymerization initiator, the monomer fine oil droplets are polymerized as they are, and magnetic particles are obtained.

The reaction conditions during the polymerization of the magnetic particles, such as the solvent, mixing ratio, temperature, reaction time, etc., are the average of the monomer used, the type of functional group for carrying the physiologically active substance, the initiator, the emulsifier, and the magnetic particles to be synthesized. It can be appropriately determined, for example, by conducting a pilot test, depending on the particle size, the amount of the physiologically active substance carried on the surface of the fine particles, and the like.

Long-chain alcan or higher alcohol soluble in the monomer is dissolved in the monomer oil droplets and added as a hydroforve to increase the osmotic pressure of the oil droplets to improve the stability of the oil droplets and the grains by Ostwald ripening. It is possible to suppress an increase in diameter non-uniformity and synthesize monodisperse latex particles.
As the hydroforb that can be used in the present invention, a hydroforb that can be used in ordinary mini-emulsion polymerization can be used, and for example, hexadecane, silsesquioxane, or a hydrophobic polymer (for example, polystyrene, polymethacrylic acid) can be used. (Methyl) can be mentioned, and hexadecane or polystyrene is preferable.

As mentioned above, in general, the particle size can be made uniform by using a hydroforve, but in addition, the present inventors use a hydroforve to cause phase separation in magnetic particles, which causes magnetism. It has been found that the magnetic material contained in the particles may not be uniform. As the particle size becomes smaller, the content of magnetic material contained in the magnetic particles may decrease. Therefore, when producing magnetic particles using Hydroforb, the magnetic material content can be adjusted by adjusting the ratio with the polymerized monomer. Can be adjusted. A person skilled in the art can evaluate the characteristics of the produced magnetic particles and set these ratios as appropriate.

Further, by carrying out the polymerization reaction in the presence of fluorescent molecules, magnetic particles having a fluorescent function can also be produced. For example, a rare earth metal chelate complex is preferable because it has a long fluorescence lifetime and a narrow spectral width, and thus noise due to the background can be avoided.

Examples of the rare earth metal constituting such a fluorescent rare earth metal chelate complex include europium, samarium, terbium, and dysprosium.
Particles having such a fluorescent function can be used in a time-resolved fluorescent immunoassay, which is one of the measuring methods for in vitro diagnostic agents. The time-resolved fluorescence immunoassay method for measuring the generated fluorescence in a time-resolved manner is preferable in that it can enable more sensitive measurement.

Analytical reagents can be prepared according to known methods, except that the magnetic particles for supporting the physiologically active substance of the present invention are used.
The analytical reagent shown in the present invention is a reagent for analyzing a physiologically active substance, which is obtained by carrying a physiologically active substance capable of reacting with the analysis target substance in a biological sample on magnetic particles for carrying the physiologically active substance.
Further, the combination of the physiologically active substance and the substance to be analyzed, and the reaction between the physiologically active substance and the functional group for supporting the physiologically active substance can be appropriately selected from known methods and used.

Considering in line with the prior art, it is possible to modify the surface of magnetic particles with a water-soluble polymer by a polymerization method in which a monomer is polymerized while dispersing magnetic particles in a liquid phase to form a polymer that encloses the particles. Even if there is, it is difficult to control the molecular weight and the like of the interparticle distance, and it is considered difficult to increase the content of the magnetic substance in the magnetic particles having a small particle size. According to the report, the fact that the magnetic substance content is relatively large and the particles in which the magnetic particles are uniformly dispersed can be produced has a remarkable effect.

Examples of the physiologically active substance that can be used in the embodiment of the present invention include substances that can react with the substance to be analyzed in the biological sample, such as antigens, antibodies, enzymes, receptors, DNA, RNA, and sugar chains. Can be mentioned.

The substances to be analyzed in the biological sample that can be used in the embodiment of the present invention include IgG, C-reactive protein (CRP), ferritin, β-2 microglobulin, α-fetoprotein (AFP), IgE, and hepatitis B. Virus (HBS antibody or HBc antibody), D dimer, fibrin-fibrinogen degradation product (FDP), soluble fibrin (SF), plasmin-α2-plasmin inhibitor complex (PPI), prostate-specific antigen (PSA), elastase 1. Elastase XDP, thrombomodulin (TM), or albumin (preferably serum albumin), thrombin-anti-thrombin complex (TAT), preceptin, myoglobin, CEA, AFP, ferritin, β2M, PIVKA2, PSA, PAP, CA19-9. , CA125, CA15-3, CA72-4, ST-439, SCC antigen, γ-SM, NSE, BCA225, pepsinogen (I, II), proGRP, cifra, HER2 / neu, thyroid stimulating hormone (TSH), thyroid hormone (T3, T4, FT3, FT4), T3UPTAKE, cortisol, luteinizing hormone (LH), follicular stimulating hormone (FSH), prolactin (PRL), growth hormone (HGH), ACTH, insulin, C peptide, DHEA-S, Estradiol (E2) Estriol (E3), testosterone, progesterone, human chorionic gonadotropin (HCG, HCGpreg, β-HCG), PTH, intact PTH, wholePTH, hANP, osteocalcin, calcitonin, procalcitonin, thyroglobulin, human brain sodium diuresis Peptide (BNP), human brain sodium diuretic peptide precursor N-end fragment (NTproBNP), PINP, urinary deoxypyridinoline, BAP, β-crossplus (urine), HCV antibody, HCV core antibody, HBs antigen, HBs antibody, HBe antigen / antibody, HBc antibody IgM type HBc antibody, hepatitis B virus core related antigen, HAV antibody, IgM type HAV antibody, IgG type HAV antibody, HIV antigen / antibody, HIV antibody 1/2, HIVp24 antigen, HTLV-1 Antibodies, IgG cytomegalo antibody, syphilis TP antibody, anti-CCP antibody, total IgE, specific IgE, anti-nuclear antibody, anti-DNA antibody, anti-ssDNA antibody, anti-ds antibody, anti-TSH receptor antibody, anti-thyroid peroxidase antibody, anti-cypress Loglobulin antibody, anti-mitanitic antibody, anti-SSA / Ro antibody, anti-SSB / La antibody, anti-Sm antibody, anti-RNP antibody, anti-galactose-deficient IgG antibody, P-ANCA, MPO-ANCA, IgG, IgA, IgM, IgD, Myoglobin, C3 / C4, Troponin T, Troponin I, α1-M, KL-6, Eosinophilic Basic Protein (ECP), Phenitoin, Phenobarbital, Carbamazepine, Valproic Acid, Tobramycin, Gentamycin, Bancomycin, Theophylline, Digoxin, Cyclosporin, tachlorimus, folic acid, CK-MB, VB12, sIL-2R, EPO and the like can be mentioned.

For example, when an antibody is used as the physiologically active substance, a monoclonal antibody or a polyclonal antibody can be used. Further, as the type of antibody, in addition to the immunoglobulin molecule itself, an antibody fragment such as Fab, Fab', F (ab') ₂ or Fv can be used.
For example, when DNA is used as the physiologically active substance, a DNA probe having about 5 to 100 bases complementary to the substance to be analyzed can be used.

The analyzable test sample that can be used in the embodiment of the present invention is not particularly limited as long as it is a sample that may contain the substance to be analyzed, and in particular, a biological sample such as blood, serum, plasma, etc. Examples include urine, plasma, or cell or tissue disruption fluid.

The analytical reagent that can be used in the embodiment of the present invention can be used by a known latex method (for example, latex agglutination method, B / F separation using latex).
In the latex agglomeration method, the degree of agglomeration (aggregation degree) generated when the analytical reagent and the test sample are brought into contact with each other in a liquid is optically analyzed (particularly measured) in the test sample. It is possible to analyze (especially measure) the amount of the substance to be analyzed. In a specific method for optically detecting the degree of aggregation of latex particles, the measurement can be performed using, for example, an optical instrument for measuring scattered light intensity, absorbance, or transmitted light intensity. The preferred measurement wavelength is 300 to 800 nm. The measuring method is to measure the increase or decrease of scattered light intensity, absorbance, or transmitted light intensity by selecting the size (average particle size) or concentration of the latex particles to be used or setting the reaction time according to a known method. Can be done by. It is also possible to use these methods together.
In B / F separation using latex, the analytical reagent and the test sample are brought into contact with each other in a liquid, and then B / F separation is performed to separate the latex particles and the liquid and bond them to the latex particles. The amount of the substance to be analyzed in the test sample can be analyzed (particularly measured) by analyzing (particularly measuring) the substance to be analyzed or the substance to be analyzed remaining in the liquid.

Hereinafter, the present invention will be specifically described with reference to Examples, but these do not limit the scope of the present invention.
<< Example 1: Synthesis of macro-RAFT agent HOOC-POEGMA ₁₄ >>

In a 100 mL eggplant flask, 7.0 g of oligo (ethylene glycol) methyl ether methyllate, OEGMA, manufactured by Shin-Nakamura Chemical Industry Co., Ltd., 4-cyano-4-[(dodecylsulfanyl) as a RAFT agent. Thiocarbonyl) sulfanyl] pentanic acid (4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanic acid, CSPA, manufactured by Sigma-Aldrich) 0.4 g, 1,4-dioxane (manufactured by Kanto Chemical Co., Ltd.) 20 mL, 2, 0.016 g of 2'-azobisisobutyronitrile (2,2'-azobisisobutyronirile, AIBN, manufactured by Tokyo Kasei Co., Ltd.) was added, and freeze degassing was performed three times. Then, it was polymerized at 70 ° C. for 9 hours under a nitrogen atmosphere. The reaction solution was removed by an evaporator, diluted with tetrahydrofuran (THF, manufactured by Kanto Chemical Co., Inc.), and then reprecipitated with hexane (manufactured by Kanto Chemical Co., Inc.) three times to obtain the macro-RAFT agent HOOC-POEGMA ₁₄ . Purified.
¹ Using H-NMR (Burker DPX300NMR, manufactured by Buruker), the average degree of polymerization of the macro RAFT agent HOOC-POEGMA ₁₄ is 14, and using gel permeation chromatography (HLC-8220GPC, manufactured by TOSOH), It was confirmed that the polydispersity of the macro-RAFT agent HOOC-POEGMA ₁₄ was 1.25.

<< Example 2: Synthesis of amphipathic block copolymer HOOC- (POEGMAN-b-PStm) >>

HOOC-POEGMA ₁₄ 6.0 g, styrene (manufactured by Kanto Chemical Co., Inc.) 0.67 g, toluene (manufactured by Kanto Chemical Co., Inc.) 6.4 mL, and AIBN 0.013 g were added to a 100 mL eggplant flask, and freeze degassing was performed three times. The mixture was polymerized at 80 ° C. for 24 hours under a nitrogen atmosphere. The reaction solution is removed by an evaporator, diluted with THF, and then reprecipitated with hexane (manufactured by Kanto Chemical Co., Inc.) three times to purify the amphipathic block copolymer PSt ₅ -b-POEGMA ₁₄ -COOH. did.

<< Example 3: Introduction of double bond to amphipathic block copolymer HOOC- (POEGMA ₁₄ -b-PSt ₅ ) >>

To a 100 mL eggplant flask, add PSt ₅ -b-POEGMA ₁₄ -COOH (1.0 g), n-hexylamine (manufactured by Tokyo Kasei Co., Ltd.) 0.25 g, and tetrahydrofuran (THF, manufactured by Kanto Chemical Co., Inc.) 4.2 mL, and freeze. Degassing was performed 3 times. The mixture was polymerized at room temperature for 24 hours under a nitrogen atmosphere. The reaction solution was removed by an evaporator, diluted with THF, and then reprecipitated with hexane three times to purify HS-PSt ₅ -b-POEGMA ₁₄ -COOH.

After removing water with a heat gun under an argon atmosphere, 0.075 g of triethylamine (manufactured by Kanto Chemical Co., Inc.), dried THF, and above in a 50 mL three-necked flask equipped with a calcium chloride tube, a dropping funnel, and an argon balloon. Purified HS-PSt ₅ -b-POEGMA ₁₄ -COOH was added and stirred. 90 μL of methacryloyl chloride and dried THF were added dropwise, and the mixture was stirred in a water bath for the first 30 minutes under argon gas bubbling, then switched to room temperature and stirred for 15 hours. The salt produced was removed with filter paper, and the reaction solution was removed with an evaporator. Subsequently, it was diluted with THF and then reprecipitated with hexane three times to purify the amphipathic block copolymer methacrylthio-PSt ₅ -b-POEGMA ₁₄ -COOH into which a double bond was introduced.
¹ Using 1 H1-NMR (Burker DPX300NMR, manufactured by Buruker), the introduction rate of the double bond is 77%, and using gel permeation chromatography (HLC-8220GPC, manufactured by TOSO), the double bond is formed. It was confirmed that the polydispersity of the introduced amphoteric block copolymer methacryloylthio-PSt ₅ -b-POEGMA ₁₄ -COOH was 1.27.

<< Example 4: Method for synthesizing magnetic particles >>
Styrene, divinylbenzene, and polystyrene as hydroform were added to the magnetic material and ultrasonically treated with an ultrasonic cleaner (BRANSON, 3510) for 3 minutes to obtain a monomer / magnetic material composite dispersion solution (solution A).
Ultrapure water, the double-bonded amphipathic block copolymer prepared in Example 3, and the water-soluble initiator potassium persulfate were dissolved in a 100 mL sample bottle and stirred with a magnetic stirrer (solution B). ..
Solution A was transferred to solution B and ultrasonically irradiated for 15 minutes under ice-cooling using an ultrasonic disperser to obtain a monomer / magnetic material composite oil droplet.
The obtained monomer / magnetic composite oil droplets were transferred to a 100 mL three-necked flask, _N2 bubbled, stirred at 200 rpm, the temperature of the water bath was raised to 70 ° C., and the mixture was stirred after 8 hours for polymerization.
After 8 hours, the reactor was cooled to terminate the polymerization.
Magnetic particles were purified by magnetizing with a magnet, removing the aqueous dispersion solvent, and redispersing in ultrapure water three times.
The amount of each raw material charged is shown in Table 1 below.

<< Example 5: Evaluation of Physical Properties of Magnetic Particles >>
The particle size of 100 magnetic particles was randomly measured from the images of the magnetic particles taken by a scanning electron microscope (JEOL-6510, manufactured by JEOL Ltd.), and the average value was calculated. Table 2 shows the results of calculating the content of the magnetic substance in the magnetic particles by a thermogravimetric measuring device (DTA-60A, manufactured by SHIMADZU).
By changing the amount of polystyrene used as the hydroforb, the particle size of the magnetic particles and the content of the magnetic substance can be controlled. According to the method of the present invention, it was found that magnetic particles having a particle size and a magnetic substance content useful for use as a diagnostic agent can be stably produced.

<< Example 6: Modification of antibody to magnetic particles >>
In Example 5, the magnetic particles produced under the conditions of Sample 1 were used to carry a physiologically active substance, and the usefulness as an in vitro diagnostic agent was evaluated. To 1.0 mL of 1.0 wt% magnetic particles, 100 μL of a 100 mg / mL EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) aqueous solution was added and stirred for 10 minutes. To this, 500 μL of 1.0 mg / mL TAT (thrombin-antithrombin complex) antibody was added, and the mixture was further stirred for 1 hour. After the stirring was completed, the magnetic particles to which the antibody was bound were collected using a magnet, and the reaction solution was washed with distilled water three times to prepare antibody-modified magnetic particles.

<< Example 7: Performance evaluation of in vitro diagnostic agent >>
Using a TAT calibrator (manufactured by LSI Medience Corporation), the amount of light emitted from the magnetic particles modified with the TAT antibody was measured. The apparatus used was the all-clinical testing system STACIA (manufactured by LSI Medience Corporation). The result of the obtained luminescence amount is shown in FIG.
Even when the TAT was as low as 20 ng / mL, a sufficient amount of light emission could be confirmed. Further, since the TAT is 0 ng / mL, that is, the so-called blank value is very low, it was considered that the magnetic particles according to the present invention are useful as an in vitro diagnostic agent.

Claims (12)

Steps to prepare and mix monomers, radical polymerization initiators, emulsifiers, and magnetic materials,
Miniemulsion polymerization including a step of shearing the obtained mixture by ultrasonic irradiation to form monomer micro oil droplets and a step of heating to the polymerization initiation temperature of the radical polymerization initiator to polymerize the monomer micro oil droplets. It is a magnetic particle for carrying a physiologically active substance produced by the above, and the emulsifier is the following general formula (1) :.

[In the equation, n is an integer of 2 or more, m is an integer of 2 or more, and
Each of R1 and R4 is independent, and at least one of them is a functional group for carrying a physiologically active substance.
_R2-1 and _R2-2 are independent hydrogen atoms, halogen atoms, and alkyl groups, respectively.
R3 is a functional group derived from a hydrophilic compound.
R5 is a functional group that imparts hydrophobicity and is
R6 is a halogen atom, an alkyl group, a hydrocarbon group containing an unsaturated bond, or a functional group derived from an initiator at the time of emulsifier synthesis]
The magnetic particles for supporting a physiologically active substance, which are amphipathic block copolymers represented by. The magnetic for supporting a physiologically active substance according to claim 1, wherein R1 or R4 is a group selected from the group consisting of a carboxyl group, a maleimide group, an amino group, a mercapto group, a hydroxyl group, an aldehyde group, and an epoxy group. particle. The hydrophilic compound in R3 is selected from the group consisting of oligoethylene glycol, polyethylene glycol, 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer, polyvinyl alcohol, polyvinylpyrrolidone, polyamino acids, polypeptides, monosaccharides, and polysaccharides. The magnetic particle for carrying a physiologically active substance according to claim 1 or 2, which is a compound to be used. The R5 is a hydrogen atom, a halogen atom, an alkyl group, a substituted or unsubstituted aromatic compound group, a carbonyl group, an amide group, an amino group, an aldehyde group, and a keto group, and an amine, an aldehyde, a ketone, and an ether. The magnetic particle for carrying a physiologically active substance according to any one of claims 1 to 3, which is one or more functional groups selected from the group consisting of functional groups derived from each compound. The magnetic particle for supporting a physiologically active substance according to any one of claims 1 to 4 , wherein the emulsifier has a molecular weight of 4 to 1,000,000. The magnetic particle for supporting a physiologically active substance according to any one of claims 1 to 5, wherein the hydrophilic segment has a molecular weight of 2 to 500,000. The magnetic particle for supporting a physiologically active substance according to any one of claims 1 to 6, wherein the hydrophobic segment has a molecular weight of 2 to 500,000. Any of claims 1 to 7 , wherein the functional group having a carboxyl group of R1 or R4 is derived from a polycarboxylic acid selected from the group consisting of polyacrylic acid, polymethacrylic acid, polyitaconic acid, polymaleic acid, and polyfumalic acid. The magnetic particles for carrying a physiologically active substance according to item 1. The magnetic particle for supporting a physiologically active substance according to any one of claims 1 to 8, wherein the R1 or R4 has one or more carboxyl groups. The magnetic particle for supporting a physiologically active substance according to any one of claims 1 to 9, wherein R6 is a functional group having a double bond introduced on the hydrophobic side. Steps to prepare and mix monomers, radical polymerization initiators, emulsifiers, and magnetic materials,
Miniemulsion polymerization including a step of shearing the obtained mixture by ultrasonic irradiation to form monomer micro oil droplets and a step of heating to the polymerization initiation temperature of the radical polymerization initiator to polymerize the monomer micro oil droplets. This is a method for producing magnetic particles for carrying a physiologically active substance.
When the mini-emulsion polymerization is performed, the emulsifier is used in the following general formula (1):

[In the equation, n is an integer of 2 or more, m is an integer of 2 or more, and
Each of R1 and R4 is independent, and at least one of them is a functional group for carrying a physiologically active substance.
_R2-1 and _R2-2 are independent hydrogen atoms, halogen atoms, and alkyl groups, respectively.
R3 is a functional group derived from a hydrophilic compound.
R5 is a functional group that imparts hydrophobicity and is
R6 is a halogen atom, an alkyl group, a hydrocarbon group containing an unsaturated bond, or a functional group derived from an initiator at the time of emulsifier synthesis]
A method for producing magnetic particles for supporting a physiologically active substance, which is characterized by being a compound represented by. The production method according to claim 11, further comprising a step of synthesizing the emulsifier by controlled / living radical polymerization or ionic polymerization.

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