JPH10270233A - Magnetic polymer particles and manufacture therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new magnetic polymer particles in which a superparamagnetic material is dispersed in superior manner and a method by which the polymer particles is surely manufactured. SOLUTION: Magnetic polymer particles contain 1-100 pts.wt. superparamagnetic material with respect to 100 pts.wt. copolymer, composed of (A) 30-99 wt.% alkyl (metha)acrylate containing a 4-20C alkyl group, (b) 1-20 wt.% unsaturated carboxylic acid, and (c) 0-69 wt.% monomer which is copolymerizable with the alkyl (metha)acrylate and unsaturated carboxylic acid and is set for a number-average particle diameter of 0.02-30 μm. In a method for manufacturing the magnetic polymer particles, a suspension is obtained by dispersing a monomer composition prepared by dispersing the superparamagnetic material in the monomer containing the alkyl (metha)acrylate at a rate of >=30 wt.% and the monomer is polymerized with the alkyl (metha) acrylate in the suspension.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a magnetic polymer particle and a method for producing the same, and more particularly, to a carrier for a diagnostic agent, a carrier for separating bacteria, a carrier for separating cells, a carrier for separating and purifying nucleic acids,
The present invention relates to magnetic polymer particles useful as a protein separation / purification carrier, an immobilized enzyme carrier, a drug delivery carrier, a magnetic toner, a magnetic ink, a magnetic paint, and the like, and a method for producing the same.

[0002]

2. Description of the Related Art A magnetic polymer latex is an excellent carrier for use as a diagnostic agent carrier, a carrier for separating and purifying cells, proteins or nucleic acids, etc. because of its advantage that latex particles can be easily separated and recovered by utilizing magnetic force. It is expected to be easy to handle. Further, the magnetic polymer latex formed into a film by drying or heating is expected to be applied to uses such as magnetic toner, magnetic ink, and magnetic paint. As a method for synthesizing magnetic polymer particles constituting such a magnetic polymer latex, the following methods (1) to (3) are known as methods for obtaining particles of a type in which a magnetic substance is present inside the particles.

(1) A method of obtaining a magnetic polymer particle by dispersing a magnetic material which is not a superparamagnetic material subjected to a magnetizing treatment and a lipophilic treatment in a polymerizable monomer, and subjecting the polymerizable monomer to suspension polymerization to obtain a magnetic polymer particle. See JP-A-59-221302).

(2) Dispersing a magnetic substance subjected to lipophilic treatment in an organic phase containing a vinyl aromatic monomer, homogenizing the dispersion in an aqueous phase using a homogenizer, and then polymerizing the dispersion. To obtain magnetic polymer particles having a relatively small particle diameter (see Japanese Patent Publication No. 4-3088).

(3) Precipitating an iron compound in the presence of porous polymer particles having a specific functional group, and oxidizing the iron compound to introduce a magnetic substance into the interior of the porous polymer particles, thereby obtaining a magnetic material of 2 μm or more. A method of obtaining magnetic polymer particles having a large particle diameter and a uniform diameter (Japanese Patent Publication No. 5-10808).

However, in the above method (1), in order to magnetize a magnetic material having a coercive force that is not a superparamagnetic material, the dispersed particles coagulate and fuse during polymerization, and the resulting magnetic polymer particles are: The magnetic polymer particles have an excessively large particle size, and there is a problem that such magnetic polymer particles easily settle in an aqueous medium. In addition, when used as an adsorbent for a specific substance, there is also a problem that a sufficient amount of a target substance cannot be adsorbed due to the small surface area of the particles. JP-A-59-2213 discloses the method (1).
In JP-A-02, a polymerizable monomer mainly composed of styrene is used. However, the styrene-based polymer has a low affinity for the magnetic substance, so that the magnetic substance that has been well dispersed in the polymerization reaction system (monomer) separates and aggregates inside the polymer particles after polymerization, There is a problem that it is deposited on the particle surface. In addition, magnetic polymer particles synthesized using a polymerizable monomer mainly composed of styrene absorb ultraviolet rays and emit fluorescent light due to an aromatic ring structure introduced in a large amount. As a result, when the magnetic polymer particles are used for identification and detection of a physiologically active substance, there is a problem that many restrictions are caused. Furthermore, magnetic polymer particles obtained by using a polymerizable monomer mainly composed of styrene have a high adsorption property to proteins and a large non-specific adsorption property, so that the diagnostic system using the magnetic polymer particles has a large background. There is.

The magnetic polymer particles obtained by the above method (2) have poor uniform dispersibility of the magnetic substance inside the particle, and the magnetic substance is localized near the particle surface. For this reason, there is a problem that iron ions are eluted from the magnetic polymer particles, or the magnetic substance dissolves and flows out under acidic conditions, thereby deteriorating the characteristics of the magnetic polymer particles.

The method (3) has a problem in that the production process of the magnetic polymer particles is complicated, and since the magnetic material is not encapsulated by the polymer, iron ions are eluted.

On the other hand, a method has been disclosed in which an iron compound is precipitated in a polymer particle emulsion to ferrite the surface of the polymer particles (Japanese Patent Laid-Open Publication No. Hei 3 (1994)).
-1155862 and JP-A-5-13809. However, the magnetic polymer particles obtained by this method have a problem that the elution of iron ions occurs remarkably due to the presence of a magnetic substance on the particle surface. When the magnetic polymer particles obtained by the conventional synthesis method as described above are used, for example, as a diagnostic drug carrier, sufficient sensitivity cannot be obtained, or good practical performance cannot be obtained due to a nonspecific enzyme reaction. . The reason is that the magnetic material is partially exposed on the particle surface of the magnetic polymer particles,
Alternatively, it is considered that a micropass is formed between the particle surface and the magnetic substance existing inside the particle, so that iron ions are eluted to the outside and adversely affect practical performance.

In order to suppress the elution of iron ions to the outside to some extent, a magnetic polymer particle containing magnetic particles is obtained by polymerizing a hydrophobic cross-linking monomer in an aqueous phase using magnetic particles as a core material. Although a technique is disclosed (see JP-A-2-286729), the elution of iron ions cannot be sufficiently suppressed by this technique. Further, a technique of forming a coating layer made of a polymer not containing a magnetic substance on the surface of magnetic polymer particles has been disclosed (see Japanese Patent Publication No. 5-16164). Only the legal and spray-dry spray methods are shown and the scope of application is limited.

Also, a technique for synthesizing magnetic polymer particles by an emulsion polymerization method has been disclosed (Japanese Patent Publication No. 3-57).
921), it is difficult to synthesize particles having a large particle size by an emulsion polymerization method, and therefore, it has not been possible to obtain magnetic polymer particles having good magnetic sedimentation properties with good reproducibility.

As described above, the magnetic polymer particles obtained by the conventional synthesis method have a problem of elution of iron ions to the outside, and therefore are applied only to applications and fields which are not affected by the elution of iron ions. It is not possible at present. Therefore, development of magnetic polymer particles which are excellent in dispersibility of a magnetic substance and do not elute iron ions to the outside is desired. Also, from a practical point of view, the magnetic polymer particles
It is necessary that the resin be hardly settled in an aqueous medium and maintain a homogeneous dispersion state.

[0013]

SUMMARY OF THE INVENTION It is a first object of the present invention to provide novel magnetic polymer particles excellent in the uniform dispersibility of a superparamagnetic substance. A second object of the present invention is to
An object of the present invention is to provide magnetic polymer particles which are unlikely to settle in an aqueous medium. A third object of the present invention is to provide magnetic polymer particles which are excellent in magnetic responsiveness, hardly elute iron ions derived from a magnetic substance to the outside, and can be widely applied to various uses and fields. It is in. A fourth object of the present invention is to provide magnetic polymer particles having a good binding property to a physiologically active substance. A fifth object of the present invention is to provide a method capable of reliably producing magnetic polymer particles having the above excellent properties.

[0014]

The magnetic polymer particles of the present invention comprise (A) a structural unit derived from an alkyl (meth) acrylate having 4 to 20 carbon atoms in the alkyl group (hereinafter referred to as "structural unit A"). 30 to 99% by weight, a structural unit derived from an unsaturated carboxylic acid (B) (hereinafter referred to as “structural unit B”)
(C) 0 to 69% by weight of a structural unit derived from a vinyl monomer copolymerizable with the alkyl (meth) acrylate and the unsaturated carboxylic acid (hereinafter referred to as "structural unit C"). 100 parts by weight of a copolymer (hereinafter referred to as "specific polymer") comprising 1 to 100 parts by weight of a superparamagnetic substance and having a number average particle diameter of 0.02 to 30 m. And Further, magnetic polymer particles obtained by immobilizing an oligonucleotide or a physiologically active protein on the above particles are preferable.

The method for producing magnetic polymer particles of the present invention comprises:
Alkyl (meth) having 4 to 20 carbon atoms in the alkyl group
A superparamagnetic substance is dispersed in a monomer containing 30% by weight or more of acrylate to prepare a monomer composition, and the monomer composition is dispersed in an aqueous medium to obtain a suspension. Wherein the monomer is polymerized.

[0016]

[Action]

(1) The superparamagnetic substance is uniformly dispersed in a polymer (specific polymer), and can form magnetic polymer particles having a suitable particle diameter. (2) The magnetic polymer particles formed by dispersing the superparamagnetic material are less likely to cause agglomeration and fusion of the dispersed particles during the synthesis thereof, and have a suitable particle diameter. Therefore, the magnetic polymer particles are unlikely to settle in the aqueous medium, and can exist in a homogeneously dispersed state in the aqueous medium. (3) Since the magnetic polymer particles of the present invention contain a superparamagnetic substance in a specific ratio, excellent magnetic responsiveness is exhibited, and metal ions derived from the superparamagnetic substance elute outside. Is prevented. (4) The specific polymer comprising the specific structural unit can provide the magnetic polymer particles of the present invention with good binding properties to a physiologically active substance.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION

<Magnetic polymer particles of the present invention> Hereinafter, the present invention will be described in detail. The magnetic polymer particles of the present invention are configured such that a superparamagnetic substance is dispersed and contained (encapsulated) in particles made of a specific copolymer.

<Specific Polymer> The specific polymer constituting the magnetic polymer particles of the present invention contains the structural unit A and the structural unit B as essential components, and contains the structural unit C as an optional component. It is a polymer.

The alkyl group in the alkyl (meth) acrylate (hereinafter also referred to as "monomer (A)") constituting the structural unit A generally has 4 to 20, preferably 6 to 18 carbon atoms. You. The alkyl group may be linear, branched, or cyclic. Specific examples of such a monomer (A) include 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate, n-
Butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, pentyl acrylate, pentyl methacrylate, stearyl acrylate, stearyl methacrylate, lauryl acrylate, lauryl methacrylate; cyclohexyl acrylate, cyclohexyl methacrylate, cyclohexyl ethylene glycol acrylate, cyclohexyl ethylene glycol methacrylate, cyclohexyl diacrylate Hydrogen atom of "cyclohexyl group" such as propylene glycol acrylate, cyclohexyl dipropylene glycol methacrylate, cyclohexene diacrylate, cyclohexene dimethacrylate; methyl-substituted cyclohexyl acrylate, methyl-substituted cyclohexyl methacrylate Part substituted cycloalkyl acrylates substituted by alkyl group having 1 to 5 carbon atoms, include such substituted cycloalkyl methacrylates, these can be used alone or in combination of two or more kinds. Of these, 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate, cyclohexyl acrylate, and cyclohexyl methacrylate are preferred.

The content of the structural unit A in the specific polymer is usually 30 to 99% by weight, preferably 50 to 99% by weight.
It is 99% by weight. When the content ratio of the structural unit A is less than 30% by weight, the polymerization stability and the dispersibility of the magnetic substance are poor, and the resulting magnetic polymer particles have poor adsorption to bioactive proteins such as antibodies. In addition, the non-specific adsorption phenomenon becomes remarkable. On the other hand, when the content ratio of the structural unit A is 9
If it exceeds 9% by weight, the resulting copolymer will have excessively high hydrophobicity, and the surface of the polymer particles of the copolymer will have low physical adsorbability to proteins, and will have physiological properties such as proteins, enzymes and nucleic acids. Poor chemical bonding to the active substance.

The unsaturated carboxylic acid (hereinafter also referred to as "monomer (B)") constituting the structural unit B is a polymerizable monomer having a radically polymerizable unsaturated bond and a carboxyl group in the molecule. is there. Specific examples of the monomer (B) include acrylic acid, methacrylic acid, crotonic acid, itaconic acid, fumaric acid, and maleic acid. The content ratio of the structural unit B in the specific polymer is usually 1 to 20% by weight, and preferably 2 to 15% by weight. When the content ratio of the structural unit B is less than 1% by weight, the obtained copolymer becomes excessively hydrophobic, and the surface of the polymer particles by the copolymer becomes less physically adsorbable to the protein, and It also has poor chemical bonding to physiologically active substances such as proteins, enzymes and nucleic acids.
On the other hand, when the content ratio of the structural unit B is more than 20% by weight, the hydrophilicity of the obtained copolymer becomes excessive, and the surface of the polymer particles by the copolymer obtains good binding property to protein. Can not.

Specific examples of the vinyl monomer (hereinafter also referred to as "monomer (C)") constituting the structural unit C include:
Aromatic vinyl compounds such as styrene and α-methylstyrene; alkyl (meth) acrylates having an alkyl group having 1 to 3 carbon atoms such as methyl acrylate, methyl methacrylate, ethyl acrylate and ethyl methacrylate; vinyl cyanide compounds such as acrylonitrile Ethylene glycol diacrylate, ethylene glycol dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, diallyl phthalate, trimethylolpropane triacrylate, Multifunctional (such as trimethylolpropane trimethacrylate) Data) acrylate; there may be mentioned polyfunctional vinyl compounds such as polyfunctional aromatic vinyl compounds such as divinylbenzene, these may be used alone or in combination of two or more. Among these, an alkyl (meth) acrylate in which the alkyl group has 1 to 3 carbon atoms is preferable. When only 2-ethylhexyl (meth) acrylate is used as the monomer (A), it is preferable to use a polyfunctional vinyl compound as the monomer (C).

The content of the structural unit C in the specific polymer is usually 0 to 69% by weight. The content of the structural unit C derived from the aromatic vinyl compound is preferably from 0 to 49% by weight, more preferably from 0 to 20% by weight. When only 2-ethylhexyl (meth) acrylate is used as the monomer (A), the content of the structural unit C derived from the polyfunctional vinyl compound preferably used is 0.5 to 30% by weight. Preferably, the content is more preferably 1 to 20% by weight.
When the ratio of the structural unit derived from the polyfunctional vinyl compound is 0.5
When the amount is less than the weight percentage, the obtained magnetic polymer particles do not have sufficient hardness, which may cause deformation of the particles and fusion of the particles. On the other hand, when the ratio of the structural unit derived from the polyfunctional vinyl compound exceeds 30% by weight, the obtained magnetic polymer particles may become brittle and may not have practical strength.

The molecular weight (weight average molecular weight in terms of polystyrene) of the specific polymer constituting the magnetic polymer particles of the present invention
Is usually 10,000 or more, preferably 20,000
00 to 1,000,000.

<Superparamagnetic Material> The magnetic polymer particles of the present invention are characterized in that the magnetic material dispersed and contained in the particles is a “superparamagnetic material” and contains a superparamagnetic material. Accordingly, the particles have good magnetic attraction, and even if the magnetic attraction is repeated, substantially no magnetic history remains in the particles, and good redispersibility can be maintained. It should be noted that the superparamagnetic substance in the present invention means that 50
A magnetic material whose magnetization (residual magnetization) when returning to a zero magnetic field after applying a strong magnetic field of 00 Oersteds is 1/3 or less of magnetization (saturation magnetization) when a magnetic field of 5000 Oersteds is applied. Examples of the superparamagnetic material constituting the magnetic polymer particles of the present invention include triiron tetroxide (Fe ₃ O ₄ ),
Examples include γ-iron trioxide (γ-Fe ₂ O ₃ ), various ferrites, metals such as iron, manganese, cobalt, and chromium, and alloys such as cobalt, nickel, and manganese. Iron and ferrite are particularly preferred. The primary particles of the superparamagnetic material constituting the magnetic polymer particles of the present invention preferably have a particle size of 40 to 300 °, more preferably 50 to 200 °, and particularly preferably 60 to 150 °. When the primary particle diameter of the superparamagnetic material is less than 40 °, a large amount of a treating agent (fatty acid, silane coupling agent, titanium coupling agent) for lipophilizing the superparamagnetic material is required. In addition, an excessive amount of the treating agent may impair the good magnetic responsiveness of the superparamagnetic material. On the other hand, when the primary particle diameter of the superparamagnetic substance exceeds 300 °, the obtained magnetic polymer particles become magnetized, and the magnetic polymer particles aggregate with each other and are likely to settle in the aqueous medium. The particle diameter of the primary particles of the non-spherical superparamagnetic material is an average value of the longest diameter and the shortest diameter of each superparamagnetic material.

The content of the superparamagnetic material constituting the magnetic polymer particles of the present invention is usually 1 to 100 parts by weight, preferably 5 to 100 parts by weight, per 100 parts by weight of the specific polymer.
80 parts by weight, more preferably 10 to 60 parts by weight. When the content ratio of the superparamagnetic material is less than 1 part by weight, the obtained particles cannot exhibit sufficient magnetic responsiveness. On the other hand, when the content ratio of the superparamagnetic material exceeds 100 parts by weight, However, the superparamagnetic substance is exposed on the particle surface of the obtained magnetic polymer particles to cause problems such as elution of metal ions, and the magnetic polymer particles become brittle and do not have practical strength.

In the magnetic polymer particles of the present invention,
It is preferable that the superparamagnetic substance is uniformly dispersed in the specific polymer constituting the inside of the particle, and it is preferable that the superparamagnetic substance hardly exists on the surface of the particle and in the vicinity of the surface. Here, as a method for judging whether or not a superparamagnetic substance is almost present on the particle surface and near the surface, 0.1 g of the magnetic polymer particle is immersed in 10 ml of pure water heated to 70 ° C. for 2 hours. After that, the metal content eluted in the pure water is measured. When the metal content is 10 ppm or less, the magnetic polymer particles can be determined to be "a state in which a superparamagnetic substance hardly exists on the particle surface and near the surface".

<Number average particle diameter of magnetic polymer particles> The number average particle diameter of the magnetic polymer particles of the present invention is usually 0.02.
To 30 μm, preferably 0.1 to 20 μm, more preferably 0.2 to 15 μm, particularly preferably 0.3 to
It is 10 μm. When the number average particle diameter is less than 0.02 μm, sufficient magnetic responsiveness cannot be exhibited. On the other hand, when the number average particle diameter exceeds 30 μm, the magnetic polymer particles are likely to settle in an aqueous medium, and the magnetic polymer particles have a small particle surface area. When used, a sufficient amount of the target substance cannot be adsorbed.

The magnetic polymer particles of the present invention hardly sediment in an aqueous medium and can exist in a homogeneously dispersed state in the aqueous medium (hereinafter, this property is referred to as “stationary stability”). Therefore, the magnetic polymer particles of the present invention are excellent also from the viewpoint of handleability. Here, as a method of evaluating the standing stability, a magnetic polymer particle is dispersed in an aqueous medium to prepare a magnetic polymer latex having a solid content of 1% by weight, and the magnetic polymer latex has a liquid phase height of 2 cm. The container is shaken until the magnetic polymer particles are uniformly dispersed, and then allowed to stand for 10 minutes. When the container is allowed to stand for 10 minutes, the solid content of the upper layer of the liquid phase from the liquid surface to 1 cm below the liquid surface Measure the concentration. When the solid concentration is 0.1% by weight or more, it is considered that the static stability is excellent. In the magnetic polymer particles of the present invention, the solid content concentration is usually 0.1% by weight or more, preferably 0.3% by weight or more, and more preferably 0.6% by weight or more.

The magnetic polymer particles of the present invention preferably have a layer made of a polymer containing no superparamagnetic substance (hereinafter, also referred to as “surface coating layer”) formed on the surface thereof. With this configuration, elution of iron ions from the magnetic polymer particles can be reliably prevented without impairing the excellent handling properties of the superparamagnetic substance present inside the particles. Here, the thickness of the surface coating layer is preferably larger than the longest diameter of the superparamagnetic material used, for example, 100 ° or more.

<Method for producing magnetic polymer particles of the present invention>
In the production method of the present invention, a superparamagnetic substance is dispersed in a monomer containing 30% by weight or more of an alkyl (meth) acrylate having 4 to 20 carbon atoms in the alkyl group (the monomer (A)). To prepare a monomer composition, and the monomer composition is emulsified and dispersed in an aqueous medium (particulation).
Thus, a suspension which is an oil droplet dispersion is obtained, and the monomer is polymerized in the suspension. According to the above method, a dispersion (magnetic polymer latex) in which the magnetic polymer particles of the present invention are dispersed in an aqueous medium can be reliably produced.

In the production method of the present invention, a lipophilic superparamagnetic material can be suitably used.
When magnetic polymer particles are manufactured using a superparamagnetic material that has not been subjected to lipophilic treatment, the superparamagnetic material is unevenly distributed near the particle surface of the magnetic polymer particles, and further, the superparamagnetic material is exposed on the particle surface. Thus, iron ions may be eluted into the aqueous medium. Examples of the method for lipophilizing the superparamagnetic substance include a silane coupling agent, a method of treating with a surface treatment agent such as a titanium coupling agent, and a method of adsorbing a fatty acid salt or the like to the superparamagnetic substance. Absent. A superparamagnetic material obtained by removing a dispersion medium from a commercially available magnetic fluid can also be used.

In the production method of the present invention, the monomer used as the dispersion medium of the superparamagnetic substance is a hydrophobic monomer. By using a hydrophobic monomer, the superparamagnetic substance subjected to the lipophilic treatment can be uniformly dispersed in the monomer. And, in the monomer composition thus obtained, coarse particles and the like due to aggregation of the superparamagnetic substance are not generated, and therefore, the monomer composition can be uniformly emulsified and dispersed in an aqueous medium. .

As used herein, the term "hydrophobic monomer" refers to a monomer having a solubility in water at 20 ° C. of less than 0.1% by weight, and having a solubility of 0.1% by weight or more. Is hereinafter referred to as “hydrophilic monomer”. When the monomer used as the dispersion medium of the superparamagnetic substance is a mixture of two or more kinds, if 60% by weight or more of the monomers is a hydrophobic monomer, the mixed monomer contains a hydrophilic monomer. Is also good.

Here, as the hydrophilic monomer constituting a part of the dispersion medium, for example, acrylic acid, methacrylic acid,
Unsaturated carboxylic acids such as crotonic acid, itaconic acid and fumaric acid [the above-mentioned monomer (B)], methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, diethylene glycol diacrylate, Examples thereof include diethylene glycol dimethacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, diallyl phthalate, and vinyl acetate.

As a disperser used for emulsifying and dispersing the monomer composition in an aqueous medium, a conventionally known ultrasonic disperser, a high-pressure homogenizer, a high-shear rate disperser and the like can be used. The type of the ultrasonic dispersing machine is not particularly limited. For example, a dispersing machine having a horn-type oscillator, a dispersing machine having a plate-type oscillator, and a continuous dispersing machine for flowing a liquid through an oscillating unit. And the like. Examples of the high shear rate disperser include a homomixer, a colloid mill, a jet homogenizer, a high-pressure homogenizer, and the like.These dispersers may be appropriately selected according to the target dispersed particle diameter. Can be. Further, a membrane emulsification method in which the monomer composition is emulsified and dispersed in an aqueous medium by extruding the monomer composition into an aqueous medium through a porous membrane or a porous filter, or pressure, vibration or high voltage is applied from a nozzle. A nozzle method of discharging the monomer composition by using the nozzle method.

In the production method of the present invention, it is preferable to use water as the aqueous medium which is the dispersion medium of the monomer composition. Further, in the aqueous medium, a surfactant such as an anionic surfactant, a nonionic surfactant, a water-soluble polymer, or an inorganic suspension protectant, an emulsifier, or a suspension protectant may be added. . Among these, partially saponified polyvinyl alcohol is preferred in that it can be applied to obtain particles having a wide range of particle diameters, and that particles having both positive and negative surface charges can be synthesized.

In the method for producing magnetic polymer particles of the present invention, the polymerization initiator for polymerizing the monomer includes:
There is no particular limitation, and conventionally known radical polymerization initiators such as organic peroxides, azo-based initiators, and persulfate-based initiators can be used. Of these,
It is preferable to use an oil-soluble polymerization initiator. In particular, when preparing a monomer composition by dispersing a superparamagnetic substance in a hydrophobic monomer, it is preferable to add an oil-soluble polymerization initiator, thereby preventing the formation of polymer particles containing no superparamagnetic substance. can do.

The polymerization reaction of the monomer is preferably carried out in an oxygen-free atmosphere. The reaction temperature varies depending on the decomposition temperature of the polymerization initiator to be used. For example, when benzoyl peroxide is used, the polymerization reaction can suitably proceed at 75 to 85 ° C.

In the production method of the present invention, after preparing the magnetic polymer particles, the particles may be washed in order to remove the surfactant or suspension protective agent used in the polymerization. On this occasion,
It is preferable to use water or a water-soluble solvent such as alcohol or acetone, and it is also preferable to add a small amount of a surfactant to this. Further, if necessary, the polymerized magnetic polymer particles can be classified by means such as sedimentation separation, coagulation separation, centrifugation, or magnetic separation to adjust the particle diameter and the particle size distribution to the desired values.

In the production method of the present invention, any one of the following steps (a) to (c) is carried out in order to surely prevent the superparamagnetic substance from flowing out of the obtained magnetic polymer particles. Is preferred. In addition, the following steps (a) to
The magnetic polymer particles of the present invention to be used in (c) are hereinafter also referred to as “particles before modification”.

Step (a): a step of polymerizing a vinyl monomer in the presence of the unmodified particles. Step (b): a step of bringing an iron dissolving agent into contact with the particles before modification to elute and remove the superparamagnetic substance existing near the surface of the particles. Step (c): a step of bringing an organic base and / or a water-soluble solvent into contact with the particles before modification.

<Step (a)> This step (a) is a step of polymerizing a vinyl monomer in the presence of the particles before modification. By this step (a), a surface coating layer containing no superparamagnetic substance is formed on the surface of the magnetic polymer particles by polymerization. Then, magnetic polymer particles obtained through this step (a) (hereinafter also referred to as “surface-modified particles (a)”).
According to the, elution of iron-based superparamagnetic material is reliably suppressed,
As a result, the performance as magnetic polymer particles is improved (expansion of applications), and the stability of performance over time (improved reliability)
Can be achieved.

The vinyl monomer used in the step (a) can be appropriately selected according to the use of the finally obtained magnetic polymer particles (the surface-modified particles (a)) and the like. Preferred compounds include vinyl compounds, (meth) acrylate compounds, and unsaturated carboxylic acids. In addition, from the viewpoint of efficiently forming the nonmagnetic polymer component (surface coating layer) present on the surface of the surface-modified particles (a), the polymer constituting the particles before modification is used as the vinyl monomer. It is preferable to use a monomer which is hardly absorbed by the copolymer), for example, a water-soluble vinyl monomer.

The amount of the monomer used in the step (a) is
Usually 10 to 1000 with respect to 100 parts by weight of the particles before modification.
Parts by weight, preferably 20 to 500 parts by weight, more preferably 30 to 200 parts by weight. If the amount of the monomer used is too small, it is difficult to form a sufficient surface coating layer to suppress the outflow of the superparamagnetic material,
On the other hand, if the amount of the monomer used is excessive, polymer particles having no magnetism are likely to be generated after the end of this step.

The specific method for polymerizing the vinyl monomer in the presence of the magnetic polymer particles (particles before modification) is not particularly limited.
A monomer absorption polymerization method in which the vinyl monomer is absorbed by the unmodified particles, and then the monomer in the unmodified particles is polymerized. (B) The vinyl is added to the magnetic polymer latex in which the unmodified particles are dispersed in water. A seed polymerization method of continuously or intermittently supplying a system monomer and polymerizing the monomer with the unmodified particles as seed particles, (c) forming an adsorbed phase on the particle surface of the unmodified particles, Among them, a capsule polymerization method in which a vinyl monomer is polymerized can be exemplified.

Among these methods, the above methods (a) and (b) are preferable from the viewpoint that the surface coating layer can be reliably formed even if the particles before modification are small particles. In the method (b), after the polymerization reaction for obtaining the polymer constituting the unmodified particles is substantially completed (specifically, when the polymerization conversion rate becomes 85% or more). It is preferable to add a vinyl monomer to the polymerization reaction system to continue the polymerization reaction of the monomer.

<Step (b)> This step (b) is a step in which an iron dissolving agent is brought into contact with the particles before modification to elute and remove the superparamagnetic substance existing near the surface of the particles in advance. Here, the “iron dissolving agent” refers to a substance that can dissolve the iron component (superparamagnetic substance) that constitutes the particles before modification and that does not substantially dissolve the polymer that constitutes the particles before modification. Shall be referred to.

As a method for bringing the iron-solubilizing agent into contact with the particles before the modification, a method for directly contacting the iron-solubilizing agent with the particles before the modification, and a method for dispersing the particles before the modification in water. The method of contacting both, specifically,
A method in which an aqueous solution of an iron dissolving agent is added to a magnetic polymer latex in which the particles before modification are dispersed and contained, and the like, and the above method is preferable.

By bringing the iron particles into contact with the particles before modification, the iron component existing on the surface and near the surface of the particles before modification is preferentially dissolved and removed. Then, this step (b)
The magnetic polymer particles (hereinafter, also referred to as “surface-modified particles (b)”) obtained through the above-mentioned process are suppressed to a level at which the elution amount of the iron-based superparamagnetic material does not pose a practical problem.
Improvement of performance as magnetic polymer particles (expansion of applications),
It is possible to improve the temporal stability of performance (improve reliability).

Examples of the iron dissolving agent that can be used in the step (b) include an inorganic acid, an organic acid, and a complexing agent with iron.

Here, examples of inorganic acids that can be used as iron dissolving agents include sulfuric acid, hydrochloric acid, and nitric acid. Since inorganic acids have a high dissolution rate of iron components, when using inorganic acids as iron dissolving agents, not only the surface and near the surface of the unmodified particles, but also the superparamagnetic material contained inside the particles It may be dissolved and removed. Therefore, it is preferable to control the dissolved amount of the iron component by adjusting the concentration of the inorganic acid, the treatment time, and the like so that the iron component is not dissolved and removed more than necessary (the superparamagnetic substance inside the particle is not dissolved). .

Here, when the inorganic acid is used, the preferred amount of dissolution and removal of the iron component is as follows: the weight of the iron component (superparamagnetic substance) contained in the particles before modification is “x”, the surface modification is When the weight of the iron component (superparamagnetic substance) contained in the porous particles (b) is "y", the amount of [(xy) / x] is 0.05 to 0.6. It is preferred that
If this value is less than 0.05 (the amount of elution removed is too small), the iron component (superparamagnetic substance) remaining on the surface of the magnetic polymer particles and in the vicinity thereof elutes during use, and It may not have sufficient practical performance depending on the field of application, such as typical use. on the other hand,
When the value of [(xy) / x] exceeds 0.6 (when the elution removal amount is excessive), the superparamagnetic substance inside the particles is eluted, and the magnetic sedimentation speed is reduced. In some cases, it may not have high magnetic responsiveness.

The processing conditions in the case of using an inorganic acid as an iron dissolving agent are selected so as to secure the dissolution removal amount (xy) of the iron component as a guide. Preferred treatment conditions vary depending on the type of iron-based superparamagnetic material or inorganic acid, but from the viewpoint of controlling the amount of dissolution and removal, the treatment temperature is 0 to
100 ° C. and the treatment time are 0.5 to 20 hours. In a mode in which sulfuric acid is brought into contact with the particles before modification in a dispersion (magnetic polymer latex) in which the particles before modification are dispersed in an aqueous medium, a preferred example of the processing conditions is as follows. The concentration of the raw particles is 4 g / liter, the sulfuric acid concentration is 2 N, the processing temperature is 25 ° C., and the processing time is 2 hours. After the treatment, the magnetic polymer particles [surface-modified particles (b)] are washed and redispersed, and then neutralized by adding an alkaline compound to the dispersion of the surface-modified particles (b). Is preferred.

Examples of the organic acid which can be used as the iron dissolving agent include oxalic acid, citric acid, chloroacetic acid, mercaptoacetic acid, sulfosalicylic acid and the like. Of these, mercaptoacetic acid and sulfosalicylic acid are preferred. Complexing agents usable as iron dissolving agents include thiocyanates such as potassium thiocyanate, sodium thiocyanate and guanidine thiocyanate;
-Mercapto compounds such as mercaptoethanol, dithioglycerin, dithiotrimethylolpropane, and dithioerythritol; nitrogen-based complexing agents such as o-phenanthroline, 2,2'-bipyridyl, 8-quinolinol, and ethylenediaminetetraacetic acid. it can. Of these, potassium thiocyanate, guanidine thiocyanate, 2-mercaptoethanol, dithioerythritol, o-phenanthroline, and 2,2′-bipyridyl are preferred. These iron dissolving agents may be used alone, but are preferably used in combination of two or more.
In this case, it is preferable to use different types of iron solubilizers, such as a combination of an organic acid and a thiocyanate, or a combination of a mercapto compound and a thiocyanate.

The organic acid and the complex forming agent used as the iron dissolving agent can selectively dissolve and remove the iron component existing on the surface of the pre-modified particles and in the vicinity of the surface. When an organic acid is used as the iron dissolving agent, the preferred amount of dissolution and removal of the iron component varies depending on the amount of the superparamagnetic substance existing on the surface and near the surface of the unmodified particles. In the case where the amount of the superparamagnetic material to be modified is small, the weight of the iron component (superparamagnetic material) contained in the particles before modification is “x”, and the iron contained in the surface modified particles (b) is “x”. When the weight of the component (superparamagnetic substance) is “z”, the amount is preferably such that the value of [(x−z) / x] is 0.01 to 0.4.

The treatment conditions when using an organic acid and / or a complex forming agent do not need to be controlled as strictly as when using an inorganic acid. Dispersion in which particles before modification are dispersed in an aqueous medium (magnetic polymer latex)
Guanidine thiocyanate (complexing agent) and 2-mercaptoethanol (complexing agent)
In a mode in which the particles are brought into contact with each other, a preferred example of the processing conditions is as follows.
Liter, the concentration of guanidine thiocyanate is 1 mol / l, the concentration of 2-mercaptoethal is 1% by weight, the treatment temperature is 25 ° C, and the treatment time is 12 hours.

In the surface-modified particles (b) obtained through the step (b), the amount of the iron-based superparamagnetic material remaining on the particle surface and in the vicinity of the surface is preferably as small as possible. Specifically, the surface-modified particles subjected to the cleaning treatment (b)
After immersing 0.1 g in 10 ml of pure water heated to 70 ° C. for 2 hours, the metal content eluted in the pure water is preferably 10 ppm or less, more preferably 1 ppm or less. You.

<Step (c)> This step (c) is a step of bringing an organic base and / or a water-soluble solvent into contact with the particles before modification.

Specific examples of the organic base that can be used in step (c) include organic amine compounds such as ammonia, ethanolamine, diethanolamine, triethanolamine, dimethylamine, diethylamine, and trimethylamine.

The water-soluble solvent that can be used in the step (c) is an organic solvent having a solubility in water at 25 ° C. of 1% by weight or more and having an affinity for a polymer. Specific examples of such a water-soluble solvent include acetone, mercaptoethanol, erythritol, dithioerythritol, ethyl acetate, butyl carbitol acetate, and phenyl cellosolve.

In this step (c), an organic base and / or an aqueous base of magnetic polymer particles (particles before modification) are added to the aqueous dispersion.
Alternatively, it is performed by adding a water-soluble solvent. Here, the amount of the organic base used is usually 0.1 to 20% by volume of the aqueous dispersion of the particles before modification. The amount of the water-soluble solvent used is usually from 1 to 95 of the aqueous dispersion of the particles before modification.
% By volume. The concentration of the particles before modification in the aqueous dispersion is not particularly limited, but may be 0.1 to 20.
% By weight. In addition, when the particles before modification settle, it is preferable to perform the treatment while stirring appropriately. The pH of the aqueous dispersion is usually 9 or more, and preferably 11 or more. The treatment temperature is not particularly limited, but is preferably about 50 ° C to 80 ° C. The processing time is not particularly limited, but is preferably 1 to 40 hours.

The magnetic polymer particles obtained through this step (c) (hereinafter also referred to as “surface-modified particles (c)”) are particles having a large surface charge, specifically, having a surface charge of 0.
It becomes particles of more than 05 meq / g. Here, the “surface charge amount” is a value obtained by measuring the acid present on the surface of the magnetic polymer particles by conductivity titration, and is equivalent to milliequivalent /
g (the number of milliequivalents of carboxyl groups present on the particle surface per 1 g of magnetic polymer particles). References describing this measurement method include “J. Electroa
nal. Chem. , Vol. 37 p. 161 (19
72)). Particles having such a large surface charge can be used as carboxyl group-modified particles without practical problems. In addition, particles having such a large surface charge have good dispersion stability even in a dispersion medium having a high electrolyte concentration in the use of the particles, and also have excellent redispersibility after the particles are settled, and also have a functional surface property. Using the group, a nucleic acid or a physiologically active substance such as an antigen / antibody or an enzyme can be chemically bonded to the surface.
As a method for obtaining a predetermined amount of surface charge, a commonly used method can be used as it is. For example,
A method of directly adding an acid monomer that can be copolymerized at the time of polymerization, a method of forming a particle by adding an ester monomer that can be copolymerized, and then obtaining a surface charge by hydrolysis, or a highly reactive function such as glutar methacrylate. It may be copolymerized with a copolymerizable monomer having a group.

The magnetic polymer particles according to the present invention (the magnetic polymer particles of the present invention and the magnetic polymer particles obtained by the method of the present invention) include a diagnostic drug carrier, a bacterium separation carrier, a cell separation carrier, a nucleic acid separation and purification carrier, and a protein separation. It can be suitably used as a purification carrier, an immobilized enzyme carrier, a drug delivery carrier, a magnetic toner, a magnetic ink, a magnetic paint, and the like. In addition, the magnetic polymer particles according to the present invention include DN.
Physiologically active substances such as nucleic acids such as A and RNA, antigens / antibodies, and enzymes can be easily bonded (chemically or physically bonded).

The nucleic acid-immobilized magnetic polymer particles can be obtained by immobilizing oligonucleotides on the surface of the magnetic polymer particles of the present invention. The length of the oligonucleotide is 10 to 100 bases, preferably 15 to 70 bases.
Is a base. If necessary, the immobilized oligonucleotide may be a single-stranded DNA, and may be a double-stranded DNA.
Or RNA. Such a nucleic acid can be prepared using a commercially available nucleic acid synthesizer. In the present invention, as a method of immobilizing the nucleic acid-containing carrier particles, for example, the methods described in JP-A-7-75599 and JP-A-6-335380 can be applied. That is, specifically, one equivalent of the nucleic acid probe and one equivalent of the DNA complementary to the hybridization site of the nucleic acid probe are mixed in an annealing buffer, heated at 80 ° C. for 5 minutes, and then gradually returned to room temperature. Form a double strand. This double-stranded DNA
Is purified by a hydroxyapatite column, and then added to the particle dispersion together with a dehydrating condensing agent, and by heating, the amino group in the base sequence of the immobilized site of the nucleic acid probe and the carboxylic acid group on the particle surface are bonded by amide bond Can be fixed. Here, examples of the dehydrating condensing agent include carbodiimides such as 1-ethyl-3- (N, N'-dimethylamino) propylcarbodiimide, N-ethyl-5-phenylisoxazolium-3 'sulfonate, -Ethoxycarbonyl {2} ethoxy {1, 2}
Although a water-soluble dehydrating condensing agent such as dihydroquinoline is preferred, an oil-soluble dehydrating condensing agent can also be used. The amount of the particles used is usually 0.5 to 500 g, preferably 5 to 5 g per 1 mmol of the nucleic acid probe.
0 g is appropriate. The reaction of immobilizing the nucleic acid probe on the particles may be carried out usually in an aqueous medium having a pH of about 3 to 11 at 4 to 70 ° C. for 5 minutes to overnight.

Next, the nucleic acid probe particle of the present invention can be obtained by releasing the complementary base sequence bound to the hybridization site in the nucleic acid probe immobilized on the particle. At this time, the detachment of the complementary base sequence,
The denaturation can be performed by heat or alkali denaturation, and the complementary base sequence can be removed by means such as centrifugation and filtration. The nucleic acid-immobilized magnetic polymer particles prepared in this way can be used for gene diagnosis and other genetic engineering in general. Specifically, messenger RNA can be directly recovered from a cell lysate using, for example, magnetic polymer particles to which oligo dT is bound. Or, if a nucleic acid sequence complementary to the AIDS virus nucleic acid is used, the AIDS virus is recovered from the biological specimen,
It can be detected using a DNA probe method or a polymerase chain reaction method. From the composition of the magnetic polymer particles of the present invention, non-specific adsorption of proteins is significantly lower than that of conventional styrene-based particles, so that when used for the above diagnostic and research purposes, it is easy to realize a low background. Therefore, detection with high sensitivity can be performed, which is preferable. In addition, in the enzymatic reaction, the activity decrease due to the adsorption of the enzyme protein is small, and compared with the conventional carrier,
A highly efficient enzyme reaction can be realized. The bioactive protein can be immobilized on the magnetic polymer particles of the present invention to prepare protein-immobilized magnetic polymer particles carrying the bioactive protein. The bioactive protein includes a protein having general bioactivity, or a complex thereof. Specific examples include immunoglobulins, various enzymes, and avidin proteins. Such a protein can be bound to the surface of the magnetic polymer particles in the same manner as in the above-described nucleic acid immobilization.
Specifically, it can be reacted with a carboxyl group on the particle surface by utilizing the amino group of the protein. Various measures are taken to increase the reaction efficiency. For example, the reaction may be carried out by first reacting the dehydration condensing agent with the carboxyl group of the particles and then adding the protein. The antibody-bound particles, avidin-bound particles, and enzyme-bound particles thus prepared can be used in the fields of diagnosis and research. In particular, the background is extremely low because the amount of protein physically adsorbed by the magnetic polymer particles of the present invention is small. Therefore, a very small amount of an object to be measured can be detected with high sensitivity.

[0067]

EXAMPLES Examples of the present invention will be described below, but the present invention is not limited to these examples.
In the following, “parts” means “parts by weight”.

Example 1 Acetone was added to an oil-based magnetic fluid "Fericolloid HC50" (manufactured by Taiho Kogyo KK) to precipitate and precipitate particles, which were then dried to obtain a lipophilic treatment. A ferrite-based superparamagnetic material having a surface (particle size: 0.01 μm) was obtained. According to the recipe shown in Table 1, 75 parts of cyclohexyl methacrylate, 5 parts of methacrylic acid, 20 parts of methyl methacrylate and 3 parts of benzoyl peroxide (polymerization initiator) are added to 40 parts of the superparamagnetic material, and the system is mixed and stirred. To uniformly disperse the superparamagnetic material to prepare a monomer composition. On the other hand, an aqueous medium was prepared by dissolving 10 parts of polyvinyl alcohol, 0.05 part of sodium laurate and 0.1 part of polyethylene oxide nonylphenyl ether in 1000 parts of water. The above-mentioned monomer composition is added to the obtained aqueous medium (aqueous phase), the system is preliminarily stirred with a homomixer, and then subjected to a dispersion treatment with an ultrasonic disperser to obtain an oil having an average particle diameter of 2 μm. A suspension (oil droplet dispersion) in which droplets (oil phase) were dispersed in an aqueous medium was prepared. Then, the obtained suspension was charged into a two-liter flask with a stirrer having a capacity of 2 liters, and the temperature of the system was increased to 75 ° C., and the monomer in the oil droplets was stirred for 5 hours under a nitrogen atmosphere. Was polymerized (suspension polymerization) to prepare a dispersion (magnetic polymer latex) in which the magnetic polymer particles of the present invention were dispersed in an aqueous medium. The magnetic polymer particles of the present invention dispersed in the magnetic polymer latex [hereinafter, also referred to as “magnetic polymer particles (1)”. ] Can be easily attracted and settled by a magnet and collected, and the collected magnetic polymer particles (1)
Was easily redispersible in water. Therefore,
The operation of washing and purifying the magnetic polymer particles (1) by performing magnetic precipitation in distilled water was performed three times.

Example 2 Same as Example 1 except that the monomer composition in the monomer composition was changed to 75 parts of cyclohexyl acrylate, 3 parts of methacrylic acid and 2 parts of acrylic acid according to the formulation shown in Table 1. Then, a monomer composition and a suspension are prepared, and suspension polymerization is performed, whereby the magnetic polymer particles of the present invention [hereinafter, also referred to as “magnetic polymer particles (2)”. ] Was dispersed in an aqueous medium to prepare a dispersion (magnetic polymer latex).

Example 3 Except that the monomer composition in the monomer composition was changed to 10 parts of 2-ethylhexyl methacrylate, 50 parts of cyclohexyl methacrylate, 5 parts of methacrylic acid and 35 parts of styrene according to the formulation shown in Table 1. By preparing a monomer composition and a suspension in the same manner as in Example 1, and performing suspension polymerization,
The magnetic polymer particles of the present invention [hereinafter also referred to as “magnetic polymer particles (3)”. ] Was dispersed in an aqueous medium to prepare a dispersion (magnetic polymer latex).

Comparative Example 1 A monomer composition was prepared in the same manner as in Example 1 except that the monomer composition in the monomer composition was changed to 5 parts of methacrylic acid and 95 parts of styrene in accordance with the recipe shown in Table 1. By preparing a suspension and performing suspension polymerization, magnetic polymer particles for comparison [hereinafter also referred to as “magnetic polymer particles (C1)”. ] Was dispersed in an aqueous medium to prepare a dispersion (magnetic polymer latex).

Comparative Example 2 According to the recipe shown in Table 1, except that the monomer composition in the monomer composition was changed to 10 parts of 2-ethylhexyl methacrylate, 5 parts of methacrylic acid, 10 parts of methyl methacrylate and 75 parts of styrene. In the same manner as in Example 1, a monomer composition and a suspension were prepared, and suspension polymerization was carried out to obtain magnetic polymer particles for comparison [hereinafter, “magnetic polymer particles (C
2) ". ] Was dispersed in an aqueous medium to prepare a dispersion (magnetic polymer latex).

For each of the magnetic polymer latexes obtained in Examples 1 and 2 and Comparative Examples 1 and 2, the polymerization conversion, the solid content, and the content of the superparamagnetic substance in the magnetic polymer particles were measured. Further, according to the evaluation method described below, polymerization stability, dispersibility of the superparamagnetic material in the magnetic polymer particles, to evaluate the static stability, according to the measurement method described below, the shape of the magnetic polymer particles,
The number average particle diameter and the coefficient of variation were measured. The results are shown in Table 1.

(A) Evaluation of polymerization stability: After the completion of the polymerization reaction, the reaction product was passed through a 200-mesh stainless steel wire mesh, and the amount of aggregates remaining on the wire mesh was less than 10% by weight of the total solids. The case was judged as "good", and the case where the amount of aggregates was 10% by weight or more of the total solids was judged as "poor".

(B) Evaluation of dispersibility of superparamagnetic substance: After dyeing with uranyl acetate, the state of dispersion of the superparamagnetic substance in the magnetic polymer particles was observed by a transmission electron microscope, and the superparamagnetic substance was uniformly dispersed throughout the polymer particles. Or, when the polymer particles were uniformly dispersed in the center of the polymer particles, it was judged as "good", and when the polymer particles were localized in a part of the vicinity of the surface or in the entire vicinity of the surface, it was judged as "poor".

(C) Evaluation of static stability: The magnetic polymer latex was diluted to adjust the solid content concentration (c ₀ ) to 1% by weight, and the diluted liquid was adjusted to have a liquid phase height of 2 cm. After being contained in a sample bottle having a capacity of 20 ml and shaking the container until the magnetic polymer particles are uniformly dispersed, 1
After leaving still for 0 minutes, only the upper layer portion from the liquid level to 1 cm below the liquid level was taken out, and the solid content concentration (c) was measured.
The value calculated by c ₀ ) × 100 was determined as a static stability index (% by weight).

(D) Measurement of number average particle size and variation coefficient of magnetic polymer particles: After staining with uranyl acetate, electron micrographs of the magnetic polymer particles were taken with a transmission electron microscope, and 500 randomly selected magnetic polymer particles were taken. The number average particle diameter and the coefficient of variation thereof were determined by measuring the particle diameter of the magnetic polymer particles.

Comparative Example 3 A ferromagnetic material was prepared by subjecting magnetite fine particles "magnetite EPT500" (manufactured by Toda Kogyo Co., Ltd.) having a particle diameter of 3000% to lipophilic treatment with sodium oleate. According to the formulation shown in Table 1, a monomer composition and a suspension were prepared in the same manner as in Example 1 except that the ferromagnetic material was used instead of the superparamagnetic material, and a suspension polymerization was carried out. Polymer particles [hereinafter, also referred to as “magnetic polymer particles (C3)”. ] Was dispersed in an aqueous medium to prepare a dispersion (magnetic polymer latex). About the obtained magnetic polymer latex, polymerization conversion, solid content, ferromagnetic substance content ratio in magnetic polymer particles, polymerization stability, ferromagnetic substance dispersibility in magnetic polymer particles, static stability, magnetic polymer Table 1 also shows the results of the particle shape, the number average particle diameter, and the coefficient of variation. The magnetic particles of Comparative Example 3 had residual magnetization, and the particles collected by magnetic suction attracted each other and had poor redispersibility.

[0079]

[Table 1]

Example 4 After 3 hours had elapsed since the start of suspension polymerization, 19 parts of methyl methacrylate and 1 part of methacrylic acid were added to the reaction system, followed by suspension polymerization for 5 hours. Except for the above, a dispersion (magnetic polymer latex) of the magnetic polymer particles of the present invention was produced in the same manner as in Example 1. The polymerization conversion of the magnetic polymer latex thus obtained is 98% by weight, and the magnetic polymer particles of this example constituting the magnetic polymer latex include:
A surface coating layer containing no superparamagnetic material was formed.

<Example 5> 100 parts of the magnetic polymer latex (1) obtained in Example 1 was added to 1000 parts of 0.1 N hydrochloric acid, and the extraordinary particles present near the surface of the magnetic polymer particles (1) were added. The magnetic substance was eluted and removed. Here, the processing time was one hour. Then, the treated magnetic polymer particles are washed and purified by magnetic separation,
A dispersion (magnetic polymer latex) of the magnetic polymer particles of the present invention was produced. A surface coating layer containing no superparamagnetic material was formed on the magnetic polymer particles of this example.

<Example 6> To 8.0 g (1 g in solid content) of the magnetic polymer latex obtained in Example 1, 250 ml of 2N sulfuric acid (iron dissolving agent) was added. The particles before modification [magnetic polymer particles (1)] were subjected to acid treatment (treatment for elution and removal of superparamagnetic substances existing near the surface) by gentle stirring for 3 hours. Next, the treated magnetic polymer particles are separated and collected by magnetic sedimentation, and the collected magnetic polymer particles are mixed with polyoxyethylene nonylphenyl ether “Emulgen 910”.
Washed twice with 200 ml of an aqueous solution (manufactured by Kao Corporation) (0.01% by weight), redispersed in 10 ml of distilled water, and adjusted the pH of the resulting dispersion to an aqueous sodium hydroxide solution (0.5%). (% By weight). By such an operation, 0.75 g of magnetic polymer particles [surface-modified particles (b)] from which the superparamagnetic substance near the surface was eluted and removed.
(Magnetic polymer latex) was obtained. In the obtained surface-modified particles (b), the ferrite content measured by a thermobalance was 22.5% by weight, and 20% by weight of the ferrite (superparamagnetic material) contained in the particles before modification was dissolved and removed. It was done. Further, the surface-modified particles (b)
Was 1.8 μm, which was not much different from the particle diameter of the particles before modification. Furthermore, ion-exchanged water was added to 1 g (0.1 g in solid content) of the dispersion of the magnetic polymer particles [surface-modified particles (b)] obtained in this example, and the magnetic sedimentation treatment was performed twice to obtain a surface. The modified particles (b) were collected, and the surface modified particles (b) were sufficiently washed with water. Next, the surface-modified particles (b) were added to 10 ml of ion-exchanged water, and the system was stirred at 70 ° C. for 24 hours. Thereafter, the supernatant portion of this system was sampled and the iron concentration was measured by atomic absorption spectrometry. The iron concentration was 1 ppm or less, indicating that the iron was present on the surface of the surface-modified particles (b) and near the surface. It was confirmed that the amount of the superparamagnetic material was extremely small. For comparison, when the same operation was performed on 0.8 g (0.1 g in solid content) of the dispersion of the magnetic polymer particles (2) obtained in Example 2, the iron concentration in the supernatant was 73 ppm. there were.

<Example 7> To 0.8 g (0.1 g in solid content) of the magnetic polymer latex obtained in Example 1,
1 g of 1% by weight aqueous ammonia (organic base) was added. At this time, the pH of the system (magnetic polymer latex) was 12.5. The system is then heated at 60 ° C for 1
The particles before modification [magnetic polymer particles (1)] were alkali-treated by gentle shaking for 0 hour. Thereafter, the system was cooled and neutralized with 0.5N sulfuric acid to adjust the pH to 7.5. Next, the magnetic polymer particles (the surface-modified particles (c)) that had been subjected to the alkali treatment were washed and re-dispersed four times with pure water. The content ratio of the superparamagnetic material in the surface-modified particles (c) thus obtained was 28% by weight, and the surface-modified particles (c)
Was 1.8 μm, which was not much different from the content ratio and the particle size of the particles before modification. When the surface charge of the surface-modified particles (c) was measured, it was 0.125 meq / g, which was compared with the surface charge of the magnetic polymer particles (1) (0.002 meq / g). It was confirmed that a large value was obtained.

<Examples 8 to 16> Particles before modification were prepared in the same manner as in Example 7 except that an organic base or a water-soluble solvent shown in Table 2 below was used in place of 1% by weight of aqueous ammonia. [Magnetic polymer particles (1)] were treated to obtain surface-modified particles (c). In Examples using only a water-soluble solvent as a treating agent, the treatment was performed after adjusting the pH of the system to 12 with a 0.5 N aqueous sodium hydroxide solution. Table 2 also shows the value of the surface charge measured for each of the obtained surface-modified particles (c) in the same manner as in Example 7.

<Example 17> The procedure of Example 7 was repeated, except that 0.8 g (0.1 g in solid content) of the magnetic polymer latex obtained in Example 2 was used and 1 g of acetone was used in combination as a treating agent. The particles before modification [magnetic polymer particles (2): surface charge of 0.005 meq / g] were treated to obtain surface modified particles (c). With respect to the surface-modified particles (c) thus obtained, the value of the surface charge measured in the same manner as in Example 7 is also shown in Table 2.

Example 18 The procedure of Example 7 was repeated except that 0.8 g (0.1 g in solid content) of the magnetic polymer latex obtained in Example 3 was used and 1 g of acetone was used as a treating agent. The particles before modification [magnetic polymer particles (3): surface charge amount 0.003 meq / g] were treated to obtain surface modified particles (c). With respect to the surface-modified particles (c) thus obtained, the value of the surface charge measured in the same manner as in Example 7 is also shown in Table 2.

[0087]

[Table 2]

<Test Example 1> Carrying out immunodiagnostic carrier particles (carrier for enzyme immunoassay) by conducting the following tests
Was evaluated. 10 mg of each of the magnetic polymer latexes obtained in Examples 1 to 5, Example 7, Example 9, Example 14, and Comparative Examples 1 to 3 was collected in terms of solid content, and magnetic polymer particles were collected from the collected magnetic polymer latex. Was magnetically separated, and the magnetic polymer particles were dispersed in 1 ml of a phosphate buffered saline solution (pH 7.5). Next, 200 μl of anti-human IgM antibody was added to this dispersion.
g of the polymer particles and gently shaking at room temperature for 1 hour to adsorb the antibody on the particle surface to sensitize the magnetic polymer particles. The magnetic polymer particles after sensitization were magnetically separated,
Whether or not the antibody remained in the supernatant after separation was confirmed by measuring the absorbance at 280 nm, and the presence of the antibody was not recognized in any of the supernatants. Was confirmed to be adsorbed on the magnetic polymer particles. 0.5% bovine serum albumin was added to each of the magnetically separated magnetic polymer particles (anti-human IgM antibody-sensitized particles).
1 ml of a phosphate buffered saline solution (pH 7.5) containing 1% polyethylene glycol was added, and the mixture was vibrated with a vibrator and shaken gently for 30 minutes at room temperature to adsorb the antibody. By performing a blocking treatment on the surface of the unreacted particles with albumin, diagnostic drug particles for the enzyme immunoassay (EIA method) were prepared. The following operation was performed using each of the diagnostic agent particles prepared as described above. 0 ng / ml, 50 ng / ml, 100 ng
100 μl of a phosphate buffer solution containing human IgM antigen at a concentration of 500 μg / ml and 500 ng / ml, 20 μl of a dispersion containing particles of the diagnostic agent was added to each, and the mixture was allowed to stand at room temperature for 30 minutes. Subsequently, 200 μl of a solution containing 100 μg of an anti-human IgM antibody in which acetylcholinesterase was bound to magnetic polymer particles magnetically separated was used.
The mixture was added and dispersed by vibration with a vibrator, and then allowed to stand at 25 ° C for 1 hour. Then, "Ellman's reagent"(Cayman's reagent) which is a substrate of acetylcholinesterase was used.
100 μl), and the mixture was gently shaken at room temperature for 10 minutes to allow the enzyme reaction to develop color. After adding 50 μl of 2N sulfuric acid as a reaction stopping solution, the absorbance at 412 nm was measured. As a control test, 20 μl of a dispersion containing particles not sensitized was added to 100 μl of a phosphate buffered saline containing no human IgM antigen, and the same operation was performed to measure the absorbance. Table 3 shows the results.

[0089]

[Table 3]

<Test Example 2> Streptavidin (manufactured by Wako Pure Chemical Industries, Ltd.) was bound in place of anti-human IgM in the same manner as in Test Example 1, and the magnetic polymer particles were washed after the reaction in the same manner. Processed. Finally, a dispersion of 5% by weight was obtained. A biotin-bound acetylcholinesterase solution (1 μg / ml) was added to 0 μl, 2 μl, of 10 μl (0.5 mg) of the streptavidin-bound magnetic polymer particles.
5 μl or 100 μl was added, a 5M NaCl solution was added so that the final salt concentration was 1M NaCl solution, and the reaction volume was adjusted to 0.5 ml with distilled water. The reaction was allowed to stand for 10 minutes. Next, a substrate was added in the same manner as in Test Example 1, reacted under the same conditions, and the absorbance was measured. Table 4 shows the results.

[0091]

[Table 4]

<Test Example 3> K, which is a human leukocyte cancer cell
PRM1-16 containing 562 cells containing 1% by weight fetal calf serum
The cells were cultured in 40 media. When the number of cells in the culture suspension reaches 500,000 cells / mL, 1 mL of the culture suspension
Was taken in a sampling tube, and centrifuged at 500 rpm for 5 minutes to collect cells. After adding 1 mL of potassium phosphate buffer (pH 7.2) to the collected cells,
Cell-free by sonication. This cell-free K5
62 cell lysate in 3 centrifuge tubes (2 mL volume)
0.5 mL each in 10 mM phosphate buffer (pH
Diluted to 2 mL by 5). Then, HIV-1DN taken from human leukocytes (NY10 strain) cultured by incorporating AIDS virus DNA into each of the centrifuge tubes.
A was added for 0, 10, and 50 molecules, respectively. After vortexing, place each of the centrifuge tubes in Example 1
Was added thereto, and 2 μL of a dispersion having a solid content of 10% by weight, in which the cationic magnetic polymer particles obtained by the above were dispersed, was added, and the mixture was rotationally stirred at room temperature at 10 rpm for 5 minutes. Next, the magnetic polymer particles were magnetically separated, and a PCR reaction solution shown in Table 5 below was added to each of the separated magnetic polymer particles for 2 minutes.
A PCR reaction was performed by adding 5 μL each.

[0093]

[Table 5]

In the above Table 5, “Primer SK145
The sequence of “A” is “5′CCCACAAGATTTAAA”
ACCCA 3 ′ ”and“ primer SK451
The sequence of “A” is “5′TGAAGGGTACTAGTA”
GTTTC 3 '", which was synthesized using a DNA synthesizer" type 381A "(manufactured by Applied Biosystems) in accordance with the manufacturer's manual.
In C, a purified product was obtained. The PCR reaction was performed using “Thermal cycler model JP2000” (PERKIN EL
MER CETUS) (94 ° C. × 0.5
Min, 55 ° C x 1.0 min, 72 ° C x 1.5 min)
The amplification reaction was performed with 0 cycles and a program of 72 ° C. × 7 minutes. Take 5 μL of the product of the amplification reaction by the first PCR method and use “Primer SK145” (manufactured by Takara) in place of “Primer SK145A”
"Primer SK451A"
The reaction of the nested PCR method was performed in the same manner except that SK451 ”(manufactured by Takara) was used. The reaction amplification products of the PCR method and the nested PCR method were combined with a 2% by weight agarose gel “Agarose 1600”.
It was electrophoresed in a TBE buffer (buffer consisting of 50 mM boric acid, pH 8.2) with a Mupid-type electrophoresis apparatus using [manufactured by Wako Pure Chemical Industries, Ltd.]. Table 6 shows the results.

[0095]

[Table 6]

According to the results shown in Table 6, the use of the magnetic polymer particles of the present invention makes it possible to recover DNA using magnetic separation instead of centrifugation.
It turned out that it can be used for the R method.

[0097]

According to the present invention, it is possible to provide novel magnetic polymer particles having excellent superparamagnetic dispersibility. The magnetic polymer particles of the present invention are excellent in magnetic responsiveness and, in a dispersion of the magnetic polymer particles (magnetic polymer latex), do not cause a problem that iron ions are eluted from the magnetic polymer particles into the aqueous medium. The magnetic polymer latex can be suitably used in various applications and technical fields where the application of the magnetic polymer latex has been limited due to this problem. In addition, the magnetic polymer particles of the present invention hardly settle in an aqueous medium, and can exist in a homogeneous dispersion state in the aqueous medium. According to the production method of the present invention, it is possible to reliably produce magnetic polymer particles having excellent properties as described above and a dispersion thereof.

The magnetic polymer particles of the present invention can be widely applied to various uses and fields that require magnetic responsiveness due to the above excellent properties. For example, antigens,
By physically or chemically adsorbing an antibody, protein, nucleic acid, or the like, it can be applied to a wide range as a diagnostic agent. In addition, when used as a diagnostic agent for enzyme immunoassay, non-specific coloring caused by elution of iron ions from magnetic polymer particles is suppressed, so that the practicality and reliability as a diagnostic agent are further enhanced. I have. Also,
Since the superparamagnetic material is not exposed on the surface of the magnetic polymer particles, it has excellent electrostatic properties and film-forming properties, magnetic toner,
It is also useful as a magnetic ink and a magnetic paint.

Claims (3)

[Claims] (A) 30 to 99% by weight of a structural unit derived from an alkyl (meth) acrylate having 4 to 20 carbon atoms in an alkyl group, and (B) a structural unit of 1 to 20 derived from an unsaturated carboxylic acid. % By weight and (C) 100 parts by weight of a copolymer composed of 0 to 69% by weight of a structural unit derived from a vinyl monomer copolymerizable with the alkyl (meth) acrylate and the unsaturated carboxylic acid. Magnetic polymer particles comprising 1 to 100 parts by weight of a body and having a number average particle size of 0.02 to 30 μm. 2. A superparamagnetic substance is dispersed in a monomer containing an alkyl (meth) acrylate having an alkyl group having 4 to 20 carbon atoms in a proportion of 30% by weight or more to prepare a monomer composition. A method for producing magnetic polymer particles, comprising dispersing a composition in an aqueous medium to obtain a suspension, and polymerizing the monomer in the suspension. 3. A magnetic polymer particle obtained by immobilizing an oligonucleotide or a physiologically active protein on the particle of claim 1.