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

Abstract

To provide magnetic beads capable of binding a larger amount of the target substance. [Solution] 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 an alkynyl group and an azide group.

Description

[Technical Field]

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

[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 to the surface via a coupling agent. 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 also having excellent heat resistance, wear resistance, etc., and being hard and resistant to cracking, making them suitable for use as carriers and the like. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 6-102708 [Overview of the project] [Problems that the invention aims to solve]

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

[0006] According to the present invention, the following magnetic beads, diagnostic reagents, and magnetic beads are provided.

[0007] [1] Magnetic beads comprising a polymer and magnetic particles, The surface of the magnetic beads contains surface functional groups, Magnetic beads, wherein 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 includes a structural unit represented by the following formula (1). [ka] (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 beads according to [2], wherein the number of atoms in the main chain in X in formula (1) is 6 or more. [4] The magnetic beads according to [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 [1] to [4], wherein the surface functional group further comprises a carboxyl group. [6] Magnetic beads according to any of [1] to [5] above, 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 [1] to [6], wherein the surface functional group is capable of bonding with biomimetic substances. [8] Magnetic beads according to any one of the above [1] to [7], which can be used in diagnostic agents. [9] It further contains bio-related substances, The magnetic beads according to any one of [1] to [8], wherein the bio-related substance is bonded to the surface functional group.

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

[11] A magnetic bead intermediate containing a polymer and magnetic particles, and step (A) of preparing a magnetic bead intermediate containing a carboxy group on the surface of the magnetic bead intermediate. A method for producing magnetic beads, comprising step (B) of converting the carboxy group of the magnetic bead intermediate into a group containing a surface functional group containing at least one selected from the group consisting of an alkynyl group and an azide group.

[12] Step (A) includes a step of polymerizing the monomeric substances (a) in a suspension containing the monomeric substances (a) and the magnetic particles. The method for producing magnetic beads according to

[11] , wherein the monomeric substances (a) include monomeric substances (a1) containing a carboxy group.

[13] The monomeric substances (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. The method for producing magnetic beads according to

[12] .

[14] Step (B) includes a step of reacting the carboxy group of the magnetic bead intermediate with a compound (b) having the surface functional group. The method for producing magnetic beads according to any one of

[11] to

[13] .

[15] The compound (b) includes at least one selected from the group consisting of amines having the surface functional group and alcohols having the surface functional group. The method for producing magnetic beads according to

[14] .

[16] The 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-propin-1-ol, 3-butin-1-ol, 3-butin-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. [Effects of the Invention]

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

[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 toners, magnetic inks, magnetic paints, and the like. 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). This invention provides magnetic beads 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 group in this embodiment includes 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 group, it becomes possible to bond a larger amount of the target substance.

[0015] The alkynyl group is, for example, a substituted or unsubstituted chain-like alkynyl group having 2 to 30 carbon atoms, and specifically includes at least one selected from the group consisting of an ethynyl group, a propargyl group, and a 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 may include at least one selected from the group consisting of, for example, a cyclooctinyl group, an azacyclooctinyl group, and a 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 in this embodiment preferably further comprises a carboxyl group. By further comprising a carboxyl group in the surface functional group, the dispersibility of the magnetic beads in a dispersion containing magnetic beads can be further improved.

[0018] The surface functional groups of this embodiment are preferably capable of binding to biomimetic substances. The surface functional groups capable of binding to biomimetic substances preferably include at least one selected from the group consisting of alkynyl groups and azide groups.

[0019] <polymer> The magnetic beads of this embodiment contain a polymer. The polymer in this embodiment is not particularly limited, but preferably includes a (meth)acrylic polymer. The polymer in this embodiment may be a homopolymer or a copolymer, but is preferably a copolymer.

[0020] The polymer of this embodiment preferably contains the surface functional groups of this embodiment.

[0021] The polymer of this embodiment preferably contains structural units represented by formula (1).

[0022] [ka]

[0023] In formula (1), R 1 ~R 3Each is 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 Each is 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, R 3 is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, more preferably, R 1 and R 2 are hydrogen atoms, 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, R 3 is a hydrogen atom or a methyl group.

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

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

[0027]

Chemical formula

[0028] In formula (1a), the atoms in the main chain in X are R 3The numbers in the chemical formula refer to atoms involved in the bond between the carbon atom (methyl group) and X and the atom (ethynyl group) Y. In other words, formula (1a) shows that the number of atoms in the main chain of X is 11.

[0029] Here, if Y is a substituted or unsubstituted linear alkynyl group, the boundary between X and Y in formula (1) is defined such that the atoms adjacent to the triple bond in Y are included in X. Therefore, in formula (1a), Y is defined as an ethynyl group, rather than defining Y as a propagyl group by combining the structure (-CH2-) at the point marked "11" with the terminal triple bond.

[0030] Here, for example, in formula (1a), the structure at the point marked "3" is "-CH2-", but we define that the only atoms in the main chain of X are carbon atoms, and hydrogen atoms do not belong to the main chain of X. This is because only carbon atoms are involved in the bond between the second and fourth adjacent atoms.

[0031] 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 5th and 7th 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 equation (1), the atoms constituting the main chain in X are not particularly limited and can be any atoms with a valency of two 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 the position of R in the aromatic ring. 3 This refers to the orthogonal position relative to the group involved in the bond 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, the target substance may not bond well. 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 well. Therefore, the inventors hypothesize that by not including an aromatic ring in which X contains a Y group only at the o position, 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 enabling 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, if Y is a substituted or unsubstituted cyclic alkynyl group, unlike a substituted or unsubstituted linear 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] [ka]

[0043] The polymer of this embodiment may further contain other structural units other than the structural unit represented by formula (1). 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 manufacturing magnetic beads described later. The polymer of this embodiment preferably comprises a structural unit represented by formula (1) and a structural unit 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 contain magnetic particles. The magnetic particles in this embodiment are not particularly limited as long as they are magnetic particles, but for example, they include at least one selected from the group consisting of magnetite (Fe3O4), maghemite (γ-Fe2O3), hematite (α-Fe2O3), manganese ferrite (MnFe2O4), cobalt ferrite (CoFe2O4), nickel ferrite (NiFe2O4), copper ferrite (CuFe2O4), 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 in this embodiment may be modified with, for example, a coupling agent or the like. The magnetic particles of this embodiment preferably 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 size 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 size of magnetic particles refers to the value measured by a transmission electron microscope (TEM), specifically, the value obtained by performing image analysis on a TEM image and calculating the arithmetic mean of the particle sizes of any 10 magnetic particles.

[0051] In this embodiment, the content of magnetic particles in the magnetic beads 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 content of the 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 ingredients> 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 magnetic beads refers to the volume-average particle size 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 1% by mass aqueous solution of magnetic beads in which the solvent is water.

[0054] The applications of the magnetic beads of this embodiment are not particularly limited and can be applied to, for example, diagnostic agents, bacterial isolation, cell culture, drug delivery, magnetic toner, magnetic ink, magnetic paint, etc. The magnetic beads of this embodiment are preferably magnetic beads that can be used in diagnostic reagents because they can bind a larger amount of the target substance. The applications of the magnetic beads in 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 bonded to the surface functional groups of the magnetic beads of this embodiment. The bio-related substance in 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 includes, for example, at least one selected from the group consisting of water, methanol, ethanol, isopropyl alcohol, propylene glycol monomethyl ether, and propylene glycol monomethyl ether acetate, and preferably includes 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 drug] The diagnostic agent of this embodiment includes the magnetic beads of this embodiment.

[0059] [Manufacturing method for 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] <Process (A)> The method for manufacturing magnetic beads according to this embodiment comprises a polymer, magnetic particles, and a magnetic bead intermediate containing these particles, and includes step (A) of preparing a magnetic bead intermediate containing carboxyl groups on its surface.

[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 monomers (a) preferably include monomers (a1) containing a carboxyl group. The monomers (a1) preferably further include, in addition to a carboxyl group, at least one selected from the group consisting of a vinyl group, a vinylidene group, and a (meth)acryloyl 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, and more preferably at least one selected from the group consisting of methacrylic acid, 2-methacryloyloxyethyl succinic acid, and 2-acryloyloxyethyl succinic acid, and even more preferably, from the viewpoint of enabling the resulting magnetic beads to bind even more of the target substance, it comprises at least one selected from the group consisting of 2-methacryloyloxyethyl succinic acid and 2-acryloyloxyethyl succinic acid.

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

[0067] [ka]

[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] [ka]

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

[0072] The content 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 content 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] The monomer (a) preferably further comprises monomer (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 vinyl group, vinylidene group, and (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). Monomers (a3) ​​are monomers that do not contain a carboxyl group and contain two or more polymerizable groups. Since monomers (a3) ​​function as crosslinking agents, including monomers (a3) ​​in monomers (a) can further improve the heat resistance of the resulting magnetic beads.

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

[0087] The method for preparing the suspension of this embodiment is not particularly limited, but preferably involves sequentially performing (i) and (ii) below. (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] <Process (B)> The method for producing 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 comprising 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 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 be reacted 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 group of a magnetic bead intermediate through condensation. Epoxy compounds having surface functional groups can be reacted with carboxyl groups in magnetic bead intermediates through addition reactions.

[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, the term "alcohols" is not limited to alcohols, but also includes alcohol derivatives that can be subjected to condensation reactions 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-propyno-1-ol, 3-butyno-1-ol, 3-butyno-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-butyno-2-ol, (S)-(-)-3-butyno-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 comprises 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-propin-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 can be any known condensation or addition reaction conditions. One 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 processes> The method for manufacturing magnetic beads according to this embodiment may further include other steps besides steps (A) and (B). Other processes include, for example, the process 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] The embodiments of the present invention have been described above, but these are merely examples, and various other configurations can also 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. [Examples]

[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] <Manufacturing of magnetic particles modified with silane coupling agents> 60 g of FeCl2·4H2O (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 135 g of FeCl3·6H2O (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a separable flask. Then, 1500 mL of pure water, which had been pre-purged with nitrogen by nitrogen bubbling, was added to dissolve the FeCl2·4H2O and FeCl3·6H2O. 400 mL of 28 wt% aqueous ammonia was then poured into the mixture, 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 the process of adding water 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. After that, the magnetic particles were 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 intermediates> 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 a monomer 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 silane coupling agent-modified magnetic particles 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 92.8 parts by mass of deionized water and 2.0 parts by mass of polyvinyl alcohol) 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 (manufactured by 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 again 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 (manufactured by AS ONE Corporation, product name: AVO-250NS-D). The dryer was then vacuum dried under reduced pressure using a vacuum pump (manufactured by 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 (manufactured by 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 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] <Manufacturing 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 under reduced pressure using a vacuum pump at 50°C overnight to obtain 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. Then, the supernatant was discarded after centrifugation and deionized water was added twice. Finally, deionized water was added after centrifugation once to wash the magnetic beads, and a suspension of magnetic beads containing alkynyl groups as surface functional groups was obtained. 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] [ka]

[0116] [Example 2] Except for the points described below, the magnetic beads of Example 2 were obtained using the same method as the manufacturing method of the magnetic beads of Example 1. 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.) as the monomer (a1) containing a carboxyl group.

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

[0118] [ka]

[0119] [Example 3] Except for the points described below, the magnetic beads of Example 3 were obtained using the same method as the magnetic beads of Example 1. In Example 1, the production of the magnetic bead intermediate was changed to 2.4 parts by mass of methyl methacrylate and 2.4 parts by mass of ethylene glycol dimethacrylate. In addition, 0.25 parts by mass of methacrylic acid (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 the monomer (a1) containing a carboxyl group. In Example 1, <Production of magnetic beads containing an alkynyl group as a surface functional group>, instead of 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), 0.27 mL of a 0.27 mol / L aqueous solution of propargyl-PEG2-amine (manufactured by Tokyo Chemical Industry Co., Ltd.) was used.

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

[0121] [ka]

[0122] [Example 4] Except for the points described below, the magnetic beads of Example 4 were obtained using the same method as the magnetic beads of Example 1. In Example 1, the production of the magnetic bead intermediate was changed to 2.4 parts by mass of methyl methacrylate and 2.4 parts by mass of ethylene glycol dimethacrylate. In addition, 0.25 parts by mass of methacrylic acid (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 the monomer (a1) containing a carboxyl group.

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

[0124] [ka]

[0125] [Example 5] Except for the points described below, the magnetic beads of Example 5 were obtained using the same method as the magnetic beads of Example 1. 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 monomer (a1) containing a carboxyl group.

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

[0127] [ka]

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

[0129] <Manufacturing of magnetic beads containing azide groups as surface functional groups> A purified suspension containing a magnetic bead intermediate, prepared in the same manner as in Example 1, was subjected to magnetic separation using a magnet. The material to be dried was transferred to a 50 mL glass bottle and placed in a vacuum dryer. The mixture was then vacuum dried under reduced pressure using a vacuum pump at 50°C overnight 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 deionized 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 a 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. Then, the supernatant was discarded after centrifugation and deionized water was added twice. Finally, deionized water was added after centrifugation once to wash the magnetic beads, and a suspension of magnetic beads containing alkynyl groups as surface functional groups was obtained. 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] [ka]

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

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

[0134] [ka]

[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 1-6 and Comparative Example 1 was collected using a pipette, and deionized water was added to prepare a 1% by mass aqueous solution of magnetic beads, which was used as the measurement sample. The particle size distribution was measured for the sample using a laser diffraction particle size distribution analyzer (Beckman Coulter, product name: LS 13 320), and the volume-average particle size (MV) was obtained.

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

[0138] <Evaluation of fluorescence intensity> (Addition of fluorescent molecules) Examples 1-5 and Comparative Example 1 To the magnetic bead (suspension) of Examples 1-5 and Comparative Example 1, ion-exchanged water was added to obtain a diluted magnetic bead suspension so that the concentration of the magnetic beads was 2% by mass. 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 then 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. This 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 containing fluorescein on its surface was obtained by the same method as in Examples 1-5 and Comparative Example 1, except that a 20 μM aqueous solution of oligoonic acid having fluorescein at the 5' end and an azede 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 azede 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, where the maximum fluorescence signal of the magnetic beads (non-oligonucleotide-contaminated magnetic beads) in Examples 1-6 and Comparative Example 1 was 10. 2 The settings were configured accordingly. Note that the fluorescence signals of the magnetic beads (non-oligonucleotide-contaminated magnetic beads) in Examples 1-6 and Comparative Example 1 were obtained from fluorescence signals obtained separately when the non-oligonucleotide-contaminated magnetic bead suspension was measured using the same method. The following evaluation criteria were used to assess the fluorescence intensity detected when the sample was passed through a flow cytometer. When the fluorescence intensity of magnetic beads without oligonucleotides was denoted as F1 and the fluorescence intensity of magnetic beads with oligonucleotides was denoted as F2, those with an F2 / F1 ratio exceeding 200 were evaluated as "AA (particularly good)". Those with an F2 / F1 ratio between 100 and 200 were evaluated as "A (good)", those between 30 and 100 were evaluated as "B (fairly good)", those between 10 and 30 were evaluated as "C (average)", and those with an F2 / F1 ratio of 10 or less were evaluated as "D (poor)".

[0141] <Analysis of surface functional groups> We investigated analytical methods to determine whether carboxyl groups remain on the surface functional groups of the magnetic beads in Examples 1 to 6, but we were unable to establish an analytical method. In other words, it remains unclear whether carboxyl groups remain on the surface functional groups of the magnetic beads in Examples 1 to 6. However, the inventors suspect that trace amounts of carboxyl groups remain in the magnetic beads of Examples 1 to 6. The reasons are as follows: In Examples 1-6, the magnetic beads maintained their dispersion stability in water even after washing. The inventors hypothesize that the magnetic beads in Examples 1-6 were able to maintain their dispersion stability because they contained trace amounts of carboxyl groups. Furthermore, considering the manufacturing method of the magnetic beads, it is presumed that trace amounts of carboxyl groups originating from the magnetic bead intermediate remain in the magnetic beads of Examples 1 to 6. In other words, 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] [Table 1]

[0143] Table 1 shows that the magnetic beads in 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.

Claims

1. Magnetic beads comprising a polymer and magnetic particles, The surface of the magnetic beads contains surface functional groups, Magnetic beads, wherein 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 includes a structural unit represented by the following formula (1). 【Chemistry 1】 (In formula (1) above, R １ ~R ３ Each of these 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 group is capable of bonding with biomolecules.

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

9. It further contains bio-related substances, The magnetic beads according to any one of claims 1 to 3, 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 step (A) of preparing a magnetic bead intermediate containing a polymer and magnetic particles, wherein the magnetic bead intermediate contains a carboxyl group on its surface, A method for producing magnetic beads, comprising the step (B) of converting the carboxyl group of the magnetic bead intermediate to a group having a surface functional group including at least one selected from the group consisting of an alkynyl group and an azide group.

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

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. The 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 the 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. The 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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