JP4683225B2 - Magnetic polymer particles for diagnostic agents and method for producing the same - Google Patents

Description

  The present invention relates to a magnetic polymer particle for a diagnostic agent that is excellent in magnetic separation and has a large amount of binding with a biological substance and a method for producing the same.

  In recent years, magnetic polymer particles can provide an excellent reaction field in the immune reaction between an antigen and an antibody and in the hybridization between DNAs or between DNA and RNA. (For example, JP 2004-205481 A).

  In general, the smaller the particle size, the larger the surface area per unit weight of the magnetic polymer particle, and the larger the surface area per unit weight, the greater the amount of binding of biological substances per unit weight. That is, in the magnetic polymer particles, generally, the smaller the particle size, the greater the amount of binding of biological substances per unit weight.

However, in general, as the particle size of the magnetic polymer particles decreases, the magnetic separation property decreases. On the other hand, when magnetic polymer particles having a large particle size are used with an emphasis on magnetic separation, the larger the particle size, the smaller the amount of binding of the biological substance per unit weight. That is, it is difficult to achieve both excellent magnetic separation properties and high binding ability with biological materials.
JP 2004-205481 A

  The present invention provides a magnetic polymer particle for a diagnostic agent that is excellent in magnetic separation and has a large amount of binding with a biological substance and a method for producing the same.

  The inventors of the present invention obtained magnetic polymer particles having a major axis / minor axis = 1.1 to 2.0 by thermally and / or mechanically deforming the spherical magnetic mother particles. The present inventors have found that the particles have excellent magnetic separation properties and high binding ability with biological materials, and have reached the present invention.

  The magnetic polymer particles for diagnostic agents according to the first aspect of the present invention have a major axis / minor axis = 1.1 to 2.0.

  In the present invention, the “major diameter” of a particle refers to the largest diameter of the cross section passing through the center of the particle, and the “minor diameter” of the particle refers to the smallest diameter of the cross section diameter passing through the center of the particle. Say.

  The diagnostic magnetic polymer particles may include a core including magnetic fine particles and a shell including a polymer portion. In this case, the core may include non-magnetic core particles and a magnetic layer provided on the surface of the core particles. Furthermore, in this case, the major axis / minor axis of the core particle can be 1.1 to 2.0.

  The method for producing diagnostic magnetic polymer particles according to the second aspect of the present invention is the above magnetic polymer producing method including a step of thermally deforming spherical magnetic polymer particles.

  A method for producing diagnostic magnetic polymer particles according to the third aspect of the present invention is the method for producing a magnetic polymer, comprising a step of mechanically deforming spherical magnetic polymer particles.

A method for producing diagnostic magnetic polymer particles according to the fourth aspect of the present invention comprises:
Forming a magnetic layer on the surface of the core particle having a major axis / minor axis of 1.1 to 2.0;
Forming a polymer portion on the outside of the magnetic layer;
including.

  The method for producing diagnostic magnetic polymer particles further includes a step of thermally deforming spherical non-magnetic core particles to form core particles having a major axis / minor axis of 1.1 to 2.0. Can do.

  The method for producing magnetic polymer particles for diagnostic agents further includes the step of mechanically deforming spherical non-magnetic core particles to form core particles having a major axis / minor axis of 1.1 to 2.0. Can do.

  According to the magnetic polymer particles for diagnostic agents, it is possible to achieve both a high biological substance binding ability and an excellent magnetic separation property. Therefore, the magnetic polymer particles for diagnostic agents can be used in a wide range of fields such as biochemistry, paints, paper, electrophotography, cosmetics, pharmaceuticals, agricultural chemicals, foods, and catalysts. The magnetic polymer particles for diagnostic agents are particularly useful as biochemical carriers such as medical diagnostic carriers and automatic measuring instrument compatible particles because they can achieve both high binding ability of biological substances and excellent magnetic separation properties.

  According to the method for producing diagnostic magnetic polymer particles, the magnetic polymer particles can be produced simply and efficiently by including a step of thermally and / or mechanically deforming the spherical magnetic polymer particles. be able to.

  Further, according to the method for producing diagnostic magnetic polymer particles, a step of forming a magnetic layer on the surface of the core particles having a major axis / minor axis of 1.1 to 2.0, and on the outside of the magnetic layer Including the step of forming the polymer part, the magnetic polymer particles of the diagnostic magnetic polymer particles can be easily and efficiently produced.

1. Magnetic polymer particles and production method thereof 1.1. Magnetic polymer particle The magnetic polymer particle which concerns on one Embodiment of this invention has a flat shape which is long diameter / short diameter = 1.1-2.0. Here, if the major axis / minor axis is less than 1.1, high biomaterial binding ability and magnetic separation cannot be achieved at the same time, while if the major axis / minor axis exceeds 2.0, redispersibility is achieved. Inferior.

  The major axis and minor axis of the magnetic polymer particles according to an embodiment of the present invention can be measured from a photograph of the particles taken with a scanning electron microscope (SEM). This also applies to the major axis and minor axis of the core particles described later. For example, in the examples described later, the major axis and minor axis of a particle are measured from a photograph of 300 particles obtained by a scanning electron microscope (SEM), and the number average and minor axis of the major axis in 300 particles are measured. The number average is obtained, and the ratio of the number average of the major axis to the number average of the minor axis is defined as the major axis / minor axis.

  In the magnetic polymer particles according to one embodiment of the present invention, the major axis / minor axis can be adjusted by the heat treatment temperature and mechanical treatment conditions described later, the type of polymer used, and the molecular weight.

  The major axis of the magnetic polymer particles according to one embodiment of the present invention is preferably 0.05 to 10 μm, and more preferably 0.1 to 5 μm. When the particle size is less than 0.05 μm, sufficient magnetic separation is not exhibited, it takes a considerably long time to separate the particles, and a considerably large magnetic force is necessary to separate them. There is a case to become. On the other hand, when the particle size exceeds 10 μm, the particles are likely to settle in the dispersion medium, so that an operation of stirring the dispersion medium is necessary when binding to the biological substance, and the surface area relative to the weight of the particles is large. Since the ratio is small, it may be difficult to bind to a sufficient amount of the biological substance.

  The magnetic polymer particles according to an embodiment of the present invention preferably include magnetic fine particles (M) and a polymer portion (P).

The magnetic fine particles (M) are not particularly limited, but iron oxide-based substances are typical, and MFe ₂ O ₄ (M = Co, Ni, Mg, Cu, Li _0.5 Fe _0.5, etc.) Or ferrite represented by Fe ₃ O ₄ , or γFe ₂ O ₃ . In particular, γFe ₂ O ₃ and Fe ₃ O ₄ are preferable as magnetic materials having high saturation magnetization and low residual magnetization. The magnetic fine particles (M) are preferably made of a superparamagnetic magnetic material from the viewpoint of good redispersibility after separation and purification by magnetism because there is little residual magnetization, for example, a particle diameter of about 5 to 20 nm. Ferrite and / or magnetite fine particles can be suitably used.

  The polymer part (P) is preferably made of a radical polymerizable polymer such as a (meth) acrylate polymer or a styrene polymer. When the polymer part (P) constitutes the outermost surface of the particle, the polymer part (P) is preferably a polymer having a functional group capable of binding to a biological substance, for example, hydrophobic such as polystyrene or polycyclohexyl methacrylate. More preferably, the polymer comprises a polymer having a functional group or a polymer having a surface functional group such as a carboxyl group or a tosyl group.

  As a specific structure of such magnetic polymer particles including the magnetic particles (M) and the polymer part (P), for example, the magnetic particles (M) are dispersed in the continuous phase of the polymer part (P). Particles (structure I), particles having a secondary aggregate of magnetic fine particles (M) as a core and a polymer portion (P) as a shell (structure II), non-magnetic core particles such as organic polymers, A core particle having a secondary aggregate layer (magnetic layer) of magnetic fine particles (M) provided on the surface of the core particle is used as a core, and a polymer portion (P) which is an outer layer of the base particle is used as a shell. Particles (structure III) to be used. Among these, particles of structure II or III (that is, magnetic polymer particles including a core including magnetic fine particles and a shell including a polymer portion) are preferable, and particles of structure III are more preferable. In the particles of structure III, the major axis / minor axis of the core particle is preferably 1.1 to 2.0. A specific polymerization method for the particles of the structure III is as disclosed in, for example, Japanese Patent Application Laid-Open No. 2004-205481.

  The magnetic polymer particles according to an embodiment of the present invention can be used by being dispersed in a dispersion medium. Examples of the dispersion medium include an aqueous medium. The aqueous medium is not particularly limited, and examples thereof include water and water containing an aqueous solvent. Examples of the aqueous solvent include alcohols (for example, ethanol, alkylene glycol, monoalkyl ether, etc.).

1.2. Method for Producing Magnetic Polymer Particles Examples of a method for producing magnetic polymer particles according to an embodiment of the present invention include (i) a method of polymerizing monomers in an aqueous dispersion of flat magnetic material, and (ii) spherical magnetic polymer particles. (Iii) a method of mechanically deforming spherical magnetic polymer particles, (iv) a flat non-magnetic core particle, and magnetic fine particles (M) provided on the surface of the core particle Examples thereof include a method in which a mother particle having a secondary aggregate layer (magnetic material layer) is used as a core and a shell of a polymer portion (P) is formed outside the mother particle.

  As the flat magnetic material in the method (i), for example, acicular maghematite, plate-like barium ferrite, or the like can be used. Here, the flat magnetic body preferably has a major axis / minor axis of 1.1 to 2.0. The aqueous dispersion of the flat magnetic material can be obtained by dispersing the flat magnetic material in a dispersant aqueous solution. By polymerization of the monomer, a core made of a flat magnetic material and a shell made of a polymer part (P) can be formed. As the monomer, for example, a radical polymerizable monomer is preferable.

  As the polymer part (P) of the spherical magnetic polymer particles used in the above methods (ii) and (iii), for example, thermoplastic resins such as (meth) acrylate polymers and styrene polymers can be suitably used. In order to prevent fusion between particles, a crosslinked thermoplastic resin can be more suitably used as the polymer part (P).

  Examples of the method of thermally deforming the spherical magnetic polymer particles (ii) include a method of deforming the particles by the thermoplasticity of the particles by heating the spherical magnetic polymer particles in a gravitational field. .

  Examples of the method of (iii) deforming the spherical magnetic polymer particles mechanically include a method of deforming the spherical magnetic polymer particles by applying a physically strong force from the outside. As a method of applying a physically strong force, for example, a mortar, an automatic mortar, a ball mill, a method using a mechanochemical effect such as a blade pressure type powder compression method, a mechanofusion method, a jet mill, a hybridizer The thing using the impact method in high-speed air currents, such as, is mentioned. In order to efficiently deform the spherical magnetic polymer particles, it is desirable that the force applied is strong. As a method for deforming the spherical magnetic polymer particles with a strong force, more specifically, in a vessel equipped with a stirring blade, The spherical magnetic polymer particles may be deformed at a peripheral speed of preferably 15 m / second or more, more preferably 30 m / second or more, and still more preferably 40 to 150 m / second. When the peripheral speed of the stirring blade is lower than 15 m / sec, the particles may not be deformed. In addition, although there is no restriction | limiting in particular about the upper limit of the peripheral speed of a stirring blade, It determines automatically from points, such as an apparatus to be used and energy efficiency.

  As the flat non-magnetic core particles used in the above method (iv), for example, known rod-like and plate-like particles can be used. For example, rod-like microcrystalline cellulose colloidal particles, bowl-shaped crosslinked styrene type And particles. Alternatively, the flat nonmagnetic core particles obtained by deforming the spherical nonmagnetic core particles may be used as the flat nonmagnetic core particles used in the method (iv). More specifically, the flat non-magnetic core particles are formed such that the major axis / minor axis becomes 1.1 to 2.0 by deforming spherical core particles thermally and / or mechanically. It may be. In this case, after forming the magnetic layer on the surface of the flat non-magnetic core particle to form the core (mother particle), the polymer part (P) is formed outside the core, whereby the major axis / minor axis can be reduced. Preferably, magnetic polymer particles of 1.1 to 2.0 are formed.

  One or both of (ii) the step of thermally deforming the spherical magnetic polymer particles and (iii) the step of mechanically deforming the spherical magnetic polymer particles, according to one embodiment of the present invention. It is more preferable to contain.

1.3. Applications The magnetic polymer particles according to an embodiment of the present invention can be used in a wide range of fields such as biochemistry, paints, paper, electrophotography, cosmetics, pharmaceuticals, agricultural chemicals, foods, and catalysts.

  The magnetic polymer particles according to an embodiment of the present invention include, in particular, a diagnostic drug carrier, a bacteria separation carrier, a cell separation carrier, a nucleic acid separation purification carrier, a protein separation purification carrier, an immobilized enzyme carrier, a drug delivery, and the like. It is useful for applications. More specifically, the antigens or antibodies such as proteins are bound to the magnetic polymer particles, and quantification using the turbidity change of the solution due to passive agglutination reaction based on the antigen-antibody reaction with the target antibody or antigen. For qualitative detection, an antibody is bound to the magnetic polymer particles, and antigens (organisms (for example, viruses, bacteria, cells, etc.), biological substances (for example, hormones), chemical substances (for example, dioxins)) Use for collecting and concentrating by binding to the antibody, binding a nucleic acid analog such as DNA to the magnetic polymer particle, binding the nucleic acid to the nucleic acid analog using hybridization, and collecting and detecting the nucleic acid. Use for binding and collecting chemical substances such as proteins and dyes that bind to the nucleic acid analog, For binding and collecting biotins (or biotins) with molecules containing biotins (or avidins), and collecting and detecting antibodies or antigens on the magnetic polymer particles Examples thereof include use of the magnetic polymer particles as a carrier for binding and enzyme immunoassay utilizing colorimetry or chemiluminescence.

  Further, using the magnetic polymer particles according to one embodiment of the present invention, a carrier such as a 96-well plate can be replaced with an automatic analyzer using magnetism. For example, anti-plasmin antibody for antiplasmin test, anti-D dimer antibody for D-dimer test, anti-FDP antibody for FDP test, anti-tPA antibody for tPA test, anti-thrombin = antithrombin complex antibody for TAT test, anti-antithrombin complex antibody for FPA test Coagulation / fibrinolysis-related antigen or antibody for FPA antibody, etc .; BFP anti-BFP antibody, CEA anti-CEA antibody, AFP anti-AFP antibody, ferritin anti-ferritin antibody, CA19-9 anti-CA19- Anti-apolipoprotein antibody for testing apolipoprotein, anti-apolipoprotein antibody for testing β2-microblobrin, anti-α1-microglobulin antibody for testing α1-microglobulin, immunity Anti-immunoglobulin antibody for globulin testing, anti-CRP antibody for CRP testing, etc. Antigen or antibody for serum protein-related test; antigen or antibody for endocrine function test such as anti-HCG antibody for HCG test; anti-HBs antibody for HBs antigen test, HBs antigen for HBs antibody test, HCV antigen for HCV antibody test, HIV-1 HIV-1 antigen for antibody, HIV-2 antigen for HIV-2 antibody test, HTLV-1 antigen for HTLV-1 test, mycoplasma antigen for mycoplasmosis test, toxoplasma test for toxoplasma test, streptolysin O antigen for ASO test, etc. Antigens or antibodies for infectious disease-related tests; DNA antigens for testing anti-DNA antibodies, antigens or antibodies for autoimmunity-related tests such as heat-modified human IgG for RF tests; drugs such as anti-digoxin antibodies for testing digoxin, anti-lidocaine antibodies for testing lidocaine Examples include, but are not limited to, antigens for analysis or antibodies. It is not a thing. As the antibody, either a polyclonal antibody or a monoclonal antibody may be used.

  In the magnetic polymer particles according to one embodiment of the present invention, the substances to be tested are biological substances, chemical substances, and organisms contained in the immunological test reagent and the sample to be tested. In the present invention, “biologically related substance” refers to all substances related to a living body. Examples of the biological substance include substances contained in the living body, substances derived from the substance contained in the living body, and substances that can be used in the living body.

  The biological substance to be examined is not particularly limited. For example, proteins (eg, enzymes, antibodies, aptamers, receptors, etc.), peptides (eg, glutathione), nucleic acids (eg, DNA or RNA), carbohydrates, Lipids and other cells or substances (for example, various blood-derived substances including various blood cells such as platelets, red blood cells, white blood cells, hormones (for example, luteinizing hormone, thyroid stimulating hormone, etc.), viruses, bacteria, fungi, protozoa, Examples include proteins and nucleic acids that are constituent elements of parasites, etc. More specifically, examples of proteins include proteins that are cancer markers such as biologically derived proteins, prostate-specific markers, and bladder cancer markers.

  The chemical substance to be inspected is not particularly limited, and examples thereof include environmental pollutants such as dioxins and pharmaceuticals (for example, antibiotics, anticancer agents, antiepileptic agents, etc.).

  The organism to be examined is not particularly limited. For example, various cancer cells, various floating cells, viruses (for example, hepatitis B virus, hepatitis C virus, herpes simplex virus, HIV virus, rubella virus, influenza virus, etc.), Examples include bacteria (for example, Neisseria gonorrhoeae, MRSA, E. coli, etc.), fungi (for example, Candida, ringworm, Cryptocox, alpergillus, etc.), protozoa and parasites (for example, Toxoplasma, malaria, etc.) and the like.

  As described above, the magnetic polymer particles according to an embodiment of the present invention can be used as bio-related substance binding particles. In this case, for example, a substance for binding a biological substance may be solidified on the magnetic polymer particles according to the embodiment of the present invention. For example, biotins are exemplified as such biological substances, and avidins are exemplified as substances for binding biological substances. As a method for producing such bio-related substance binding particles (biotin binding particles), for example, a known method for binding biotins to avidins is applied, and the magnetic polymer according to one embodiment of the present invention is applied. A method of binding avidin to the particles can be mentioned.

  For example, in a phosphate buffer or a phosphate buffer containing 1M sodium chloride, the biotins binding particles and a protein or oligonucleotide modified with biotins (biotins binding probe) are allowed to stand for 10 minutes to 1 at room temperature. After mixing for a period of time, unreacted protein or oligonucleotide is removed by a solid-liquid separation operation, whereby probe-bound particles on which the protein or oligonucleotide is immobilized can be prepared. Needless to say, biotins can be bound to avidins immobilized on the surface of the particles for binding biotins by this method.

  Here, as biotins, for example, biotin-ε-N-lindine, biocytin hydrazide, 2-iminobiotin, biotinyl-ε-N-aminocaproic acid-N-hydroxysuccinimide ester, amino or sulfhydryl derivatives, sulfosuccinimide imino Biotin derivatives such as diotine, biotin bromoacetyl hydrazide, p-diazobenzoylbiotin, and 3- (N-maleimidopyropionyl) biotin can be used.

  Examples of a method for modifying a protein or oligonucleotide with biotins include, for example, (i) reacting an ester of biotins with N-hydroxyimides (for example, biotin-N-hydroxysuccinimide) with an amino group of a protein molecule. (Ii) After binding phthalimidotriethylene glycol to the 5 ′ end of the oligonucleotide, it is hydrolyzed with ammonium hydroxide to form a primary amino group. Examples of the method include, for example, a method of modifying the 5 ′ end of an oligonucleotide with biotins by binding biotin-N-hydroxysuccinimide to the amino group. Use various methods It is possible to, by an appropriate method, it is also possible to modify the 3 'end of the oligonucleotide by biotin.

  The magnetic polymer particles of one embodiment of the present invention can be suitably used for biochips using the particles, for example, biochips disclosed in JP-A-2005-148048.

  In addition, the use of the magnetic polymer particles according to an embodiment of the present invention is not limited to the use as a biochemical carrier, and can be used in, for example, the above-described fields.

2. Examples Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

2.1. Evaluation Method The flat magnetic polymer particles obtained in Examples and Comparative Examples described later were evaluated by the following methods. Further, the particle diameter of the flat magnetic polymer particles was measured by the following method unless otherwise specified.

2.1.1. Evaluation of Biotin Binding Amount Using biotin as a biological substance, the biotin binding amount of the flat magnetic polymer particles obtained in each Example and Comparative Example was evaluated.

  After 2 mg of biotin-binding particles obtained in each Example and Comparative Example were dispersed in 1.0 ml of water, 8000 pmol of biotin (Lucifer yellow cadaverine biotin-X, dipotassium salt) was added and added 37 Invert mixing was carried out at 15 ° C. for 15 minutes. Next, the particles were separated by magnetic separation, and the fluorescence intensity of the supernatant was measured with a fluorescence spectrophotometer (PF-777, JASCO) to calculate the amount of unreacted fluorescently labeled biotin. Furthermore, the difference between the amount of unreacted fluorescently labeled biotin and the amount of fluorescently labeled biotin solution before binding (4000 pmol) is obtained, and this difference is divided by the mass of the particle, thereby binding biotin per mg of particle. The amount (pmol / mg) was determined.

That is, the biotin binding amount of the flat magnetic polymer particles obtained in each example or comparative example was calculated from the following formula (1).
Biotin binding amount (pmol / mg) =
{(Amount of fluorescently labeled biotin before binding (pmol)) − (Amount of unreacted fluorescently labeled biotin after binding (pmol))} / (Mass of flat magnetic polymer particles (mg))
(1)

2.1.2. Evaluation of magnetic separation time The flat magnetic polymer particles obtained in each Example and Comparative Example were diluted with water to prepare a test solution containing 0.01% by weight of magnetic particles. This test solution is well dispersed and placed in a square optical cell having an optical path length of 1 cm, set in a spectrophotometer (manufactured by JASCO Corporation, model V-550), and a surface magnetic density of 2900 gauss is placed beside this cell holder. The time (t) at which the neodymium magnet was placed was set to 0, the time until the absorbance at 550 nm was attenuated to 90% of the initial value (absorbance at t = 0) was measured, and this time was defined as the magnetic separation time.

2.1.3. Evaluation of long diameter / short diameter of flat magnetic polymer particles SEM observation of flat magnetic polymer particles was performed, and the long diameter and short diameter of 300 particles were measured using a photograph at a magnification of 5,000 times. The number average of the diameters was determined, and the value of the major axis / minor axis was determined from the obtained average values of the major axis and the minor axis.

2.2. Example 1
2.2.1. Preparation of flat magnetic mother particles using a step of thermally deforming styrene / divinylbenzene = 99/1 copolymer (average particle size 1.5 μm) with reference to the polymerization method described in JP-A-07-238105 Was polymerized and washed with water three times by centrifugation. 100 g of this hydrous slurry was dried with a dryer at 100 ° C. for 24 hours to obtain a powder of flat polymer core particles deformed by heat. The number average of the major axis / minor axis of the polymer core particles was 1.2.

  After adding acetone to an oily magnetic fluid (trade name: “EXP series”, manufactured by Ferrotec Co., Ltd.) that is a dispersion of magnetic fine particles having an average particle diameter of 10 nm, the magnetic fine particles are precipitated and precipitated. By separating and drying, a ferrite-based magnetic fine particle powder having a hydrophobized surface was obtained.

  The above-mentioned flat polymer core particle powder 15 g and the above-mentioned hydrophobized magnetic fine particle powder 15 g are thoroughly mixed with a mixer, and this mixture is mixed with a hybridization system NHS-O type (manufactured by Nara Machinery Co., Ltd.). Was used and treated at 80 ° C. for 5 minutes at a peripheral speed of a blade (stirring blade) of 100 m / second (16200 rpm) to obtain 30 g of flat magnetic mother particles.

2.2.2. Preparation of flat magnetic polymer particles An aqueous solution containing 0.5% by weight of sodium dodecylbenzenesulfonate and 0.5% by weight of a nonionic emulsifier (trade name: “Emulgen 150”, manufactured by Kao Corporation) (hereinafter referred to as “dispersant aqueous solution ( D) ") was charged into a 1 L separable flask, and then 30 g of the flat magnetic mother particles were charged, dispersed with a homogenizer, and heated to 60 ° C. Next, 50 g of the aqueous dispersant solution (D) weighed in another container, 30 g of cyclohexyl methacrylate, 7.5 g of methacrylic acid, and 1.5 g of tertiary butyl peroxy 2-ethylhexanate (manufactured by NOF Corporation; Perbutyl O) The pre-emulsion dispersed by adding was added dropwise to the 1 L separable flask controlled at 60 ° C. over 2 hours. After dripping, this liquid was heated up to 80 degreeC and made to react for 2 hours.

  The obtained aqueous dispersion of magnetic particles was subjected to magnetic purification and gravity sedimentation purification, and then the solid content concentration was adjusted to 1% to obtain an aqueous dispersion of flat magnetic polymer particles. Table 1 shows the measurement results of the major axis / minor axis value and magnetic separation time.

2.2.3. Preparation of Biotin-Binding Particles 1 mL of an aqueous dispersion of flat magnetic polymer particles having a solid content concentration of 1% was magnetically separated and replaced with 1 mL of 0.1 mM MES buffer (pH 6.0). 0.1 mL of 0.1 mM MES buffer solution (pH 6.0) in which 1 mg of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (manufactured by Dojin Chemical) was dissolved was added, and streptavidin (manufactured by Sigma) 0 Streptavidin was immobilized on the surface of the particles by adding 0.1 mL of 0.1 mM MES buffer solution (pH 6.0) in which 4 mg was dissolved and rotating and stirring at room temperature for 8 hours. Then, after magnetic separation treatment, a phosphate buffer solution (PBS, 0.1% BSA / PBS, pH = 7.2) containing 0.1% bovine serum albumin was added to the solid matter to magnetically Unreacted streptavidin was removed by repeating the separation treatment three times. The biotin-binding particles thus obtained were dispersed in a phosphate buffer solution (PBS, 0.1% BSA / PBS, pH = 7.2) containing 0.1% bovine serum albumin to obtain a solid content concentration. A 1% dispersion was prepared and the amount of biotin bound was evaluated. The results are shown in Table 1.

2.3. Example 2
2.3.1. Preparation of flat magnetic mother particles by a process of thermal and mechanical deformation With reference to the polymerization method described in JP-A-07-238105, styrene / divinylbenzene / n-butyl acrylate = 85/4/11 copolymer (Average particle size 1.5 μm) was polymerized and washed with water three times by centrifugation. 100 g of this hydrous slurry was dried with a dryer at 60 ° C. for 24 hours to obtain flat polymer core particle powder. The number average of major axis / minor axis of the flat polymer core particles was 1.1.

  Next, acetone was added to an oily magnetic fluid (trade name: “EXP series”, manufactured by Ferrotec Co., Ltd.), which is a dispersion of magnetic fine particles having an average particle diameter of 10 nm, to precipitate and precipitate the particles. By drying, a ferrite-based magnetic fine particle powder having a hydrophobized surface was obtained.

  The above-mentioned flat polymer core particle powder 15 g and the above-mentioned hydrophobized magnetic fine particle powder 15 g are thoroughly mixed with a mixer, and this mixture is mixed with a hybridization system NHS-O type (manufactured by Nara Machinery Co., Ltd.). Was used and treated at 120 ° C. for 5 minutes at a peripheral speed of a blade (stirring blade) of 100 m / second (16200 rpm) to obtain 30 g of flat magnetic mother particles.

2.3.2. Preparation of flat magnetic polymer particles An aqueous dispersion of flat magnetic polymer particles was obtained in the same manner as in 2.2.2 above. Table 1 shows the measurement results of the major axis / minor axis value and magnetic separation time.

2.3.3. Preparation of biotin-binding particles Dispersions of biotin-binding particles were prepared in the same manner as in 2.2.3, and the amount of biotin binding was evaluated. The results are shown in Table 1.

2.4. Comparative Example 1
2.4.1. Preparation of Spherical Magnetic Core Particles Styrene / divinylbenzene = 80/20 copolymer (average particle size 1.7 μm) was polymerized with reference to the polymerization method described in JP-A-07-238105 and centrifuged three times. Washed with water. 100 g of this hydrous slurry was dried with a dryer at 60 ° C. for 24 hours to obtain polymer core particle powder. The number average of major axis / minor axis of the polymer core particles was 1.0.

  Next, after adding acetone to oily magnetic fluid (trade name: “EXP series”, manufactured by Ferrotec Co., Ltd.) which is a dispersion of magnetic fine particles having an average particle diameter of 10 nm, the particles are precipitated and precipitated. Was dried to obtain a ferrite-based magnetic fine particle powder having a hydrophobized surface.

  Next, 15 g of the polymer core particle powder and 15 g of the magnetic fine particle powder are mixed well with a mixer, and this mixture is used with a hybridization system NHS-O type (manufactured by Nara Machinery Co., Ltd.). Then, treatment was performed at 80 ° C. for 5 minutes at a peripheral speed of a blade (stirring blade) of 100 m / second (16200 rpm) to obtain 30 g of magnetic mother particles.

2.4.2. Preparation of magnetic polymer particles An aqueous dispersion of magnetic polymer particles was obtained in the same manner as in 2.2.2 above. Table 1 shows the measurement results of the major axis / minor axis value and magnetic separation time.

2.4.3. Preparation of biotin-binding particles Dispersions of biotin-binding particles were prepared in the same manner as in 2.2.3, and the amount of biotin binding was evaluated. The results are shown in Table 1.

  From the results of Table 1, according to the magnetic polymer particles obtained in Examples 1 and 2, the long diameter / short diameter = 1.1 to 2.0, so that the binding amount with the biological substance (biotin) is Many and excellent in magnetic separation. In contrast, since the magnetic polymer particles obtained in Comparative Example 1 have a major axis / minor axis of less than 1.1, both the magnetic separability and the binding amount of the biological substance were found in Examples 1 and 2. It was inferior to the magnetic polymer particles obtained in 1.

Claims (10)

  Magnetic polymer particles for diagnostic agents having a major axis / minor axis = 1.1 to 2.0. The magnetic polymer particles for diagnostic agents according to claim 1, wherein the major axis / minor axis is 1.1 to 1.3. In claim 1 or 2 ,
Magnetic polymer particles for diagnostic agents, comprising a core containing magnetic fine particles and a shell containing a polymer portion. In claim 3 ,
The said core is a magnetic polymer particle for diagnostic agents containing the nonmagnetic core particle and the magnetic body layer provided in the surface of this core particle. In claim 4 ,
Magnetic polymer particles for diagnostic agents, wherein the core particles have a major axis / minor axis of 1.1 to 2.0. The manufacturing method of the magnetic polymer particle for diagnostic agents of Claim 1 or 2 including the process of thermally deforming a spherical magnetic polymer particle. The method for producing magnetic polymer particles for diagnostic agents according to claim 1 or 2 , comprising a step of mechanically deforming the spherical magnetic polymer particles. Forming a magnetic layer on the surface of the core particle having a major axis / minor axis of 1.1 to 2.0;
Forming a polymer portion on the outside of the magnetic layer;
A method for producing magnetic polymer particles for diagnostic agents, comprising: In claim 8 ,
A method for producing magnetic polymer particles for diagnostic agents, further comprising the step of thermally deforming spherical non-magnetic core particles to form core particles having a major axis / minor axis of 1.1 to 2.0. In claim 8 or 9 ,
A method for producing magnetic polymer particles for diagnostic agents, further comprising a step of mechanically deforming spherical non-magnetic core particles to form core particles having a major axis / minor axis of 1.1 to 2.0.