Magnetic particle, magnetic particle for assay, and method for producing magnetic particle

Abstract

Provided is a magnetic particle that includes a secondary particle that is an aggregate of a plurality of primary particles having a particle diameter considered to provide superparamagnetism, and that has a larger particle size. [Solution] This magnetic particle comprises a secondary particle that is an aggregate of a plurality of primary particles, and is characterized in that the average particle size of the secondary particle is 500-5000 nm, and the average particle size of the plurality of primary particles is 2-20 nm.

Description

Magnetic particles, magnetic particles for assay, and method for producing magnetic particles

[0001] The present invention relates to magnetic particles, magnetic particles for assays, and a method for producing magnetic particles.

[0002] Fe ３ O ４ Magnetic particles containing Fe are used in the biomedical field, such as immunoassays, separation and purification of biomolecules, and drug delivery. ３ O ４ The particles must be highly manipulable by an external magnetic field. ３ O ４ The main magnetic properties of the particles are remanent magnetization and saturation magnetization (hereinafter simply referred to as "magnetization"). ３ O ４ Depends on particle size.

[0003] Fe ３ O ４ In order to easily control the aggregation and dispersion of particles in a medium by an external magnetic field, the magnetization when the application of the external magnetic field is stopped is Fe ３ O ４ It is necessary to reduce the residual magnetization of the particles. ３ O ４ It is known that the remanent magnetization of particles decreases with decreasing particle size, and when the particle size is 20 nm or less, the remanent magnetization becomes zero, that is, the particles exhibit superparamagnetism.

[0004] Non-Patent Document 1 describes Fe(III) chloride hexahydrate synthesized by adding sodium acrylate to an ethylene glycol solution in order to reduce the particle size of the primary particles. ３ O ４ Non-Patent Document 2 discloses Fe clusters synthesized by adding trisodium citrate to an ethylene glycol solution of anhydrous iron (III) chloride in order to reduce the particle size of the primary particles. ３ O ４ These Fe clusters are disclosed. ３ O ４ Both clusters are superparamagnetic because the primary particle size is 20 nm or less.

[0005] S. Xuan, Y. X. J. Wang, J. C. Yu, K. C. F. Leung, Chemistry of Materials, vol. 21, p. 5079-5087 (2009). J. Liu, Z. Sun, Y. Deng, Y. Zou, C. Li, X. Guo, L. Xiong, Y. Gao, F. Li, D. Zhao, Angewandte Chemie International Edition, vol. 48, p. 5875-5879 (2009).

[0006] The present invention aims to provide magnetic particles that include secondary particles formed by aggregation of multiple primary particles having a particle size that is considered to be superparamagnetic, and the secondary particles have a larger particle size.

[0007] In order to solve the above problems, the present invention provides magnetic particles comprising secondary particles formed by aggregation of a plurality of primary particles, wherein the secondary particles have an average particle size of 500 nm or more and 5,000 nm or less, and the primary particles have an average particle size of 2 nm or more and 20 nm or less. The present invention also provides magnetic particles for assays, comprising the above magnetic particles and a resin layer on the outer surface of the magnetic particles. The present invention also provides a method for producing magnetic particles comprising secondary particles formed by aggregation of a plurality of primary particles, the method comprising the steps of obtaining an iron(III)-containing solution, aging the iron(III)-containing solution, mixing the iron(III)-containing solution obtained in the aging step with a precipitant to obtain a mixed solution, and heating the mixed solution.

[0008] According to the present invention, an Fe alloy that exhibits both superparamagnetism and large magnetization is ３ O ４ A cluster can be provided.

[0009] Magnetic particles (Fe) according to an embodiment of the present invention ３ O ４ Schematic diagram of the structure of a magnetic particle for assay according to an embodiment of the present invention. ３ O ４ Cluster synthesis flow. Fe by blending method ３ O ４Cluster synthesis flow. Fe produced in Example 1 ３ O ４ SEM image of the cluster (low magnification). ３ O ４ SEM image of the cluster (high magnification). ３ O ４ SEM image of the cluster (low magnification). ３ O ４ SEM image of the cluster (high magnification). ３ O ４ SEM image of the cluster (low magnification). ３ O ４ SEM image of the cluster (high magnification). ３ O ４ SEM image of the cluster (low magnification). ３ O ４ SEM image (high magnification) of the Fe cluster produced in Example 14. ３ O ４ SEM image (low magnification) of the Fe cluster produced in Example 14. ３ O ４ SEM image (high magnification) of the Fe cluster produced in Example 22. ３ O ４ SEM image of the cluster. Fe produced in Example 23 ３ O ４ SEM image of the cluster. Fe produced in Example 24 ３ O ４ SEM image of the cluster. Fe produced in Example 25 ３ O ４ SEM image of the Fe cluster produced in Example 26. ３ O ４ SEM image of the cluster. Fe produced in Example 27 ３ O ４ SEM image of the cluster. Fe produced in Example 28 ３ O ４ SEM image of the cluster. Fe produced in Example 29 ３ O ４ SEM image of the cluster. Fe produced in Comparative Example 2 ３ O４ SEM image of the cluster (low magnification). ３ O ４ SEM image of the cluster (high magnification). ３ O ４ SEM image of the cluster (low magnification). ３ O ４ SEM image of the cluster (high magnification). ３ O ４ SEM image of the cluster. Fe produced in Example 30 ３ O ４ SEM image of the Fe cluster produced in Example 31. ３ O ４ SEM image of the Fe cluster produced in Example 32. ３ O ４ SEM image of the cluster. Fe produced in Synthesis Example 2 ３ O ４ SEM images of clusters. XRD patterns of the produced particles. (a) Example 22, (b) Example 24, (c) Example 26, (d) Example 28. Hysteresis curves of the particles produced in Example 22. Hysteresis curves of the particles produced in Example 24. Hysteresis curves of the particles produced in Example 26. Hysteresis curves of the particles produced in Example 28.

[0010] The embodiment of the present invention will be described in detail. ３ O ４ "Cluster" and "secondary particle" are synonymous.

[0011] [First embodiment] The first embodiment of the present invention relates to magnetic particles. First, the problem in this embodiment will be described. In various tests using magnetic particles, such as an optical waveguide type test system described later, the magnetic field generated by an external magnetic field is ３ O ４ In order to enable rapid movement and transport of particles in the medium, Fe ３ O ４ The particles must have a large magnetization. Generally, the magnetization per particle of a magnetic particle increases with increasing particle size. Therefore, Fe ３ O ４If the particle size is increased, Fe ３ O ４ The magnetization per particle can be increased.

[0012] Thus, Fe, which is highly manipulable by an external magnetic field, ３ O ４ Since there is a trade-off between the requirement for superparamagnetism, which is small particle size, and the requirement for large particle size, which is large magnetization, we have developed an Fe alloy that achieves both superparamagnetism and large magnetization. ３ O ４ The particles are difficult to obtain.

[0013] Several Fe particles of several tens of nanometers ３ O ４ Fe, which is a secondary particle formed by the aggregation of primary particles ３ O ４ Clusters are known. ３ O ４ By controlling the number of clusters of primary particles while keeping the size of the clusters small, in other words, by controlling the size of the secondary particles, it is possible to obtain Fe nanoparticles that exhibit both superparamagnetism and large magnetization. ３ O ４ It is possible to obtain clusters.

[0014] On the other hand, Fe produced by the methods of Non-Patent Documents 1 and 2 ３ O ４ Both clusters are superparamagnetic because the primary particle size is 20 nm or less. However, the secondary particle size is 280 nm at most in Non-Patent Document 1 and 410 nm at most in Non-Patent Document 2. ３ O ４ The magnetization per particle of the cluster is small. ３ O ４ The clusters are not suitable for applications where magnetic particles need to be rapidly moved or transported in a medium by a magnetic field. ３ O ４ The clusters need to have a larger particle size.

[0015] The magnetic particles according to an embodiment of the present invention are magnetic particles including secondary particles formed by aggregation of a plurality of primary particles, characterized in that the average particle size of the secondary particles is 500 nm or more and 5000 nm or less, and the average particle size of the primary particles is 2 nm or more and 20 nm or less. The magnetic particles according to this embodiment are Fe particles having both superparamagnetism and large magnetization. ３ O ４ It is possible to provide clusters. When used for assays, particularly immunoassays, it is possible to collect assay magnetic particles in a solution in a shorter time than conventional products, thereby significantly improving work efficiency. It is also possible to collect assay magnetic particles even in highly viscous solutions, making immunoassays of highly viscous solutions possible. A detailed explanation is provided below.

[0016] The magnetic particles according to this embodiment are magnetic particles including secondary particles formed by aggregation of a plurality of primary particles. ３ O ４ The structure of the magnetic particle (cluster) is shown in FIG. ３ O ４ It is preferable that the Fe according to this embodiment is ３ O ４ The cluster is composed of multiple Fe ３ O ４ The secondary particles 12 are formed by aggregating primary particles 11 of the above. In the magnetic particles according to this embodiment, the secondary particles are preferably spherical in shape. ３ O ４ The shape of the clusters is preferably spherical, depending on the application. In this embodiment (FIGS. 5A, 5B, 6A, 6B, 7A, 7B, 8A, 9A, 10A to 10H, 13A to 13E, 15A, and 15B), the term "sphere" refers to a substantially spherical shape such as a "round shape" or "ball-like shape," and is not limited to a perfect sphere. It also includes anisotropic spheres and spheres with partial depressions or cracks. The Fe according to this embodiment ３ O ４ The shape of the clusters can be confirmed by observing the secondary particles with a scanning electron microscope (SEM).

[0017] As mentioned above, Fe３ O ４ The magnetic properties of the clusters are ３ O ４ It depends on the particle size of the primary particles (primary particle size) and the particle size of the secondary particles (secondary particle size) of the cluster. ３ O ４ The remanence of the clusters decreases with the decrease in the primary particle size, and when the primary particle size is 20 nm or less, the remanence is zero, i.e., the clusters exhibit superparamagnetism. ３ O ４ It correlates with the magnetization of the cluster, and Fe ３ O ４ The magnetization of the clusters increases with increasing secondary particle size. That is, magnetic particles with larger secondary particle size have larger magnetization (e.g., saturation magnetization). Fe particles where primary particles are aggregated to have a particle size of 500 nm or more ３ O ４ The clusters are highly magnetized.

[0018] In the magnetic particles according to this embodiment, the secondary particles preferably have superparamagnetic properties. ３ O ４ The magnetic properties and magnetization of the secondary particles of the clusters can be measured using, for example, a commercially available vibrating sample magnetometer (VSM) or a superconducting quantum interference device (SQUID).

[0019] Fe in this embodiment ３ O ４ The primary particle size and secondary particle size of the clusters are number-average particle sizes measured from scanning electron microscope (SEM) images. ３ O ４The average particle size of 50 primary particles randomly extracted from the SEM image of the cluster and the average particle size of the secondary particles were calculated. The primary particle size was calculated using the Scherrer formula from the half-width of the diffraction peak of the X-ray diffraction method (XRD). The median diameters D50 and D90 of the secondary particle size were calculated from the number-based particle size distribution measured by dynamic light scattering (DLS).

[0020] The magnetic particles according to this embodiment have an average secondary particle size of 500 nm or more and 5000 nm or less, preferably 500 nm or more and 3000 nm or less. The magnetic particles according to this embodiment have a secondary particle size (upper limit) of 5000 nm or less, preferably 4000 nm or less, more preferably 3000 nm or less, and even more preferably 2400 nm or less. The magnetic particles according to this embodiment also have a secondary particle size (lower limit) of preferably 500 nm or more, more preferably 700 nm or more, even more preferably 800 nm or more, and particularly preferably 1000 nm or more. By doing so, the magnetic particles can achieve large magnetization (e.g., large saturation magnetization). For example, magnetic particles having a particle size twice that of conventional particles (e.g., secondary particle size of 800 nm) can have a saturation magnetization approximately eight times that of conventional particles (e.g., secondary particle size of 400 nm), and magnetic particles having a particle size six times that of conventional particles (e.g., secondary particle size of 2400 nm) can have a saturation magnetization more than 200 times that of conventional particles.

[0021] The magnetic particles according to this embodiment have an average primary particle size (average particle size) of 2 nm or more and 20 nm or less. The magnetic particles according to this embodiment have a primary particle size (upper limit) of 20 nm or less, preferably 19 nm or less, more preferably 18 nm or less, and even more preferably 16 nm or less. The magnetic particles according to this embodiment also have a primary particle size (lower limit) of 2 nm or more, preferably 4 nm or more, more preferably 7 nm or more, even more preferably 10 nm or more, and particularly preferably 12 nm or more. In this way, the magnetic particles according to this embodiment have superparamagnetic properties and can have a remanent magnetization of 0 (zero).

[0022] The magnetic particles according to this embodiment are typically composed of Fe ３ O ４ However, other Fe may be used within the range in which the effect of this embodiment can be obtained. ２ O ３ The magnetic particles according to this embodiment may contain iron oxide such as FeO. In addition to the single substance, the magnetic particles may be particles of a mixture containing a magnetic substance such as ferrite. ３ O ４ The magnetic particles according to this embodiment are preferably Fe ３ O ４ It is preferable that the clusters consist of only γ-Fe clusters, and basically, other compositions and structures, such as γ-Fe ２ O ３ It is preferable that the magnetic particles do not contain Fe. ３ O ４ consisting only of γ-Fe ２ O ３ The absence of other substances such as those mentioned above can be confirmed by X-ray diffraction (XRD).

[0023] [Application Example] An application example of this embodiment is related to magnetic particles for assay. The magnetic particles for assay according to the embodiment of the present invention preferably have the magnetic particles according to this embodiment and a resin layer on the outside of the magnetic particles.

[0024] The magnetic particles for assay according to this embodiment will be described with reference to FIG. 2. The magnetic particles for assay 36 according to this embodiment preferably have a silica layer between the magnetic particles and the resin layer. ３ O ４ The clusters 31 are covered with an inorganic material layer 32 .

[0025] Fe ３ O ４ In order to ensure the smoothness of the surface of the cluster 31, Fe ３ O ４ The clusters are covered with an inorganic substance layer 32. The inorganic substance layer 32 is preferably a silica layer. Silica is chemically stable and can be easily formed by a wet method such as the Stöber method. ３ O ４A uniform and smooth layer can be formed on the surface of the cluster 31. The outside of the inorganic substance layer 32 is covered with a resin layer.

[0026] In the assay magnetic particles according to this embodiment, the resin layer preferably includes at least a first resin layer 33 and a second resin layer 34. In the assay magnetic particles 36 according to this embodiment, the first resin layer 33 and the second resin layer 34 are formed on the outside of the inorganic substance layer 32. The first resin layer 33 is made of Fe ３ O ４ For example, the silica layer formed by the Stöber method has a porous structure, so that Fe ３ O ４ In the magnetic particles for assay according to this embodiment, the first resin layer 33 is preferably a hydrophobic resin layer. The first resin layer 33 is preferably a styrene-based resin that has high chemical stability and hydrophobicity.

[0027] In the assay magnetic particles according to this embodiment, the second resin layer 34 is preferably disposed outside the first resin layer 33, and the second resin layer 34 is preferably a polyglycidyl methacrylate layer. The assay magnetic particles 36 according to this embodiment preferably include a ligand bound to the surface of the resin layer, and the ligand is preferably an antibody 35. The second resin layer 34 is provided to immobilize the antibody 35 on the outermost surface of the assay magnetic particles 36. Since functional groups suitable for binding the antibody 35 to the outermost surface of the assay magnetic particles 36 include carboxyl groups, amino groups, thiol groups, epoxy groups, etc., the second resin layer 34 is preferably a resin containing these functional groups.

[0028] The magnetic particles for assay according to this embodiment may further contain a fluorescent material. The method of applying the fluorescent material is not limited, and any known technique may be selected. The timing of applying the fluorescent material may be, for example, mixing the fluorescent material when forming the silica layer, forming the silica layer and then applying the fluorescent material before forming the first resin layer, mixing the fluorescent material together when forming the first resin layer, forming the first resin layer and then applying the fluorescent material before forming the second resin layer, or applying the fluorescent material after forming the second resin layer.

[0029] The fluorescent material can be applied, for example, by mixing the fluorescent material with an organic solvent after forming the resin layer and stirring the mixture while heating, or by mixing the fluorescent material together when forming the resin layer. There are no limitations on the fluorescent material to be selected, and any known fluorescent material can be used. The fluorescent material may be either non-polymerizable or polymerizable. Examples of non-polymerizable fluorescent materials include rhodamine B, rhodamine 6G, and europium complexes such as Lumisys® E-400 and Lumisys® E-460. Examples of polymerizable fluorescent materials include acryloxyethyl thiocarbamoyl rhodamine B and methacryloxyethyl thiocarbamoyl rhodamine B.

[0030] (Method of detecting antigens using magnetic particles in an optical waveguide testing system) When detecting antigens from a sample such as a specimen, an optical waveguide testing system uses magnetic particles bound to antibodies that specifically bind to the antigen, and a sensor to which the antibodies that specifically bind to the antigen are immobilized. The sensor is placed below the direction of gravity, and an electromagnet or other device that generates magnetic force is placed near the sensor. When the electromagnet is activated, the magnetic particles can be attracted to the sensor by the action of gravity and magnetic force. Then, by utilizing an antigen-antibody reaction, the magnetic particles attracted to the sensor bind to antibodies attached to the sensor surface via the antigen. Furthermore, by placing an electromagnet above the direction of gravity, magnetic particles that are not bound to antibodies attached to the sensor surface can be pulled in the direction opposite to gravity. This allows the magnetic particles that bind to the sensor to be separated from those that are not bound.

[0031] By measuring the change in the amount of emitted light when light is incident on the sensor from a light source, it is possible to determine the presence, concentration, and amount of magnetic particles bound to the sensor surface via an antigen-antibody reaction. In other words, when magnetic particles are present (a large number) on the sensor surface, the amount of emitted light is smaller than when they are not present (a small number). Therefore, the presence, concentration, and amount of a target substance such as an antigen in a sample can be determined based on the attenuation of the amount of emitted light detected before the magnetic particles bind to the sensor.

[0032] When detecting a substance to be measured using this method, it is important to reduce test errors by reducing nonspecific adsorption of proteins other than the substance to be measured to the magnetic particles and by reducing adsorption in the absence of antigens on the sensor surface. To achieve this, a resin layer is formed on the surface of the magnetic particles. The resin layer preferably has hydrophilic properties and contains functional groups that can bind to ligands such as antibodies.

[0033] Second Embodiment The second embodiment of the present invention relates to a method for producing magnetic particles. The object of the method for producing magnetic particles according to this embodiment is to facilitate control of primary and secondary particle sizes. The method for producing magnetic particles according to this embodiment is a method for producing magnetic particles including secondary particles formed by aggregation of a plurality of primary particles, and includes the steps of obtaining an iron(III)-containing solution, aging the iron(III)-containing solution, obtaining a mixed solution by mixing the iron(III)-containing solution obtained in the aging step with a precipitant, and heating the mixed solution. The production method according to this embodiment makes it easy to control the primary and secondary particle sizes, and therefore provides a method for producing magnetic particles that are superparamagnetic yet have a wide range of magnetization (a wide range of secondary particle sizes) suited to various applications. This is explained below.

[0034] The method for producing magnetic particles according to this embodiment is a method for producing magnetic particles including secondary particles formed by aggregation of a plurality of primary particles. ３ O ４ The magnetic properties of the clusters are ３ O ４Depends on the primary and secondary particle size of the clusters. ３ O ４ The remanence of the clusters decreases with the decrease in the primary particle size, and when the primary particle size is 20 nm or less, the remanence is zero, i.e., the clusters exhibit superparamagnetism. ３ O ４ It correlates with the magnetization of the cluster, and Fe ３ O ４ The magnetization of the clusters increases with increasing particle size. ３ O ４ If the particle size of the clusters can be controlled, Fe with a wide range of magnetization can be obtained. ３ O ４ However, in this embodiment, Fe clusters can be obtained. ３ O ４ When the shape of the clusters is deformed and bound together, or when they become amorphous, they are not included in the magnetic particles according to this embodiment, which are secondary particles formed by aggregation of a plurality of primary particles, because in such cases, the secondary particles are no longer in a state of further aggregation, and are no longer considered to be secondary particles according to this embodiment.

[0035] <Fe ３ O ４ Synthesis of Clusters> Fe according to this embodiment ３ O ４ The synthesis flow of the Fe cluster is shown in Figure 3. ３ O ４ The synthesis of the cluster mainly comprises an iron(III)-containing solution preparation step (a step of obtaining an iron(III)-containing solution), a mixed solution preparation step (a step of obtaining a mixed solution), a heating step (a step of heating the mixed solution), and optionally an aging step (a step of aging the iron(III)-containing solution). Each step will be described in detail below.

[0036] <Step of Obtaining Iron(III)-Containing Solution> The method for producing magnetic particles according to this embodiment includes a step of obtaining an iron(III)-containing solution containing ethylene glycol and iron(III) chloride anhydride. Preferably, the iron(III)-containing solution obtained in the step of obtaining the iron(III)-containing solution contains ethylene glycol and iron(III) chloride anhydride. First, the preparation of the iron(III)-containing solution will be described. The iron(III)-containing solution is prepared by dissolving an iron(III) compound in ethylene glycol. As the iron(III) compound, iron(III) chloride anhydride or iron(III) chloride hexahydrate is suitable.

[0037] These may be used alone or in any mixture in any ratio. In addition to iron(III) chloride, iron(III) nitrate, iron(III) acetate, iron(III) sulfate, iron(III) ammonium sulfate, iron(III) acetylacetonate, iron(III) hydroxide, iron(III) oxide, and hydrates thereof may be used alone or in a mixture of two or more. The iron(III) compound is not limited to these compounds, as long as it dissolves uniformly in ethylene glycol.

[0038] Ethylene glycol is a type of organic compound called a polyol, which has multiple OH groups in its structure. Polyols are used in the synthesis of metal microparticles (polyol method) and play two roles: a solvent and a reducing agent. In this embodiment, ethylene glycol also serves as a solvent that dissolves iron (III) chloride, and at the same time, it acts as a reducing agent that reduces a portion of Fe (III) to Fe (II). In addition to ethylene glycol, polyols that are liquid at room temperature, such as diethylene glycol, triethylene glycol, 1,2-propanediol, and 1,3-propanediol, can also be used, but these cannot be used alone and are mixed with ethylene glycol in any ratio.

[0039] Fe ３ O ４ is an iron oxide containing Fe(II) and Fe(III) in a molar ratio of 1:2. ３ O ４When obtaining Fe(III), the reducing agent ethylene glycol reduces part of the Fe(III) to Fe(II), so Fe ３ O ４ Therefore, the concentration of iron(III) chloride in ethylene glycol, which is both a solvent and a reducing agent, is important. The concentration of iron(III) chloride is preferably 0.05M or more and 0.2M or less, more preferably 0.1M or more. If the concentration of iron(III) chloride is less than 0.05M, that is, if the ethylene glycol, which is a reducing agent, is in excess relative to Fe(III), the reduction of Fe(III) is promoted, and Fe(II) increases, resulting in the formation of Fe(III). ３ O ４ However, when the concentration of iron(III) chloride exceeds 0.2 M, increasing the concentration of the precipitant to match the concentration of iron(III) chloride increases the viscosity of the mixture. This makes it difficult to prepare a uniform mixture. Furthermore, convection in the mixture is difficult to occur, making it difficult to heat the mixture uniformly.

[0040] Here, a method for producing magnetic particles according to this embodiment will be described, which includes secondary particles formed by aggregation of multiple primary particles, and includes the steps of obtaining an iron(III)-containing solution, aging the iron(III)-containing solution, obtaining a mixture by mixing the iron(III)-containing solution obtained in the aging step with a precipitant, and heating the mixture. In the method for producing magnetic particles according to this embodiment, the iron(III)-containing solution obtained in the step of obtaining the iron(III)-containing solution may contain ethylene glycol and iron(III) chloride anhydride. In the method for producing magnetic particles according to this embodiment, the iron(III)-containing solution obtained in the step of obtaining the iron(III)-containing solution may contain ethylene glycol, iron(III) chloride anhydride, and externally added water. In the method for producing magnetic particles according to this embodiment, the iron(III)-containing solution obtained in the step of obtaining the iron(III)-containing solution may contain ethylene glycol, iron(III) chloride anhydride, and iron(III) chloride hexahydrate. In addition, in the method for manufacturing magnetic particles according to this embodiment, the iron(III)-containing solution obtained in the step of obtaining the iron(III)-containing solution may contain ethylene glycol, iron(III) chloride hexahydrate, and externally added water.

[0041] <Step of Obtaining a Mixed Solution> The method for producing magnetic particles according to this embodiment includes a step of obtaining a mixed solution by mixing an iron(III)-containing solution with a precipitant. Furthermore, if the method for producing magnetic particles according to this embodiment includes an aging step, it also includes a step of obtaining a mixed solution by mixing the iron(III)-containing solution obtained in the aging step with a precipitant. When a precipitant is added to the iron(III)-containing solution, precursor particles precipitate. The dispersion in which these precursor particles are dispersed is called a mixed solution. The precursor particles are particles of an amorphous, water-soluble iron(III) compound. In the method for producing magnetic particles according to this embodiment, the precipitant is preferably an acetate. Specifically, urea, alkali metal hydroxide, or acetate can be used as the precipitant, but acetates such as sodium acetate, potassium acetate, and ammonium acetate are particularly preferred.

[0042] <Water in the Mixed Solution> In the method for producing magnetic particles according to this embodiment, water derived from at least one of iron(III) chloride hexahydrate and externally added water is added in at least one of the steps of obtaining the iron(III)-containing solution and obtaining the mixed solution. Water can be added to the mixed solution. The presence of water in the mixed solution contributes to the reduction of the amount of Fe produced. ３ O ４ Water may be added to the ethylene glycol before dissolving the iron(III) compound, to the iron(III)-containing solution, or to the mixture after adding the precipitating agent, which affects the primary particle size and shape of the clusters.

[0043] The water in the mixed solution may be hydrated water of hydrates such as iron(III) chloride hexahydrate or intentionally added water (hereinafter also referred to as "externally added water"). It is necessary that water originating from at least one of iron(III) chloride hexahydrate and externally added water is present. When no water is present in the mixed solution, for example, when the iron(III) compound is iron(III) chloride anhydride, heating a mixed solution consisting of ethylene glycol, iron(III) chloride anhydride, and sodium acetate produces Fe(II) particles with small primary particle size. ３ O ４ Clusters are formed, but they are not deformed or disintegrated. ３ O ４ The clusters may stick together.

[0044] When the molar ratio of iron(III) chloride anhydrous to iron(III) chloride hexahydrate is changed while the iron(III) chloride concentration is kept constant, the Fe ３ O ４ Although the primary particle size of the clusters increases, deformation and adhesion are suppressed and the shape becomes spherical. Also, when external water is intentionally added to a mixed solution of ethylene glycol and iron (III) chloride anhydride, the amount of external water added increases, and the above-mentioned Fe ３ O ４ The primary particles of the clusters become larger in size, and the secondary particles become more spherical.

[0045] With increasing amount of water in the mixture, Fe ３ O ４The primary particle size of the clusters increases due to the Fe ３ O ４ Since the solubility of Fe in water is higher than that in ethylene glycol, the presence of water in the mixed solution ３ O ４ This is presumably because the degree of supersaturation of Fe decreases and the number of nuclei generated decreases. ３ O ４ The shape of the cluster is Fe ３ O ４ It is thought that the surface free energy of the solvent is affected. ３ O ４ and ethylene glycol interfacial free energy and Fe ３ O ４ Since the interfacial free energy of the ethylene glycol / water mixed solvent is different from that of the Fe / Fe mixed solvent, the presence or absence of water in the mixed solution ３ O ４ It is believed that the shape of the cluster changes.

[0046] In the method for producing magnetic particles according to this embodiment, the amount of water per 30 mL of ethylene glycol in the iron(III)-containing solution is preferably more than 0 mL and not more than 10.0 mL, more preferably 0.2 mL or more and not more than 8.0 mL. ３ O ４ If the amount of water is more than 10.0 mL, the Fe ３ O ４ As mentioned above, the primary particles of Fe do not form clusters with increasing water content. ３ O ４ Although the primary particles of the clusters become larger in size, if the amount of water is 8.0 mL or less, the increase in the size of the primary particles can be suppressed by combining the operations described below, such as adding additives, stirring during heating, and aging. ３ O ４ Surprisingly, Fe ２ O ３The amount of water per 30 mL of ethylene glycol is the sum of the hydrated water of the hydrate and the externally added water. For example, when 0.9 mmol of iron(III) chloride anhydride and 3.6 mmol of iron(III) chloride hexahydrate are dissolved in 30 mL of ethylene glycol and 0.4 mL of externally added water is added, the amount of hydrated water contained in 3.6 mmol of iron(III) chloride hexahydrate is 0.39 mL, so the amount of water per 30 mL of ethylene glycol is 0.79 mL.

[0047] <Additives in the mixed solution> Fe ３ O ４ The size of the primary particles, which are the units that make up the clusters, has been reduced, and Fe ３ O ４ To improve the dispersibility of the clusters, an additive may be added to the mixture. Examples of the additive include high molecular weight compounds, low molecular weight compounds, surfactants, and chelating agents. － It is preferable that the additive has a group that becomes a carboxylate ion (-COO － ) strongly coordinates to Fe(III), so Fe ３ O ４ The growth rate of primary particles can be controlled by the addition of carboxylate ions (-COO － As the additive having a group which becomes a carboxylic acid, carboxylic acids and carboxylates can be used, but carboxylates which have a high degree of dissociation and are likely to generate carboxylate ions are preferably used. Specifically, citric acid, malic acid, acrylic acid, tartaric acid, lactic acid, oleic acid, succinic acid, maleic acid, fumaric acid, and alkali metal salts thereof are preferred, and trisodium citrate and sodium acrylate are particularly preferred.

[0048] These additives include Fe ３ O ４ In addition to the reduction of the size of the primary particles of the cluster, the Fe ３ O ４ It also has the effect of making the clusters spherical. In other words, even if there is no water in the mixture, the presence of an additive will cause the formation of spherical Fe clusters. ３ O ４Clusters can be obtained. These additives also have the effect of reducing the size of secondary particles.

[0049] <Step of Heating the Mixed Liquid> The method for producing magnetic particles according to this embodiment includes a step of heating the mixed liquid. The heating temperature of the mixed liquid is 160° C. or higher and 240° C. or lower, and preferably 180° C. or higher and 220° C. or lower. When the heating temperature is lower than 160° C., Fe ３ O ４ On the other hand, when the heating temperature exceeds 240°C, the reaction rate is fast and Fe ３ O ４ The cluster size distribution becomes larger.

[0050] The heating time of the mixed solution is 2 hours or more and 24 hours or less, preferably 4 hours or more and 12 hours or less. If the heating time is less than 2 hours, Fe may be easily removed from the precursor particles. ３ O ４ If the conversion to clusters is not complete and the heating time exceeds 24 hours, Ostwald ripening will result in the formation of Fe ３ O ４ The primary particle size of the clusters increases.

[0051] The mixed liquid may contain components whose boiling points are equal to or lower than the heating temperature, such as ethylene glycol, which has a boiling point of 197° C., or water, which has a boiling point of 100° C. Therefore, it is preferable to heat the mixed liquid using a pressure-resistant sealed container generally called an "autoclave."

[0052] The mixture may also be heated under stirring conditions using a container equipped with a stirring function. When heating under static conditions using the aforementioned sealed container, a concentration gradient occurs in the mixture as the reaction progresses, making the mixture non-uniform. On the other hand, when heating under stirring conditions, mass transfer is promoted and the mixture can be maintained in a homogeneous state at all times, which is effective in reducing the particle size of the primary particles.

[0053] After heating, the container is left at room temperature until it cools sufficiently, and then the contents are removed. ３ O ４ The clusters are washed twice with ethanol and twice with water, and then dried in a dry oven at 80°C.

[0054] <Step of Aging Iron(III)-Containing Solution> The method for producing magnetic particles according to this embodiment may include a step of aging the iron(III)-containing solution. The iron(III)-containing solution is a homogeneous solution in which an iron(III) compound is completely dissolved in ethylene glycol. Aging is an operation of maintaining this iron(III)-containing solution at a constant temperature for a certain period of time.

[0055] Aging changes the solvation state of Fe(III), for example by promoting the exchange reaction of the Fe(III) ligand from chloride ion to ethylene glycol, resulting in the final product Fe(III). ３ O ４ It is speculated that this affects the structure of the cluster. Also, although the iron (III) compound appears to be completely dissolved macroscopically, the iron (III) compound is dispersed in the ethylene glycol in a kind of oligomer-like state. The oligomer becomes a monomer by aging, which increases the activity of the dissolved species of Fe (III), resulting in the formation of Fe ３ O ４ It is believed that the primary and secondary particle sizes of the clusters change.

[0056] As described above, since aging is related to the dissolved species of Fe(III), it is essential that aging be performed before adding a precipitant to the iron(III)-containing solution. Even if a precipitant is added to the iron(III)-containing solution and aging is performed after the precursor particles are generated, Fe(III) is not dissolved. ３ O ４ The structure of the cluster remains unchanged.

[0057] As the aging temperature increases and the aging time increases, Fe ３ O ４ The primary particle size of the clusters becomes smaller and the secondary particle size becomes larger. In the method for producing magnetic particles according to this embodiment, the aging temperature in the aging step is preferably 0°C or higher and 130°C or lower, more preferably 10°C or higher and 130°C or lower. If the aging temperature is lower than 0°C, the effect of aging is small, and if it exceeds 130°C, Fe ３ O ４In the method for producing magnetic particles according to this embodiment, the aging time in the aging step is preferably 1 hour or more and 500 hours or less, more preferably 1 hour or more and 200 hours or less. Aging for less than 1 hour is preferable because Fe ３ O ４ Aging for more than 500 hours without causing any change in the structure of the clusters is excessive because the effect saturates after a certain time.

[0058] When aging an iron(III)-containing solution, Fe ３ O ４ The timing of adding additives, which affects the primary and secondary particle sizes of the clusters, is also important. Even if the type and concentration of additives are the same, the timing of adding the additives can dramatically change the secondary particle size in particular.

[0059] When an additive is added to an iron(III)-containing solution after aging, the particle size reduction effect of the additive is enhanced, and the secondary particle size is smaller than when the additive is added to an iron(III)-containing solution without aging. On the other hand, when an additive is added to an iron(III)-containing solution before aging, the particle size reduction effect of the additive is offset by the particle size increase effect of aging, and the secondary particle size is larger than when the additive is added to an iron(III)-containing solution without aging.

[0060] Furthermore, when the concentration of the additive added to the iron(III)-containing solution before aging is low (0.01 M or less), the effect of aging on increasing the particle size is further promoted by the additive, resulting in the secondary particles becoming gigantic. This is thought to be because the structure of the dissolved species of Fe(III) changes due to aging in the presence of the additive.

[0061] As mentioned above, the Fe(III)-containing solution after aging was used. ３ O ４ The secondary particle size of the clusters depends on the degree of aging. Secondary particles of 500 nm to 3000 nm can be produced. When two or more iron(III)-containing solutions with different degrees of aging are mixed in any ratio, Fe ３ O４ The secondary particle size of the clusters can be controlled. ３ O ４ The method for controlling the particle size of the clusters is called the blending method. ３ O ４ The synthesis flow of the cluster is shown in Figure 4.

[0062] For example, a weakly aged iron (III)-containing solution and a strongly aged iron (III)-containing solution were used, respectively, for short aging times and for Fe (III) particles with a particle size of 500 nm. ３ O ４ The iron(III)-containing solution from which clusters were obtained and the Fe(III)-containing solution from which clusters were obtained with a long aging time and a particle size of 1000 nm were obtained were used. ３ O ４ When the iron(III)-containing solution is used to obtain iron(III) clusters, the Fe(III) clusters can be obtained by mixing these in any ratio. ３ O ４ The particle size of the clusters can be controlled within the range of 500 nm to 1000 nm.

[0063] <Product Identification Method> The product was identified by X-ray diffraction measurement (XRD). XRD was performed using an X'PertPro manufactured by Spectris under the conditions of θ-2θ scanning, tube voltage 40 kV, tube current 45 mA, scan angle 10 to 70°, step 0.02°, and measurement time / step 1 sec.

[0064] <Fe ３ O ４ Method for measuring primary particle size of clusters> Fe ３ O ４ The primary particle size of the clusters was measured by scanning electron microscopy (SEM) or XRD. SEM was performed using an S-4800 manufactured by Hitachi High-Technologies Corporation under conditions of an acceleration voltage of 5 kV and an irradiation current of 10 μA. Using an SEM image at a magnification of 100,000 times, the particle sizes of 50 primary particles randomly extracted from the image were measured, and their average value was calculated.

[0065] XRD was performed using a Spectris X'PertPro under the conditions of θ-2θ scanning, tube voltage 40 kV, tube current 45 mA, scan angle 33-38°, step 0.01°, and measurement time / step 2 sec. The primary particle diameter D nm was calculated using Scherrer's formula (1): D = Kλ / β cos θ (1) where K is the Scherrer constant (K = 0.89), λ is the wavelength of the X-ray (λ = 0.1542 nm), and β is Fe. ３ O ４ is the half-width rad of the diffraction peak of the (311) plane, and θ is the Bragg angle (0.3091 rad).

[0066] <Fe ３ O ４ Method for measuring secondary particle size of clusters> Fe ３ O ４ The secondary particle size of the clusters was measured using SEM or dynamic light scattering (DLS). SEM was performed using the above-mentioned equipment and conditions. Using an SEM image at 10,000x magnification, the particle sizes of 50 secondary particles randomly extracted from the image were measured, and their average value was calculated.

[0067] Using a laser diffraction / scattering particle distribution analyzer Partica LA-950 manufactured by Horiba Ltd., Fe was measured by DLS. ３ O ４ The particle size distribution of the clusters was obtained, and the median diameters D50 and D90 were calculated from the particle size distribution based on the measured number of clusters.

[0068] <Fe ３ O ４ Method for measuring magnetic properties of clusters> Fe ３ O ４ The magnetic properties of the cluster were measured by measuring the hysteresis curve using a magnetic property measuring system MPMS3 manufactured by Quantum Design, at a measurement temperature of 298 K in VSM mode in a magnetic field range of −20,000 to +20,000 Oe.

[0069] <Preparation of magnetic particles for assay> Fe particles produced by controlling the particle size ３ O ４ The clusters can be further used as magnetic particles for assays by forming an inorganic or organic layer on the particle surface.３ O ４ To ensure the surface smoothness of the clusters, a silica layer may be formed by a wet method such as the Stöber method. It is then preferable to form a styrene-based resin layer with high chemical stability and hydrophobicity as the first resin layer, and then form a polyglycidyl methacrylate layer as the second resin layer. The second resin layer preferably has a surface capable of binding a ligand, and the ligand is preferably an antibody. Since carboxyl groups, amino groups, thiol groups, epoxy groups, etc. are suitable functional groups for binding antibodies to the outermost surface of the assay magnetic particles, it is preferable that the second resin layer be a resin containing these functional groups.

[0070] The assay magnetic particles may further contain a fluorescent material. The method for applying the fluorescent material is not limited, and any known technique can be selected. The timing for applying the fluorescent material may be, for example, mixing the fluorescent material when forming the silica layer, forming the silica layer and then applying the fluorescent material before forming the first resin layer, mixing the fluorescent material together when forming the first resin layer, forming the first resin layer and then applying the fluorescent material before forming the second resin layer, or even after forming the second resin layer. The fluorescent material can be applied, for example, by mixing the fluorescent material with an organic solvent after forming the resin layer and stirring under heating, or by mixing the fluorescent material together when forming the resin layer. The fluorescent material is not limited, and any known fluorescent material can be selected. The fluorescent material may be polymerizable or non-polymerizable. Examples of non-polymerizable fluorescent materials include rhodamine B, rhodamine 6G, and europium complexes such as Lumisys (registered trademark) E-400 and Lumisys (registered trademark) E-460. Examples of polymerizable fluorescent materials include acryloxyethyl thiocarbamoyl rhodamine B and methacryloxyethyl thiocarbamoyl rhodamine B.

[0071] The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples. ３ O ４ The cluster results are summarized in Table 1.

[0072] <Fe ３ O ４ Synthesis of Clusters Example 1 4.5 mmol of iron (III) chloride anhydrous (FeCl ３ (Kishida Chemical Co., Ltd.) was dissolved in 0.15 M iron(III) chloride in ethylene glycol (iron(III)-containing solution).

[0073] The iron(III)-containing solution was added with external H ２ After adding 0.4 mL of HCl, aging was performed. The aging temperature was 25°C, and the aging time was 72 hours. The iron (III)-containing solution was poured into a pressure-resistant container equipped with a Teflon (registered trademark) inner cylinder, sealed, and then placed in an incubator. The iron (III)-containing solution after aging was diluted with sodium acetate (CH ３ 2.7 g of COONa (Kishida Chemical Co., Ltd.) was added to obtain a mixture in which precursor particles were dispersed. This mixture was poured into a pressure vessel equipped with a Teflon (registered trademark) inner cylinder and sealed. The pressure vessel was heated in an oven at 180°C for 12 hours. After the reaction was completed, the pressure vessel was cooled sufficiently to around room temperature, and then the generated Fe ３ O ４ The clusters were taken out. ３ O ４ The clusters were washed twice with ethanol and twice with water, and then dried in an oven at 80°C. ３ O ４ SEM images of the clusters are shown in Figures 5A and 5B.

[0074] Example 2 Magnetic particles were produced in the same manner as in Example 1, except that the aging temperature of the iron (III)-containing solution was set to 60° C. and the aging time was set to 72 hours. ３ O ４ SEM images of the clusters are shown in Figures 6A and 6B.

[0075] Example 3 Magnetic particles were prepared in the same manner as in Example 1, except that the aging temperature of the iron (III)-containing solution was set to 100° C. and the aging time was set to 72 hours.

[0076] Example 4 Magnetic particles were prepared in the same manner as in Example 1, except that the aging temperature of the iron (III)-containing solution was set to 60° C. and the aging time was set to 24 hours.

[0077] Example 5 Magnetic particles were prepared in the same manner as in Example 1, except that the aging temperature of the iron (III)-containing solution was set to 60° C. and the aging time was set to 120 hours.

[0078] Example 6 Magnetic particles were produced in the same manner as in Example 1, except that the aging temperature of the iron (III)-containing solution was set to 60° C. and the aging time was set to 168 hours. ３ O ４ SEM images of the clusters are shown in Figures 7A and 7B.

[0079] Example 7 In Example 1, the aging temperature of the iron(III)-containing solution was set to 60°C, and the aging time was set to 168 hours. Trisodium citrate was added to the iron(III)-containing solution after aging to a concentration of 0.01 M. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0080] Example 8 In Example 1, 0.9 mmol of FeCl was added to ethylene glycol. ３ and 3.6 mmol of FeCl ３ ・6H ２ The solution containing dissolved O was used as the iron(III)-containing solution. ２ The amount of O added was 0 mL. The aging temperature of the iron (III)-containing solution was 60° C., and the aging time was 168 hours. Other than these conditions, magnetic particles were produced in the same procedure as in Example 1. ３ O ４ SEM images of the clusters are shown in Figures 8A and 8B.

[0081] Example 9 In Example 1, 0.9 mmol of FeCl was added to ethylene glycol. ３ and 3.6 mmol of FeCl ３ ・6H ２The solution in which O was dissolved was used as an iron(III)-containing solution. The aging temperature of the iron(III)-containing solution was 25°C, and the aging time was 168 hours. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0082] Example 10 In Example 1, 0.9 mmol of FeCl was added to ethylene glycol. ３ and 3.6 mmol of FeCl ３ ・6H ２ The solution containing dissolved O was used as the iron(III)-containing solution. ２ The amount of O added was 0 mL. The aging temperature of the iron(III)-containing solution was 60°C, and the aging time was 168 hours. Trisodium citrate was added to the aged iron(III)-containing solution to a concentration of 0.01 M. Other than these conditions, magnetic particles were prepared using the same procedure as in Example 1.

[0083] Example 11 In Example 1, 3.6 mmol of FeCl was added to ethylene glycol. ３ and 0.9 mmol of FeCl ３ ・6H ２ The solution in which O was dissolved was used as an iron(III)-containing solution. The aging temperature of the iron(III)-containing solution was 60°C, and the aging time was 168 hours. Sodium acrylate was added to the aged iron(III)-containing solution to a concentration of 0.01 M. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0084] Example 12 In Example 1, 4.5 mmol of FeCl was added to ethylene glycol. ３ ・6H ２ The solution containing dissolved O was used as the iron(III)-containing solution. ２ The amount of O added was 0 mL. Other than these conditions, magnetic particles were prepared in the same manner as in Example 1.

[0085] Example 13 In Example 1, 4.5 mmol of FeCl was added to ethylene glycol. ３ ・6H ２The solution in which O was dissolved was used as an iron(III)-containing solution. The aging temperature of the iron(III)-containing solution was 60° C., and the aging time was 168 hours. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0086] Example 14 In Example 1, 4.5 mmol of FeCl was added to ethylene glycol. ３ ・6H ２ The solution in which O was dissolved was used as an iron(III)-containing solution. Sodium acrylate was added to the iron(III)-containing solution before aging so that the concentration was 0.01 M. The aging temperature of the iron(III)-containing solution was 60°C, and the aging time was 168 hours. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1. The produced Fe ３ O ４ SEM images of the clusters are shown in Figures 9A and 9B.

[0087] Example 15 In Example 1, the aging temperature of the iron (III)-containing solution was set to 60°C, and the aging time was set to 168 hours. The mixed solution was heated under stirring conditions using a flask and a mechanical stirrer. Magnetic particles were produced using the same procedure as in Example 1, except for these conditions.

[0088] Example 16 In Example 1, 4.5 mmol of FeCl was added to ethylene glycol. ３ ・6H ２ The solution containing dissolved O was used as the iron(III)-containing solution. ２ The amount of O added was 0 mL. Disodium malate was added to the iron(III)-containing solution before aging to a concentration of 0.01 M. The aging temperature of the iron(III)-containing solution was 60°C, and the aging time was 168 hours. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0089] Example 17 In Example 1, 4.5 mmol of FeCl was added to ethylene glycol. ３ ・6H ２ The solution containing dissolved O was used as the iron(III)-containing solution. ２The amount of O added was 0 mL. Disodium malate was added to the iron(III)-containing solution before aging to a concentration of 0.08 M. The aging temperature of the iron(III)-containing solution was 60°C, and the aging time was 168 hours. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0090] Example 18 In Example 1, 4.5 mmol of FeCl was added to ethylene glycol. ３ ・6H ２ The solution containing dissolved O was used as the iron(III)-containing solution. ２ The amount of O added was 0 mL. Sodium oleate was added to the iron(III)-containing solution before aging to a concentration of 0.01 M. The aging temperature of the iron(III)-containing solution was 60°C, and the aging time was 168 hours. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0091] Example 19 In Example 1, 4.5 mmol of FeCl was added to ethylene glycol. ３ ・6H ２ The solution containing dissolved O was used as the iron(III)-containing solution. ２ The amount of O added was 0 mL. Sodium oleate was added to the iron(III)-containing solution before aging to a concentration of 0.08 M. The aging temperature of the iron(III)-containing solution was 60°C, and the aging time was 168 hours. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0092] Example 20 In Example 1, sodium acrylate was added to the iron(III)-containing solution before aging to a concentration of 0.04 M. The aging temperature of the iron(III)-containing solution was 60° C., and the aging time was 168 hours. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0093] Example 21 In Example 1, the aging temperature of the iron(III)-containing solution was set to 60°C, and the aging time was set to 168 hours. Sodium acrylate was added to the iron(III)-containing solution after aging to a concentration of 0.04 M. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0094] Examples 22 to 29 In Example 1, 4.5 mmol of FeCl was added to ethylene glycol. ３ ・6H ２ The solution containing dissolved O was used as the iron(III)-containing solution. ２ The amount of O added was 0 mL. The aging temperature of the iron (III)-containing solution was 60°C, and the aging time was 4 to 168 hours. Other than these conditions, magnetic particles were produced in the same procedure as in Example 1. For Examples 22 to 29, the amount of Fe produced was ３ O ４ SEM images of the clusters are shown in Figures 10A-10H.

[0095] Comparative Example 1 Magnetic particles were prepared in the same manner as in Example 1, except that the iron (III)-containing solution was not aged.

[0096] Comparative Example 2 In Example 1, the externally added H ２ Magnetic particles were prepared in the same manner as in Example 1, except that the amount of O added was 0 mL. ３ O ４ SEM images of the clusters are shown in Figures 11A and 11B.

[0097] Comparative Example 3 In Example 1, the externally added H ２ The amount of O added was 8.5 mL. The aging temperature of the iron (III)-containing solution was 60° C., and the aging time was 168 hours. Other than these conditions, magnetic particles were produced in the same procedure as in Example 1. ３ O ４ SEM images of the clusters are shown in Figures 12A and 12B. ３ O ４ No cluster formation was observed.

[0098] Comparative Example 4 Magnetic particles were prepared in the same manner as in Example 1, except that the aging temperature of the iron (III)-containing solution was changed to 140°C.

[0099] Comparative Example 5 In Example 1, 1,2-propanediol (Kishida Chemical Co., Ltd.) was added with 4.5 mmol of FeCl ３ ・6H ２The solution in which O was dissolved was used as an iron(III)-containing solution. The aging temperature of the iron(III)-containing solution was 60° C., and the aging time was 168 hours. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0100] Comparative Example 6: In Example 1, the iron (III)-containing solution was not aged. The mixed solution was heated under stirring using a flask and a mechanical stirrer. Other than these conditions, magnetic particles were produced using the same procedure as in Example 1.

[0101] <Fe by blending method> ３ O ４ Control of Cluster Particle Size> Examples 30 to 32 Solution A (weak aging solution), which was a 0.15 M iron(III)-containing solution prepared by dissolving iron(III) chloride hexahydrate in ethylene glycol, and Solution B (strong aging solution), which was a 0.15 M iron(III)-containing solution prepared by dissolving iron(III) chloride hexahydrate in ethylene glycol, aged at 60°C for 168 hours, were prepared.

[0102] Solution A and solution B were mixed in an arbitrary ratio to prepare 30 mL of solution C. The volume ratio of solution A to solution B was varied within the range of 100:0 to 0:100. Synthesis Example 1 was the case where the volume ratio of solution A to solution B was 100:0, and Synthesis Example 2 was the case where the volume ratio of solution A to solution B was 0:100.

[0103] Prepared solution C was added with CH ３ 2.7 g of COONa was added to obtain a mixture in which precursor particles were dispersed. This mixture was poured into a pressure vessel equipped with a Teflon (registered trademark) inner cylinder and sealed. The pressure vessel was heated in an oven at 180°C for 12 hours. After the reaction was completed, the pressure vessel was cooled sufficiently to around room temperature, and then the generated Fe ３ O ４ The clusters were taken out. ３ O ４ The clusters were washed twice with ethanol and twice with water, and then dried in an oven at 80°C. ３ O ４ The cluster results are summarized in Table 2. ３ O ４SEM images of the clusters are shown in Figures 13A-13E.

[0104] <Identification of the product> As a result of XRD, all particles were identified as Fe ３ O ４ The XRD patterns of the particles produced in Examples 22, 24, 26 and 28 are shown in FIG.

[0105] <Fe ３ O ４ Measurement of magnetic properties of clusters>> As a result of measuring the magnetic properties, all particles exhibited superparamagnetic behavior without hysteresis. The hysteresis curves of Examples 22, 24, 26, and 28 are shown in Figures 15A to 15D, respectively.

[0106] <<Preparation of Magnetic Particles for Assay>> <Reference Example 1> 0.5 g of the magnetic particles prepared in Example 1 was dispersed in a mixed solution of 75 mL of ethanol (Kishida Chemical Co., Ltd.) and 75 mL of pure water. Next, 1.5 mL of tetraethyl orthosilicate (Kishida Chemical Co., Ltd.) was added, and 22.5 mL of 28% aqueous ammonia (Kishida Chemical Co., Ltd.) was added as a catalyst, and the mixture was allowed to react for 1.5 hours while stirring. After the reaction, the solvent was removed by centrifugation, and the particles were washed seven times with pure water to form a silica layer on the magnetic particles.

[0107] The obtained silica-coated magnetic particles were dispersed in a mixed solution of 10 mL of ethanol and 10 mL of pure water, and 100 μL of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) was added as a silane coupling agent and mixed thoroughly. Next, 2 mL of 28% aqueous ammonia was added and stirred for 1.5 hours. Next, the magnetic particles were collected with a magnet, the solvent was removed, and the particles were thoroughly washed with pure water, and then 60 mL of nitrogen-bubbled pure water was added to obtain an aqueous dispersion.

[0108] Next, this dispersion was placed in a four-neck 200 mL flask and stirred for 15 minutes while bubbling with nitrogen at a stirring speed of 200 rpm. Subsequently, after switching from nitrogen bubbling to nitrogen flow, 50 μL of styrene (manufactured by Kishida Chemical Co., Ltd.) was added to this dispersion. Next, 0.02 g of potassium persulfate (manufactured by Sigma-Aldrich Co., Ltd.) was dissolved in 2 mL of pure water previously degassed by nitrogen bubbling, and 1 mL was added to the flask. Next, using an oil bath, the mixture was heated at 35 ° C for 30 minutes, then heated to 60 ° C and maintained for 1 hour. Subsequently, 200 μL of glycidyl methacrylate (manufactured by Kishida Chemical Co., Ltd.) was added, and the mixture was maintained for a further 12 hours to complete the polymerization. After completion of the polymerization, the solvent was removed by centrifugation and thoroughly washed with pure water.

[0109] Next, 10 mg of the obtained magnetic particles were dispersed in 5 mL of pure water. Separately from this dispersion, 350 mg of mercaptosuccinic acid (Kishida Chemical) was dissolved in 5 mL of pure water, and 0.8 mL of triethylamine (Tokyo Chemical Industry Co., Ltd.) was added to adjust the pH. 1 mL of this solution was added to the particle dispersion, thoroughly stirred, and heat-treated at 60°C for 3 hours. Thereafter, the solvent was removed by centrifugation, and the mixture was thoroughly washed with pure water to obtain a dispersion of magnetic particles having a silica layer, a first resin layer, and a second resin layer.

[0110] Reference Example 2: 0.5 g of the magnetic particles prepared in Example 1 were dispersed in a mixed solution of 75 mL of ethanol and 75 mL of pure water. Next, 1.5 mL of tetraethyl orthosilicate was added, and 22.5 mL of 28% aqueous ammonia was added as a catalyst. The mixture was allowed to react for 1.5 hours while stirring. After the reaction, the solvent was removed by centrifugation, and the particles were washed seven times with pure water to form a silica layer on the magnetic particles.

[0111] The obtained silica-coated magnetic particles were dispersed in a mixed solution of 10 mL of ethanol and 10 mL of pure water, and 100 μL of 3-methacryloxypropyltrimethoxysilane was added as a silane coupling agent and mixed thoroughly. Next, 2 mL of 28% aqueous ammonia was added and stirred for 1.5 hours. Next, the magnetic particles were collected with a magnet, the solvent was removed, and the particles were thoroughly washed with pure water, and then 60 mL of nitrogen-bubbled pure water was added to obtain an aqueous dispersion.

[0112] Next, this dispersion was placed in a four-neck 200 mL flask and stirred for 15 minutes while bubbling with nitrogen at a stirring speed of 200 rpm. Subsequently, after switching from nitrogen bubbling to nitrogen flow, 5 mg of a europium complex, Lumisys (registered trademark) E-400 (manufactured by Central Techno Co., Ltd.), 2 mg of sodium styrenesulfonate (manufactured by Tokyo Chemical Industry Co., Ltd.), and 50 μL of styrene in which 5 mg of divinylbenzene (manufactured by Tokyo Chemical Industry Co., Ltd.) had been dissolved were added to this dispersion.

[0113] Next, after heating at 35°C for 30 minutes using an oil bath, 1 mg of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in 1 mL of pure water that had been degassed in advance by nitrogen bubbling, and 500 μL of this solution was added to the flask. The temperature was raised to 60°C and maintained for 2 hours. Subsequently, 200 μL of glycidyl methacrylate was added, and the mixture was maintained for an additional 12 hours to complete the polymerization. After completion of the polymerization, the solvent was removed by centrifugation, and the mixture was thoroughly washed with pure water.

[0114] Next, 10 mg of the obtained magnetic particles were dispersed in 5 mL of pure water. Separately from this dispersion, 350 mg of mercaptosuccinic acid was dissolved in 5 mL of pure water, and 0.8 mL of triethylamine was added to adjust the pH. 1 mL of this solution was added to the particle dispersion, thoroughly stirred, and heated at 60°C for 3 hours. The solvent was then removed by centrifugation, and the mixture was thoroughly washed with pure water to obtain a dispersion of magnetic particles having a silica layer, a first resin layer containing a fluorescent material, and a second resin layer. When the obtained magnetic particle dispersion was irradiated with a black light (manufactured by Nichia Corporation) with an excitation wavelength of 375 nm, red light emission was observed.

[0115]

[0116]

[0117] The present invention is not limited to the above-described embodiments, and various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the following claims are appended to apprise the public of the scope of the present invention.

[0118] This application claims priority based on Japanese Patent Application No. 2023-145189 filed on September 7, 2023, Japanese Patent Application No. 2023-145346 filed on September 7, 2023, and Japanese Patent Application No. 2024-151501 filed on September 3, 2024, the entire contents of which are incorporated herein by reference.

[0119] 11 Fe ３ O ４ Primary particles forming clusters 12 Fe consisting of a plurality of primary particles ３ O ４ Cluster 31 Fe ３ O ４ Cluster 32 Inorganic material layer 33 First resin layer 34 Second resin layer 35 Antibody 36 Magnetic particles for assay

Claims

1. A magnetic particle comprising secondary particles which are an aggregate of a plurality of primary particles, the secondary particles having an average particle size of 500 nm or more and 5,000 nm or less, and the primary particles having an average particle size of 2 nm or more and 20 nm or less.

2. The magnetic particles according to claim 1, wherein the average particle size of the secondary particles is 500 nm or more and 3000 nm or less.

3. The secondary particles are Fe ３ O ４ The magnetic particle according to claim 1 or 2, comprising:

4. The magnetic particles according to any one of claims 1 to 3, wherein the secondary particles have a spherical shape.

5. The magnetic particles according to any one of claims 1 to 4, wherein the magnetic property of the secondary particles is superparamagnetic.

6. A magnetic particle for assay, comprising the magnetic particle according to any one of claims 1 to 5 and a resin layer on the outside of the magnetic particle.

7. The magnetic particles for use in an assay according to claim 6, which have a silica layer between the magnetic particles and the resin layer.

8. The magnetic particles for an assay according to claim 6 or 7, wherein the resin layer comprises at least a first resin layer and a second resin layer.

9. The magnetic particles for an assay according to claim 8, wherein the first resin layer is a hydrophobic resin layer.

10. The magnetic particle for assay according to claim 8 or 9, wherein the second resin layer is disposed outside the first resin layer, and the second resin layer is a polyglycidyl methacrylate layer.

11. The magnetic particles for use in an assay according to any one of claims 6 to 10, comprising a ligand bound to the surface of the resin layer.

12. The magnetic particle for use in an assay according to claim 11, wherein the ligand is an antibody.

13. A method for producing magnetic particles including secondary particles formed by an aggregation of multiple primary particles, the method comprising the steps of: obtaining an iron(III)-containing solution; aging the iron(III)-containing solution; obtaining a mixed solution by mixing the iron(III)-containing solution obtained in the aging step with a precipitant; and heating the mixed solution.

14. The method for producing magnetic particles described in claim 13, wherein the iron(III)-containing solution obtained in the step of obtaining the iron(III)-containing solution contains ethylene glycol and anhydrous iron(III) chloride.

15. The method for producing magnetic particles described in claim 13, wherein the iron(III)-containing solution obtained in the step of obtaining the iron(III)-containing solution contains ethylene glycol, anhydrous iron(III) chloride, and externally added water.

16. The method for producing magnetic particles described in claim 13, wherein the iron(III)-containing solution obtained in the step of obtaining the iron(III)-containing solution contains ethylene glycol, iron(III) chloride anhydrous, and iron(III) chloride hexahydrate.

17. The method for producing magnetic particles described in claim 13, wherein the iron(III)-containing solution obtained in the step of obtaining the iron(III)-containing solution contains ethylene glycol, iron(III) chloride hexahydrate, and externally added water.

18. The method for producing magnetic particles according to any one of claims 13 to 17, wherein the aging time in the aging step is from 1 hour to 200 hours.

19. The method for producing magnetic particles according to any one of claims 13 to 18, wherein the aging temperature in the aging step is 10°C or higher and 130°C or lower.

20. A method for producing magnetic particles described in any one of claims 14 to 17, wherein the amount of water per 30 mL of ethylene glycol in the iron (III)-containing solution is 10.0 mL or less.

21. The method for producing magnetic particles described in claim 20, wherein the amount of water per 30 mL of ethylene glycol in the iron (III)-containing solution is 0.2 mL or more and 8.0 mL or less.

22. The method for producing magnetic particles according to any one of claims 13 to 21, wherein the precipitating agent is an acetate salt.

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