JP2018132505A - Magnetic composite particles, manufacturing method therefor, and immunoassay particles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide magnetic composite particles which can be quickly separated from a sample solution using magnetism, and exhibit superior dispersion stability even in a solution containing electrolytes.SOLUTION: A magnetic composite particle comprises a core particle containing an inorganic oxide or polymer and an outer shell formed on a surface thereof, the outer shell containing magnetic nanoparticles, a silicon compound, and polymeric molecules. When average particle diameter values of the magnetic composite particles in pure water (d), in 5 mM sodium chloride solution (d), and in 10 mM sodium chloride solution (d) as measured by the dynamic light scattering method are compared, (d)/(d) shall be 3.5 or less and (d)/(d) shall be 3.5 or less.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a magnetic composite particle which can be suitably used in immunoassay and the like, and a method for producing the same, and further relates to an immunoassay particle produced using the magnetic composite particle.

In order to separate and collect target substances such as various proteins, nucleic acids, cells, etc. from sample liquids such as blood, the surface of a given particle is loaded with antibodies suitable for each target substance, and the particles after target substance supplementation An antigen-antibody measurement method for collecting and analyzing the antibody has been studied.
In recent years, in applications that require rapid measurement and analysis in clinical tests, in order to shorten the measurement time, an antibody-supported carrier with magnetism added is used to capture the target substance from the outside. A method for recovering the magnetic addition carrier rapidly by applying magnetism and recovering the target substance has been studied.

  In order to impart high magnetic separation characteristics to a fixed carrier to which magnetism is added, the proportion of the magnetic component contained in the magnetic material added to the fixed carrier is increased, (In particular, a magnetic material having a large saturation magnetization value is used).

Patent Document 1 proposes magnetic polymer particles mainly used for immunodiagnosis, etc., having an average particle diameter of 0.2 to 4.0 μm and a residual magnetization of 10 to 35% of saturation magnetization. ing.
On the other hand, in the patent document 2, the present inventors arranged an outer shell layer on the surface of a core particle having an inorganic oxide or a polymer as a fixed carrier with magnetism, and magnetite particles and sodium silicate were placed on the outer shell layer. Coexisting with each other, a magnetic inclusion particle having a volume average particle size of 10 to 500 nm, a number average particle size CV value of 8% or less, and a saturation magnetization at 25 ° C. of 15 emu / g or more is disclosed.

JP 2000-306718 A Japanese Patent No. 5419199

The magnetic body-encapsulated particles, which are fixed carriers with added magnetism disclosed in Patent Document 2, exhibited excellent magnetic separation characteristics.
However, according to further studies by the present inventors, the magnetic substance-encapsulated particles sometimes require time for separation and recovery from the sample liquid using magnetism.

  Furthermore, the present inventors have found that it is important that the magnetic substance-encapsulated particles have high spontaneous sedimentation resistance.

  If the magnetic encapsulated particles are not sufficiently dispersible in the sample solution, and if they are naturally settled, the antigen-antibody contact in the sample solution will be insufficient, and sufficient contact time will not be gained for capturing the target substance. There is a possibility that the abundance of the target substance in the sample solution is underestimated, leading to misdiagnosis.

  Furthermore, when considering application to physiological diagnosis using the magnetic substance-encapsulated particles, the sample solution contains an electrolyte, and the magnetic substance-encapsulated particles are high even in a solution containing the electrolyte. It is required to have natural sedimentation resistance.

  The present invention has been made under the above-mentioned circumstances, and the problem to be solved is that it can be separated from a sample solution in a short time by using magnetism, and further in a solution containing an electrolyte. Is to provide magnetic composite particles having excellent dispersion stability.

  In order to solve the above-described problem, a magnetic composite particle in which an outer shell is formed on the surface of a core particle containing an inorganic oxide or a polymer, the outer shell being formed of a magnetic nanoparticle, a silicon compound, and It has been conceived that, by using a polymer, magnetic composite particles that are prevented from aggregating even in a solution containing an electrolyte such as sodium chloride are obtained.

The magnetic composite particles include a predetermined amount of nano-sized magnetic material on the outer shell provided on the surface of fine particles containing inorganic oxide or polymer (in some cases, referred to as “nuclear particles” in the present invention). (It may be described as “magnetic nanoparticles” in the present invention). In particular, when the magnetic nanoparticles are formed of magnetite or γ iron oxide, high saturation magnetization is expressed. Furthermore, the outer surface of the magnetic composite particles is coated with a silicon compound so as to contain magnetic nanoparticles, whereby the separation of the magnetic nanoparticles from the surface of the magnetic composite particles can be suppressed. In addition, by adding a polymer to the outer shell portion, even in a solution containing an electrolyte, aggregation can be suppressed and particles having excellent dispersion stability can be obtained.
And if it is a magnetic composite particle which has this structure, a particle | grain can be efficiently isolate | separated and recovered by the application of the magnetic field from the outside. Further, even in a sample solution containing an electrolyte, the dispersion stability is excellent, so that the target substance can be efficiently separated and recovered even in immunoassay.

That is, the first invention for solving the above-described problem is
A magnetic composite particle in which an outer shell is formed on the surface of a core particle containing an inorganic oxide or polymer,
The outer shell comprises magnetic nanoparticles, a silicon compound and a polymer,
The average particle diameter of the magnetic composite particles measured by the dynamic light scattering method was compared in pure water (d _{0 mM} ), sodium chloride aqueous solution 5 mM (d _{5 mM} ), and sodium chloride aqueous solution 10 mM (d _{10 mM} ). When
This is a magnetic composite particle having a value of (d _{5 mM} ) / (d _{0 mM} ) of 3.5 or less and a value of (d _{10 mM} ) / (d _{0 mM} ) of 3.5 or less.
The second invention is
The polymer is the magnetic composite particle according to the first invention, which is polyethylene glycol.
The third invention is
The outer shell is the magnetic composite particle according to the first or second invention, further containing a silane coupling agent.
The fourth invention is:
The magnetic composite particles according to any one of the first to third inventions, wherein the magnetic nanoparticles contained in the outer shell are magnetite or γ iron oxide.
The fifth invention is:
The magnetic composite particle according to any one of the first to fourth inventions having a spherical or substantially spherical shape.
The sixth invention is:
The magnetic composite particles according to any one of the first to fifth inventions, wherein a saturation magnetization value is 30 Am ² / kg or more and 200 Am ² / kg or less.
The seventh invention
The magnetic composite particle according to any one of the first to sixth inventions, wherein the core particle is a polymer.
The eighth invention
An immunoassay particle comprising an antibody in the outer shell of the magnetic composite particle according to any one of the first to seventh inventions.
The ninth invention
Producing a suspension of magnetic nanoparticles;
A step of producing a core particle containing an inorganic oxide or a polymer and having a volume average particle diameter measured from a transmission electron microscope image of 20 nm or more and 200 nm or less;
Adding the core particles to the suspension of magnetic nanoparticles to obtain a suspension of magnetic nanoparticle adsorbed core particles;
Suspension of magnetic composite particles in which an aqueous solution of a silicon compound is added to a suspension of magnetic nanoparticle adsorbing core particles, and an outer shell containing the magnetic nanoparticles and the silicon compound is provided on the surface of the core particles. Producing a liquid;
This is a method for producing magnetic composite particles, comprising the step of adding a polymer after stirring the suspension of the magnetic composite particles and causing the outer shell to contain the polymer.
The tenth invention is
The method for producing magnetic composite particles according to the ninth aspect, wherein the silicon compound is silicon alkoxide.
The eleventh invention is
The aqueous solution of the silicon compound is the method for producing a magnetic composite particle according to the ninth or tenth invention, further including a silane coupling agent.
The twelfth invention
The method for producing magnetic composite particles according to any one of the ninth to eleventh inventions, wherein the polymer is polyethylene glycol.

  Since the magnetic composite particles according to the present invention are excellent in dispersion stability even in a solution containing an electrolyte, the particles can be efficiently separated and recovered by applying a magnetic field from the outside. In addition, even in a sample solution containing an electrolyte, it is excellent in dispersion stability, so that the target substance can be efficiently separated and recovered during immunoassay.

1 is a schematic cross-sectional view of a magnetic composite particle according to the present invention. 1 is a schematic cross-sectional view of magnetic composite particles according to the present invention that are aggregated in a sample solution.

The magnetic particles according to the prior art may require time for separation and recovery from the sample solution as described above. In particular, when the sample solution is a solution containing an electrolyte, the magnetic particles aggregate in a short time and spontaneously settle. However, it may be necessary to verify whether or not the obtained measurement result is appropriate, and there is a possibility that it cannot sufficiently cope with an application for which a quick and accurate measurement result is required.
The magnetic composite particles having a core-shell structure in which an outer shell containing a magnetic nanoparticle, a silicon compound, and a polymer is provided on the surface of a core particle containing an inorganic oxide or a polymer. Settled. First, the magnetic composite particles according to the present invention will be described with reference to the drawings.

FIG. 1 is a schematic cross-sectional view of a magnetic composite particle according to the present invention.
The magnetic composite particle 1 according to the present invention has a core particle 11 at the center, and magnetic nanoparticles 12 are adsorbed on the surface of the core particle 11. Further, the core particle 11 is covered with a layer 13 of silicon compound. The silicon compound layer 13 contains the magnetic nanoparticles 12 and constitutes the outer shell 14.
Here, 1. 1. magnetic nanoparticles, 2. nuclear particles; 3. layer of silicon compound; 4. Magnetic composite particles, 5. Synthesis of magnetic composite particles, The description will be made in the order of the formation of the outer shell of the magnetic composite particles.

1. Magnetic nanoparticles The magnetic nanoparticles 12 are made of magnetite (Fe ₃ O ₄ ) or γ iron oxide, and the average particle diameter is preferably 5 nm or more and 50 nm or less.
The value of the saturation magnetization of the magnetic nanoparticles 12 is 0.1 Am ² / kg or more and 200 Am ² / kg or less, more preferably 120 Am ² / kg or less, and still more preferably 80 Am ² / kg or less. If the saturation magnetization value is 0.1 Am ² / kg or more, sufficient magnetic field responsiveness can be given to the magnetic composite particle 1, and if it is 200 Am ² / kg or less, the magnetic composite particle 1 due to residual magnetization. This is because it is possible to avoid the situation of aggregation.
Moreover, it is preferable that the load of the magnetic nanoparticle 12 in the magnetic composite particle 1 is 0.095 mass% or more and 95 mass% or less. If the loading amount is 0.095% by mass or more, the magnetic field response speed of the magnetic composite particles 1 can be ensured, and if it is 95% by mass or less, the density of the magnetic composite particles 1 does not become excessive. This is because the dispersibility in the sample liquid can be ensured.

2. Core particle The core particle 11 is comprised including the inorganic oxide or the polymer. The volume average particle diameter of the core particles is preferably 20 nm or more and 200 nm or less. Examples of the polymer include polymethyl methacrylate (sometimes described as “PMMA” in the present invention) and polystyrene (sometimes described as “PSt” in the present invention).
Examples of the inorganic oxide include silica, talc, kaolinite, magnesium carbonate, calcium carbonate and the like.

The property of these polymers and inorganic oxides is that the density is 5.0 g / cm ³ or less, preferably 4.0 g / cm ³ or less, more preferably 3.0 g / cm ³ or less. By selecting the core particles having a density in this range, the buoyancy at the time of dispersion in the sample liquid and the weight of the core particles themselves are balanced, so that the dispersibility in the sample liquid can be improved. However, if the buoyancy is excessive, the core particles may float on the sample solution. From the viewpoint of avoiding this situation, depending on the specific gravity of the sample solution to be tested, it is preferable to select one having a core particle density of 0.3 g / cm ³ or more, preferably 0.5 g / cm ³ or more. good.

  In the present invention, particles in a state where the magnetic nanoparticles 12 are adsorbed and aggregated on the surface of the core particles 11 are referred to as “magnetic nanoparticle adsorbed core particles”.

3. Silicon Compound Layer The silicon compound layer 13 was formed from a silicon alkoxide such as, for example, tetraethylorthosilicate (Si (OC ₂ H ₅ ) ₄ ) (sometimes referred to as “TEOS” in the present invention). It is preferably composed of a silicon oxide polymer which is a silicon compound. Further, the silicon compound layer 13 includes a silane coupling agent such as 3-methacryloxypropyltrimethoxysilane (may be described as “MPTMS” in the present invention), and two or more kinds of silicon compound raw materials. More preferably, the polymerization is performed using This is because a polymerizable functional group can be introduced into the molecule and a hydroxyl group (—OH) that serves as an antibody scaffold can be introduced.

From the above viewpoint, the amount of the silane coupling agent in the silicon compound layer 13 is, for example, 0.001 mol /% when the concentration of the magnetic nanoparticle adsorption nucleus particles is 0.01 vol% or more and 1.0 vol% or less. m is preferably ³ or more 1 kmol / ^{m 3} or less.
If the amount of the silane coupling agent is 0.001 mol / m ³ or more, the effect as a silane coupling agent is exhibited, and if it is 1 kmol / m ³ or less, self-condensation between silane coupling agent molecules can be avoided. It is.

  The outer shell 14, which is a silicon compound layer 13 containing the magnetic nanoparticles 12, has an effect of suppressing the separation of the magnetic nanoparticles 12 from the surface of the core particles 11.

4). Magnetic composite particle The magnetic composite particle 1 preferably has a spherical or substantially spherical shape, but its volume average particle diameter is measured, for example, with a caliper from a transmission electron microscope image of the magnetic composite particle 1. Can be calculated. In the present invention, the volume average particle diameter calculated from the transmission electron microscope image is described as (d _TEM ).
Note that “the magnetic composite particle 1 has a spherical or substantially spherical shape” means that the magnetic composite particle has a circular or substantially circular cross section, and the aspect ratio in the cross section is, for example, 1.3 or less. Say something.

The magnetic composite particle 1 according to the present invention preferably has a saturation magnetization value of 30 Am ² / kg or more and 200 Am ² / kg or less. This is because, when the saturation magnetization value is 30 Am ² / kg or more, the magnetic composite particles 1 can be easily separated from the sample solution in a short time by using magnetism such as a permanent magnet. On the other hand, if it is 200 Am ² / kg or less, the situation of aggregation of the magnetic composite particles due to residual magnetization can be avoided.

FIG. 2 is a schematic cross-sectional view of the magnetic composite particles 1 according to the present invention that are aggregated in a sample solution.
In the sample solution, the magnetic composite particles 1 according to the present invention may be dispersed alone, or a plurality of two or more particles may be aggregated. FIG. 2 is a schematic cross-sectional view of a state in which, for example, three magnetic composite particles 1 are aggregated.

The particle diameter of the magnetic composite particles 1 aggregated in the sample solution can be measured and calculated by, for example, a dynamic light scattering method. In the present invention, the particle diameter calculated from the dynamic light scattering method is described as (d _DLS ). The (d _DLS ) can also be considered as a hydrodynamic diameter.

Of the magnetic composite particles 1 _{(d TEM)} is at 30nm or more 210nm or _less, the value is 2 or less which is the ratio of _{(d DLS)} and _{(d TEM) (d DLS)} / (d TEM).
The value of (d _DLS ) / (d _TEM ) being 2 or less means that the particle diameter existing as an integral part in the sample liquid and the original volume average particle diameter are substantially the same or very close to each other. Indicates. That is, the magnetic composite particles are present in the sample solution while ensuring a form close to monodisperse without aggregation. From this, it can be said that the magnetic composite particles 1 are excellent in dispersion stability in the sample solution.

  By adsorbing a desired antibody to the magnetic composite particles according to the present invention as described above, it was possible to obtain immunoassay particles having good responsiveness in a magnetic field in a sample solution. As a result, it is considered that the antigen present in the sample solution can be captured without leaving, and the risk of misdiagnosis can be reduced.

5. Synthesis of Magnetic Composite Particles Regarding the synthesis of magnetic composite particles according to the present invention, 1) synthesis of magnetic nanoparticles, 2) synthesis of nuclear particles, 3) synthesis of magnetic nanoparticle adsorbing core particles, and 4) magnetic composite particles. Will be described in the order of synthesis.

1) Synthesis of magnetic nanoparticles Magnetic nanoparticles were synthesized by a so-called coprecipitation method. This method is a synthesis method in which magnetite magnetic nanoparticles are generated simply by adding a basic solution to a mixed solution containing Fe ²⁺ and Fe ^{3+ in} a ratio of 1: 2. On the other hand, γ iron oxide (maghemite) is obtained, for example, by drying the magnetite obtained by the above-described method in the atmosphere and then heating and oxidizing at a low temperature (about 350 ° C.).
Furthermore, it is also preferable to modify the surface by adding a coupling agent in order to impart a positive charge to the surface of the generated magnetic nanoparticles to stabilize the dispersion.
In addition, the crystal structure of the substance constituting the magnetic nanoparticles can be identified by, for example, applying the above-described liquid on a preparation and drying it naturally, and then analyzing it using X-ray diffraction.

2) Synthesis of core particles As a core particle material having a volume average particle diameter of about 50 nm, polymerized particles can be prepared by adding potassium persulfate or ammonium persulfate to a mixture of St monomer and MMA monomer and polymerizing the mixture.
In addition, polymer particles of PMMA can be produced by a soap-free emulsion polymerization method using MMA monomer as a material for core particles having a volume average particle diameter of about 100 nm.
Furthermore, a polymerizable silane coupling agent MPTMS is added to the surface of the produced polymer particles after the initiation of polymerization for the purpose of increasing the affinity with the magnetic nanoparticles and introducing a functional group that can serve as a base point for the reaction. By polymerization, core particles having a volume average particle diameter of about 50 nm to about 100 nm are obtained.

3) Synthesis of magnetic nanoparticle-adsorbed core particles Magnetic nanoparticle-adsorbed core particles can be obtained by mixing the magnetic nanoparticles and the core particles with shaking.

4) Synthesis of magnetic composite particles Magnetic composite particles were synthesized by collecting magnetic nanoparticle adsorbing nuclei from a dispersion of magnetic nanoparticle adsorbing nuclei and mixing it with a silicon compound solution. Stir with shaking.

6). Formation of outer shell of magnetic composite particles The formation of the outer shell of the magnetic composite particles according to the present invention will be described in the order of 1) pretreatment, 2) a polymer to be contained, and 3) an effect of containing a polymer.

1) Pretreatment Adding a specific kind of polymer to the suspension containing magnetic composite particles described in “5. Synthesis of magnetic composite particles, 4) Synthesis of magnetic composite particles” above. Can be implemented.
At this time, it is preferable to allow sufficient shaking and stirring time, that is, aging time, before the addition of the polymer from the mixing of the magnetic nanoparticle adsorbing core particles and the silicon compound liquid described in the above section. The specific aging time depends on the kind of polymer to be added, but is preferably 5 hours or longer, more preferably 12 hours or longer, and most preferably 18 hours or longer. Furthermore, considering the productivity, the upper limit of the aging time is considered.
This is because it is possible to prevent the polymer to be added from being excessively absorbed in the silicon compound layer constituting the outer shell by taking a sufficient aging time before adding the polymer.

  Further, after the addition of the polymer, the obtained suspension can be refluxed at a temperature exceeding 60 ° C. This is for fixing the polymer to the outer shell while avoiding the volatilization of the polymer.

2) Polymers to be included Various polymers can be included, but the magnetic composite particles according to the present invention are intended for adaptation to living bodies, and are excellent in dispersion stability even in an electrolyte solution. Polyethers are preferable from the viewpoint of being required, and polyethylene glycol (sometimes referred to as “PEG” in the present invention) is particularly preferable because of easy handling and availability.

3) Effect of containing polymer The surface modification by adding a polymer to the outer shell of the magnetic composite particle according to the present invention is performed between the particles when the magnetic composite particle is dispersed in the electrolyte-containing solution. The effect of suppressing the occurrence of agglomeration is demonstrated. Details will be described in the Examples section. However, the magnetic composite particles in which the polymer is introduced into the outer shell portion of the particles according to the present invention are added at a concentration of 10 mol / L. When dispersed in an electrolyte-containing solution, the average degree of aggregation is 2.0 to 3.2, whereas in the case of magnetic composite particles not introduced with a polymer, the average degree of aggregation is 4.7. It becomes a piece.

From the above, the introduction of the polymer into the outer shell of the magnetic composite particle confirms the aggregation suppressing effect of the magnetic composite particle in the electrolyte-containing solution.
Furthermore, regarding the zeta potential of the magnetic composite particles depending on whether or not the polymer is introduced into the outer shell, there is a difference between the zeta potential in water and the data potential in the electrolyte when the polymer is introduced into the outer shell. can not see. On the other hand, in the case where the polymer is not introduced, the numerical value of the absolute value changes small, and it can be confirmed that aggregation is likely to proceed.

  As a result, the magnetic composite particles according to the present invention are excellent in dispersion stability even in a solution containing an electrolyte, and can be separated from a sample solution using magnetism in a short time because it has an appropriate magnetization. Turned out to be.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples.

Example 1
In Example 1, first, polymer particles having a volume average particle diameter of 192.5 nm are used as core particles, Fe ₃ O ₄ nanoparticles as magnetic nanoparticles are formed on the outer shell formed on the surface, and TEOS and MPTMS are used as silicon compounds. The magnetic composite particles were synthesized, and PEG was further introduced into the outer shell to obtain the magnetic composite particles according to Example 1, and the characteristics thereof were evaluated.
Hereinafter, for the magnetic composite particles according to Example 1, 1. 1. Synthesis of magnetic nanoparticles 2. synthesis of nuclear particles; 3. Synthesis of magnetic nanoparticle adsorption core particles 4. Synthesis of magnetic composite particles 5. Introduction of PEG into the outer shell of magnetic composite particles, The magnetic composite particles will be described in the order of characteristic evaluation.

1. Synthesis of Magnetic Nanoparticles The synthesis of magnetic nanoparticles according to Example 1 of the present invention will be described in the order of 1) raw materials for magnetic nanoparticles and 2) synthesis of magnetic nanoparticles.

1) Raw material of magnetic nanoparticles FeCl ₂ (manufactured by High Purity Chemical Laboratory, purity 99.9%) was prepared as one of Fe sources of Fe ₃ O ₄ nanoparticles, which are magnetic nanoparticles according to Example 1. Moreover, FeCl ₃ (manufactured by Wako Pure Chemical Industries) was prepared as another Fe source.
As a dispersion stabilizer for the magnetic nanoparticles and a coupling agent for imparting a positive charge, N-trimethoxysilylpropyl-N, N, N, -trimethylammonium chloride (manufactured by GELEST Inc., 50% methanol solution) ( In the present invention, it may be described as “TSA”).
Aqueous ammonia (manufactured by Wako Pure Chemical Industries, Ltd., 25% by mass reagent special grade aqueous solution) was prepared as a pH adjuster for the synthesis of the magnetic nanoparticles.
Furthermore, deionized water having an electric resistance value of 18.2 MΩcm was prepared as a reaction solvent for synthesizing the magnetic nanoparticles.

2) Synthesis of magnetic nanoparticles As a reaction vessel, a separable flask having an inner diameter of 7.5 cm and a height of 15 cm was used. A four-pitch paddle with a blade diameter of 5 cm and an inclination angle of 45 ° was used as the stirrer, and the stirring speed was 300 rpm.

186 cm ³ of deionized water from which dissolved oxygen was removed by nitrogen bubbling was added to the reaction vessel, and 5 cm ^{3 of} ₂ kmol / m ³ FeCl ₂ aqueous solution and 20 cm ³ of 1 kmol / m ³ FeCl ₃ aqueous solution were added and stirred at 35 ° C. Started. The reaction vessel was filled with nitrogen and nitrogen bubbling was performed for 30 minutes.
Ammonia water (11.7 cm ³⁾ was charged into the reaction vessel to initiate the reaction. 30 seconds after the start of the reaction, 2.1 cm ³ of TSA was added and stirring was continued for 3 hours.

  After adding 2 times weight ethanol to the suspension of the obtained magnetic nanoparticles, the mixture is thoroughly mixed, centrifuged (12000 rpm, 15 minutes) to remove the supernatant, and then deionized. Water was added to obtain a suspension having a magnetic nanoparticle concentration of 1.0% by mass according to Example 1. The volume average particle diameter of the magnetic nanoparticles in the obtained suspension was 9 nm. The saturation magnetization of the magnetic nanoparticles was 63 emu / g.

2. Synthesis of Core Particles The synthesis of the core particles according to Example 1 of the present invention will be described in the order of 1) raw material of the core particles and 2) synthesis of the core particles.

1) Raw material of core particles As a monomer for forming the core particles according to Example 1, methyl methacrylate (MMA) (manufactured by Wako Pure Chemical Industries, reagent grade, purity 98%) was prepared.
In addition, sodium octadecyl sulfate (SOS) (Ward Hill MA, USA) was prepared as an anionic activator.
At that time, in order to remove the hydroquinone of the polymerization inhibitor contained in the MMA, the St and MMA were passed through a glass column filled with a polymerization inhibitor remover (manufactured by Aldrich).

  As one of ionic comonomers that form the core particles according to Example 1, sodium p-styrenesulfonate (NaSS) (manufactured by Wako Pure Chemical Industries, reagent grade, purity 80%) was prepared.

  As a polymerization initiator at the time of synthesizing the core particles according to Example 1, potassium persulfate (KPS) (manufactured by Wako Pure Chemical Industries, reagent special grade, purity 95%) was prepared.

  3-methacryloxypropyltrimethoxysilane (MPTMS) (manufactured by Shin-Etsu Chemical Co., Ltd., reagent grade, purity 95%) was prepared as a polymerizable silane coupling agent during synthesis of the core particles according to Example 1.

  Deionized water was prepared as a reaction solvent when synthesizing the core particles according to Example 1.

2) Synthesis of core particles A cylindrical closed glass reactor having an internal volume of 110 cm ³ was used for the reaction, and a magnetic stirrer was used for stirring.
First, deionized water was added to the reactor and nitrogen bubbling was performed for 30 minutes, and then the flow was switched to nitrogen flow. To this, MMA and NaSS were added and stirred for 20 minutes, and KPS was added to initiate polymerization. While continuing stirring, a polymerization reaction was carried out at 65 ° C. for 2 hours to obtain a suspension containing core particles. At this time, MPTMS was added 40 minutes after the start of polymerization.

  After completion of the polymerization reaction, the obtained suspension is centrifuged (12000 rpm, 15 minutes), the supernatant is removed, and the core particles are collected, and this is redispersed in deionized water by ultrasonic irradiation, and washed by centrifugation. Went. The centrifugal washing was performed three times, and the core particles according to Example 1 were dispersed in deionized water.

3. Synthesis of magnetic nanoparticle adsorbing core particles The core particles synthesized in “2. Synthesis of nuclear particles” and the magnetic nanoparticles according to Example 1 synthesized in “1. Synthesis of magnetic nanoparticles” into a centrifuge tube with a volume of 50 cm ³ . The suspension was added, and stirring was performed for 1 minute to synthesize magnetic nanoparticles adsorbing core particles.
At this time, the volume of the suspension was 20 cm ³ .

  The suspension containing the magnetic nanoparticle adsorbing core particles was centrifuged at 12000 rpm for 10 minutes to remove the supernatant and floating particles. The magnetic nanoparticle adsorbing core particles were washed three times by the centrifugation.

4). Synthesis of Magnetic Composite Particles The obtained suspension of magnetic nanoparticle adsorption core particles was centrifuged (under the same conditions). The obtained magnetic nanoparticle adsorbing core particles were collected, water was further added, and ultrasonic dispersion was performed for 1.5 hours to obtain a suspension. Here, ethanol, 25% aqueous ammonia 0.41 cm ³ , TEOS, and MPTMS were added in this order, and the mixture was stirred for 24 hours at room temperature to provide a silicon compound layer on the magnetic nanoparticle-adsorbed core particles.
At this time, magnetic nanoparticles adsorbed core particles 0.12 volume%, TEOS is 5 mol ^/ m 3, MPTMS is 5 mol / ^{m 3,} water 3~9kmol / ^{m 3,} the ammonia was 0.3 mol / ^{m 3.}

5. Introduction of PEG into the outer shell The suspension of magnetic composite particles prepared in “4. Synthesis of magnetic composite particles” with a silicon compound layer on the magnetic nanoparticle adsorbing core particles was added to TEOS and MPTMS. Then, the mixture was aged by continuing stirring for 6 hours.
To this suspension, PEG was added as a surface modification so as to have a concentration of 1 mmol / L, and the mixture was aged for 6 hours after the addition of the PEG. The introduction operation was performed to obtain a suspension containing magnetic composite particles containing a polymer in the outer shell.

  The obtained suspension was centrifuged at 12,000 rpm for 10 minutes, and the precipitate was collected and redispersed in pure water containing no electrolyte, whereby the magnetic composite particles of Example 1 were collected. A suspension was obtained.

6). Characteristic Evaluation of Magnetic Composite Particles (d _DLS ) in pure water of the obtained magnetic composite particles containing a polymer in the outer shell according to Example 1 was measured by a dynamic light scattering photometer described later. . In the present invention, (d _DLS ) in pure water is “(d _{0 mM} )”.

Here, a method for measuring (d _{0 mM} ) of magnetic composite particles in a suspension of magnetic composite particles containing a polymer in the outer shell according to Example 1 will be described.
The (d _{0 mM} ) measurement of the magnetic composite particles in the suspension of the magnetic composite particles _subjected to the surface modification according to Example 1 was performed using a dynamic light scattering photometer.
Specifically, the suspension of the magnetic composite particles subjected to surface modification according to Example 1 was diluted to adjust the concentration of the magnetic composite particles to 0.001% by volume (d _{0 mM} ). A suspension sample was obtained, and a sample irradiated with ultrasonic waves for 100 minutes was loaded into a dynamic light scattering photometer (ELSZ-2, manufactured by Otsuka Electronics Co., Ltd.), and (d _{0 mM} ) was measured.

The precipitate was re-dispersed in an aqueous sodium chloride solution having a concentration of 5 mmol / L at room temperature, and then (d _DLS ) was measured by a dynamic light scattering photometer (this value is referred to as “(d _{5 mM} )”. To do).
The precipitate was redispersed in an aqueous sodium chloride solution having a concentration of 10 mmol / L at room temperature, and then (d _DLS ) was measured with a dynamic light scattering photometer (this value was expressed as “(d _{10 mM} ) ”).

The ratios of (d _{5 mM} ) / (d _{0 mM} ), (d _{10 mM} ) / (d _{0 mM} ), (d _{10 mM} ) / (d _{5 mM} ) were determined and listed in Table 1.

(Example 2)
In the same manner as in Example 1, except that the aging time after addition of the silicon compound was 18 hours, a suspension of magnetic composite particles according to Example 2 was obtained and measured in the same manner. did. The obtained results are also shown in Table 1.

(Example 3)
A suspension of magnetic composite particles according to Example 3 was obtained in the same manner as in Example 1 except that the concentration of PEG to be added was changed to 3 mmol / L, and the suspension was measured in the same manner. The obtained results are also shown in Table 1.

Example 4
A suspension of magnetic composite particles according to Example 4 was obtained in the same manner as in Example 2 except that the concentration of PEG to be added was changed to 3 mmol / L, and this was measured in the same manner. The obtained results are also shown in Table 1.

(Example 5)
A suspension of magnetic composite particles according to Example 5 was obtained in the same manner as in Example 1 except that the concentration of PEG to be added was changed to 5 mmol / L, and the suspension was measured in the same manner. The obtained results are also shown in Table 1.

(Example 6)
A suspension of magnetic composite particles according to Example 6 was obtained in the same manner as in Example 2 except that the concentration of PEG to be added was changed to 5 mmol / L, and the suspension was measured in the same manner. The obtained results are also shown in Table 1.

(Comparative Example 1)
Stirring is not continued in “Suspension of magnetic composite particles in which a silicon compound layer is provided on the magnetic nanoparticle adsorbing core particles” described in the column of “5. Introduction of PEG into outer shell” of Example 1. The magnetic composite particles according to Comparative Example 1 were used as they were.
The same characteristic evaluation as in Example 1 was performed on the obtained magnetic composite particles according to Comparative Example 1.

(Summary)
From the value of (d _{0 mM} ) in Examples 1 to 6 and Comparative Example 1, (d _DLS ) of the magnetic composite particles according to Examples 1 to 6 in which a polymer is contained in the outer shell is a polymer in the outer shell. It was found that (d _DLS ) is equal to or larger than that of the magnetic composite particles according to Comparative Example 1 containing no. This tendency was remarkable when the treatment concentration at the time of inclusion on the surface was 3 mmol / L.

In particular, the magnetic composite particles according to Examples 1 to 6 in which a polymer is contained in the outer shell and the magnetic composite particles according to Comparative Example 1 in which no polymer is contained in the outer shell are added at a concentration of 10 mmol / L. When redispersed in an aqueous NaCl solution, it was found that (d _DLS ) increased and aggregation occurred. Although such a tendency was confirmed even when added to a 5 mmol / L NaCl solution, the effect is remarkably confirmed particularly when added to a high concentration electrolyte solution.
In particular, in the magnetic composite particles according to Comparative Example 1, it was found that an average of 4.7 magnetic composite particles caused aggregation.
In contrast, in the magnetic composite particles according to Examples 1 to 6, it was found that an average of 2.0 to 3.2 magnetic composite particles were agglomerated, and the outer shell contained a polymer. This proved that aggregation was greatly suppressed.

1. Magnetic composite particles 11. Nuclear particle 12. Magnetic nanoparticles 13. Layer of silicon compound 14. shell

Claims (12)

A magnetic composite particle in which an outer shell is formed on the surface of a core particle containing an inorganic oxide or polymer,
The outer shell comprises magnetic nanoparticles, a silicon compound and a polymer,
The average particle diameter of the magnetic composite particles measured by the dynamic light scattering method was compared in pure water (d _{0 mM} ), sodium chloride aqueous solution 5 mM (d _{5 mM} ), and sodium chloride aqueous solution 10 mM (d _{10 mM} ). When
Magnetic composite particles having a value of (d _{5 mM} ) / (d _{0 mM} ) of 3.5 or less and a value of (d _{10 mM} ) / (d _{0 mM} ) of 3.5 or less.   The magnetic composite particle according to claim 1, wherein the polymer is polyethylene glycol.   The magnetic composite particle according to claim 1, wherein the outer shell further contains a silane coupling agent.   The magnetic composite particle according to any one of claims 1 to 3, wherein the magnetic nanoparticles contained in the outer shell are magnetite or γ iron oxide.   5. The magnetic composite particle according to claim 1, wherein the magnetic composite particle has a spherical shape or a substantially spherical shape. 6. The magnetic composite particle according to claim 1, wherein the saturation magnetization has a value of 30 Am ² / kg or more and 200 Am ² / kg or less.   The magnetic composite particle according to claim 1, wherein the core particle is a polymer.   The particle | grain for immunoassay in which the antibody exists in the outer shell of the magnetic body composite particle in any one of Claim 1 to 7. Producing a suspension of magnetic nanoparticles;
A step of producing a core particle containing an inorganic oxide or a polymer and having a volume average particle diameter measured from a transmission electron microscope image of 20 nm or more and 200 nm or less;
Adding the core particles to the suspension of magnetic nanoparticles to obtain a suspension of magnetic nanoparticle adsorbed core particles;
Suspension of magnetic composite particles in which an aqueous solution of a silicon compound is added to a suspension of magnetic nanoparticle adsorbing core particles, and an outer shell containing the magnetic nanoparticles and the silicon compound is provided on the surface of the core particles. Producing a liquid;
A method for producing magnetic composite particles, comprising a step of adding a polymer after stirring the suspension of the magnetic composite particles and causing the outer shell to contain the polymer.   The method for producing magnetic composite particles according to claim 9, wherein the silicon compound is silicon alkoxide.   The method for producing magnetic composite particles according to claim 9 or 10, wherein the aqueous solution of the silicon compound further contains a silane coupling agent.   The method for producing magnetic composite particles according to claim 9, wherein the polymer is polyethylene glycol.

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