JP2015044956A - Magnetic particles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide biomolecule-trapped particles which inhibit nonspecific adsorption and can be easily isolated and collected by magnetic force.SOLUTION: A magnetic particle comprises a superparamagnetic nanoparticle, a hydrophobic polymer, and a copolymer A having the following structural units (a) to (c): (a) a structural unit represented by the specified general formula (1); (b) a structural unit represented by the specified general formula (2); and (c) a structural unit represented by the specified general formula (3).

Description

  The present invention relates to a magnetic particle that can be easily separated and recovered by magnetic force, and non-specific adsorption is suppressed.

  Capturing biomolecules in specific particles is very useful as a means for separating, purifying, measuring, detecting, etc. biomolecules. By capturing biomolecules in specific particles, for example, detecting and diagnosing test target substances such as biomarkers and hormones for infectious diseases and cancer from patients, separating specific biomolecules such as nucleic acids and proteins By quantifying, specific bacteria and cells can be isolated.

  For this reason, particles that can trap biomolecules (biomolecule trapping particles) have been widely studied. For example, Patent Document 1 discloses a magnetic polymer particle obtained by introducing a polymerization initiating group on the surface of a magnetic particle, conducting a living polymerization reaction using the polymerization initiating group as a polymerization initiating source, and grafting the polymer onto the surface of the magnetic particle. Has been. By coating the magnetic particles with a polymer, various functions of the polymer can be imparted to the magnetic particles.

  By the way, what becomes a problem with such biomolecule-capturing particles is nonspecific adsorption that captures non-objects that are not intended to be captured by the biomolecule-capturing particles. Nonspecific adsorption is a phenomenon that occurs due to physical and chemical interaction between a non-object and a biomolecule-capturing particle.

  Such non-specific adsorption may result in insufficient purification and separation and inaccurate measurement results. In order to reduce nonspecific adsorption, a method called blocking is conventionally used. Blocking is a method in which a biomolecule-capturing substance is immobilized on a particle, and then the surface of the particle is coated with a biomolecule-based blocking agent such as albumin or casein with a small amount of adsorbed impurities. However, there may be a case where the coating effect of the blocking agent cannot be obtained sufficiently, or the action changes with time due to alteration of the blocking agent and nonspecific adsorption occurs. For this reason, the reduction of non-specific adsorption by the blocking agent was insufficient.

  Against this background, in order to further reduce nonspecific adsorption, a technique for treating particles with various functional polymers has been developed.

  For example, in Patent Document 2, a nonmagnetic core particle is coated with a magnetic layer, and the magnetic layer is further coated with a polymer containing 2,3-dihydroxypropyl group (a part of the hydroxyl group is a tosyloxy group or a carboxyalkyl group). Magnetic particles coated with a layer (converted to carbonyloxy groups) are disclosed. Non-specific adsorption is suppressed by the presence of the 2,3-dihydroxypropyl group. Furthermore, a biomolecule capturing function can be introduced into the magnetic particles by combining various functional groups in the polymer layer with a biomolecule capturing substance. Magnetic particles are widely used in this field because they can be easily separated and collected by magnetic force.

  It is also known that a polymer having a phosphorylcholine group has an action of suppressing nonspecific adsorption. For example, Patent Document 3 discloses a microparticle in which a biomolecule-capturing substance is immobilized, in which a first unit having a 2-methacryloyloxyethyl phosphorylcholine group and an electron-attracting substituent are bonded to a carbonyl group. There is disclosed a microparticle characterized in that a polymer containing a second unit having a carboxylic acid derivative group is coated on the surface.

  Further, as a technique using a polymer having a phosphorylcholine group, the surface of the titanium oxide particle is coated with a phosphorylcholine group-containing polymer which is represented by the following formula (I) and may contain a structural unit represented by the following formula (II). Further, polymer-coated particles have been developed (Patent Document 4).

  The triethoxysilane group in the formula (I) causes a silane coupling reaction with titanium oxide particles having a hydroxyl group, whereby the phosphorylcholine group-containing polymer is fixed to the titanium oxide. Since such polymer-coated particles are particles whose core particles have a large specific gravity, they can be collected and analyzed by natural sedimentation after being combined with a substance to be separated, or instead of natural sedimentation or by natural sedimentation. Subsequently, it is said that centrifugation can be performed. However, natural sedimentation requires time for recovery, and centrifugation requires a large apparatus.

JP 2006-328309 A JP 2007-288133 A JP 2006-201091 A Japanese Patent Laid-Open No. 2007-22886

  An object of the present invention is to provide biomolecule-capturing particles that can suppress non-specific adsorption and can be easily separated and collected by magnetic force.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has used superparamagnetic nanoparticles, a hydrophobic polymer, and a copolymer having a specific structural unit, for example, by using emulsification, and these are interlinked. It was found that the magnetic particles linked by the action can be used as biomolecule-capturing particles, in which non-specific adsorption is suppressed and can be easily separated and recovered by magnetic force, and the present invention has been completed. .

That is, the gist of the present invention is as follows.
1. Magnetic particles comprising superparamagnetic nanoparticles, a hydrophobic polymer, and a copolymer A having the following structural units (a) to (c):
(A) Structural unit represented by the following general formula (1):
(B) Structural unit represented by the following general formula (2):
(C) Structural unit represented by the following general formula (3):

(Wherein R _1a is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and m is an integer of 2 to 6);

(Wherein R _1b is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R _2b is a hydrogen atom or OR (R represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group), and n Is an integer from 2 to 18);

(In the formula, R _1c is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, q is an integer of 2 to 10, and R _2c is a functional group capable of immobilizing a biomolecule).

  2. 2. The magnetic particle according to 1 above, wherein the superparamagnetic nanoparticles are fine particles containing iron oxide.

3. _3. The magnetic particles according to 1 or 2 above, wherein the superparamagnetic nanoparticles include at least one kind of nanoparticles selected from Fe ₃ O ₄ and γ-Fe ₂ O ₃ .

  4). 4. The magnetic particle according to any one of 1 to 3, wherein the monomer constituting the hydrophobic polymer is styrene, divinylbenzene, or a mixture thereof.

  5. The constituent ratio of the structural unit (a) in the copolymer A is 35 to 95 mol%, the constituent ratio of the structural unit (b) is 1 to 50 mol%, and the constituent ratio of the structural unit (c) is 1 to The magnetic particle according to any one of 1 to 4 above, which is 30 mol%.

  6). 6. The magnetic particle according to any one of 1 to 5, wherein the functional group capable of immobilizing a biomolecule is a carboxyl group or an active ester group thereof, an epoxy group, a tosyl group, an amino group, a thiol group, or a bromoacetamide group.

7). A solvent containing a hydrophobic polymer and superparamagnetic nanoparticles and insoluble in water and water containing a copolymer A having the following structural units (a) to (c) are emulsified:
(A) Structural unit represented by the following general formula (1):
(B) Structural unit represented by the following general formula (2):
(C) Structural unit represented by the following general formula (3):

(Wherein R _1a is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and m is an integer of 2 to 6);

(Wherein R _1b is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R _2b is a hydrogen atom or OR (R represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group), and n Is an integer from 2 to 18);

(Wherein R _1c is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, q is an integer of 2 to 10, and R _2c is a functional group capable of immobilizing a biomolecule);
Magnetic particles obtained by removing a solvent that does not dissolve in water from the obtained emulsion.

  8). 8. The magnetic particle according to 7 above, wherein the solvent insoluble in water dissolves the hydrophobic polymer.

  9. In the emulsification, the magnetic particles according to 7 or 8 above, wherein 0.01 to 2000 parts by weight of the hydrophobic polymer and 2 to 2000 parts by weight of the copolymer A are used with respect to 100 parts by weight of the superparamagnetic nanoparticles.

  According to the present invention, it is possible to provide magnetic particles that can be easily separated and collected by magnetic force, can immobilize (capture) biomolecules, and suppress nonspecific adsorption of biomolecules. .

FIG. 1 shows a scanning electron micrograph of the magnetic particles obtained in Example 1.

  Hereinafter, the magnetic particles of the present invention will be described in detail.

≪Magnetic particles≫
As described above, the magnetic particles of the present invention include superparamagnetic nanoparticles, a hydrophobic polymer, and a copolymer A having specific structural units (a) to (c). Hereinafter, each of these components and the method for producing the magnetic particles of the present invention will be described.

<Superparamagnetic nanoparticles>
The superparamagnetic nanoparticles are typically fine particles containing iron oxide having a particle size of 20 nm or less, preferably 5 to 20 nm, and more specific examples include MFe ₂ O ₄ (M = Co, Ni , Mg, Cu, Li _0.5 Fe _0.5, etc.), magnetite represented by Fe ₃ O ₄ , or γ-Fe ₂ O ₃ . Among these, at least one kind of nanoparticles selected from γ-Fe ₂ O ₃ and Fe ₃ O ₄ having strong saturation magnetization and low residual magnetization is preferable. Further, such a magnetic material may be treated with a known hydrophobizing agent such as a titanium coupling agent, a silane coupling agent or a higher fatty acid.

  Due to the presence of such superparamagnetic nanoparticles, the particles of the present invention have magnetism and can be easily separated and recovered by magnetic force.

  The content of superparamagnetic nanoparticles in the magnetic particles (in 100% by weight) of the present invention is preferably 10 to 90% by weight from the viewpoint of imparting sufficient magnetism to the particles. The content of the superparamagnetic nanoparticles is calculated by performing TG-DTA measurement on the magnetic particles and subtracting the weight loss (corresponding to the weight of the polymer component) when the temperature is raised to 600 ° C. from the weight of the magnetic particles. be able to.

<Hydrophobic polymer>
The hydrophobic polymer is a polymer having poor affinity with an aqueous solvent, and mechanical strength and heat resistance can be imparted to the magnetic particles of the present invention by using the hydrophobic polymer for the core. Examples of monomers constituting the hydrophobic polymer include aromatic vinyl monomers such as styrene, α-methylstyrene and divinylbenzene, nitrile group-containing monomers such as (meth) acrylonitrile, and methacrylic acid having an alkyl group having 4 or more carbon atoms. Examples include esters, α-olefins such as ethylene and propylene, and dienes such as butadiene. Styrene, divinylbenzene or a mixture thereof is preferable. The hydrophobic polymer may be a homopolymer composed of one kind of monomer or a copolymer composed of two or more kinds of monomers.

  In the production of the magnetic particles of the present invention, it is considered that the hydrophobic polymer covers at least a part of the superparamagnetic nanoparticles and contributes to the formation of the particle form. From this point, the hydrophobic polymer is usually used in an amount of 0.01 to 2000 parts by weight with respect to 100 parts by weight of superparamagnetic nanoparticles in the production of magnetic particles.

<Copolymer A having structural units (a) to (c)>
The magnetic particles of the present invention include a copolymer A having the specific structural units (a) to (c).
The phosphorylcholine structure in the structural unit (a) leads to a reduction in nonspecific adsorption,
The structural unit (b) imparts hydrophobicity to the copolymer A, and during the production of the magnetic particles of the present invention, the copolymer A interacts with superparamagnetic nanoparticles or a hydrophobic polymer, The magnetic particles of the present invention, which is a composite of these three components, are easily formed,
The structural unit (c) has a functional group capable of immobilizing a biomolecule, and can impart a biomolecule capturing ability to the magnetic particles of the present invention. Hereinafter, each of these structural units will be described.

(Structural unit (a))
The structural unit (a) is represented by the following general formula (1):

In the formula, R _1a means a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group having 1 to 4 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group and tert-butyl group. R _1a is preferably a hydrogen atom, a methyl group or an ethyl group, more preferably a hydrogen atom or a methyl group.

  m means the carbon number of an alkylene group, and is an integer of 2-6.

  Examples of the monomer serving as the structural unit represented by the formula (1) include 2-acryloyloxyethyl phosphorylcholine, 2-methacryloyloxyethyl phosphorylcholine, 2- (2-ethylacryloyloxy) ethyl phosphorylcholine, 2- (2-propylacryloyloxy). ) Ethyl phosphorylcholine, 2- (2-butylacryloyloxy) ethyl phosphorylcholine and the like. From the viewpoint of the effect of suppressing nonspecific adsorption of the magnetic particles of the present invention, 2-methacryloyloxyethyl phosphorylcholine (MPC) is preferably used. For example, MPC is prepared by a known method (for example, see Polymer Journal, Vol. 22, No. 5, (May 1990), pp. 355-360), or supplied by a reagent such as Tokyo Chemical Industry Co., Ltd. It can be obtained from a supplier.

  In the copolymer A in the magnetic particle of the present invention, the structural unit (a) may be one type or two or more types having different structures may exist.

(Structural unit (b))
The structural unit (b) is represented by the following general formula (2):

In the formula, R _1b means a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group having 1 to 4 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group and tert-butyl group. R _1b is preferably a hydrogen atom, a methyl group or an ethyl group, more preferably a hydrogen atom or a methyl group.

R _2b means a hydrogen atom or an OR group (R represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group). Examples of the aliphatic hydrocarbon group include an alkyl group and an alkenyl group, and examples of the aromatic hydrocarbon group include a phenyl group.

n means the carbon number of an alkylene group, and is an integer of 2-18. R _2b is not necessarily bonded to the methylene group farthest from the ester group in the alkylene group represented by (CH ₂ ) _n , and may be bonded to any methylene group constituting the alkylene group. Good.

  Examples of the monomer to be the structural unit represented by the formula (2) include ethyl methacrylate, propyl methacrylate, butyl methacrylate, pentyl methacrylate, hexyl methacrylate, octyl methacrylate, dodecyl methacrylate, 2-ethoxyethyl methacrylate. , 2-ethoxypropyl methacrylate, 2-phenoxyethyl methacrylate, 2-butoxyethyl methacrylate and the like are preferable.

  In the copolymer A in the magnetic particle of the present invention, the structural unit (b) may be one type or two or more types having different structures may be present.

(Structural unit (c))
The structural unit (c) is represented by the following general formula (3):

In the formula, R _1c means a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group having 1 to 4 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group and tert-butyl group. R _1c is preferably a hydrogen atom, a methyl group or an ethyl group, more preferably a hydrogen atom or a methyl group.

  q means the number of repeating units of the ethyleneoxy group and is an integer of 2 to 10.

R _2c is a functional group capable of immobilizing a biomolecule, and is not particularly limited as long as it is a group that specifically interacts with a target biomolecule. Specific examples thereof include a carboxyl group or an active ester group thereof, an epoxy group, a tosyl group, an amino group, a thiol group, or a bromoacetamide group.

  The active ester group is an ester group formed by bonding an electron-withdrawing group or a bulky group to a carbonyloxy group. Such an ester bond is easily subjected to a reaction such as a nucleophilic substitution reaction by an amino group, a thiol group, a hydroxyl group, and the like. By reacting a biomolecule with the active ester group portion, the magnetic particles of the present invention The molecule can be immobilized. Similarly, a carboxyl group and an epoxy group are highly reactive groups, and a biomolecule can be reacted with this portion. If the biomolecule has no site capable of reacting with an active ester group, carboxyl group, epoxy group, etc., a group capable of reacting with these groups such as an amino group is introduced into the biomolecule by a known method. do it.

  The active ester group is, for example, a group represented by the structure of the following formula (c) or (d).

  In the formula (c) and the formula (d), R ′ and R ″ are each independently a monovalent organic group, and may be any of linear, branched or cyclic. ), Two R ″ may form a ring together with N to which they are bonded.

Specific examples of such an active ester group include groups having the following structure.

  In addition to these, as an active ester group, an ester group of pentafluorophenol, an ester group of thiophenol; 5-norbornene-2,3-dicarboximide group and N-hydroxy-5-norbornene-2,3-dicarboxy An ester group of a heterocyclic hydroxy compound such as an imide group; and a cyanomethyl ester group.

  Examples of the monomer serving as the structural unit represented by the formula (3) include p-nitrophenyloxycarbonyl (poly) oxyethylene methacrylate (MEONP), N-succinimidyloxycarbonyl di (ethylene glycol) methacrylate (N-succinimidyloxycarbonyldi). (Ethylenylcol) methacrylate, PENHS) and the like are preferable. For example, MEONP can be prepared by known methods (see, for example, Biomacromolecules 2004, 5, 342-347), and PENHS is prepared by known methods (see, for example, Biosensors and Bioelectronics 38 (2012) 209-214). be able to.

  In the copolymer A in the magnetic particle of the present invention, the structural unit (c) may be one type or two or more types having different structures may exist.

(Copolymer A)
The copolymer A used in the present invention is a copolymer having the various structural units (a) to (c) described above, and even these block copolymers are random copolymers. May be.

  Regarding the structural units (a) to (c) in the copolymer A, the structural unit (a) is preferably 35 to 95 mol% of the structural units (b), and the structural units (b) ) Is preferably 1 to 50 mol%, and the structural unit (c) is 1 to 30 mol% (however, the total of the constituent ratios of the structural units (a) to (c) is 100 mol%). In addition, the composition ratio of each structural unit in the copolymer A can be measured by nuclear magnetic resonance spectroscopy.

  The copolymer A described above may be used alone or in combination of two or more in the present invention. Further, the copolymer A imparts good nonspecific adsorption reduction function and biomolecule capture function to the magnetic particles of the present invention, and interacts with the hydrophobic polymer and superparamagnetic nanoparticles in the production of the magnetic particles. From the viewpoint of appropriately forming the particle morphology, it is preferable to use 2 to 2000 parts by weight for 100 parts by weight of the superparamagnetic nanoparticles in the production of the magnetic particles.

(Method for producing copolymer A)
The copolymer A used in the present invention is a mixture of the monomer compounds forming the structural units (a) to (c) described above, preferably in an amount that constitutes the structural proportions of the structural units described above, and radical polymerization or the like. It can manufacture by the well-known polymerization method. When the copolymer A is produced by radical polymerization, for example, solution polymerization can be performed at 30 to 90 ° C. in an inert gas atmosphere such as argon.

  The solvent used for the solution polymerization is appropriately selected. For example, alcohols such as methanol, ethanol and isopropanol, ethers such as diethyl ether, and organic solvents such as chloroform can be used alone or in combination.

  Moreover, as a radical polymerization initiator used for radical polymerization reaction, what is normally used in radical polymerization can be used. For example, azo initiators such as azobisisobutyronitrile (AIBN), azobisvaleronitrile; and lauroyl peroxide, benzoyl peroxide, t-butylperoxyneodecanoate, t-butylperoxypivalate, etc. Oil-soluble organic peroxides can be used.

  The copolymer A obtained by the radical polymerization reaction can be recovered, for example, by a reprecipitation method using a poor solvent for the copolymer.

[Method for producing magnetic particles]
The magnetic particles of the present invention can be produced, for example, by the following method.

  A hydrophobic polymer is dissolved in a solvent that does not dissolve in water, and superparamagnetic nanoparticles treated with a known hydrophobizing agent are dispersed in the polymer solution. The oil phase thus obtained is mixed with water containing a water-soluble emulsifier, and emulsified using a known emulsifier, whereby an oil-in-water type (O / W type) in which oil droplets are dispersed in water. ) Form an emulsion. Next, the magnetic particles of the present invention can be obtained by slowly evaporating a solvent that does not dissolve in water under vacuum. At this time, at least a part of the water may evaporate.

  In addition to being insoluble in water, the solvent can further dissolve the hydrophobic polymer for emulsification. Examples of such a solvent include chlorinated aliphatic hydrocarbons such as chloroform and dichloromethane, and organic solvents such as aromatic hydrocarbon solvents such as toluene, xylene, and benzene.

  As the emulsifier, a disper mixer, a homomixer, a high-pressure homogenizer, an ultrasonic disperser, or the like can be used.

  Copolymer A is used as the water-soluble emulsifier. Copolymer A has a hydrophilic part of the structural unit (a) and a hydrophobic part of the structural unit (b), and functions as an emulsifier.

  By adjusting the amount of the hydrophobic polymer, superparamagnetic nanoparticles and copolymer A to be subjected to the emulsification, the content of these three components in the magnetic particles of the present invention obtained can be adjusted. For example, the amount of each component in the emulsification is preferably 0.01 to 2000 parts by weight of the hydrophobic polymer and 2 to 2000 parts by weight of the copolymer A with respect to 100 parts by weight of the superparamagnetic nanoparticles. Magnetic particles can be obtained.

[Characteristics of magnetic particles]
For example, in the magnetic particles of the present invention obtained by utilizing emulsification as described above, when forming an emulsion, the copolymer A has a hydrophilic structural unit (a) on the outside and a hydrophobic structural unit (b) on the outside. It is thought that the inside micelle is formed. In the magnetic particles obtained in the present invention, the structural unit (a) of the copolymer A is arranged on or near the surface, and the structural unit (b) interacts with the hydrophobic polymer and superparamagnetic nanoparticles. It is considered that the polymer A is in the form of particles covering at least a part of the hydrophobic polymer and the superparamagnetic nanoparticles.

  Further, the copolymer A is sufficiently fixed in the magnetic particles and exhibits its function without undergoing a coupling reaction with titanium oxide as in the invention of Patent Document 4.

  The magnetic particles of the present invention containing such various components have magnetism due to the presence of superparamagnetic nanoparticles and can be easily separated and recovered by magnetic force. Specifically, in a scale in which about 2 mg of magnetic particles are dispersed in 1 mL of solvent, by applying a magnetic field using a neodymium-iron-boron magnet having a surface magnetic density of about 4300 gauss, it takes about 10 to 60 seconds. The magnetic particles can be collected at a specific location in water and can be easily recovered.

[Use of magnetic particles]
Since the magnetic particle of the present invention has a functional group capable of immobilizing a biomolecule on the structural unit (c) of copolymer A on or near the surface of the magnetic particle, the biomolecule can be immobilized. This biomolecule-fixing functional group is not particularly limited as long as it is a group that specifically interacts with a target biomolecule. The specific interaction may be a physical interaction or a chemical interaction. In addition, if the biomolecule immobilized with the biomolecule-immobilizable functional group is a molecule that specifically interacts with another biomolecule, another biomolecule is passed through the immobilized biomolecule. Can be immobilized.

  In the magnetic particle of the present invention, it is considered that nonspecific adsorption is suppressed because the copolymer A containing a phosphorylcholine structure and a functional group capable of immobilizing a biomolecule is disposed on the surface of the particle or in the vicinity thereof. When the magnetic particles are used, it is considered that some functional groups remain without reacting with biomolecules. However, the remaining functional groups are deactivated (for example, treated with an alkali compound or an amino compound). ), The effect of reducing nonspecific adsorption can be pierced.

  The magnetic particle of the present invention can be used as an excellent biomolecule-capturing particle that hardly causes non-specific adsorption of non-target biomolecules. More specifically, the magnetic particles of the present invention are used to diagnose a diagnosis target substance such as an infectious disease or cancer biomarker or hormone from a patient, or a specific biomolecule such as a nucleic acid or protein. Separation and quantification, and specific bacteria and cells can be isolated.

  Specific examples of such biomolecules include nucleic acids, aptamers, proteins, peptides, sugar chains and glycoproteins. Examples of the protein include enzymes, antibodies, and antigens.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these at all.

<Preparation of copolymer A>
Copolymers A1 and A2 used in the following examples were produced as follows.

(Method for producing copolymer A1)
In a glass tube containing 15 mL of ethanol as a solvent, after substitution with argon, the monomers 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA) and p-nitrophenyloxycarbonyl-polyoxyethylene methacrylate ( MEONP, the number of repeating units of oxyethylene groups was 4.5 (average value), a radical polymerization initiator (azobisisobutyronitrile (AIBN)) was added, and radical polymerization was performed at 60 ° C. for 24 hours. .

  The polymerization is performed by sealing the glass tube so that oxygen in the air is not mixed to prevent oxygen inhibition, and the total monomer concentration in radical polymerization is 0.5 mol / L (MPC: 1.8106 g, BMA: 0.1140 g). , MEONP: 0.3218 g), and the initiator concentration was 0.0025 mol / L (AIBN: 5.9 mg).

  The polymerization solution obtained by the above radical polymerization was subjected to reprecipitation using a chloroform-ether mixed solvent (2: 8). Subsequently, the solvent was removed by vacuum drying to obtain a copolymer A1. The composition in copolymer A1 was MPC: BMA: MEONP = 92: 7: 1 (molar ratio) with respect to the charged composition MPC: BMA: MEONP = 80: 10: 10 (molar ratio).

(Method for producing copolymer A2)
MEONP was changed to N-succinimidyloxycarbonyldi (ethylene glycol) methacrylate (N-succinimidyloxycarbonyl dimethacrylate, PENHS), and the total monomer concentration in radical polymerization was 1.0 mol / L (MPC: 3.5890 g, BMA: 0.2152 g, PENHS: 0.4694 g) and the initiator concentration was changed to 0.005 mol / L (AIBN: 12.1 mg). A2 was produced.

  The composition in copolymer A2 was MPC: BMA: PENHS = 79: 16: 5 (molar ratio) with respect to the charged composition MPC: BMA: PENHS = 80: 10: 10 (molar ratio).

[Example 1] Production of magnetic particles Magnetic particles of Example 1 were produced by the method described below.

  A solution in which 200.0 mg of hydrophobic coated iron oxide (particle size: about 10 nm) and 203.9 mg of polystyrene (manufactured by Sigma Aldrich) were added to 4 mL of chloroform, and a solution of 21.1 mg of copolymer A1 dissolved in 20 mL of water; Were mixed and emulsified with an ultrasonic disperser. The magnetic particles of Example 1 were obtained by removing chloroform from the obtained emulsion under reduced pressure.

  FIG. 1 shows a scanning electron micrograph of the obtained magnetic particles. Generation of particles having a diameter of 50 to 500 nm can be confirmed.

[Example 2] Production of magnetic particles The magnetic particles of Example 2 were the same as Example 1 except that the amount of polystyrene used was changed to 200.0 mg and the copolymer A1 was changed to the copolymer A2. Manufactured.

[Comparative Example 1]
As the magnetic particles of Comparative Example 1, Dynabeads (MyOne Carboxylic Acid, Life Technologies Co., hydrophilic polymer-coated magnetic particles) was prepared.

<Evaluation of specific capture>
(Binding of protein A to the magnetic particles of Examples 1 and 2)
2 mg of the magnetic particles of Examples 1 and 2 were each dispersed in 1.2 M dipotassium hydrogen phosphate buffer (pH 8.5), and the magnetic particles were recovered by magnetic separation to remove the supernatant, and again 1.2 M phosphoric acid. Dispersion in dipotassium hydrogen buffer (pH 8.5), collection by magnetic separation, and supernatant removal were repeated twice.

  1 mg / mL protein A solution (manufactured by Sigma Aldrich, Protein A from Staphylococcus aureus Soluble, Cowan strain, recombinant, expressed in E. coli, aqueous ≧ 95%) And diluted with a dipotassium hydrogen phosphate buffer (pH 8.5)) and stirred overnight at 37 ° C. with a rotary stirrer.

  The magnetic particles are recovered by magnetic separation, dispersed again in 1.2M dipotassium hydrogen phosphate buffer (pH 8.5), and recovery and supernatant removal by magnetic separation are repeated twice, and then 0.1M 2-amino Ethanol (solvent: 0.1 M Tris-HCl buffer (pH 9.5)) was added, and the mixture was stirred at room temperature for 1 hour with a rotary stirrer, and MEONP in copolymer A (A1 and A2) constituting magnetic particles. Alternatively, PENHS was inactivated. The supernatant was removed by magnetic separation, and the magnetic particles were washed with PBS (containing 0.1% Tween 20) and further washed with PBS (containing 0.02% Tween 20).

(Binding of Protein A to Magnetic Particles of Comparative Example 1)
2 mg of the magnetic particles of Comparative Example 1 were dispersed in a 15 mM MES-NaOH buffer solution (pH 6.0), the magnetic particles were recovered by magnetic separation, the supernatant was removed, and again in a 15 mM MES-NaOH buffer solution (pH 6.0). Dispersion and collection by magnetic separation / supernatant removal were repeated twice.

  40 mg / mL water-soluble carbodiimide was added to the magnetic particles separated from the supernatant by magnetic separation, and the mixture was stirred at room temperature with a rotary stirrer for 20 minutes. After recovering the magnetic particles by magnetic separation and removing the supernatant, a 1 mg / mL protein A solution (manufactured by Sigma Aldrich, Protein A from Staphylococcus aureus Soluble, Cowan strain, recombinant, expressed in E. coli, aquase, 95 % (HPLC) was diluted with 15 mM MES-NaOH buffer (pH 6.0), and the mixture was stirred overnight at room temperature with a rotary stirrer.

(Evaluation of specific capture of rabbit antibody IgG)
The magnetic particles of Examples 1 and 2 subjected to the above treatment (binding of protein A to the magnetic particles) and the magnetic particles of Comparative Example 1 were recovered by magnetic separation, and after removing the supernatant, the magnetic particles were multiplied by 5 times. Diluted rabbit serum (VECTOR LABORATIES. INC. Sei Serum, Rabbit, Normal diluted with PBS (containing 0.02% Tween 20), containing rabbit antibody IgG) was added, and the mixture was stirred at room temperature with a rotary stirrer for 1 hour. did.

  The magnetic particles were collected by magnetic separation, washed with PBS (containing 0.02% Tween 20), 0.1 M glycine-HCl buffer (pH 2.8) was added, and the mixture was stirred at room temperature with a rotary stirrer for 15 minutes. . Since the buffer solution is acidic, the bond between IgG and protein A is cleaved, and when nonspecific adsorption occurs on the magnetic particles, the nonspecifically adsorbed substance is also separated from the magnetic particles. It is thought that.

Thereafter, the magnetic particles and the reaction solution (mainly the glycine-HCl buffer solution, which contains IgG and nonspecifically adsorbed substances if present) are separated magnetically, and 280 nm in the reaction solution. The absorbance was measured. A spectrophotometer (U-3500) manufactured by Hitachi High-Tech Fielding Co., Ltd. was used for measuring the absorbance. This absorbance mainly reflects the amount of rabbit antibody IgG specifically captured by the magnetic particles. From the absorbance and the molar extinction coefficient of rabbit antibody IgG (A ₂₈₀ (1 mg / mL) ≈1.4), the amount of rabbit antibody IgG in the reaction solution was calculated. The calculated amounts of rabbit antibody IgG (amount captured by the magnetic particles) are shown in Table 1 below.

<Evaluation of non-specific adsorption>
Further, in the above <Evaluation of specific capture>, 5 μL of each reaction solution obtained for the magnetic particles of Examples 1 and 2 and the magnetic particles of Comparative Example 1 and a sample buffer (4% SDS, 20% glycerol, 100 mM DTT). , 0.01% BPB, 0.125M Tris-HCl, pH 6.8) was mixed well with a vortex mixer and heated with a tube heater at 90 ° C. for 3 minutes. Biocraft Co., Ltd. slab electrophoresis apparatus, Wako Pure Chemical Industries, Ltd. precast polyacrylamide gel "Supersep TM Ace, 12.5%, 17 well", and electrophoresis buffer (Tris 3g, Glycine 14.3g, SDS 1g) 10 μl per lane of the gel was applied using a sample diluted to 1 L with pure water) and subjected to electrophoresis. The staining was performed by a general CBB (Coomassie Brilliant Blue) staining method. The stained gel was scanned and imaged with a scanner.

  In the stained gel images, in Examples 1 and 2, bands are clearly confirmed in the vicinity of molecular weights of about 25 kDa and 50 kDa corresponding to the protein constituting the subunit of the rabbit antibody IgG, and bands are confirmed in other parts. However, in Comparative Example 1, in addition to these bands, bands were also observed in the vicinity of 60 to 75 kDa, and it was confirmed that nonspecific adsorption occurred. The above results are summarized in Table 1 below.

<Magnetic response>
The time from when the magnetic particles of Examples 1 and 2 and 2 mg of each of the magnetic particles of Comparative Example 1 are dispersed in 1 mL of PBS buffer solution and the magnet is brought close to the color of the liquid becoming transparent (the magnetic particles are completely recovered) Time). The results are shown in Table 1 below. As the magnet, a neodymium-iron-boron magnet having a surface magnetic density of about 4300 gauss was used.

Claims (9)

Magnetic particles comprising superparamagnetic nanoparticles, a hydrophobic polymer, and a copolymer A having the following structural units (a) to (c):
(A) Structural unit represented by the following general formula (1):
(B) Structural unit represented by the following general formula (2):
(C) Structural unit represented by the following general formula (3):

(Wherein R _1a is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and m is an integer of 2 to 6);

(Wherein R _1b is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R _2b is a hydrogen atom or OR (R represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group), and n Is an integer from 2 to 18);

(In the formula, R _1c is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, q is an integer of 2 to 10, and R _2c is a functional group capable of immobilizing a biomolecule).   The magnetic particles according to claim 1, wherein the superparamagnetic nanoparticles are fine particles containing iron oxide. The magnetic particles according to claim 1 or 2, wherein the superparamagnetic nanoparticles include at least one kind of nanoparticles selected from Fe ₃ O ₄ and γ-Fe ₂ O ₃ .   The magnetic particle according to any one of claims 1 to 3, wherein the monomer constituting the hydrophobic polymer is styrene, divinylbenzene, or a mixture thereof.   The constituent ratio of the structural unit (a) in the copolymer A is 35 to 95 mol%, the constituent ratio of the structural unit (b) is 1 to 50 mol%, and the constituent ratio of the structural unit (c) is 1 to The magnetic particle according to any one of claims 1 to 4, which is 30 mol%.   The magnetic particle according to any one of claims 1 to 5, wherein the functional group capable of immobilizing a biomolecule is a carboxyl group or an active ester group thereof, an epoxy group, a tosyl group, an amino group, a thiol group, or a bromoacetamide group. A solvent containing a hydrophobic polymer and superparamagnetic nanoparticles and insoluble in water and water containing a copolymer A having the following structural units (a) to (c) are emulsified:
(A) Structural unit represented by the following general formula (1):
(B) Structural unit represented by the following general formula (2):
(C) Structural unit represented by the following general formula (3):

(Wherein R _1a is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and m is an integer of 2 to 6);

(Wherein R _1b is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R _2b is a hydrogen atom or OR (R represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group), and n Is an integer from 2 to 18);

(Wherein R _1c is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, q is an integer of 2 to 10, and R _2c is a functional group capable of immobilizing a biomolecule);
Magnetic particles obtained by removing a solvent that does not dissolve in water from the obtained emulsion.   The magnetic particle according to claim 7, wherein the solvent insoluble in water dissolves the hydrophobic polymer.   The magnetic particles according to claim 7 or 8, wherein in emulsification, 0.01 to 2000 parts by weight of hydrophobic polymer and 2 to 2000 parts by weight of copolymer A are used with respect to 100 parts by weight of superparamagnetic nanoparticles.