Magnetic beads

Abstract

Provided are magnetic beads capable of improving the reliability of spectra obtained by measurement using a flow cytometer. [Solution] A magnetic bead comprising a polymer and magnetic particles, wherein D obtained by the following method 10 , D 50 and D 90 The span value calculated from D 90 -D 10 ) / D 50 is 1.00 or less. [Method] A measurement sample of an aqueous magnetic bead solution having a concentration of 1% by mass is subjected to ultrasonic dispersion treatment for 3 minutes using an ultrasonic cleaner, and the measurement sample after the ultrasonic dispersion treatment is measured for volume-based particle size distribution using a laser diffraction particle size distribution analyzer, and D 10 particle diameter (D 10 ), D 50 particle diameter (D 50 ) and D 90 particle diameter (D 90 ) are obtained respectively.

Description

[Technical Field]

[0001] This invention relates to magnetic beads. [Background technology]

[0002] As a technology relating to magnetic beads containing polymers and magnetic particles, for example, the technology described in Patent Document 1 is known.

[0003] Patent Document 1 describes a method for producing magnetic polymer particles, characterized by mixing 100 parts by weight of a radically polymerizable vinyl monomer containing 1 to 20% by weight of an unsaturated carboxylic acid with 5 to 200 parts by weight of a magnetic material, dispersing the mixture in water, polymerizing it with an oil-soluble initiator, and then treating the resulting particles with an organic base and / or a water-soluble solvent. According to the method for producing magnetic polymer particles described in Patent Document 1, it is stated that particles with a large number of useful carboxyl groups on their surface and a high surface charge can be obtained. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 10-87711 [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention provides magnetic beads that can improve the reliability of spectra obtained by measurements using a flow cytometer. [Means for solving the problem]

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

[0007] [1] Magnetic beads comprising a polymer and magnetic particles, D obtained by the following method10 , D 50 and D 90 calculated from, the span value (D 90 -D 10 ) / D 50 is 1.00 or less, magnetic beads. [Method] Using an aqueous magnetic bead solution with a concentration of 1 mass% as a measurement sample, the measurement sample is subjected to ultrasonic dispersion treatment for 3 minutes using an ultrasonic cleaner, and the volume-based particle size distribution is measured for the measurement sample after the ultrasonic dispersion treatment using a laser diffraction particle size distribution analyzer, D 10 particle diameter (D 10 ), D 50 particle diameter (D 50 ) and D 90 particle diameter (D 90 ) are obtained respectively. [2] D obtained by the above method 10 , D 50 and D 90 calculated from (D 90 -D 50 ) / D 50 when the value of is defined as α, α is 0.50 or less, the magnetic beads according to the above [1]. [3] D obtained by the above method 10 , D 50 and D 90 calculated from (D 90 -D 50 ) / D 50 the value of is defined as α, and (D 50 -D 10 ) / D 50 when the value of is defined as β, the value of α / β is 0.50 or more and 1.20 or less, the magnetic beads according to the above [1] or [2]. [4] D obtained by the above method 50 is 3 µm or more and 50 µm or less, the magnetic beads according to any one of the above [1] to [3]. [5] D obtained by the above method 10 is 1 µm or more and 40 µm or less, the magnetic beads according to any one of the above [1] to [4]. [6] D obtained by the above method 90 Magnetic beads according to any one of the above [1] to [5], wherein the diameter is 5 μm or more and 60 μm or less. [7] The magnetic beads according to any one of [1] to [6], wherein the polymer contains structural units derived from (meth)acrylate monomers. [8] The (meth)acrylate monomer comprises monomer (a), The magnetic beads according to [7], wherein the monomer (a) comprises at least one selected from the group consisting of n-butyl (meth)acrylate, t-butyl (meth)acrylate, dodecyl (meth)acrylate, stearyl (meth)acrylate, isobornyl (meth)acrylate, cyclohexyl (meth)acrylate, glycidyl (meth)acrylate, and trifluoroethyl (meth)acrylate. [9] The magnetic beads according to [7] or [8], wherein the (meth)acrylate monomer comprises methyl (meth)acrylate.

[10] The magnetic beads according to any one of the above [1] to [9], wherein the surface of the magnetic beads contains at least one selected from the group consisting of a carboxyl group, an alkynyl group, an azide group, a hydroxyl group, an amino group, and an epoxy group.

[11] The magnetic beads according to any one of [1] to

[10] , wherein the content of the magnetic particles in the magnetic beads is 1% by mass or more and 40% by mass or less when the total mass of the magnetic beads is 100% by mass.

[12] Magnetic beads according to any one of [1] to

[11] , wherein the particle diameter of the magnetic particles is 1 nm or more and 5000 nm or less.

[13] The magnetic beads according to any one of the above [1] to

[12] , wherein the magnetic particles include at least one selected from the group consisting of magnetite and maghemite.

[14] Magnetic beads according to any of the above [1] to

[13] , which can be used in diagnostic agents. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide magnetic beads that can improve the reliability of spectra obtained by measurements using a flow cytometer. [Modes for carrying out the invention]

[0009] 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)acrylate" represents a concept that encompasses both methacrylate and acrylate. The same applies to similar notations such as "(meth)acrylic acid". Furthermore, in this specification, "reliability of the spectrum obtained by measurement using a flow cytometer" may be abbreviated as "reliability of the spectrum."

[0010] [Magnetic beads] 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. Some of these magnetic beads are measured using a flow cytometer (hereinafter also referred to as FCM). In addition, in FCM measurements, magnetic beads of multiple particle sizes may be used. In such cases, if the spectra from magnetic beads of a certain particle size and the spectra from magnetic beads of similar particle sizes overlap, the reliability of the spectra may decrease.

[0011] The inventors diligently studied how to improve the reliability of spectra obtained by measurements using FCM. As a result, the inventors determined that the span value (D) of the magnetic beads is 90 -D 10 ) / D 50We arrived at the present invention by believing that by keeping this value below a certain upper limit, we can reduce the overlap between spectra and improve the reliability of the spectra.

[0012] The magnetic beads of this embodiment are magnetic beads comprising a polymer and magnetic particles, wherein the span value (D 90 -D 10 ) / D 50 It is 1.00 or less. The span value (D) of the magnetic beads in this embodiment 90 -D 10 ) / D 50 From the viewpoint of further improving spectral reliability, the value is preferably 0.90 or less, more preferably 0.80 or less, even more preferably 0.75 or less, even more preferably 0.70 or less, and even more preferably 0.65 or less. The lower limit is not particularly limited, but may be, for example, 0.40 or more, 0.45 or more, 0.50 or more, or 0.55 or more. Furthermore, the span value (D) of the magnetic beads in this embodiment 90 -D 10 ) / D 50 From the viewpoint of further improving spectral reliability, the value is preferably 0.40 to 1.00, more preferably 0.40 to 0.90, even more preferably 0.40 to 0.80, even more preferably 0.45 to 0.75, even more preferably 0.50 to 0.70, and even more preferably 0.55 to 0.65.

[0013] Here, D in this specification 10 , D 50 and D 90 The following terms represent the values ​​obtained by the methods described below.

[0014] [method] A 1% by mass aqueous solution of magnetic beads was used as the measurement sample. The measurement sample was subjected to ultrasonic dispersion treatment using an ultrasonic cleaner for 3 minutes. After ultrasonic dispersion treatment, the volume-based particle size distribution of the measurement sample was measured using a laser diffraction particle size distribution analyzer. 10 Particle size (D 10 ), D50 Particle size (D 50 ) and D 90 Particle size (D 90 ) are obtained respectively.

[0015] The span value of magnetic beads can be set to a desired value by adjusting the manufacturing method of the magnetic beads. Specifically, the span value of magnetic beads can be reduced by manufacturing magnetic beads by suspension polymerization, preparing a suspension by membrane emulsification, adjusting the type and content of the monomers used as raw materials, using monomer (a) described later as the monomer used as a raw material, or manufacturing magnetic beads by a method that includes a classification step.

[0016] The magnetic beads of this embodiment are (D 90 -D 50 ) / D 50 When the value of is α, from the viewpoint of further improving the reliability of the spectrum, α is preferably 0.50 or less, more preferably 0.45 or less, even more preferably 0.40 or less, even more preferably 0.35 or less, and even more preferably 0.32 or less, and the lower limit is not particularly limited, but may be, for example, 0.15 or more, 0.20 or more, or 0.25 or more.

[0017] The magnetic beads of this embodiment are (D 50 -D 10 ) / D 50 When the value of is denoted as β, β is preferably 0.50 or less, more preferably 0.45 or less, even more preferably 0.40 or less, and even more preferably 0.38 or less, from the viewpoint of further improving the reliability of the spectrum, and the lower limit is not particularly limited, but may be, for example, 0.15 or more, 0.20 or more, or 0.25 or more.

[0018] The magnetic beads of this embodiment are (D 90 -D 50 ) / D 50 Let the value of be α, (D 50 -D 10 ) / D 50When the value of is denoted as β, from the viewpoint of further improving the reliability of the spectrum, the value of α / β is preferably 0.50 or more and 1.20 or less, more preferably 0.55 or more and 1.10 or less, even more preferably 0.60 or more and 1.00 or less, even more preferably 0.65 or more and 0.95 or less, and even more preferably 0.70 or more and 0.90 or less.

[0019] D of the magnetic beads of this embodiment 50 Preferably, the particle size is 3 μm to 50 μm, more preferably 5 μm to 48 μm, even more preferably 10 μm to 45 μm, even more preferably 20 μm to 42 μm, even more preferably 25 μm to 40 μm, and even more preferably 28 μm to 38 μm.

[0020] D of the magnetic beads of this embodiment 10 Preferably, the particle size is 1 μm to 40 μm, more preferably 3 μm to 38 μm, even more preferably 5 μm to 35 μm, even more preferably 10 μm to 30 μm, and even more preferably 15 μm to 25 μm.

[0021] D of the magnetic beads of this embodiment 90 Preferably, the particle size is 5 μm to 60 μm, more preferably 8 μm to 55 μm, even more preferably 15 μm to 50 μm, even more preferably 30 μm to 48 μm, and even more preferably 35 μm to 45 μm.

[0022] The coefficient of variation (CV) in the volume-based particle size distribution obtained by the laser diffraction-scattering method of the magnetic beads of this embodiment is preferably 40% or less, more preferably 35% or less, even more preferably 30% or less, and even more preferably 28% or less, from the viewpoint of further improving spectral reliability, and the lower limit is not particularly limited, but may be, for example, 10% or more, or 15% or more. The volume-based particle size distribution obtained by laser diffraction-scattering to determine the coefficient of variation (CV) is D 10 , D 50 and D 90This refers to something obtained using a method similar to that used for obtaining volume-based particle size distribution.

[0023] The magnetic beads of this embodiment include a polymer and magnetic particles. The magnetic beads of this embodiment preferably contain magnetic particles inside the polymer.

[0024] The magnetic beads of this embodiment have at least one selected from the group consisting of carboxyl groups, alkynyl groups, azide groups, hydroxyl groups, amino groups, and epoxy groups on their surface, more preferably at least one selected from the group consisting of carboxyl groups, alkynyl groups, and azide groups, and even more preferably a carboxyl group.

[0025] The alkynyl group may be a substituted or unsubstituted linear alkynyl group, or a substituted or unsubstituted cyclic alkynyl group. The alkynyl group includes, for example, at least one selected from the group consisting of ethynyl group, propargyl group, trimethylsilylethynyl group, cyclooctinyl group, azacyclooctinyl group, and dibenzocyclooctinyl group, and preferably includes an ethynyl group.

[0026] When magnetic beads contain functional groups as described above on their surface, it becomes possible to immobilize a target substance (hereinafter referred to as the target substance) by reacting it with a substance that can bond to the functional groups. Magnetic beads containing such functional groups are preferable from the viewpoint of enabling applications in the medical and bio-fields, such as diagnostic agents.

[0027] The components included in the magnetic beads of this embodiment will be described in detail below.

[0028] <polymer> The magnetic beads of this embodiment contain a polymer. The polymer of this embodiment is not particularly limited, but preferably contains at least one monomer-derived structural unit selected from the group consisting of (meth)acrylate monomers, styrene monomers, and vinyl monomers, and more preferably contains a structural unit derived from a (meth)acrylate monomer.

[0029] The (meth)acrylate monomer includes, for example, at least one selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate, and aminoalkyl (meth)acrylate.

[0030] The (meth)acrylate monomer preferably contains monomer (a). Monomer (a) comprises at least one selected from the group consisting of n-butyl (meth)acrylate, t-butyl (meth)acrylate, dodecyl (meth)acrylate, stearyl (meth)acrylate, isobornyl (meth)acrylate, cyclohexyl (meth)acrylate, glycidyl (meth)acrylate, and trifluoroethyl (meth)acrylate, preferably comprising at least one selected from the group consisting of n-butyl methacrylate, t-butyl methacrylate, dodecyl methacrylate, stearyl methacrylate, isobornyl methacrylate, cyclohexyl methacrylate, glycidyl methacrylate, and trifluoroethyl methacrylate, more preferably comprising at least one selected from the group consisting of isobornyl methacrylate and t-butyl methacrylate, and even more preferably comprising isobornyl methacrylate.

[0031] The (meth)acrylate monomer preferably contains methyl (meth)acrylate, and more preferably contains methyl methacrylate.

[0032] The polymer of this embodiment preferably includes structural units derived from monomer (b) having a carboxyl group. The monomer (b) having a carboxyl group preferably further comprises at least one selected from the group consisting of a vinyl group, a vinylidene group, and a (meth)acryloyl group, and more preferably further comprises a (meth)acryloyl group.

[0033] Monomer (b) comprises, for example, at least one selected from the group consisting of (meth)acrylic acid, 2-(meth)acryloyloxyethyl succinic acid, mono-2-(methacryloyloxy)ethyl phthalate, and mono-2-(acryloyloxy)ethyl phthalate, preferably comprising 2-(meth)acryloyloxyethyl succinic acid, and more preferably comprising 2-methacryloyloxyethyl succinic acid.

[0034] From the viewpoint of further improving spectral reliability, the polymer of this embodiment preferably contains structural units derived from monomer (a) and structural units derived from methyl (meth)acrylate, and more preferably contains structural units derived from monomer (a), structural units derived from methyl (meth)acrylate, and structural units derived from monomer (b).

[0035] <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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The content of magnetic particles 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, from the viewpoint of improving the magnetic properties of the magnetic beads and increasing the functional groups on the surface of the magnetic beads, when the total content of the magnetic beads is considered as 100% by mass.

[0041] The magnetic beads of this embodiment may contain components other than polymers and magnetic particles.

[0042] [Uses of magnetic beads] 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 improve the reliability of the spectrum. 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.

[0043] [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.

[0044] 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.

[0045] [Manufacturing method for magnetic beads] The method for manufacturing the magnetic beads of this embodiment is not particularly limited, but preferred embodiments are described below. The method for producing magnetic beads according to this embodiment preferably includes the steps of (A) dispersing a monomer mixture containing monomers in an aqueous medium to obtain a suspension, and (B) suspend polymerization of the monomers in the suspension. The method for producing magnetic beads according to this embodiment more preferably includes, in addition to steps (A) and (B), a step of preparing a monomer mixture and a step of preparing an aqueous medium.

[0046] The following describes in detail each step of the manufacturing method for magnetic beads according to this embodiment.

[0047] <Steps for preparing a monomer mixture> The method for manufacturing magnetic beads in this embodiment preferably includes a step of preparing a monomer mixture. The step of preparing the monomer mixture is performed before step (A).

[0048] The monomer mixture of this embodiment contains monomers. The monomer in this embodiment is not particularly limited as long as it contains a polymerizable group. The polymerizable group includes, for example, at least one selected from the group consisting of vinyl group, vinylidene group, and (meth)acryloyl group.

[0049] The preferred embodiments of the monomers included in the monomer mixture of this embodiment are the same as the preferred embodiments of the monomers in the "monomer-derived structural units" in the polymer of the magnetic beads of this embodiment. Monomers (a) and (b) included in the monomer mixture described later are also synonymous with monomers (a) and (b) in the "monomer-derived structural units".

[0050] The monomer mixture of this embodiment preferably contains monomer (a). If step (A) is a step in which a suspension is obtained by a membrane emulsification method, the monomer mixture of this embodiment contains monomer (a), which can reduce the span value of the resulting magnetic beads. The reason for this is not clear, but the inventors speculate that monomer (a) is a highly hydrophobic monomer, and that when monomer (a) is included in the monomer mixture, irregular fragmentation of the monomer mixture within the pores of the porous membrane used in step (A) is suppressed, and the particle size of the monomer mixture droplets in the suspension can be made more uniform, thus reducing the span value of the resulting magnetic beads.

[0051] The content of monomer (a) in the monomer mixture of this embodiment is preferably 0.01% to 15% by mass, more preferably 0.1% to 14% by mass, and even more preferably 0.5% to 12% by mass, when the total content of all components in the monomer mixture is taken as 100% by mass, from the viewpoint of reducing the span value of the resulting magnetic beads.

[0052] The monomer mixture of this embodiment preferably contains methyl (meth)acrylate. The content of methyl (meth)acrylate in the monomer mixture of this embodiment is preferably 30% to 75% by mass, more preferably 40% to 70% by mass, and even more preferably 45% to 63% by mass, when the total content of all components in the monomer mixture is taken as 100% by mass, from the viewpoint of reducing the span value of the resulting magnetic beads.

[0053] The content of monomer (a) in the monomer mixture 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 25 parts by mass or less, and even more preferably 1 part by mass or more and 20 parts by mass or less, from the viewpoint of reducing the span value of the magnetic beads, when the content of methyl (meth)acrylate in the monomer mixture is 100 parts by mass.

[0054] The monomer mixture of this embodiment preferably includes monomer (b) having a carboxyl group. The content of monomer (b) in the monomer mixture of this embodiment is preferably 1% by mass or more and 50% by mass or less, more preferably 5% by mass or more and 45% by mass or less, and even more preferably 10% by mass or more and 40% by mass or less, when the total content of all components in the monomer mixture is taken as 100% by mass.

[0055] The monomers in this embodiment preferably include polyfunctional monomers. A polyfunctional monomer is a monomer containing two or more polymerizable groups. Since polyfunctional monomers function as crosslinking agents, including a polyfunctional monomer in the monomer mixture of this embodiment can further improve the heat resistance of the resulting magnetic beads.

[0056] The polymerizable group of the polyfunctional monomer 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.

[0057] The polyfunctional monomer includes, 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 including ethylene glycol di(meth)acrylate, and more preferably including ethylene glycol dimethacrylate.

[0058] The content of the polyfunctional monomer in the monomer mixture of this embodiment is preferably 1% by mass or more and 30% by mass or less, more preferably 3% by mass or more and 25% by mass or less, and even more preferably 5% by mass or more and 20% by mass or less, when the total content of all components in the monomer mixture is taken as 100% by mass.

[0059] The monomer mixture of this embodiment preferably contains monomer (a) and methyl (meth)acrylate, from the viewpoint of reducing the span value of the resulting magnetic beads. The monomer mixture of this embodiment more preferably comprises monomer (a), methyl (meth)acrylate, and monomer (b) from the viewpoint of reducing the span value of the resulting magnetic beads and further improving heat resistance, etc., and even more preferably comprises monomer (a), methyl (meth)acrylate, monomer (b), and a polyfunctional monomer.

[0060] The monomer mixture of this embodiment preferably contains magnetic particles. The content of magnetic particles in the monomer mixture of this embodiment is preferably 0.05% by mass or more and 5% by mass or less, more preferably 0.1% by mass or more and 4% by mass or less, and even more preferably 0.5% by mass or more and 3% by mass or less, when the total content of all components in the monomer mixture is taken as 100% by mass.

[0061] The monomer mixture of this embodiment preferably further comprises a polymerization initiator. The polymerization initiator preferably includes a thermal polymerization initiator, and more preferably includes a thermal radical polymerization initiator. The amount of polymerization initiator in the monomer mixture is appropriate.

[0062] The monomer mixture of this embodiment may also contain organic solvents, hydrophobic polymers, and the like.

[0063] The method for preparing the monomer mixture is not particularly limited, but is preferably a method in which the following steps (i) to (iii) are carried out sequentially. (i) Mix the monomer components. (ii) Dissolve the polymerization initiator in the mixed monomer components. (iii) Magnetic particles are added to the monomer components, and the magnetic particles are dispersed to obtain a monomer mixture.

[0064] <Steps for preparing an aqueous medium> The method for manufacturing magnetic beads according to this embodiment preferably includes a step of preparing an aqueous medium. The step of preparing the aqueous medium is performed before step (A).

[0065] The aqueous medium in this embodiment is not particularly limited as long as it is an aqueous medium that can be used in suspension polymerization. The aqueous medium in this embodiment contains water, preferably deionized water.

[0066] The aqueous medium of this embodiment may contain components such as dispersion stabilizers, surfactants, and polymerization inhibitors as appropriate. The amount of each component is appropriate.

[0067] The method for preparing the aqueous medium is not particularly limited; for example, each component can be mixed in any way.

[0068] <Step (A) to obtain the suspension> The method for producing magnetic beads in this embodiment preferably includes the step (A) of dispersing a monomer mixture containing monomers in an aqueous medium to obtain a suspension.

[0069] Step (A) is preferably a step of obtaining a suspension by membrane emulsification. If step (A) is a step in which a suspension is obtained by a membrane emulsification method, the span value of the resulting magnetic beads can be reduced.

[0070] Membrane emulsification is a method of preparing suspensions using porous membranes. The membrane emulsification method in this embodiment may be, for example, a direct membrane emulsification method or a membrane emulsification method accompanied by pre-emulsification. However, from the viewpoint of further simplifying the manufacturing process by omitting the preparation step of the pre-emulsified emulsion, a direct membrane emulsification method is preferred. Direct membrane emulsification is a method of obtaining a suspension by directly dispersing a monomer mixture in an aqueous medium through a porous membrane. The membrane emulsification method with pre-emulsification is a method in which a pre-emulsified emulsion, prepared by mixing a monomer mixture with an aqueous medium under mild conditions, is passed through a porous membrane to obtain a suspension.

[0071] The porous membrane is not particularly limited, but it includes at least one selected from the group consisting of, for example, glass porous membranes (e.g., SPG membranes) and PTFE membranes, and preferably includes glass porous membranes.

[0072] The pore size of the porous membrane is not particularly limited, but may be, for example, 1 μm to 50 μm, 2 μm to 30 μm, or 3 μm to 10 μm.

[0073] In the direct membrane emulsification method, the flow rate when directly dispersing a monomer mixture in an aqueous medium through a porous membrane is, for example, 1 μL / (min·cm).2 ) or more 500μL / (min cm 2 ) or less, and 2 μL / (min·cm 2 ) or more 500μL / (min cm 2 ) or less, and 5 μL / (min·cm 2 ) or more 200μL / (min cm 2 ) or less, and 10 μL / (min·cm 2 ) or more 100μL / (min cm 2 ) The following are also acceptable. Here, the above flow rate is given by the unit area [cm²] of the porous membrane. 2 This refers to the flow rate of the monomer mixture per minute per unit area. The porous membrane area refers to the area of ​​the porous membrane in contact with the aqueous medium during membrane emulsification.

[0074] Step (A) may be, for example, a step in which a monomer mixture is added to an aqueous medium, stirred using a homogenizer or the like, and a suspension is obtained.

[0075] The mixing ratio of the monomer mixture to the aqueous medium is expressed as a mass ratio, for example, monomer mixture:aqueous medium = 1:99 to 25:75.

[0076] <Step (B) of suspension polymerization of monomers> The method for producing magnetic beads in this embodiment preferably includes a step (B) of suspension polymerization of monomers in a suspension.

[0077] In step (B), the polymerization conditions are not particularly limited, but for example, they can be set to a temperature of 50°C or higher and 90°C or lower, and a time of 1 hour or higher and 30 hours or lower.

[0078] <Classification process> The method for manufacturing magnetic beads according to this embodiment preferably includes a classification step. The manufacturing method for magnetic beads in this embodiment includes a classification step, which makes it possible to reduce the span value of the resulting magnetic beads.

[0079] The classification method in the classification process may be dry classification or wet classification, but wet classification is preferred from the viewpoint of further improving classification accuracy. Specific methods of wet classification include, for example, classifying magnetic beads using a cell strainer in an aqueous dispersion of magnetic beads prepared at a predetermined concentration.

[0080] <Other processes> The method for manufacturing magnetic beads according to this embodiment may further include other steps. Other processes include, for example, separating the magnetic beads from the suspension and acid washing the magnetic beads. Methods for separating magnetic beads from a suspension include, for example, centrifugal separation and magnetic separation.

[0081] 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]

[0082] 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.

[0083] First, we will explain the manufacturing process of magnetic particles modified with the silane coupling agent used as a raw material.

[0084] 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. 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.

[0085] [Example 1] <Steps for preparing a monomer mixture> 1.6 parts by mass of methyl methacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 0.03 parts by mass of isobornyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) as monomer (a), 0.33 parts by mass of ethylene glycol dimethacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.89 parts by mass of 2-methacryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd., product name: Light Ester HOMS(N)) as monomer (b) having a carboxyl group were mixed, and 0.06 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, 0.04 parts by mass of magnetic particles (median diameter 146 nm) modified with a silane coupling agent were added and treated for 15 minutes in an ultrasonic bath (Sonic Bio Co., Ltd., sealed ultrasonic disruptor, product name: BIORUPTOR II (TYPE24)) under an ice bath to completely disperse the magnetic particles modified with the silane coupling agent. Furthermore, considering subsequent processes, aggregates of undispersed magnetic particles were removed using a microsyringe filter (Tokyo Glass Instruments Co., Ltd., hydrophobic PTFE, 1.0 μm) to obtain a monomer mixture in which the magnetic particles were uniformly dispersed.

[0086] <Steps for preparing an aqueous medium> Separately, 94.6 parts by mass of deionized water was added, and 0.03 parts by mass of sodium nitrite (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 0.2 parts by mass of sodium dodecyl sulfate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 2.3 parts by mass of 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 were dissolved in a 300 mL glass beaker to create an aqueous medium.

[0087] <Steps to obtain a suspension by membrane emulsification> The above monomer mixture was introduced into a 5 mL gastight syringe (TRAJAN / SGE) using a VAN metal needle (manufactured by Tsubasa Kogyo Co., Ltd., 90° cut tip, 16G x 40, locking base, 61-9093-07). The aqueous medium was stirred at 150 rpm in a general-purpose rotary homogenizer and then mixed using a syringe pump (Harvard, product name: PUMP 11 ELITE) at a flow rate of 10 μL / min (26 μL / min·cm). 2 The above monomer mixture was emulsified through a microglass filter (S.P.G. Techno Co., Ltd., DS08-050N, hydrophilic, pore size: 5 μm) to obtain a magnetic composite emulsion containing magnetic particles as a suspension.

[0088] <Process of suspension polymerization of monomers> The obtained suspension (magnetic composite emulsion) was transferred to a four-necked separable flask, 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 rotated at 150 rpm to replace oxygen with nitrogen. The water bath temperature was then raised to 70°C, and suspension polymerization was carried out for 24 hours to obtain magnetic beads.

[0089] <Purification process> After polymerization, the suspension was passed through a filter (manufactured by Yotoriyama Co., Ltd., 200 mesh) to remove aggregates. Next, the magnetic beads were washed by centrifugation followed by the addition of water three times, followed by the addition of ethanol once, and followed by the addition of water once. Furthermore, magnetic separation was performed using a magnet, and the dried material was transferred to a 50 mL glass bottle. The bottle was then placed in a vacuum apparatus (manufactured by AS ONE Corporation, product name: AVO-250NS-D) and evacuated under reduced pressure using a vacuum pump (manufactured by Sato Vacuum Co., Ltd., product name: P135D, 0.67 Pa or less). Under reduced pressure, the material was vacuum-dried 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 the mixture was exposed to the acid for 60 minutes. Subsequently, the treated magnetic beads were washed and purified by magnetic separation, and finally, water was added and ultrasonic irradiation was performed to obtain a purified suspension. The obtained purified suspension was used as the magnetic beads of Example 1.

[0090] [Example 2] Magnetic beads of Example 2 were obtained in the same manner as in Example 1, except that the monomer mixture and aqueous medium were formulated as shown in Table 1.

[0091] [Example 3] <Steps for preparing a monomer mixture> 3.8 parts by mass of methyl methacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 0.45 parts by mass of ethylene glycol dimethacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.75 parts by mass of 2-methacryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd., product name: Light Ester HOMS(N)) as a monomer (b) having a carboxyl group were mixed, and 0.31 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 treated in an ultrasonic bath (manufactured by Yamato Scientific Co., Ltd., tabletop ultrasonic cleaner, product name: CPX5800H-J) to completely dissolve the polymerization initiator. 0.16 parts by mass of magnetic particles (median diameter 146 nm) modified with a silane coupling agent were added, and the mixture was treated in the ultrasonic bath for 15 minutes to completely disperse the magnetic particles modified with the silane coupling agent, obtaining a monomer mixture.

[0092] <Steps for preparing an aqueous medium> 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 92.5 parts by mass and the amount of polyvinyl alcohol was 2.1 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.

[0093] <Steps to obtain a suspension> 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 5 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.

[0094] <Process of suspension polymerization of monomers> The obtained suspension was subjected to the same procedure as in Example 1, <the process of suspending and polymerizing monomers>, to obtain magnetic beads.

[0095] <Purification process> The polymerized suspension obtained in the above-mentioned <step of suspension polymerization of monomers> was subjected to the same procedure as the <purification step> in Example 1 to obtain a purified suspension.

[0096] <Classification process> A cell strainer with a mesh size of 20 μm was placed on a 50 mL conical tube. A purified suspension prepared to a concentration of 10% by mass was passed through the cell strainer with a mesh size of 20 μm to obtain permeate 1. Next, permeate 1 was passed through a cell strainer with a mesh size of 10 μm to obtain permeate 2. Then, permeate 2 was passed through a cell strainer with a mesh size of 5 μm, and the magnetic beads that did not pass through the cell strainer with a mesh size of 5 μm were thoroughly washed with pure water. The washed magnetic beads that did not pass through the cell strainer with a mesh size of 5 μm were used as the magnetic beads of Example 3. The cell strainers with mesh sizes of 20 μm, 10 μm, and 5 μm were manufactured by pluriSelect Life Science UG & Co.KG (PLS), and were registered trademarks called pluriStrainer. Mesh size refers to the size of the mesh opening of the cell strainer.

[0097] [Comparative Examples 1-2] The magnetic beads (purified suspensions) of Comparative Examples 1 and 2 were obtained in the same manner as in Example 3, except that the monomer mixture and aqueous medium were formulated as shown in Table 1, and the <classification step> in Example 3 was omitted.

[0098] [Measurement and Evaluation] The magnetic beads obtained in each example and comparative example were subjected to the following measurements and evaluations. The results are shown in Table 1.

[0099] <Particle size of magnetic beads> The magnetic beads from each example and comparative example were prepared as a 1% by mass aqueous solution of magnetic beads and used as the measurement sample. The measurement samples were subjected to ultrasonic dispersion treatment for 3 minutes using a tabletop ultrasonic cleaner (manufactured by Yamato Scientific Co., Ltd., product name: CPX5800H-J). For the sample after ultrasonic dispersion treatment, the volume-based particle size distribution was measured using a laser diffraction particle size distribution analyzer (Beckman Coulter, product name: LS 13 320), and the coefficient of variation (CV) and D were determined.10 particle diameter (D 10 ), D 50 particle diameter (D 50 ), D 90 particle diameter (D 90 ) were obtained respectively. From the obtained D 10 , D 50 and D 90 values, the span value ((D 90 -D 10 ) / D 50 ), the value of α ((D 90 -D 50 ) / D 50 ), the value of β ((D 50 -D 10 ) / D 50 ), and α / β were calculated respectively.

[0100] <Content of Magnetic Particles in Magnetic Beads> A portion of the magnetic beads from each example and each comparative example was collected and vacuum-dried to obtain a dried powder. The dried powder was subjected to thermogravimetric thermal analysis (TG-DTA). The content of magnetic particles in the magnetic beads was determined under the following conditions. Measuring apparatus: 2000SR (manufactured by NETZSCH) Atmosphere: Nitrogen Heating rate: 10°C / min Sample measurement container: Platinum 5 mg of the dried powder was placed in a platinum cell, and the cell was set in the measurement section. Thereafter, the measurement section was heated to 800°C while injecting nitrogen. After holding the temperature of the measurement section 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.

[0101] <Spectrum Reliability> For the magnetic beads of each example and each comparative example, the reliability of the spectrum obtained by measurement using FCM was evaluated based on the following criteria. A: In measurement using FCM, the spectrum is less likely to overlap with spectra of other magnetic bead groups having different particle size levels B: In measurement using FCM, the spectrum is likely to overlap with spectra of other magnetic bead groups having different particle size levels

[0102] Table 1

Claims

1. Magnetic beads comprising a polymer and magnetic particles, D obtained by the following method １０ , D ５０ and D ９０ The span value (D) is calculated from this. ９０ -D １０ ) / D ５０ Magnetic beads in which the coefficient is 1.00 or less. [method] Using a 1% by mass concentration aqueous magnetic bead solution as a measurement sample, the measurement sample is subjected to ultrasonic dispersion treatment for 3 minutes using an ultrasonic cleaner, and the volume-based particle size distribution of the measurement sample after the ultrasonic dispersion treatment is measured using a laser diffraction particle size distribution analyzer to obtain D １０ particle diameter (D １０ ), D ５０ particle diameter (D ５０ ) and D ９０ particle diameter (D ９０ ) are respectively obtained.

2. D obtained by the above method １０ , D ５０ and D ９０ (D) ９０ -D ５０ ) / D ５０ The magnetic beads according to claim 1, wherein when the value of is α, α is 0.50 or less.

3. D obtained by the above method １０ , D ５０ and D ９０ (D) ９０ -D ５０ ) / D ５０ Let the value of be α, (D ５０ -D １０ ) / D ５０ The magnetic beads according to claim 1 or 2, wherein when the value of is β, the value of α / β is 0.50 or more and 1.20 or less.

4. D obtained by the above method ５０ The magnetic beads according to claim 1 or 2, wherein the diameter is 3 μm or more and 50 μm or less.

5. D obtained by the above method １０ The magnetic beads according to claim 1 or 2, wherein the diameter is 1 μm or more and 40 μm or less.

6. D obtained by the above method ９０ The magnetic beads according to claim 1 or 2, wherein the diameter is 5 μm or more and 60 μm or less.

7. The magnetic beads according to claim 1 or 2, wherein the polymer comprises structural units derived from (meth)acrylate monomers.

8. The (meth)acrylate monomer comprises monomer (a), The magnetic beads according to claim 7, wherein the monomer (a) comprises at least one selected from the group consisting of n-butyl (meth)acrylate, t-butyl (meth)acrylate, dodecyl (meth)acrylate, stearyl (meth)acrylate, isobornyl (meth)acrylate, cyclohexyl (meth)acrylate, glycidyl (meth)acrylate, and trifluoroethyl (meth)acrylate.

9. The magnetic beads according to claim 7, wherein the (meth)acrylate monomer comprises methyl (meth)acrylate.

10. The magnetic beads according to claim 1 or 2, wherein the surface of the magnetic beads contains at least one selected from the group consisting of a carboxyl group, an alkynyl group, an azide group, a hydroxyl group, an amino group, and an epoxy group.

11. The magnetic beads according to claim 1 or 2, wherein the content of the magnetic particles in the magnetic beads is 1% by mass or more and 40% by mass or less when the total mass of the magnetic beads is 100% by mass.

12. The magnetic beads according to claim 1 or 2, wherein the particle diameter of the magnetic particles is 1 nm or more and 5000 nm or less.

13. The magnetic beads according to claim 1 or 2, wherein the magnetic particles include at least one selected from the group consisting of magnetite and maghemite.

14. Magnetic beads according to claim 1 or 2, which can be used in diagnostic agents.

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