Magnetic bead, diagnostic agent, and method for producing magnetic bead

Abstract

This magnetic bead includes a polymer and magnetic particles. The surface of the magnetic bead includes a surface functional group. The surface functional group includes at least one selected from the group consisting of an alkynyl group and an azido group.

Description

Magnetic beads, diagnostic reagents, and methods for manufacturing magnetic beads

[0001] This invention relates to magnetic beads, diagnostic reagents, and methods for producing magnetic beads.

[0002] Magnetic beads containing polymers and magnetic particles are known. Regarding technologies related to magnetic beads, for example, the technology described in Patent Document 1 is known.

[0003] Patent Document 1 describes magnetic particles characterized by containing magnetic powder on which polymer chains are bonded via a coupling agent to the surface. The magnetic particles described in Patent Document 1 are described as having good dispersibility, dispersion stability, and affinity with binder resins, being small in size with a narrow particle size distribution, and having excellent heat resistance, wear resistance, etc., and being hard and resistant to cracking, making them suitable for use as carriers and the like.

[0004] Japanese Unexamined Patent Publication No. 6-102708

[0005] This invention provides magnetic beads capable of binding a larger amount of the target substance.

[0006] The present invention provides magnetic beads, a diagnostic agent, and a method for manufacturing magnetic beads as described below.

[0007] [1] Magnetic beads comprising a polymer and magnetic particles, wherein the surface of the magnetic beads includes a surface functional group, and the surface functional group comprises at least one selected from the group consisting of alkynyl groups and azide groups. [2] The magnetic beads according to [1], wherein the polymer comprises a structural unit represented by the following formula (1). (In formula (1) above, R １ ~R ３[1] The magnetic bead according to [2], wherein each of the elements is independently a hydrogen atom or an organic group having 1 to 20 carbon atoms, X is a divalent organic group including a main chain, the number of atoms in the main chain is 3 or more, and Y is an alkynyl group or an azide group. [3] The magnetic bead according to [2], wherein the number of atoms in the main chain of X in formula (1) is 6 or more. [4] The magnetic bead according to [2] or [3], wherein the main chain of X in formula (1) is composed of one or more atoms selected from the group consisting of carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms, and silicon atoms, and at least one atom is an oxygen atom. [5] The magnetic bead according to any one of [1] to [4], wherein the surface functional group further comprises a carboxyl group. [6] The magnetic bead according to any one of [1] to [5], wherein the volume average particle diameter (MV) is 10 μm or more and 50 μm or less. [7] The magnetic bead according to any one of [1] to [6], wherein the surface functional group is capable of bonding with bio-related substances. [8] Magnetic beads according to any one of [1] to [7] that can be used in a diagnostic agent. [9] Magnetic beads according to any one of [1] to [8], further comprising a bio-related substance, wherein the bio-related substance is bonded to the surface functional group.

[10] A diagnostic agent comprising the magnetic beads according to any one of [1] to [9].

[11] A method for producing magnetic beads, comprising the steps of: (A) preparing a magnetic bead intermediate comprising a polymer, magnetic particles, and a magnetic bead intermediate, wherein the magnetic bead intermediate has a carboxyl group on its surface; and (B) converting the carboxyl group of the magnetic bead intermediate to a group having a surface functional group comprising at least one selected from the group consisting of alkynyl groups and azide groups.

[12] A method for producing magnetic beads according to

[11] , wherein step (A) comprises polymerizing monomers (a) in a suspension comprising monomers (a) and the magnetic particles, wherein monomers (a) include monomers (a1) having a carboxyl group.

[13] The method for producing magnetic beads according to

[12] , wherein the monomers (a1) include at least one selected from the group consisting of methacrylic acid, acrylic acid, 2-methacryloyloxyethyl succinic acid, 2-acryloyloxyethyl succinic acid, 2-methacryloyloxyethyl hexahydrophthalic acid, 2-acryloyloxyethyl hexahydrophthalic acid, 2-carboxyethyl methacrylate, 2-carboxyethyl acrylate, itaconic acid, 4-[[6-(acryloyloxy)hexyl]oxy]benzoic acid, 2-carboxyethyl acrylate oligomer, and 2-carboxyethyl methacrylate oligomer.

[14] The method for producing magnetic beads according to any one of

[11] to

[13] , wherein step (B) includes a step of reacting the carboxyl group of the magnetic bead intermediate with the compound (b) having the surface functional group.

[15] The method for producing magnetic beads according to

[14] , wherein compound (b) comprises at least one selected from the group consisting of amines having the surface functional group and alcohols having the surface functional group.

[16] Compound (b) is propargylamine, 2-(2-propynyloxy)ethylamine, 2-[2-(2-propynyloxy)ethoxy]ethylamine, 4-ethynylaniline, 4-(2-propynyloxy)aniline, triethylene glycol 2-aminoethylpropargyl ether, 2-propyne-1-ol, 3-butyne-1-ol, 3-butyne-2-ol, (S)-1-octin-3-ol, 6-heptin-1-ol, 1-pentin-3-ol, 4-pentin-1-ol, 4-pentin-2-ol, 1-hexyn-3-ol, 5-hexyn-1-ol, 9-decine-1-ol, 10-undecine-1-ol, (R)-(+) A method for producing magnetic beads according to

[14] , comprising at least one selected from the group consisting of -3-butyne-2-ol, (S)-(-)-3-butyne-2-ol, 2-methyl-3-butyne-2-ol, 3-methyl-1-pentin-3-ol, 1-ethynyl-1-cyclohexanol, 1-phenyl-2-propyne-1-ol, diethylene glycol mono(2-propyne-1-yl) ether, triethylene glycol mono(2-propynyl) ether, 3-azidopropylamine, 6-azidohexylamine, azido-PEG3-amine, azido-PEG4-amine, 6-azidohexane-1-ol, and glycidylpropargyl ether.

[0008] According to the present invention, magnetic beads capable of binding a larger amount of the target substance can be provided.

[0009] The embodiments of the present invention will be described below. Unless otherwise specified, the numerical range "A to B" represents A or greater and B or less. In this embodiment, the notation "(meth)acryloyl group" represents a concept that encompasses both methacryloyl group and acryloyl group. The same applies to similar notations such as "(meth)acrylate".

[0010] Magnetic beads containing polymers and magnetic particles are known. Magnetic beads are used, for example, in diagnostic agents, bacterial isolation, cell culture, drug delivery, magnetic toner, magnetic ink, magnetic paint, etc. For example, in applications such as diagnostic agents, magnetic beads are required to have the ability to bind a larger amount of the target substance (e.g., bio-related substances). The present invention provides magnetic beads that are capable of binding a larger amount of the target substance.

[0011] [Magnetic Beads] The magnetic beads of this embodiment are magnetic beads comprising a polymer and magnetic particles, wherein the surface of the magnetic beads includes a surface functional group, and the surface functional group includes at least one selected from the group consisting of alkynyl groups and azide groups.

[0012] The magnetic beads of this embodiment preferably contain magnetic particles inside the polymer.

[0013] The components of the magnetic beads in this embodiment will be described in detail below.

[0014] <Surface Functional Groups> The magnetic beads of this embodiment include surface functional groups on the surface of the magnetic beads. The surface functional groups of this embodiment include at least one selected from the group consisting of alkynyl groups and azide groups. By including at least one selected from the group consisting of alkynyl groups and azide groups in the surface functional groups, it becomes possible to bond a larger amount of the target substance.

[0015] The alkynyl group is, for example, a substituted or unsubstituted linear alkynyl group having 2 to 30 carbon atoms, and specifically includes at least one selected from the group consisting of ethynyl group, propargyl group, and trimethylsilylethynyl group, and preferably includes an ethynyl group. Furthermore, the alkynyl group in the surface functional group of this embodiment may be a substituted or unsubstituted cyclic alkynyl group, and for example includes at least one selected from the group consisting of cyclooctinyl group, azacyclooctinyl group, and dibenzocyclooctinyl group.

[0016] The surface functional group of this embodiment preferably includes at least one selected from the group consisting of ethynyl groups and azide groups.

[0017] The surface functional group of the present embodiment preferably further contains a carboxy group. By further containing a carboxy group in the surface functional group, the dispersibility of the magnetic beads can be further improved in a dispersion liquid containing the magnetic beads.

[0018] The surface functional group of the present embodiment is preferably capable of binding to a biomolecule. The surface functional group capable of binding to a biomolecule preferably contains at least one selected from the group consisting of an alkynyl group and an azide group.

[0019] <Polymer> The magnetic beads of the present embodiment contain a polymer. The polymer of the present embodiment is not particularly limited, but preferably contains a (meth)acrylic polymer. Further, the polymer of the present embodiment may be a homopolymer or a copolymer, but is preferably a copolymer.

[0020] The polymer of the present embodiment preferably contains the surface functional group of the present embodiment.

[0021] The polymer of the present embodiment preferably contains a structural unit represented by formula (1).

[0022]

[0023] In formula (1), R １ ~ R ３ are each independently a hydrogen atom or an organic group having 1 to 20 carbon atoms, X is a divalent organic group containing a main chain, the number of atoms in the main chain is 3 or more, and Y is an alkynyl group or an azide group.

[0024] In formula (1), R １ ~ R ３ are each independently, preferably a hydrogen atom or an alkyl group having 1 to 20 carbon atoms, more preferably a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, still more preferably a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and still more preferably a hydrogen atom or a methyl group. In formula (1), preferably, R １ and R ２ are hydrogen atoms, and R ３is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, more preferably, R １ and R ２ is a hydrogen atom, R ３ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, still more preferably, R １ and R ２ are hydrogen atoms, and R ３ is a hydrogen atom or a methyl group.

[0025] In formula (1), the number of atoms in the main chain of X is 3 or more. The number of atoms in the main chain of X means the number of carbon atoms between R ３ and X and the atoms involved in the bond with Y.

[0026] Regarding the definition of the atoms in the main chain of X, formula (1a) will be exemplified and specifically explained.

[0027]

[0028] In formula (1a), the atoms in the main chain of X mean the carbon atom between R ３ (methyl group) and X and the atoms in the chemical formula that are involved in the bond with Y (ethynyl group) and are numbered. That is, in formula (1a), the number of atoms in the main chain of X is 11.

[0029] Here, when Y is a substituted or unsubstituted chain-like alkynyl group, in formula (1), the boundary between X and Y is defined such that the atom adjacent to the triple bond contained in Y is contained in X. Therefore, formula (1a) does not define that Y is a propargyl group by combining the structure (—CH ２ —) at the position marked with “11” and the terminal triple bond, but defines that Y is an ethynyl group.

[0030] Here, for example, in formula (1a), the structure at the position marked with “3” is “—CH<00​​​Furthermore, for example, in formula (1a), the structure at the point marked "6" is "-CO-", but the only atoms in the main chain of X are carbon atoms, and oxygen atoms are not considered to be part of the main chain of X. This is because the only atom involved in the bond between the adjacent fifth and seventh atoms is carbon.

[0032] Furthermore, for example, in formula (1a), the structure at the point marked "10" is "-NH-", but the only atom in the main chain of X is nitrogen, and hydrogen atoms are not considered to be part of the main chain of X. This is because the only atom involved in the bond between the adjacent 9th and 11th atoms is nitrogen.

[0033] In formula (1), the number of atoms in the main chain of X is preferably 6 or more, more preferably 8 or more, and even more preferably 10 or more, and the upper limit is not particularly limited, but may be, for example, 25 or less, 20 or less, or 18 or less. In formula (1), if the number of carbon atoms in the main chain of X is greater than or equal to the lower limit, more of the target substance can be bonded. The reason for this is not clear, but the inventors speculate that if the number of carbon atoms in the main chain of X is greater than or equal to the lower limit, the alkynyl group or azide group that functions as a surface functional group can be positioned further out on the magnetic bead, making it easier to bring it into contact with the target substance, and thus more of the target substance can be bonded.

[0034] In formula (1), the atoms constituting the main chain in X are not particularly limited and may be any atoms with a valency of 2 or higher. In formula (1), the main chain in X is preferably composed of one or more atoms selected from the group consisting of carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms, and silicon atoms, and more preferably composed of one or more atoms selected from the group consisting of carbon atoms, nitrogen atoms, and oxygen atoms. In formula (1), the main chain in X preferably contains at least one oxygen atom.

[0035] In formula (1), X preferably does not contain an aromatic ring having a group containing Y only at the o position. Here, the o position is R in the aromatic ring. ３This refers to the o-position of the group involved in bonding with the carbon atom between X and X. In formula (1), if X contains an aromatic ring having a group containing Y only at the o-position, it may be difficult for the target substance to bond. The reason for this is not clear, but the inventors speculate that if X contains an aromatic ring having a group containing Y only at the o-position, the alkynyl group or azide group that functions as a surface functional group may be located on the inside of the magnetic bead (for example, as in formula (A5) in Example 5), making it difficult to bring the target substance into contact with it, thus making it difficult for the target substance to bond. Therefore, the inventors speculate that by not including an aromatic ring in X having a group containing Y only at the o-position, the alkynyl group or azide group that functions as a surface functional group can be located further outside the magnetic bead, making it easier to bring the target substance into contact with it, and thus allowing for the bonding of even more of the target substance.

[0036] In formula (1), Y is an alkynyl group or an azide group.

[0037] In formula (1), the alkynyl group in Y is, for example, a substituted or unsubstituted linear alkynyl group having 2 to 30 carbon atoms, and is specifically selected from the group consisting of an ethynyl group and a trimethylsilylethynyl group, and is preferably an ethynyl group.

[0038] In formula (1), the alkynyl group in Y may be a substituted or unsubstituted cyclic alkynyl group, and may include at least one selected from the group consisting of, for example, a cyclooctinyl group, an azacyclooctinyl group, and a dibenzocyclooctinyl group.

[0039] Furthermore, if Y is a substituted or unsubstituted cyclic alkynyl group, the boundary between X and Y is defined such that atoms adjacent to the cyclic alkyne structure are included in X, so that X becomes a divalent organic group. In other words, unlike substituted or unsubstituted linear alkynyl groups, if Y is a substituted or unsubstituted cyclic alkynyl group, the boundary between X and Y is not defined such that atoms adjacent to the triple bond in Y are included in X.

[0040] In formula (1), Y is preferably an ethynyl group or an azide group.

[0041] The structural unit represented by formula (1) preferably includes at least one selected from the group consisting of structural units represented by the following formulas (1-1) to (1-8).

[0042]

[0043] The polymer of this embodiment may further contain other structural units other than the structural unit represented by formula (1). The other structural units are not particularly limited, but preferably include structural units derived from monomers (a2). Monomers (a2) are synonymous with monomers (a2) in the method for producing magnetic beads described later. The polymer of this embodiment preferably contains the structural unit represented by formula (1) and structural units derived from monomers (a2).

[0044] The polymer content in the magnetic beads of this embodiment is preferably 60% to 99% by mass, more preferably 70% to 98% by mass, even more preferably 75% to 97% by mass, and even more preferably 85% to 92% by mass, when the total mass of the magnetic beads is considered to be 100% by mass, in order to further improve the balance of performance such as the magnetic properties of the magnetic beads and the bonding properties with the target material.

[0045] The method for synthesizing the polymer in this embodiment is not particularly limited, but suspension polymerization is preferred. The detailed synthesis conditions may be, for example, those described in the method for manufacturing magnetic beads described later.

[0046] <Magnetic Particles> The magnetic beads of this embodiment include magnetic particles. The magnetic particles of this embodiment are not particularly limited as long as they are magnetic particles, but for example, magnetite (Fe ３ O ４ ), Maghemite (γ-Fe ２ O ３ ), hematite (α-Fe ２ O ３ ), manganese ferrite (MnFe ２ O ４ ), cobalt ferrite (CoFe ２ O４ ), nickel ferrite (NiFe ２ O ４ ), copper ferrite (CuFe ２ O ４ The material comprises at least one selected from the group consisting of Mn-Zn ferrite, Ni-Zn ferrite, Mn-Mg ferrite, Li ferrite, Cu-Zn ferrite, and goethite (FeO(OH)), preferably at least one selected from the group consisting of magnetite and maghemite, and more preferably magnetite.

[0047] The magnetic particles of this embodiment may be modified with, for example, a coupling agent. Preferably, the magnetic particles of this embodiment include magnetic particles modified with at least one selected from the group consisting of fatty acids and silane coupling agents, and more preferably include magnetic particles modified with a silane coupling agent.

[0048] The fatty acid of this embodiment includes, for example, at least one selected from the group consisting of oleic acid, stearic acid, palmitic acid, linoleic acid, and palmitoleic acid.

[0049] The silane coupling agent of this embodiment includes, for example, at least one selected from the group consisting of 3-methacryloxypropyltrimethoxysilane, 3-acryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, p-styryltrimethoxysilane, vinyltrimethoxysilane, and vinyltriethoxysilane.

[0050] The particle diameter of the magnetic particles in this embodiment is preferably 1 nm to 5000 nm, more preferably 3 nm to 1000 nm, and even more preferably 10 nm to 300 nm. Here, the particle diameter of the magnetic particles refers to a value measured by a transmission electron microscope (TEM), and specifically refers to a value obtained by performing image analysis on a TEM image and calculating the arithmetic mean of the particle diameters of any 10 magnetic particles.

[0051] The magnetic particle content in the magnetic beads of this embodiment is preferably 1% to 40% by mass, more preferably 2% to 30% by mass, even more preferably 3% to 20% by mass, and even more preferably 8% to 15% by mass, when the total amount of magnetic beads is considered as 100% by mass, in order to further improve the balance of performance such as the magnetic properties of the magnetic beads and the bonding properties with the target material.

[0052] <Other Components> The magnetic beads of this embodiment may further contain components other than polymers and magnetic particles.

[0053] The volume-average particle size (MV) of the magnetic beads in this embodiment is preferably 10 μm or more and 50 μm or less, more preferably 15 μm or more and 40 μm or less, and even more preferably 20 μm or more and 30 μm or less. The volume-average particle size (MV) of the magnetic beads refers to the volume-average particle size (MV) obtained when the particle size distribution is measured using a laser diffraction particle size distribution analyzer for a magnetic bead dispersion with a concentration of 1% by mass. Here, the magnetic bead dispersion with a concentration of 1% by mass can be, for example, a magnetic bead aqueous solution with a concentration of 1% by mass in which the solvent is water.

[0054] The applications of the magnetic beads of this embodiment are not particularly limited and can be applied, for example, to diagnostic agents, bacterial isolation, cell culture, drug delivery, magnetic toner, magnetic ink, magnetic paint, etc. The magnetic beads of this embodiment are preferably suitable for use in diagnostic agents because they can bind a larger amount of the target substance. The applications of the magnetic beads of this embodiment preferably do not include at least one application selected from the group consisting of magnetic toner, developer, and electrophotographic carrier.

[0055] The magnetic beads of this embodiment preferably further contain a bio-related substance, the bio-related substance being a magnetic bead bonded to the surface functional group of this embodiment. The bio-related substance of this embodiment is not particularly limited, but for example, it includes at least one selected from the group consisting of antibodies, nucleotides, nucleosides, oligonucleotides, nucleic acids, proteins, DNA, avidin, streptavidin, biotin, aptamers, and sugar chains, and preferably includes at least one selected from the group consisting of oligonucleotides, nucleic acids, and DNA.

[0056] [Dispersion] The dispersion of this embodiment comprises the magnetic beads of this embodiment and a solvent. The solvent comprises at least one selected from the group consisting of, for example, water, methanol, ethanol, isopropyl alcohol, propylene glycol monomethyl ether, and propylene glycol monomethyl ether acetate, and preferably contains water.

[0057] The concentration of the dispersion in this embodiment is, for example, 0.01% by mass or more and 20% by mass or less, preferably 0.1% by mass or more and 15% by mass or less, and more preferably 0.5% by mass or more and 10% by mass or less.

[0058] [Diagnostic agent] The diagnostic agent of this embodiment includes the magnetic beads of this embodiment.

[0059] [Method for Manufacturing Magnetic Beads] The method for manufacturing magnetic beads according to this embodiment comprises a polymer, magnetic particles, and a magnetic bead intermediate, and includes the steps of (A) preparing a magnetic bead intermediate containing carboxyl groups on its surface, and (B) converting the carboxyl groups of the magnetic bead intermediate into groups containing surface functional groups including at least one selected from the group consisting of alkynyl groups and azide groups.

[0060] The following provides a detailed explanation of each step.

[0061] <Step (A)> The method for manufacturing magnetic beads according to this embodiment includes a polymer, magnetic particles, and a magnetic bead intermediate containing these, and includes step (A) of preparing a magnetic bead intermediate containing carboxyl groups on the surface of the magnetic bead intermediate.

[0062] Step (A) preferably includes a step of polymerizing monomers (a) in a suspension containing monomers (a) and magnetic particles.

[0063] The monomers (a) are not particularly limited as long as they include at least one selected from the group consisting of monomers containing polymerizable groups and oligomers containing polymerizable groups. The polymerizable group is not particularly limited, but preferably includes at least one selected from the group consisting of vinyl groups, vinylidene groups, and (meth)acryloyl groups.

[0064] The monomer (a) preferably includes monomer (a1) containing a carboxyl group. The monomer (a1) preferably further includes at least one selected from the group consisting of a vinyl group, a vinylidene group, and a (meth)acryloyl group, in addition to the carboxyl group.

[0065] Monomers (a1) include, for example, at least one selected from the group consisting of methacrylic acid, acrylic acid, 2-methacryloyloxyethyl succinic acid, 2-acryloyloxyethyl succinic acid, 2-methacryloyloxyethyl hexahydrophthalic acid, 2-acryloyloxyethyl hexahydrophthalic acid, 2-carboxyethyl methacrylate, 2-carboxyethyl acrylate, itaconic acid, 4-[[6-(acryloyloxy)hexyl]oxy]benzoic acid, 2-carboxyethyl acrylate oligomer, and 2-carboxyethyl methacrylate oligomer, mono-2-(methacryloyloxy)ethyl phthalate, mono-2-(acryloyloxy)ethyl phthalate, mono-2-(methacryloyloxy)propyl phthalate, mono-2-(acryloyloxy)propyl phthalate, and p-vinylbenzoic acid, preferably methacrylic acid, acrylic acid, 2-methacryloyloxy The material comprises at least one selected from the group consisting of ethyl succinic acid, 2-acryloyloxyethyl succinic acid, 2-methacryloyloxyethyl hexahydrophthalic acid, 2-acryloyloxyethyl hexahydrophthalic acid, 2-carboxyethyl methacrylate, 2-carboxyethyl acrylate, itaconic acid, 4-[[6-(acryloyloxy)hexyl]oxy]benzoic acid, 2-carboxyethyl acrylate oligomer, and 2-carboxyethyl methacrylate oligomer, more preferably comprising at least one selected from the group consisting of methacrylic acid, 2-methacryloyloxyethyl succinic acid, and 2-acryloyloxyethyl succinic acid, and even more preferably comprising at least one selected from the group consisting of 2-methacryloyloxyethyl succinic acid and 2-acryloyloxyethyl succinic acid, from the viewpoint that the resulting magnetic beads can bind even more of the target substance.

[0066] Here, 2-carboxyethyl acrylate oligomer refers to the oligomer represented by the following formula (a1-1).

[0067]

[0068] In equation (a1-1), n ​​is between 1 and 3.

[0069] Furthermore, 2-carboxyethyl methacrylate oligomer refers to the oligomer represented by the following formula (a1-2).

[0070]

[0071] In equation (a1-2), n is between 1 and 3.

[0072] The amount of monomers (a1) in the suspension of this embodiment is preferably 1 to 30 parts by mass, more preferably 2 to 25 parts by mass, and even more preferably 4 to 20 parts by mass, when the amount of monomers (a) in the suspension of this embodiment is 100 parts by mass.

[0073] The content of monomers (a1) in the suspension of this embodiment is preferably 1 mole to 25 moles, more preferably 3 moles to 20 moles, and even more preferably 5 moles to 15 moles, when the content of monomers (a) in the suspension of this embodiment is set to 100 moles.

[0074] Monomers (a) preferably further comprises monomers (a2). Monomers (a2) are monomers that do not contain a carboxyl group and contain one polymerizable group. The polymerizable group of monomers (a2) preferably comprises at least one selected from the group consisting of a vinyl group, a vinylidene group, and a (meth)acryloyl group, and more preferably comprises a (meth)acryloyl group.

[0075] The monomers (a2) preferably include at least one selected from the group consisting of alkyl (meth)acrylates, hydroxyalkyl (meth)acrylates, aminoalkyl (meth)acrylates, etc.

[0076] The monomers (a2) preferably include at least one selected from the group consisting of methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, t-butyl (meth)acrylate, cyclohexyl (meth)acrylate, and isobornyl (meth)acrylate, more preferably include at least one selected from the group consisting of methyl (meth)acrylate, n-butyl (meth)acrylate, t-butyl (meth)acrylate, cyclohexyl (meth)acrylate, and isobornyl (meth)acrylate, and even more preferably include methyl methacrylate.

[0077] The content of monomers (a2) in the suspension of this embodiment is preferably 10 parts by mass or more and 70 parts by mass or less, more preferably 20 parts by mass or more and 60 parts by mass or less, and even more preferably 30 parts by mass or more and 50 parts by mass or less, when the content of monomers (a) in the suspension of this embodiment is 100 parts by mass.

[0078] The monomer (a) preferably includes monomer (a3). Monomer (a3) ​​is a monomer that does not contain a carboxyl group and contains two or more polymerizable groups. Since monomer (a3) ​​functions as a crosslinking agent, including monomer (a) in monomer (a) can further improve the heat resistance of the resulting magnetic beads.

[0079] The polymerizable group of monomers (a3) ​​preferably includes at least one selected from the group consisting of vinyl group, vinylidene group, and (meth)acryloyl group, and more preferably includes a (meth)acryloyl group.

[0080] The monomers (a3) ​​include, for example, at least one selected from the group consisting of ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, 1,5-pentanediol di(meth)acrylate, triethylene glycol di(meth)acrylate, and N,N'-methylenebis(acrylamide), preferably containing ethylene glycol di(meth)acrylate, and more preferably containing ethylene glycol dimethacrylate.

[0081] The amount of monomers (a3) ​​in the suspension of this embodiment is preferably 10 parts by mass or more and 70 parts by mass or less, more preferably 20 parts by mass or more and 60 parts by mass or less, and even more preferably 30 parts by mass or more and 50 parts by mass or less, when the amount of monomers (a) in the suspension of this embodiment is 100 parts by mass.

[0082] The content of monomers (a) in the suspension of this embodiment is preferably 0.1% to 20% by mass, more preferably 0.5% to 15% by mass, and even more preferably 1% to 10% by mass, when the total amount of the suspension is considered to be 100% by mass.

[0083] The configuration of the magnetic particles in this embodiment is the same as the configuration of the magnetic particles contained in the magnetic beads of this embodiment.

[0084] The magnetic particle content in the suspension of this embodiment is preferably 0.01% by mass or more and 3.00% by mass or less, more preferably 0.05% by mass or more and 1.00% by mass or less, and even more preferably 0.10% by mass or more and 0.50% by mass or less, when the total amount of the suspension is considered to be 100% by mass.

[0085] The content of magnetic particles in the suspension of this embodiment is preferably 0.1 parts by mass or more and 30 parts by mass or less, more preferably 0.5 parts by mass or more and 20 parts by mass or less, and even more preferably 1 part by mass or more and 10 parts by mass or less, when the content of monomers (a) in the suspension of this embodiment is 100 parts by mass.

[0086] The suspension of this embodiment may further contain other components besides monomers (a) and magnetic particles. These other components include, for example, reagents commonly used in suspension polymerization, specifically one or more selected from the group consisting of water, polymerization initiators, dispersion stabilizers, surfactants, polymerization inhibitors, etc. The amount of these other components is appropriate.

[0087] The method for preparing the suspension of this embodiment is not particularly limited, but preferably involves sequentially performing the following (i) and (ii): (i) Prepare a monomer mixture containing monomers (a) and magnetic particles. (ii) Disperse the monomer mixture in an aqueous medium containing water to prepare a suspension.

[0088] The conditions for polymerizing monomers (a) are not particularly limited, but for example, they can be 50°C to 90°C and for a period of 1 hour to 30 hours.

[0089] <Step (B)> The method for manufacturing magnetic beads according to this embodiment preferably includes step (B) of converting the carboxyl group of the magnetic bead intermediate into a group that includes a surface functional group which includes at least one selected from the group consisting of alkynyl groups and azide groups.

[0090] The configuration of the surface functional groups in the method for manufacturing magnetic beads of this embodiment is the same as the configuration of the surface functional groups contained in the magnetic beads of this embodiment.

[0091] Step (B) preferably includes a step of reacting the carboxyl group of the magnetic bead intermediate with a compound (b) having a surface functional group. The step of reacting the carboxyl group of the magnetic bead intermediate with the compound (b) having a surface functional group may be a condensation reaction or an addition reaction, but a condensation reaction is preferred.

[0092] Compound (b) is not particularly limited as long as it is a raw material that can react with the carboxyl group of the magnetic bead intermediate. Compound (b) may be a monomer or an oligomer, but is preferably a monomer.

[0093] Compound (b) preferably comprises at least one selected from the group consisting of amines having surface functional groups, alcohols having surface functional groups, and epoxy compounds having surface functional groups, more preferably comprises at least one selected from the group consisting of amines having surface functional groups and alcohols having surface functional groups, and even more preferably comprises amines having surface functional groups.

[0094] Amines and alcohols having surface functional groups can be reacted with the carboxyl groups of the magnetic bead intermediate through condensation. Epoxy compounds having surface functional groups can be reacted with the carboxyl groups of the magnetic bead intermediate through addition.

[0095] Here, "amines" are not limited to amines, but also include amine derivatives that can be condensed with the carboxyl group of the magnetic bead intermediate. Furthermore, "alcohols" are not limited to alcohols, but also include alcohol derivatives that can be condensed with the carboxyl group of the magnetic bead intermediate.

[0096] The amines having an alkynyl group include, for example, at least one selected from the group consisting of propargylamine, 2-(2-propynyloxy)ethylamine, 2-[2-(2-propynyloxy)ethoxy]ethylamine, 4-ethynylaniline, 4-(2-propynyloxy)aniline, and triethylene glycol 2-aminoethylpropargyl ether, and preferably at least one selected from the group consisting of propargylamine, dibenzocyclooctinamine, N-(1R,8S,9s)-bicyclo[6.1.0]nona-4-in-9-ylmethyloxycarbonyl-1,8-diamino-3,6-dioxaoctane, and 2-[2-(2-propynyloxy)ethoxy]ethylamine.

[0097] The amines having an azido group include, for example, at least one selected from the group consisting of 3-azidopropylamine, 6-azidohexylamine, azido-PEG3-amine, and azido-PEG4-amine, preferably at least one selected from the group consisting of 3-azidopropylamine and 6-azidohexylamine, and more preferably 3-azidopropylamine.

[0098] Alcohols having an alkynyl group include, for example, 2-propyne-1-ol, 3-butyne-1-ol, 3-butyne-2-ol, (S)-1-octin-3-ol, 6-heptin-1-ol, 1-pentin-3-ol, 4-pentin-1-ol, 4-pentin-2-ol, 1-hexyn-3-ol, 5-hexyn-1-ol, 9-decine-1-ol, 10-undecine-1-ol, (R)-(+)-3-butyne-2-ol, (S)-(-)-3-butyne-2-ol, 2 It comprises at least one selected from the group consisting of -methyl-3-butyne-2-ol, 3-methyl-1-pentin-3-ol, 1-ethynyl-1-cyclohexanol, 1-phenyl-2-propyne-1-ol, diethylene glycol mono(2-propyne-1-yl) ether, and triethylene glycol mono(2-propynyl) ether, and preferably at least one selected from the group consisting of 3-butyne-1-ol and 1-ethynyl-1-cyclohexanol.

[0099] Alcohols having an azide group include, for example, at least one selected from the group consisting of 3-azido-1-propanol, 4-azido-1-butanol, 5-azido-1-pentanol, 6-azidohexane-1-ol, azido-PEG3-alcohol, azido-PEG4-alcohol, azido-PEG8-alcohol, and preferably 6-azidohexane-1-ol.

[0100] Epoxy compounds having an alkynyl group include, for example, glycidylpropargyl ether.

[0101] Compound (b) is preferably propargylamine, 2-(2-propynyloxy)ethylamine, 2-[2-(2-propynyloxy)ethoxy]ethylamine, 4-ethynylaniline, 4-(2-propynyloxy)aniline, triethylene glycol 2-aminoethylpropargyl ether, 2-propyne-1-ol, 3-butyne-1-ol, 3-butyne-2-ol, (S)-1-octin-3- ol, 6-heptin-1-ol, 1-pentin-3-ol, 4-pentin-1-ol, 4-pentin-2-ol, 1-hexyn-3-ol, 5-hexyn-1-ol, 9-decine-1-ol, 10-undecine-1-ol, (R)-(+)-3-butyn-2-ol, (S)-(-)-3-butyn-2-ol, 2-methyl-3-butyn-2-ol, 3-methyl-1-pentyn-3-ol It comprises at least one selected from the group consisting of 1-ethynyl-1-cyclohexanol, 1-phenyl-2-propyne-1-ol, diethylene glycol mono(2-propyne-1-yl) ether, triethylene glycol mono(2-propynyl) ether, 3-azidopropylamine, 6-azidohexylamine, azido-PEG3-amine, azido-PEG4-amine, 6-azidohexane-1-ol, and glycidylpropargyl ether, and more preferably at least one selected from the group consisting of propargylamine, 2-[2-(2-propynyloxy)ethoxy]ethylamine, 3-butyne-1-ol, 1-ethynyl-1-cyclohexanol, 3-azidopropylamine, 6-azidohexylamine, 6-azidohexane-1-ol, and glycidylpropargyl ether.

[0102] The method for reacting the carboxyl group of the magnetic bead intermediate with compound (b) is not particularly limited, and any known condensation or addition reaction conditions may be used. For example, a method for condensing the carboxyl group of the magnetic bead intermediate with compound (b) is to condense the magnetic bead intermediate and compound (b) using a condensing agent under acidic conditions.

[0103] <Other steps> The method for manufacturing magnetic beads according to this embodiment may further include other steps besides steps (A) and (B). Examples of other steps include a step of acid washing the magnetic beads.

[0104] The preferred embodiment of the magnetic beads obtained by the manufacturing method of the magnetic beads of this embodiment is the same as the preferred embodiment of the magnetic beads of this embodiment.

[0105] Although embodiments of the present invention have been described above, these are merely examples, and various other configurations can be adopted. Furthermore, the present invention is not limited to the embodiments described above, and any modifications, improvements, etc., that can achieve the objectives of the present invention are included in the present invention.

[0106] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples unless there is a change in its essence.

[0107] [Example 1] <Production of magnetic particles modified with a silane coupling agent> In a separable flask, FeCl ２ 4H ２ O (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) 60g, FeCl ３ 6H ２ Add 135g of O (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), then add 1500mL of pure water that has been pre-purged with nitrogen by nitrogen bubbling, and add FeCl ２ 4H ２ O and FeCl ３ 6H ２ O was dissolved. 400 mL of 28% by weight ammonia water was poured into this solution, and the mixture was stirred at 200 rpm for 30 minutes in a 30°C oil bath to obtain magnetic particles. The obtained magnetic particles were recovered magnetically, the supernatant was removed, and water was added. This process was repeated five times to wash the magnetic particles.

[0108] The magnetic particles obtained above were magnetically recovered, and water was added to prepare a magnetic particle aqueous slurry so that when the slurry was 100 parts by mass, the amount of magnetic particles in the slurry was 20 parts by mass. 320 g of diethylamine and 64 g of the above magnetic particle aqueous slurry were added to a reaction vessel and treated in an ultrasonic bath for 15 minutes. 9.1 g of distilled water and 31.4 g of 3-methacryloxypropyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd.) as a silane coupling agent were added and treated in an ultrasonic bath for 30 minutes. The reaction vessel was then placed in a shaker and stirred at 100 rpm for 24 hours. The magnetic particles were then recovered by centrifugation. The recovered magnetic particles were washed by adding acetone and repeating the centrifugation process a total of three times. By air drying, magnetic particles modified with the silane coupling agent were obtained.

[0109] <Manufacturing of Magnetic Bead Intermediate> 1.9 parts by mass of methyl methacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 1.9 parts by mass of ethylene glycol dimethacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.9 parts by mass of 2-methacryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd., product name: Light Ester HOMS (N)) as monomers containing a carboxyl group (a1) were mixed, and 0.28 parts by mass of 2,2'-azobis(2,4-dimethylvaleronitrile) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added as a polymerization initiator. The mixture was then treated in an ultrasonic bath (manufactured by Yamato Scientific Co., Ltd., tabletop ultrasonic cleaner, product name: CPX5800H-J) to completely dissolve the polymerization initiator. To this, 0.28 parts by mass of magnetic particles (median diameter 146 nm) modified with the silane coupling agent obtained above were added and treated in an ultrasonic bath for 15 minutes to completely disperse the magnetic particles modified with the silane coupling agent and obtain a monomer mixture.

[0110] Separately, 0.03 parts by mass of sodium nitrite (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was placed in a four-neck separable flask. Next, an aqueous solution of deionized water and partially saponified polyvinyl alcohol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., degree of saponification: 88 mol%, degree of polymerization: 1500) as a dispersion stabilizer (an aqueous solution prepared so that the amount of deionized water was 92.8 parts by mass and the amount of polyvinyl alcohol was 2.0 parts by mass) was placed in the four-neck separable flask, and the mixture was processed by rotating the stirring blade at 150 rpm using a Three One Motor (manufactured by AS ONE Corporation) to obtain an aqueous medium. The monomer mixture obtained above was added to the resulting aqueous medium, and the mixture was stirred in an ice bath at 3000 rpm for 10 minutes using a rotor / stator homogenizer (manufactured by IKA Japan Co., Ltd., product name: T18 digital ULTRA-TURRAX, shaft generator: S18N-19G) to obtain a suspension in which the monomer mixture was dispersed as droplets in the aqueous medium.

[0111] Next, a four-necked separable flask was equipped with a stirring blade, a flux condenser, a nitrogen purge tube, and a thermometer. The flask was immersed in a water bath, and nitrogen gas was blown in for 30 minutes while the stirring blade was rotated at 150 rpm to perform nitrogen purging. After that, the temperature of the water bath was raised to 70°C, and suspension polymerization was carried out for 24 hours to obtain a magnetic bead intermediate.

[0112] After polymerization, the suspension was passed through a filter (Yotoriyama Co., Ltd., 200 mesh) to remove aggregates. Next, the suspension was centrifuged and water was added three times, followed by centrifugation and ethanol once, and then centrifugation and water once to wash the magnetic bead intermediate. Furthermore, magnetic separation was performed using a magnet, and the material to be dried was transferred to a 50 mL glass bottle and placed in a vacuum dryer (AS ONE Corporation, product name: AVO-250NS-D). The dryer was then vacuum dried under reduced pressure using a vacuum pump (Sato Vacuum Co., Ltd., product name: P135D, 0.67 Pa or less) at 50°C overnight to obtain a dried powder. 10 parts by mass of 1 N hydrochloric acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to 0.5 parts by mass of the obtained dried powder and exposed to the acid for 60 minutes. Subsequently, the treated magnetic bead intermediate was washed and purified by magnetic separation, and finally, water was added and ultrasonic irradiation was performed to obtain a purified suspension containing the magnetic bead intermediate.

[0113] <Production of magnetic beads containing alkynyl groups as surface functional groups> The purified suspension containing the magnetic bead intermediate obtained above was subjected to magnetic separation using a magnet, and the material to be dried was transferred to a 50 mL glass bottle and placed in a vacuum dryer. The material was then vacuum dried overnight at 50°C under reduced pressure by evacuating with a vacuum pump to obtain an acid-washed magnetic bead intermediate (dried powder). 0.12 g of the obtained acid-washed magnetic bead intermediate (dried powder) was mixed with 0.11 g of 1-hydroxybenzotriazole monohydrate (manufactured by Tokyo Chemical Industry Co., Ltd.) and 2.4 g of ion-exchanged water, and the magnetic bead intermediate was dispersed by ultrasonic irradiation. Furthermore, 0.72 mL of 0.1 N hydrochloric acid, 0.27 mL of a 0.27 mol / L aqueous solution of propargylamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as compound (b) having a surface functional group (alkynyl group), and 0.014 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (manufactured by Tokyo Chemical Industries, Ltd.) were added, and the mixture was shaken overnight at room temperature using a shaker (manufactured by AS ONE Corporation). Next, the reaction solution was centrifuged, the supernatant was discarded, and 0.1 N hydrochloric acid was added once. This procedure was repeated twice: centrifuged, the supernatant was discarded, and deionized water was added. This procedure was repeated once: centrifuged, and deionized water was added once. The magnetic beads were then washed to obtain a suspension of magnetic beads containing an alkynyl group as a surface functional group. The obtained suspension was used as the magnetic beads of Example 1.

[0114] The polymer in the magnetic beads of Example 1 contains a structural unit represented by the following formula (A1).

[0115]

[0116] [Example 2] Magnetic beads of Example 2 were obtained in the same manner as the method for producing magnetic beads of Example 1, except for the points described below. In the <Production of Magnetic Bead Intermediate> of Example 1, 0.9 parts by mass of 2-acryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd., product name: Light Acrylate HOA-MS(N)) was used instead of 0.9 parts by mass of 2-methacryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd., product name: Light Acrylate HOA-MS(N)) as monomers containing a carboxyl group (a1).

[0117] The polymer in the magnetic beads of Example 2 contains a structural unit represented by the following formula (A2).

[0118]

[0119] [Example 3] Magnetic beads of Example 3 were obtained in the same manner as the method for producing magnetic beads of Example 1, except for the points described below. In the <Production of Magnetic Bead Intermediate> of Example 1, the content of methyl methacrylate and the content of ethylene glycol dimethacrylate were changed to 2.4 parts by mass. Also, as monomers containing a carboxyl group (a1), 0.9 parts by mass of 2-methacryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd.) was replaced with 0.25 parts by mass of methacrylic acid (manufactured by Tokyo Chemical Industry Co., Ltd.). In the <Production of Magnetic Beads Containing an Alkynyl Group as a Surface Functional Group> of Example 1, as compound (b) having a surface functional group (alkynyl group), 0.27 mL of a 0.27 mol / L aqueous solution of propargylamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was replaced with 0.27 mL of a 0.27 mol / L aqueous solution of propargyl-PEG2-amine (manufactured by Tokyo Chemical Industry Co., Ltd.).

[0120] The polymer in the magnetic beads of Example 3 contains a structural unit represented by the following formula (A3).

[0121]

[0122] [Example 4] Magnetic beads of Example 4 were obtained in the same manner as the method for producing magnetic beads of Example 1, except for the points described below. In the <Production of Magnetic Bead Intermediate> of Example 1, the content of methyl methacrylate and the content of ethylene glycol dimethacrylate were changed to 2.4 parts by mass. Also, as monomers containing a carboxyl group (a1), 0.9 parts by mass of 2-methacryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd.) was replaced with 0.25 parts by mass of methacrylic acid (manufactured by Tokyo Chemical Industry Co., Ltd.).

[0123] The polymer in the magnetic beads of Example 4 contains a structural unit represented by the following formula (A4).

[0124]

[0125] [Example 5] Magnetic beads of Example 5 were obtained in the same manner as the method for producing magnetic beads of Example 1, except for the points described below. In the <Production of Magnetic Bead Intermediate> of Example 1, 0.9 parts by mass of mono-2-(methacryloyloxy)ethyl phthalate (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of 0.9 parts by mass of 2-methacryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd.) as monomers containing a carboxyl group (a1).

[0126] The polymer in the magnetic beads of Example 5 contains a structural unit represented by the following formula (A5).

[0127]

[0128] [Example 6] Except for the points described below, the magnetic beads of Example 6 were obtained in the same manner as the method for manufacturing magnetic beads of Example 1. Instead of the <production of magnetic beads containing alkynyl groups as surface functional groups> of Example 1, the <production of magnetic beads containing azide groups as surface functional groups> described below was performed.

[0129] <Production of magnetic beads containing azide groups as surface functional groups> A purified suspension containing a magnetic bead intermediate produced by the same method as in <Production of magnetic bead intermediate> in Example 1 was subjected to magnetic separation using a magnet, and the material to be dried was transferred to a 50 mL glass bottle and placed in a vacuum dryer. The material was then vacuum dried overnight at 50°C under reduced pressure by evacuating with a vacuum pump to obtain an acid-washed magnetic bead intermediate (dried powder). 0.12 g of the obtained acid-washed magnetic bead intermediate (dried powder) was mixed with 0.11 g of 1-hydroxybenzotriazole monohydrate (manufactured by Tokyo Chemical Industry Co., Ltd.) and 2.4 g of ion-exchanged water, and the magnetic bead intermediate was dispersed by ultrasonic irradiation. Furthermore, 0.72 mL of 0.1 N hydrochloric acid, 0.27 mL of a 0.27 mol / L aqueous solution of 3-azidopropylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) as compound (b) having a surface functional group (azide group), and 0.014 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added, and the mixture was shaken overnight at room temperature using a shaker (manufactured by AS ONE Corporation). Next, the reaction solution was centrifuged, the supernatant was discarded, and 0.1 N hydrochloric acid was added once. This procedure was repeated twice: centrifuged, the supernatant was discarded, and deionized water was added. This procedure was repeated once: centrifuged, and deionized water was added once. The magnetic beads were then washed to obtain a suspension of magnetic beads containing an alkynyl group as a surface functional group. The obtained suspension was used as the magnetic beads of Example 6.

[0130] The polymer in the magnetic beads of Example 6 contains a structural unit represented by the following formula (A6).

[0131]

[0132] [Comparative Example 1] The purified suspension containing the magnetic bead intermediate obtained in the <Production of Magnetic Bead Intermediate> of Example 1 was used as the magnetic beads of Comparative Example 1.

[0133] The polymer in the magnetic beads of Comparative Example 1 contains structural units represented by the following formula (B1).

[0134]

[0135] [Evaluation and Measurement] Measurements and evaluations were performed on the magnetic beads obtained in each example and comparative example. The results are shown in Table 1.

[0136] <Volume-average particle size (MV) of magnetic beads> A portion of the magnetic beads (suspension) from Examples 1 to 6 and Comparative Example 1 was pipettered, and deionized water was added to create a 1% by mass aqueous solution of magnetic beads, which was used as the measurement sample. The particle size distribution of the measurement sample was measured using a laser diffraction particle size distribution analyzer (Beckman Coulter, product name: LS 13 320) to obtain the volume-average particle size (MV).

[0137] <Magnetic Particle Content in Magnetic Beads> A portion of the magnetic beads (suspensions) from Examples 1-6 and Comparative Example 1 was pipettered and vacuum-dried to obtain dried powder. The dried powder was subjected to thermogravimetric analysis (TG-DTA). The magnetic particle content in the magnetic beads was determined under the following conditions: Measuring device: 2000SR (NETZSCH) Atmosphere: Nitrogen Heating rate: 10°C / min Sample measuring container: Platinum 5 mg of the purified suspension dried powder was placed in a platinum cell and set in the measuring section. Then, the measuring section was heated to 800°C while injecting nitrogen. After holding the temperature of the measuring section at 800°C for 2 minutes, the weight of the residue was taken as the weight of the magnetic particles, and the magnetic particle content in the magnetic beads was determined.

[0138] <Evaluation of fluorescence intensity> (Impeding of fluorescent molecules) - Examples 1-5 and Comparative Example 1 Deionized water was added to the magnetic beads (suspensions) of Examples 1-5 and Comparative Example 1 so that the concentration of magnetic beads was 2% by mass, to obtain diluted magnetic bead suspensions. 12.5 μL of a 20 μM aqueous solution of oligonucleotides having fluorescein at the 5' end and an azide group at the 3' end, and 5.0 μL of the above diluted magnetic bead suspension were reacted in a click reaction solution (0.2 M NaCl (Promega, Cat: V4221), 1.38 mM THPTA (Sigma, Cat: 752342-500MG), 0.4 mM copper sulfate (Nacalai Tesque, Cat: 09605-04), 2 mM sodium ascorbate (Nacalai Tesque, Cat: 11692-52), total reaction volume: 250 μL) at room temperature with stirring for 2 hours. 5 μL of Tween-20 (Nacalai Tesque, Cat: 28353-14) was added to achieve a final concentration of 0.1%, mixed, and the magnetic beads were collected using a magnetic stand. The supernatant was discarded, and 250 μL of PBS containing 0.05% Tween-20, pH 7.4 (137 mM sodium chloride (Nacalai Tesque, Cat: 31333-45), 2.7 mM potassium chloride (Nacalai Tesque, Cat: 28538-75), 10 mM disodium hydrogen phosphate (Nacalai Tesque, Cat: 31738-55), 1.8 mM potassium dihydrogen phosphate (Nacalai Tesque, Cat: 28736-75)) was added to resuspend the beads, and the magnetic beads were collected again using a magnetic stand. The same procedure was repeated twice. The beads were resuspended in 300 μL of PBS, pH 7.4 (137 mM sodium chloride (Nacalai Tesque Co., Ltd., Cat: 31333-45), 2.7 mM potassium chloride (Nacalai Tesque Co., Ltd., Cat: 28538-75), 10 mM disodium hydrogen phosphate (Nacalai Tesque Co., Ltd., Cat: 31738-55), 1.8 mM potassium dihydrogen phosphate (Nacalai Tesque Co., Ltd., Cat: 28736-75)) to obtain a magnetic bead suspension (oligonucleotide-coated magnetic bead suspension) on which oligonucleotides containing fluorescein were attached to the surface.

[0139] Example 6 A magnetic bead suspension (oligonic acid-conjugated magnetic bead suspension) having oligoonic acid having fluorescein on its surface was obtained by the same method as in Examples 1 to 5 and Comparative Example 1, except that a 20 μM aqueous solution of oligoonic acid having fluorescein at the 5' end and an azide group at the 3' end was used instead of a 20 μM aqueous solution of oligoonic acid having fluorescein at the 5' end and an azide group at the 3' end.

[0140] (Evaluation of fluorescence intensity) The oligonucleotide-contaminated magnetic bead suspension was subjected to a flow cytometer (Becton Dickinson, product name: BD LSRFortessa X-20). The measurement voltage was set to 10 for the maximum fluorescence signal of the magnetic beads (non-oligonucleotide-contaminated magnetic beads) of Examples 1-6 and Comparative Example 1. ２ The settings were adjusted accordingly. For Examples 1-6 and Comparative Example 1, the fluorescence signals of the magnetic beads (non-oligonucleotide magnetic beads) were based on the fluorescence signal values ​​obtained when the non-oligonucleotide magnetic bead suspension was measured separately using the same method. The following evaluation criteria were used based on the fluorescence intensity detected when flowed through a flow cytometer. When the fluorescence intensity of the non-oligonucleotide magnetic beads was denoted as F1 and the fluorescence intensity of the oligonucleotide magnetic beads as F2, a ratio of F2 / F1 exceeding 200 was evaluated as "AA (particularly good)". A ratio of F2 / F1 between 100 and 200 was evaluated as "A (good)", a ratio between 30 and 100 was evaluated as "B (fairly good)", a ratio between 10 and 30 was evaluated as "C (average)", and a ratio of 10 or less was evaluated as "D (poor)".

[0141] <Analysis of Surface Functional Groups> Regarding the magnetic beads of Examples 1 to 6, we investigated methods for analyzing whether carboxyl groups remain on the surface functional groups of the magnetic beads, but we were unable to establish an analytical method. That is, it is unclear whether carboxyl groups remain on the surface functional groups of the magnetic beads of Examples 1 to 6. However, the inventors speculate that trace amounts of carboxyl groups remain on the magnetic beads of Examples 1 to 6. The reasons are as follows: In Examples 1 to 6, the magnetic beads maintained dispersion stability in water even after washing. The inventors speculate that the magnetic beads of Examples 1 to 6 were able to maintain dispersion stability because trace amounts of carboxyl groups remained. Furthermore, considering the manufacturing method of the magnetic beads, we speculate that trace amounts of carboxyl groups originating from the magnetic bead intermediate remain on the magnetic beads of Examples 1 to 6. That is, the inventors speculate that the magnetic beads of Examples 1 to 5 contain alkynyl groups and carboxyl groups as surface functional groups, and the magnetic beads of Example 6 contain azide groups and carboxyl groups as surface functional groups.

[0142]

[0143] Table 1 shows that the magnetic beads of the example had a fluorescence intensity evaluation result of "C (normal)" or higher. In other words, it can be understood that the magnetic beads of this embodiment can bind a larger amount of the target substance.

[0144] This application claims priority based on Japanese Patent Application No. 2025-017391, filed on 5 February 2025, and incorporates all of its disclosures herein.

Claims

1. Magnetic beads comprising a polymer and magnetic particles, wherein the surface of the magnetic beads includes a surface functional group, and the surface functional group comprises at least one selected from the group consisting of alkynyl groups and azide groups.

2. The magnetic beads according to claim 1, wherein the polymer comprises a structural unit represented by the following formula (1). (In formula (1) above, R １ ~R ３ Each of the elements is independently a hydrogen atom or an organic group having 1 to 20 carbon atoms, X is a divalent organic group including a main chain, the main chain having 3 or more atoms, and Y is an alkynyl group or an azide group.

3. The magnetic bead according to claim 2, wherein the number of atoms in the main chain of X in formula (1) is 6 or more.

4. The magnetic bead according to claim 2 or 3, wherein the main chain in X in formula (1) is composed of one or more atoms selected from the group consisting of carbon atoms, nitrogen atoms, oxygen atoms, sulfur atoms, and silicon atoms, and at least one atom is an oxygen atom.

5. The magnetic beads according to any one of claims 1 to 3, wherein the surface functional group further comprises a carboxyl group.

6. Magnetic beads according to any one of claims 1 to 3, wherein the volume-average particle diameter (MV) is 10 μm or more and 50 μm or less.

7. The magnetic beads according to any one of claims 1 to 3, wherein the surface functional groups are capable of bonding with bio-related substances.

8. Magnetic beads according to any one of claims 1 to 3, which can be used in a diagnostic agent.

9. Magnetic beads according to any one of claims 1 to 3, further comprising a bio-related substance, wherein the bio-related substance is bonded to the surface functional group.

10. A diagnostic agent comprising magnetic beads according to any one of claims 1 to 3.

11. A method for producing magnetic beads, comprising: (A) preparing a magnetic bead intermediate containing a polymer, magnetic particles, and a magnetic bead intermediate having a carboxyl group on its surface; and (B) converting the carboxyl group of the magnetic bead intermediate to a group containing a surface functional group comprising at least one selected from the group consisting of alkynyl groups and azide groups.

12. The method for producing magnetic beads according to claim 11, wherein step (A) comprises polymerizing monomers (a) in a suspension containing monomers (a) and magnetic particles, and the monomers (a) include monomers (a1) containing carboxyl groups.

13. The method for producing magnetic beads according to claim 12, wherein the monomers (a1) include at least one selected from the group consisting of methacrylic acid, acrylic acid, 2-methacryloyloxyethyl succinic acid, 2-acryloyloxyethyl succinic acid, 2-methacryloyloxyethyl hexahydrophthalic acid, 2-acryloyloxyethyl hexahydrophthalic acid, 2-carboxyethyl methacrylate, 2-carboxyethyl acrylate, itaconic acid, 4-[[6-(acryloyloxy)hexyl]oxy]benzoic acid, 2-carboxyethyl acrylate oligomer, and 2-carboxyethyl methacrylate oligomer.

14. A method for producing magnetic beads according to any one of claims 11 to 13, wherein step (B) includes a step of reacting the carboxyl group of the magnetic bead intermediate with the compound (b) having the surface functional group.

15. The method for producing magnetic beads according to claim 14, wherein compound (b) comprises at least one selected from the group consisting of amines having the surface functional group and alcohols having the surface functional group.

16. Compound (b) is propargylamine, 2-(2-propynyloxy)ethylamine, 2-[2-(2-propynyloxy)ethoxy]ethylamine, 4-ethynylaniline, 4-(2-propynyloxy)aniline, triethylene glycol 2-aminoethylpropargyl ether, 2-propyne-1-ol, 3-butyne-1-ol, 3-butyne-2-ol, (S)-1-octin-3-ol, 6-heptin-1-ol, 1-pentin-3-ol, 4-pentin-1-ol, 4-pentin-2-ol, 1-hexyn-3-ol, 5-hexyn-1-ol, 9-decine-1-ol, 10-undecine-1-ol, (R)-(+) A method for producing magnetic beads according to claim 14, comprising at least one selected from the group consisting of -3-butyne-2-ol, (S)-(-)-3-butyne-2-ol, 2-methyl-3-butyne-2-ol, 3-methyl-1-pentin-3-ol, 1-ethynyl-1-cyclohexanol, 1-phenyl-2-propyne-1-ol, diethylene glycol mono(2-propyne-1-yl) ether, triethylene glycol mono(2-propynyl) ether, 3-azidopropylamine, 6-azidohexylamine, azido-PEG3-amine, azido-PEG4-amine, 6-azidohexane-1-ol, and glycidylpropargyl ether.

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