Iron-based magnetic metal particle for magnetic bead, and magnetic bead

Abstract

To provide an iron-based magnetic metal particle for a magnetic bead and the magnetic bead which are suitable for achieving both high corrosion resistance and an improvement in magnetic collecting force while maintaining an ability to capture a biological substance.SOLUTION: An iron-based magnetic metal particle is an iron-based magnetic metal particle in which a coating layer of an oxide having a standard free energy of Gibbs formation per 1 mol of oxygen smaller than that of iron oxide is formed on a metal core particle containing iron as a main component. The present invention provides an iron-based magnetic metal particle for a magnetic bead, in which an average particle size of the iron-based magnetic metal particle is 0.1 μm or more and 1 μm or less, a d90 based on a number ratio of circumscribed circles of the metal core particle is 0.7 μm or less, and a mass ratio of the coating layer to a total mass of the metal core particle and the coating layer is 20% or more and 55% or less.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to iron-based magnetic metal particles for magnetic beads and magnetic beads. [Background technology]

[0002] In the fields of medicine and biology, multiple magnetic particles known as magnetic beads are used to detect biological substances present in liquid media for diagnosis and testing. Examples of biological substances that can be captured from liquid media by magnetic beads include nucleic acids, proteins such as antigens, and cells. For example, one method for detecting specific diseases involves amplifying and detecting nucleic acids purified and extracted from blood, etc. Using magnetic beads in this detection method allows for automation of the purification and extraction process, reducing human error and enabling large-scale testing. Furthermore, this testing involves a magnetic collection process in which magnetic beads are collected with a magnet, and high magnetization is desirable for rapid collection.

[0003] Patent Document 1 proposes particles for immunoassays in which core particles made of polystyrene or the like having a particle size of several hundred nanometers are coated with ferrite particles made of iron oxide.

[0004] Furthermore, Patent Document 2 proposes magnetic particles in which iron oxide particles with a particle size of approximately 200 nm are used as core particles and coated with a polymer, with the aim of improving the magnetic separation speed. Meanwhile, Patent Document 3 proposes magnetic beads that are obtained by solid-phase reduction of iron oxide, which is an oxide of magnetic metal element M1, with element M2 such as titanium (Ti), and that include core particles made of magnetic metal element M1 coated with the oxide of element M2. To achieve solid-phase reduction, element M2 is limited to those whose oxide has a lower standard free energy of formation than the oxide of magnetic metal element M1, but the particle structure required to fully function as magnetic beads has not been elucidated. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 6-231957 [Patent Document 2] Japanese Patent Application Laid-Open No. 2013-167528 [Patent Document 3] Japanese Patent Application Laid-Open No. 2009-102748 Summary of the Invention [Problem to be solved by the invention]

[0006] The core particles in Patent Documents 1 and 2 both use iron oxide as a magnetism source. Iron oxide is chemically stable, so there is no risk of iron ions eluting even when used in an aqueous solution, and it is considered suitable for magnetic bead applications. However, the theoretical saturation magnetization of iron oxide is approximately 80 Am 2 / kg is the upper limit, for example, metallic iron (218Am 2 / kg), the saturation magnetization is less than half that of the magnetic beads. Therefore, if metals such as iron, cobalt, or nickel could be used as the magnetic source, the magnetic collecting force (the ability to collect magnetic beads using magnetic force) could be further increased. However, these magnetic metal particles have poor corrosion resistance and are easily oxidized in the atmosphere. In addition, the elution of ions in aqueous solutions must be avoided, and for magnetic metal particles to be put to practical use, they must be corrosion-resistant enough to suppress oxidation and elution. On the other hand, in Patent Document 3, a reduction reaction occurs in the core particles after coating, causing them to change from iron oxide to iron with a lower oxidation degree, and therefore the value of saturation magnetization is higher than that of the core particles disclosed in Patent Documents 1 and 2. However, even if the individual saturation magnetization is high, when magnetic beads made up of a large number of particles are used, a preferred particle structure for improving the ability to efficiently capture target biological substances has not been clarified, and there has been no discussion about what particle structure is suitable for magnetic beads made up of multiple particles used together.

[0007] In particular, to efficiently capture target biological substances from blood, etc., it is effective to reduce the particle size of each magnetic bead particle and increase the specific surface area (surface area per mass). However, reducing the particle size of each particle reduces the magnetization per particle, which causes a problem of reducing the magnetic collecting force, which is the ability of magnetic beads to collect multiple particles together using magnetic force.

[0008] Based on the above, the present invention aims to provide iron-based magnetic metal particles for magnetic beads and magnetic beads that can obtain the ability to capture biological materials, i.e., the ability to efficiently capture biological materials present in a liquid medium using magnetic beads consisting of multiple particles, while minimizing the elution of materials that constitute the magnetic source and enabling improved recovery of the target biological materials from the liquid medium in the magnetic collection process. [Means for solving the problem]

[0009] The present invention provides iron-based magnetic metal particles for magnetic beads, which are metal core particles primarily composed of iron and coated with a coating layer of an oxide having a Gibbs standard free energy of formation per mole of oxygen that is smaller than that of iron oxide, characterized in that the iron-based magnetic metal particles have an average particle size of 0.1 to 1 μm, a d90 based on the number rate of the circumscribed circle of the metal core particles of 0.7 μm or less, and the mass ratio of the coating layer to the total mass of the metal core particles and the coating layer is 20 to 55%.

[0010] First, by coating with an oxide whose standard Gibbs free energy of formation is smaller than that of iron oxide, oxidation of the iron-based metal core particle is suppressed while maintaining sufficient corrosion resistance. Note that "iron-based" refers to a state in which each metal core particle contains 50% or more iron in atomic ratio, and includes not only pure iron but also iron carbides and nitrides, as well as iron-based soft magnetic alloys and intermetallic compounds containing cobalt, nickel, silicon, aluminum, etc.

[0011] Furthermore, when the average particle size of iron-based magnetic metal particles is 0.1 to 1 μm, the individual particles contain non-spherical, irregularly shaped particles, resulting in a larger specific surface area than their apparent volume. This allows for highly efficient capture of biological materials. By setting the d90 (based on the number ratio of the circumscribed circles of the metal core particles) to 0.7 μm or less, the volume of the metal core particles relative to the volume of a single iron-based magnetic metal particle is kept low, ensuring sufficient coating. By setting the mass ratio of the oxide that forms the coating layer to a range of 20 to 55%, the specific gravity of each particle is lighter than that of magnetic particles composed of only one or more metal core particles. This reduces the sedimentation rate in solution, ensuring sufficient reaction time with biological materials and enabling highly efficient capture.

[0012] Furthermore, it is preferable that the oxide coating layer does not exhibit activity in living organisms. Furthermore, it is preferable that the oxide coating layer is made of titania. Titania is a bioinert ceramic and is known to be non-toxic even in animal bodies, making it suitable for this application.

[0013] In addition, the average diameter of the circumscribed circle of the metal core particle of the iron-based magnetic metal particle in the observation photograph is preferably 0.05 μm or more and 0.4 μm or less. By setting the average diameter of the metal core particles contained in the plurality of iron-based magnetic metal particles within this range, the particle size of the metal core particle can be kept low and sufficient coating can be achieved.

[0014] In addition, the saturation magnetization is 100Am 2 As described above, by setting the mass ratio of the oxide forming the coating layer in the iron-based magnetic metal particles to 20 to 55%, it is possible to increase the saturation magnetization to 100 Am while maintaining corrosion resistance. 2 / kg or more, allowing for rapid magnetization.

[0015] Furthermore, when 25 mg of the iron-based magnetic metal particles are added to 1 mL of a 6 M aqueous solution of guanidine hydrochloride and immersed at 25°C for 24 hours, the amount of iron ions eluted is preferably 17 mg / L or less. The elution of iron ions from the iron-based magnetic metal particles reduces the ability to capture the target biological material, causing fluctuations in the pH of the solution containing the biological material and the surface potential of the magnetic particles. Therefore, it is necessary to thoroughly coat the metal particles with an oxide to improve corrosion resistance. Because of this high corrosion resistance, even when used in magnetic beads, the capture reaction of biological materials is not inhibited and excellent capture ability is achieved.

[0016] Furthermore, by coating each of the iron-based magnetic metal particles of the present invention with silica, magnetic beads suitable for purifying biological substances can be obtained. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide iron-based magnetic metal particles for magnetic beads and magnetic beads that are suitable for achieving both high corrosion resistance and improved magnetic collecting force while obtaining the ability to efficiently capture biological materials using magnetic beads consisting of multiple particles. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic cross-sectional view of a single magnetic bead according to one embodiment of the present invention. [Figure 2] 1 is a flowchart showing an example of a method for producing iron-based magnetic metal particles and magnetic beads according to an embodiment of the present invention. [Figure 3] 1 is a graph showing the X-ray reflection intensity (au) for each reflection angle obtained from the X-ray reflection diffraction (XRD) pattern of the iron-based magnetic metal particles of Example 1. [Figure 4] 1 is a graph showing the X-ray reflection intensity (au) for each reflection angle obtained from the X-ray reflection diffraction (XRD) pattern of the iron-based magnetic metal particles of Example 2. [Figure 5] 1 is a graph showing the X-ray reflection intensity (au) for each reflection angle obtained from the X-ray reflection diffraction (XRD) pattern of the iron-based magnetic metal particles of Comparative Example 1. [Figure 6] 1A is a scanning electron microscope (SEM) image of the iron-based magnetic metal particles of Example 1, and FIG. 1B is a Fe mapping image. [Figure 7] FIG. 2 is a graph showing the cumulative particle size distribution of the metal core particles in the iron-based magnetic metal particles of Example 1. [Figure 8] 1A is a scanning electron microscope (SEM) image of the iron-based magnetic metal particles of Example 2, and FIG. 1B is a Fe mapping image. [Figure 9] FIG. 10 is a graph showing the cumulative particle size distribution of the metal core particles in the iron-based magnetic metal particles of Example 2. [Figure 10] 1A is a scanning electron microscope (SEM) image of the iron-based magnetic metal particles of Comparative Example 1, and FIG. 1B is a Fe mapping image. [Figure 11] FIG. 2 is a graph showing the cumulative particle size distribution of metal core particles in the iron-based magnetic metal particles of Comparative Example 1. [Figure 12] 1 is a graph showing the magnetic recovery amount (%) for each recovery start time when silica magnetic beads of Example 3, Example 4, and Reference Example 2 are recovered using a magnet. DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, iron-based magnetic metal particles for magnetic beads according to one embodiment of the present invention will be described together with their manufacturing method, followed by a description of the structure and properties of the iron-based magnetic metal particles and magnetic beads. In this specification, all descriptions of particles and beads refer to a group of particles or a group of beads consisting of multiple particles. In particular, descriptions that explicitly state that a single particle is used, such as a single particle, or descriptions of the detailed structure of a particle or bead, refer to a group of particles or beads consisting of multiple particles. Structural features that exhibit advantageous effects when using magnetic beads, such as the particle size of the magnetic beads and iron-based magnetic metal particles for magnetic beads according to an embodiment of the present invention, are shown as statistical values ​​for a group of 30 or more particles.

[0020] [Method of manufacturing iron-based magnetic metal particles] The structure of each iron-based magnetic metal particle 1 according to an embodiment of the present invention is as shown in FIG. 2. Each iron-based magnetic metal particle 1 is composed of one or more metal core particles 2 and a coating layer 3 that coats the one or more metal core particles 2. A plurality of these iron-based magnetic metal particles 1 can be obtained by the method described below. This method involves mixing an oxide powder primarily composed of iron oxide with a powder of element X alone or a powder of a compound containing element X (e.g., titanium carbide, an element described below) that forms an oxide with a lower Gibbs standard free energy of formation per mole of oxygen than iron oxide, and then heat-treating the resulting raw material powder mixture in a non-oxidizing atmosphere. During this heat treatment, the iron-based oxide powder undergoes solid-state reduction with element X to form metal core particles 2, and the surfaces of each of the metal core particles 2 are coated with a coating layer 3 made of an oxide of element X. Each step will be described in more detail below.

[0021] (Oxide powder mainly composed of iron oxide) The oxide powder material primarily composed of iron oxide may contain iron oxide as the primary component and also contain cobalt oxide, nickel oxide, etc. Examples of iron oxide include Fe2O3, Fe3O4, FeO, and various ferrite compounds, and examples of cobalt oxide and nickel oxide include CoO, Co3O4, and NiO. The oxide powder can be selected according to the target particle size of the iron-based magnetic metal particles to be finally obtained, but it is preferable that the average particle size be within the range of 0.01 to 5 μm. If the average particle size is less than 0.01 μm, secondary aggregation becomes significant, making handling during the manufacturing process difficult. On the other hand, if the average particle size exceeds 5 μm, the specific surface area becomes small due to the coarse particles, and the reduction reaction during heat treatment becomes insufficient, which is undesirable. Note that the average particle size in this embodiment was measured using a wet particle size analyzer using laser diffraction. The particle size value d50 at the 50% cumulative value in the cumulative distribution curve obtained from the particle size distribution (volume basis) was taken as the particle size value d50.

[0022] (Element X whose oxide has a Gibbs standard free energy of formation per mole of oxygen that is smaller than that of iron oxide) The element X is preferably an element X that forms an oxide with a Gibbs standard free energy of formation (ΔG) per mole of oxygen smaller than that of iron oxide in the Ellingham diagram. Specifically, (4 / 3)Fe + O2 = (2 / 3)Fe2O3 + ΔG (1) Element X is preferred, and forms an oxide characterized by a Gibbs standard free energy of formation per mole of oxygen that is smaller than the standard free energy of formation represented by formula (1). Examples of element X include Zn (zinc), Cr (chromium), Nb (niobium), Mn (manganese), V (vanadium), Si (silicon), Ti (titanium), Al (aluminum), Zr (zirconium), Mg (magnesium), and Ca (calcium). Preferred are Ti, Al, and Zr, which form bioinert oxides. Ti and Zr are more preferred, as they can be stably handled in powder form during the manufacturing process, and Ti is even more preferred. Furthermore, the raw material used in the manufacturing process may be a powder in which these metals are partially oxidized. When Ti oxide is converted into titania (TiO2: titanium oxide), it not only exhibits bioinertness but also exhibits hydrophilicity and photocatalytic properties, making it suitable for use in aqueous solutions.

[0023] As described above, the structure suitable for use in aqueous solutions is more preferable because when further silica coating is applied for use as magnetic beads, not only does it facilitate coating using a wet sol-gel method or the like, but even if there are areas that are insufficiently coated, there is no problem in use as long as hydrophilicity and photocatalytic function are expressed.

[0024] Examples of compound powders containing element X that can yield oxides with a smaller Gibbs standard free energy of formation per mole of oxygen than iron oxide include carbides and nitrides of element X. In particular, considering that pulverization and mixing are performed in the manufacturing process, carbides and nitrides that are stable even when microparticulated are preferred. The particle size of the compound powder containing element X is preferably 0.01 to 5 μm in average. If the particle size is less than 0.01 μm, it will not only be difficult to handle in the manufacturing process due to significant aggregation, but will also be very expensive, which is undesirable. On the other hand, if the particle size exceeds 5 μm, the specific surface area will be small due to the coarse particles, and the reduction reaction by heat treatment will be insufficient, which is undesirable. In order to suppress oxidation degradation in the atmosphere while sufficiently promoting the reduction reaction, the average particle size is preferably 0.1 to 5 μm.

[0025] (Mixing raw powder) When oxide powder (raw material powder 10) containing iron oxide as a main component is mixed with compound powder 20 containing element X, which forms an oxide with a smaller Gibbs standard free energy of formation per mole of oxygen than iron oxide, the compound powder 20 is preferably mixed in an amount that allows sufficient reduction of the compound powder 20 from the prepared raw material powder 10 to a magnetic metal relative to the amount of the prepared raw material powder 10. For example, when the raw material powder 10 is Fe2O3 and the compound powder 20 is TiC, the following reduction reaction formula (2) proceeds.

[0026] Fe2O3+TiC → 2Fe+TiO2+CO(gas) ···(2) In this case, the stoichiometric ratio of TiC (compound powder 20%) / Fe2O3 (raw material powder 10%) in the raw material powder blending ratio expressed as a molar ratio is 1.0, and the preferred range of this raw material blending ratio (molar ratio) is 1.0 or more and 2.0 or less. Considering that the reduction reaction is a solid-phase reaction between powders, in order to sufficiently proceed with the reduction reaction, the raw material blending ratio is preferably in the range of "stoichiometric ratio to twice the stoichiometric ratio." If the raw material blending ratio is less than the stoichiometric ratio, the reduction reaction will be insufficient, which is not preferred. On the other hand, if the raw material blending ratio exceeds twice the stoichiometric ratio, the reduction reaction will proceed sufficiently, but the content of iron-based magnetic metal particles in the product will be low, which is not preferred. If the raw material blending ratio is within the preferred range, the content of iron-based magnetic metal particles can be increased, and by setting the mass ratio of the oxide in the coating layer, described below, to 20 to 55%, the saturation magnetization can be increased to 100 Am 2 / kg or more.

[0027] The raw material powders are mixed using a mixer such as a mortar and pestle mixer, a V-shaped mixer, a vibration mill, a ball mill, an attritor, or a bead mill. Media mills such as a ball mill, an attritor, or a bead mill are more preferred. When using these media mills, the raw material powder mixture is pulverized into fine particles, making it possible to obtain iron-based magnetic metal particles with an average particle size of 0.1 to 1 μm.

[0028] (Reduction reaction) When a raw material powder mixture of an oxide powder (raw material powder 10) whose main component is iron and a compound powder 20 containing an element X whose oxide has a smaller Gibbs standard free energy of formation per mole of oxygen than iron oxide is heat-treated in a non-oxidizing atmosphere, a redox reaction occurs. As a result, iron-based magnetic metal particles coated with an oxide of element X are produced.

[0029] Non-oxidizing atmospheres include, but are not limited to, inert gases such as Ar and He, and gases such as nitrogen (N2), carbon dioxide (CO2), and ammonia (NH3). The heat treatment temperature is preferably 650°C to 900°C. If the temperature is lower than 650°C, the reduction reaction proceeds extremely slowly. If the temperature exceeds 900°C, the reduced metal particles sinter together and grow, resulting in coarsening. The heat treatment temperature is preferably 700°C to 850°C, more preferably 750°C to 850°C. The heat treatment time is preferably the time required for the iron oxide starting material to be reduced, and the preferred range varies depending on the heat treatment temperature. For example, if the temperature is 750°C to 850°C, the reduction proceeds sufficiently within 1 hour to 30 hours. If the reduction reaction proceeds sufficiently through heat treatment, the oxide powder of the raw material powder 10 is reduced to iron-based magnetic metal particles, and a coating layer made of the oxide of element X, which is a by-product, is formed.

[0030] (Magnetic Separation) The iron-based magnetic metal particles obtained after heat treatment may contain excess non-magnetic components depending on the raw material composition ratio, so it is preferable to perform magnetic separation operations multiple times using a permanent magnet as necessary to remove the non-magnetic components from the particles containing the non-magnetic components and recover the iron-based magnetic metal particles.

[0031] Through these steps, iron-based magnetic metal particles can be obtained that have an average particle size of 0.1 μm to 1 μm, an average diameter of the circumscribed circle of the metal core particles of 0.05 μm to 0.4 μm, and a d90 (based on the number ratio of the circumscribed circle of the metal core particles) of 0.7 μm or less. Therefore, the particle size of the metal core particles can be kept low, resulting in sufficient coating. By setting the mass ratio of the oxide that forms the coating layer to a range of 20% to 55%, the particle specific gravity becomes lighter than that of the iron-based magnetic metal particles alone, thereby suppressing the sedimentation rate in solution, ensuring sufficient reaction time with biological substances and enabling highly efficient capture of biological substances. In addition, by setting the mass ratio of the oxide that forms the coating layer to the total mass of the metal core particle and coating layer to 20% or more and 55% or less for the entire iron-based magnetic metal particles obtained, it is possible to increase the saturation magnetization to 100 Am while maintaining corrosion resistance. 2 / kg or more, allowing for rapid magnetic collection. Furthermore, by keeping the iron ion elution amount extremely low at 17 mg / L or less, the magnetic beads do not inhibit the capture reaction of biological materials and exhibit excellent capture ability, even when used in magnetic beads. The aforementioned "d90 based on the number rate of the circumscribed circle of the metal core particle" will be discussed later.

[0032] [Structure and properties of iron-based magnetic metal particles] As shown in FIG. 1 , the iron-based magnetic metal particle 1 according to an embodiment of the present invention is composed of a metal core particle 2 primarily composed of iron and a coating layer 3 that coats the metal core particle 2. As described above, the material of the metal core particle 2 is pure iron, an iron-based alloy, an intermetallic compound containing iron, or an iron carbide or nitride. Because soft magnetic properties are desirable for magnetic bead applications, alloying elements such as Co, Ni, Si, and Al are preferred, but not limited to these. Although iron-based metal core particles 2 exhibit higher saturation magnetization than iron oxide, they are easily oxidized in the air or in aqueous solutions. Therefore, they are coated with a coating layer 3 to provide oxidation resistance. The coating layer 3 is an oxide with a lower Gibbs standard free energy of formation per mole of oxygen than iron oxide. As described above, examples of the element X that constitutes the coating layer 3 include Zn, Cr, Nb, Mn, V, Si, Ti, Al, Zr, Mg, and Ca. By selecting element X as the element that constitutes the coating layer 3, it is possible to perform solid-phase reduction of iron oxide powder containing iron as the main component, and to form the coating layer 3 on the metal core particle 2. The element X is preferably Ti, Al, or Zr, which form bioinert oxides. Ti and Zr are more preferred, as they can be stably handled in powder form during the manufacturing process, and Ti is even more preferred. When Ti oxide becomes titania (TiO), it not only becomes bioinert but also exhibits hydrophilic and photocatalytic functions, making it suitable for use in aqueous solutions. A suitable configuration for use in aqueous solutions is that when a silica coating is applied for use as magnetic beads, coating can be easily achieved using a wet process such as the sol-gel method. Even if there are areas with insufficient coating, this is preferable as long as hydrophilic and photocatalytic functions are exhibited, as this does not hinder use. Bioinertness means that when administered to humans or animals, it does not cause significant health changes, i.e., it is non-toxic to living organisms.

[0033] The average particle size of the iron-based magnetic metal particles 1 is preferably 0.1 μm or more and 1 μm or less. If the average particle size is within this range, the target biological material can be extracted and purified as magnetic beads. More preferably, it is 0.4 μm or more and 1 μm or less. If the average particle size is within this range, the magnetic collecting force of the iron-based magnetic metal particles 1 can be improved, and the target biological material can be captured more efficiently.

[0034] Here, the average diameter of the circumscribing circle of the metal core particle 2 is preferably 0.05 μm or more and 0.4 μm or less. If it is 0.05 μm, the specific surface area is reduced, making it easier to coat all of the metal core particles 2 incorporated into one iron-based magnetic metal particle 1 with the coating layer 3 described below. If it exceeds 0.4 μm, the size of each metal core particle 2 becomes large, resulting in incomplete coating with the coating layer 3. Therefore, in order to easily coat multiple particles of the metal core particle 2 with the coating layer 3, a diameter of 0.4 μm or less is preferred. Therefore, in order to sufficiently coat the metal core particles 2 with the coating layer 3, the average diameter of the circumscribing circle of the metal core particle 2 is preferably 0.05 μm or more and 0.4 μm or less. More preferably, it is 0.2 μm or more and 0.4 μm or less. The average diameter of the circumscribing circle of such metal core particles 2 can be determined by drawing circumscribing circles for multiple particles corresponding to the metal core particles in an image of multiple metal core particles and simply averaging the diameters.

[0035] Furthermore, the d90 based on the number ratio of the circumscribing circles of the metal core particles 2 is set to 0.7 μm or less. If the diameter exceeds 0.7 μm and the particles become coarse, the coating with the coating layer 3 will be incomplete. Therefore, the d90 on the coarse particle side is limited to lower the particle size. From the above, if the average diameter of the circumscribing circles of the metal core particles 2 is within the range of 0.05 μm or more and 0.4 μm or less, and the d90 based on the number ratio of the circumscribing circles of the metal core particles 2 is 0.7 μm or less, the coating layer 3 can sufficiently cover the metal core particles 2. It is preferably 0.6 μm or less.

[0036] Here, the mass ratio of the coating layer 3 is set to 20% or more and 55% or less. The mass ratio refers to the ratio of the total mass of the oxides forming the coating layer 3 for a plurality of iron-based magnetic metal particles 1 to the total mass of the plurality of iron-based magnetic metal particles 1. For example, the mass ratio of titania (TiO2), a titanium oxide, is set to 20% or more and 55% or less. If it is less than 20%, the metal core particles 2 will not be sufficiently coated or the proportion of particles with insufficient metal core particles 2 will increase, which is undesirable. On the other hand, if it exceeds 55%, the coating will be sufficient, but the content of the metal core particles 2 serving as the magnetism source will decrease or the proportion of iron-based magnetic metal particles 1 with a low content of metal core particles 2 will increase, resulting in a decrease in the saturation magnetization of the plurality of iron-based magnetic metal particles 1 as a whole. If the mass ratio of the coating layer 3 is within this range, the iron-based magnetic metal particles 1 can exhibit rapid magnetic collecting force and excellent capturing ability while maintaining corrosion resistance.

[0037] In addition, the saturation magnetization of the iron-based magnetic metal particles 1 at this time is 100 Am 2 / kg or more is preferable. The saturation magnetization of iron oxide is about 80Am 2 / kg (Source: Scientific Chronology, National Astronomical Observatory, 2000, Vol. 73, Maruzen Co., Ltd.), so 100Am 2 / kg or more is considered a significant difference. 2 / kg or more 180Am 2 / kg or less. 180Am 2 If the mass ratio exceeds 1 / kg, the mass ratio of the coating layer 3 will be less than 20%, which may result in a decrease in corrosion resistance. Note that if a vibrating sample magnetometer (VSM) is used to measure the saturation magnetization, it is relatively easy to measure even if the test sample is in powder form.

[0038] The corrosion resistance exhibited by the coating layer 3 is evaluated by measuring the amount of iron ions eluted when 25 mg of the iron-based magnetic metal particles 1 are placed in 1 mL of a 6 M aqueous solution of guanidine hydrochloride and immersed at 25°C for 24 hours. The guanidine hydrochloride aqueous solution is an aqueous solution used when extracting nucleic acids using magnetic beads (see, for example, the SDS for TOYOBO MagExtractor (registered trademark) -Genome-). The amount of iron ions eluted in this case is preferably 17 mg / L or less. If the amount is 17 mg / L or less, the proportion of metal core particles 2 in the aqueous solution in which corrosion has progressed is not particularly high, and the overall deterioration of the capture ability and magnetic collecting force of the iron-based magnetic metal particles due to corrosion is practically negligible. The amount of iron ions eluted from the iron-based magnetic metal particles 1 is more preferably 15 mg / L or less.

[0039] The iron-based magnetic metal particles according to the embodiment of the present invention are iron-based magnetic metal particles in which a coating layer of an oxide having a Gibbs standard free energy of formation per mole of oxygen smaller than that of iron oxide is formed on a metal core particle mainly composed of iron. The iron-based magnetic metal particles have an average particle size of 0.1 μm to 1 μm, an average diameter of the circumscribed circle of the metal core particle of 0.05 μm to 0.4 μm, and a d90 based on the number ratio of the circumscribed circle of the metal core particle of 0.7 μm or less, and a mass ratio of the coating layer to the total mass of the metal core particle and the coating layer of 20% to 55%. Furthermore, the saturation magnetization of the iron-based magnetic metal particles is 100 Am 2 / Kg or more, and when 25 mg of the iron-based magnetic metal particles were added to 1 mL of a 6 M aqueous solution of guanidine hydrochloride and immersed at 25°C for 24 hours, the amount of iron ions eluted was 17 mg / L or less.

[0040] [Magnetic beads] (silica coated) To provide the iron-based magnetic metal particles 1 with the properties required for nucleic acid extraction, a silica coating layer primarily composed of silicon oxide (silica) is formed on the surface of the coating layer 3 of each iron-based magnetic metal particle 1, and a group of iron-based magnetic metal particles with the silica coating layer formed thereon constitutes a magnetic bead. Such an outer silica coating layer can be formed using a sol-gel method utilizing the hydrolysis of a metal alkoxide, such as a silicon alkoxide. Specific examples of silicon alkoxides include tetramethoxysilane, tetraethoxysilane, and aminopropyltrimethoxysilane. Silica can be obtained, for example, by the hydrolysis reaction of tetraethoxysilane, and can be produced with good reproducibility by controlling this hydrolysis reaction. It is sufficient for the silica coating to cover at least a portion of the surface of each iron-based magnetic metal particle 1, and it is more preferable for the silica coating to cover the entire surface of each particle. Furthermore, even when the silica coating is applied, rapid magnetic collection force and excellent capture ability can be maintained.

[0041] The silica-coated magnetic beads preferably have a zeta potential in a 0.01M potassium chloride (KCl) aqueous solution of -40 mV or more and -10 mV or less. Zeta potential refers to the surface potential of each particle of the magnetic beads, and while it has traditionally been used as an index to evaluate particle aggregation and dispersion, here it is treated as an index for efficiently binding biological materials to magnetic beads in an aqueous solution of a given salt concentration. Within this zeta potential range, biological materials can be efficiently bound to the magnetic beads, improving their ability to capture biological materials. [Example]

[0042] Below, examples and comparative examples of iron-based magnetic metal particles will be described, followed by examples of magnetic beads.

[0043] [Evaluation of the physical properties of iron-based magnetic metal particles] Example 1 Iron oxide (Fe2O3) powder with a particle size of 0.3 μm (listed value) and titanium carbide (TiC) powder with a particle size of 0.7 μm (listed value) were weighed out in a mass ratio of 6.2:3.8 and milled and mixed using a bead mill. The slurry concentration was adjusted using purified water as a solvent to achieve a solids ratio of 20 mass% for the two powders. The mixture was mixed until the change in particle size after milling reached saturation, resulting in a raw material mixed powder slurry. The resulting slurry was then dried, loaded into an alumina boat, and heat-treated at 800°C for 10 hours in a nitrogen gas stream. Because the raw material mixed powder exhibited some agglomeration after heat treatment, the raw material mixed powder and ethanol were adjusted to a solids ratio of 10 mass% and placed in a glass container. After ultrasonic irradiation, a permanent magnet was used to fix the magnetic metal particles to the inner wall of the container, and the supernatant was discarded. This process was repeated until the supernatant became clear. Finally, the magnetic metal particles were taken out of the glass container to obtain iron-based magnetic metal particles 1 of Example 1.

[0044] Example 2 Iron oxide (FeO) powder with a particle size of 0.3 μm (listed in the catalog) and titanium carbide (TiC) powder with a particle size of 0.7 μm (listed in the catalog) were weighed out in a mass ratio of 6.6:3.4, and then pulverized and mixed using a bead mill. The process and conditions were the same as in Example 1, and iron-based magnetic metal particles 1 of Example 2 were obtained.

[0045] (Comparative Example 1) Next, iron-based magnetic metal particles 1 of Comparative Example 1 were obtained using the same mixing ratio, steps and conditions as in Example 1, except that a ball mill was used to mix the two types of raw material powders used in Example 1.

[0046] The iron-based magnetic metal particles 1 thus obtained in Examples 1 and 2 and Comparative Example 1 were measured for various average particle sizes, X-ray diffraction patterns, saturation magnetization, and amount of eluted Fe. The methods for measuring the characteristic values ​​are as follows.

[0047] (average particle size of iron-based magnetic metal particles) The average particle size of the iron-based magnetic metal particles was measured using a wet particle size analyzer based on laser diffraction. The particle size value d50 at 50% of the cumulative distribution curve obtained from the particle size distribution (volume basis) was taken as the average particle size.

[0048] (average diameter of the circumscribed circle of the metal core particle) The average diameter of the circumscribed circle of the metal core particles is measured by observing them in a scanning electron microscope (SEM) image. In an SEM image taken at a magnification that allows the particle size of each metal core particle to be distinguished, a circumscribed circle is drawn for each particle (e.g., 30 particles) corresponding to the metal core particle, and the diameters are simply averaged to determine the average diameter of the circumscribed circle. The "circumscribed circle of the metal core particle" refers to a circle that is fitted to the image so as to encompass one of the metal core particles being measured, and the diameter is reduced to a critical value at which the outer shell of the metal core particle protrudes. Furthermore, metal core particles can be easily identified by using elemental mapping images obtained by energy dispersive X-ray spectroscopy (EDS).

[0049] (d90 based on the number rate of circumscribed circles of metal core particles) Based on the information on the number of particles corresponding to each diameter of the circumscribing circle of the metal core particle, an integrated distribution curve was created based on the number rate, and the particle size value at 90% of the integrated value was taken as d90 based on the number rate of the circumscribing circle. Images of the metal core particle can be taken using either a scanning electron microscope, a transmission electron microscope, or the like.

[0050] (X-ray diffraction pattern) X-ray diffraction (XRD) patterns in X-ray powder diffraction measurements of iron-based magnetic metal particles (the entire aggregate of multiple particles) were measured using an X-ray diffractometer "X'Pert PRO MPD" (PANalytical) under the following conditions: radiation source CuKα, tube voltage 45 kV, tube current 40 mA, sampling interval 0.02° / step, divergence slit 0.5°, scattering slit 0.5°, receiving slit 0.15 mm, and scanning range 15°≦2θ≦80°.

[0051] (Saturation magnetization) The magnetization when 0.8 MA / m was applied to the entire assembly of multiple particles was measured as saturation magnetization using a vibrating sample magnetometer (VSM).

[0052] (mass ratio of coating layer) The mass ratio of the coating layer is calculated by determining the Fe mass ratio of the metal core particle from the value of saturation magnetization, and the remainder is calculated as the oxide mass ratio of the coating layer. That is, the ratio of the above saturation magnetization to the saturation magnetization of pure iron (α-Fe) is calculated, and this is regarded as the Fe mass ratio. The remainder is then taken as the mass ratio of the coating layer. Note that the saturation magnetization of pure iron (α-Fe) is 218 Am 2 / kg (Source: Scientific Chronology, National Astronomical Observatory, 2000, Vol. 73, Maruzen Co., Ltd.).

[0053] (Fe elution amount) An immersion experiment was performed by adding 25 mg of iron-based magnetic metal particles to 1 mL of a 6 M guanidine hydrochloride aqueous solution (a dissolution and adsorption solution in TOYOBO's MagExtractor® -Genome-), mixing uniformly using a vortex mixer, and then leaving the mixture to stand at 25°C for 24 hours. After the immersion experiment, the sample powder was immobilized using a permanent magnet, and the supernatant was collected by pipetting. The concentration of iron ions dissolved in this supernatant was analyzed by ICP.

[0054] The measurement results are described below. First, the X-ray diffraction patterns of the iron-based magnetic metal particles 1 of Examples 1 and 2 obtained after heat treatment are shown in Figures 3 and 4, respectively. α-Fe, TiO2, and TiC were identified, indicating that the iron oxide in the starting material was reduced by titanium, producing titanium oxide as a by-product. The X-ray diffraction pattern of Comparative Example 1 is shown in Figure 5. As in Example 1, α-Fe, TiO2, and TiC were identified.

[0055] Next, the average particle size of the iron-based magnetic metal particles 1 after the magnetic separation operation was measured using a laser diffraction particle size distribution analyzer with pure water as a solvent. The results are shown in Table 1. The average particle size of the iron-based magnetic metal particles 1 in Example 1 was 0.7 μm, the average particle size in Example 2 was 0.96 μm, and the average particle size in Comparative Example 1 was 0.2 μm.

[0056] Next, the particle structure of Example 1 was observed using a transmission electron microscope. The results are shown in Figures 6(a) and 6(b). As can be seen from Figure 6(a), the surface of the metal core particle 2 was coated with TiO2 (titania), which constitutes the coating layer 3. Furthermore, Figure 6(b) shows a mapping image of iron elements obtained by EDS. As described above, to determine the average particle size of the metal core particles, this mapping image was used to apply a circumscribed circle to each of the multiple metal core particles 2, measure the diameters of 30 particles, and calculate the average value as the average particle size. As a result, the average particle size was 0.31 µm. Furthermore, the number of metal core particles 2 was determined for each measured circumscribed circle diameter, and the number was added up starting with the smallest circumscribed circle diameter. The diameter of the circumscribed circle corresponding to 90% of the total was calculated as d90. The relationship between the cumulative number rate and particle size at this time is shown in Figure 7. In this example, we were unable to observe particles with a particle size corresponding to a cumulative total of 90% of the number rate. However, the particle size at d90 was determined using the graph shown in Figure 7. For convenience, we connected the two particle size values ​​closest to the cumulative total of 90% of the observed particle size values ​​with a straight line, and the value at which the cumulative total was 90% on the graph in Figure 7 was taken as the particle size at d90. The particle size at d90 determined in this way was in the range of 0.4 μm to 0.5 μm. The results are shown in Table 1.

[0057] Similarly to Example 1, the particle structure of Example 2 was also observed using a transmission electron microscope. The results are shown in Figures 8(a) and 8(b). Similar to Figure 6(a), Figure 8(a) confirms that the surface of the metal core particle 2 is coated with TiO2, which is the coating layer 3. Furthermore, Figure 8(b) shows a mapping image of iron elements obtained by EDS. As described above, to determine the average particle size of the metal core particles, this mapping image was used to fit a circumscribed circle to multiple metal core particles 2, and the diameters of 30 particles were measured and the average value was calculated as the average particle size. As a result, the average particle size was 0.38 µm. Furthermore, d90 was calculated similarly to Example 1. The relationship between the cumulative number rate and particle size at this time is shown in Figure 9. The particle size at d90 was in the range of 0.6 µm to 0.7 µm. The results are shown in Table 1.

[0058] Similarly to Example 1, the particle structure of Comparative Example 1 was also observed using a transmission electron microscope. The results are shown in Figures 10(a) and 10(b). Similar to Figures 6(a) and 8(a), Figure 10(a) confirms that the surface of the metal core particle 2 is coated with TiO2, which constitutes the coating layer 3. Furthermore, Figure 10(b) shows a mapping image of iron elements obtained by EDS. As described above, to determine the average particle size of the metal core particles, this mapping image was used to fit a circumscribed circle to multiple metal core particles 2, and the diameters of 30 particles were measured and the average value was calculated as the average particle size. As a result, the average particle size was 0.48 μm. Similarly to Example 1, the d90 particle size was also determined for Comparative Example 1. The relationship between the cumulative number rate and particle size is shown in Figure 11. The particle size at d90 was in the range of 0.8 μm to 0.9 μm. The results are shown in Table 1.

[0059] 6(a), 8(a), and 10(a), it can be seen that when the metal core particles are coarse, the coating with TiO2 (titania)3 tends to be non-uniform. Furthermore, the mass ratio of TiO2, which constitutes the coating layer, is between 20% and 55%, and when this coating state is evaluated in terms of the amount of eluted iron ions, the amount of eluted iron ions is significantly less in Examples 1 and 2, which have fine metal core particles, than in Comparative Example 1. In other words, when the metal core particles are coarse, the coating with titanium oxide becomes non-uniform, resulting in poor corrosion resistance, whereas when fine metal core particles are obtained, the coating becomes uniform and corrosion resistance improves.

[0060] Furthermore, the saturation magnetization of the iron-based magnetic metal particles 1 of Examples 1 and 2 and Comparative Example 1 was measured, and the amount of Fe dissolved was also measured by an immersion experiment. The results are shown in Table 1. The saturation magnetization of the iron oxide was about 80 Am 2 / kg, it can be seen that Examples 1 and 2 and Comparative Example 1, which use pure iron for the metal core particles, have improved magnetic collecting power compared to conventional magnetic beads.

[0061] Furthermore, the results of the immersion experiment showed that the amount of eluted Fe was greater in Comparative Example 1 than in Examples 1 and 2. It is believed that Comparative Example 1, in which the metal core particles were coarse, had an uneven titanium oxide coating, which prevented sufficient corrosion resistance from being ensured, resulting in the greater amount of eluted Fe. Therefore, it was found that in Examples 1 and 2, in which the particle size was appropriately controlled, a uniform titanium oxide coating was formed, ensuring sufficient corrosion resistance.

[0062] [Table 1]

[0063] The following was found from Examples 1 and 2 and Comparative Example 1. It can be seen from Figures 6, 8, and 10 that all iron oxide powders were reduced by titanium, producing titanium oxide (TiO2: titania) as a by-product. Furthermore, Table 1 shows that the average particle size of the iron-based magnetic metal particles was coarse at 2.0 μm in Comparative Example 1, whereas it was 0.70 μm in Example 1 and 0.96 μm in Example 2, both of which were within the range of 0.1 to 1 μm. The average diameter of the circumscribed circle of the metal core particles was within the range of 0.05 to 0.4 μm in Examples 1 and 2, but was coarse at 0.48 μm in the Comparative Example. Furthermore, Table 1 shows that d90 was 0.4 to 0.5 μm in Example 1 and 0.6 to 0.7 μm in Example 2, whereas it was coarse at 0.8 to 0.9 μm in Comparative Example 1. From the above results, it can be seen that by setting the average particle size of the iron-based magnetic metal particles to 0.1 to 1 μm, the average particle size and d90 of the metal core particles can be kept fine.

[0064] [Physical properties of silica-coated iron-based magnetic metal particles] (Examples 3 and 4) Next, an example relating to silica-coated iron-based magnetic metal particles (hereinafter referred to as silica magnetic beads) will be described. In order to make the iron-based magnetic metal particles of Example 2 into magnetic beads, silica coating was carried out under the following coating conditions.

[0065] (Covering conditions) 5 g of the iron-based magnetic metal particles of Example 2 obtained above were dispersed in 100 ml of ethanol solvent, to which 3.0 g of tetraethoxysilane (TEOS) and 0.15 g of aluminum isopropoxide (AIP) were added. While stirring the solvent, a mixed solution (67.6 g of purified water, 13.7 g of 28% ammonia water, and 0.09 g of potassium chloride (KCl)) was added dropwise over 5 minutes. Then, the mixture was stirred for 1 hour while hydrolyzing the TEOS and AIP. After the reaction was completed, the mixture was washed three times with isopropyl alcohol (IPA). The mixture was then filtered to separate the solid and liquid, and dried by heating to 30°C or higher in the air. The iron-based magnetic metal particles 1 were coated with silica to obtain silica magnetic beads. The silica magnetic beads obtained in this manner are designated Example 3. In addition, the same procedure as in Example 3 was used, but the amounts of each chemical were changed to 2.3 g of tetraethoxysilane (TEOS), 0.11 g of aluminum isopropoxide (AIP), 50.7 g of purified water, 10.3 g of 28% ammonia water, and 0.07 g of potassium chloride (KCl). The resulting silica magnetic beads are designated as Example 4.

[0066] (Powder characteristics of silica magnetic beads) The powder properties of the silica magnetic beads of Examples 3 and 4 were measured. The powder properties of the silica magnetic beads of Examples 3 and 4 were the average particle size d50, which is the particle size value at 50% of the cumulative value in the cumulative distribution curve obtained from the particle size distribution (volume basis) measured by laser diffraction, the specific surface area measured by the BET method, the zeta (ζ) potential in a 0.01 M KCl aqueous solution, and the saturation magnetization (applied magnetic field 0.8 MA / m) measured by a vibrating sample magnetometer (VSM). The results are shown in Table 2.

[0067] [Table 2]

[0068] Next, the DNA extraction performance from whole blood was evaluated for the silica magnetic beads of Example 3. A commercially available nucleic acid extraction kit ("MagExtractor (registered trademark) - Genome-" manufactured by TOYOBO) was used for DNA extraction.

[0069] (DNA extraction procedure) (1) First, 100 μL of bovine whole blood was dispensed into a 1.5 mL microtube, and 750 μL of the lysis / adsorption solution and 150 μL of silica magnetic bead solution included in the kit were added. The mixture was then stirred for 10 minutes in a tube mixer to allow the DNA to adsorb to the silica magnetic beads.

[0070] (2) Next, the silica magnetic beads were collected by magnetic separation and the supernatant was removed, followed by adding 900 μL of the washing solution provided with the kit, stirring for 5 seconds with a vortex mixer, and then magnetically separating the beads again to remove the supernatant. The same washing step was then performed once more. (3) The same washing step as above was repeated twice using 900 μL of 70% ethanol. After that, 100 μL of sterile water was added to the collected silica magnetic beads and stirred for 10 minutes to elute the DNA from the silica magnetic beads. The supernatant containing the DNA was then recovered through magnetic separation, and the DNA was extracted.

[0071] (Example of DNA extraction) For Example 3, DNA was extracted using two types of magnetic bead solutions a and b according to the above extraction procedure. Magnetic bead solution a was a silica magnetic bead solution in which 7.5 mg of silica magnetic beads were dispersed in 150 μL of Tris-EDTA buffer (TE buffer), and magnetic bead solution b was a silica magnetic bead solution in which 15.0 mg of silica magnetic beads were dispersed in 150 μL of Tris-EDTA buffer (TE buffer). DNA extraction using the silica magnetic beads included in the above nucleic acid extraction kit was also performed as a reference example.

[0072] The amount of DNA extracted using each silica magnetic bead was determined by measuring the DNA concentration in 100 μL of sterilized water using a spectrophotometer (NanoDrop2000, Nucleic Acid mode). The results are shown in Table 3. Extraction was performed three times for each silica magnetic bead, and the average values ​​are shown in Table 3. The amount of DNA extracted is expressed as the concentration in 100 μL of sterilized water.

[0073] [Table 3]

[0074] As described above, we prepared silica magnetic beads with different silica coating layer formation conditions and different magnetic bead solutions, and evaluated their DNA extraction capabilities. As a result, Example 3 demonstrated sufficient extraction capability, exceeding 60% of that of the Reference Example (commercially available silica magnetic beads (TOYOBO beads)), demonstrating their effectiveness as silica magnetic beads. This is due, first, to the use of metallic Fe particles as the magnetic core, which increased the saturation magnetization of the silica magnetic beads (see Table 3), enabling reliable magnetic capture of the silica magnetic beads during DNA extraction due to their excellent magnetic field response. Second, by using metallic Fe particles in submicron sizes, we achieved silica magnetic beads of 1-2 μm in size (see Table 3), which increased the specific surface area and thus the reaction area with DNA. Therefore, we found that the magnetic beads of the present invention are suitable for achieving both high corrosion resistance, which prevents Fe elution, and improved magnetic collecting force, while maintaining the ability to capture biological materials.

[0075] Next, the DNA extraction performance of the silica magnetic beads of Examples 3 and 4 was evaluated using another commercially available nucleic acid extraction kit (MagMAX (registered trademark) DNA Multi-Sample Ultra 2.0 Kit, manufactured by Thermo Fisher Scientific Inc.). The DNA solution was prepared by using DNA extracted and purified from whole blood, and adjusting the concentration to 0.5 μg / μL in 100 μL of sterile water. Meanwhile, silica magnetic bead solutions were prepared as follows. Both the magnetic bead solution using the silica magnetic beads of Example 3 (hereinafter referred to as the magnetic bead solution of Example 3) and the magnetic bead solution using the silica magnetic beads of Example 4 (hereinafter referred to as the magnetic bead solution of Example 4) were silica magnetic bead solutions prepared by dispersing 7.5 mg of silica magnetic beads in 40 μL of Tris-EDTA buffer (TE buffer). In addition, as Reference Example 2, a silica magnetic bead solution included in a nucleic acid extraction kit was also prepared, and the DNA capture ability of each of the three types of magnetic bead solutions, namely the magnetic bead solution using the silica magnetic beads of Example 3, the magnetic bead solution using the silica magnetic beads of Example 4, and the silica magnetic bead solution included in the kit, was compared and evaluated.

[0076] The DNA extraction procedure was as follows: (1) First, 100 μL of purified DNA solution (equivalent to 50 μg of purified DNA) adjusted to a concentration of 0.5 μg / μL was dispensed into three 1.5 mL microtubes. Next, 10 μL of Enhancer Solution and 10 μL of Proteinase K, both included in the kit, were added to the purified DNA solution in each microtube. Three microtubes containing purified DNA solution, each containing 10 μL of Enhancer Solution and 10 μL of Proteinase K, were prepared in this way. Each was mixed for at least 30 seconds and then allowed to stand at 65°C for 20 minutes.

[0077] (2) Next, 400 μL of the binding solution included in the nucleic acid extraction kit was added to 40 μL each of the magnetic bead solutions of Examples 3 and 4 and the silica magnetic bead solution of Reference Example 2, and the mixture was suspended by pipetting. 440 μL each of the suspended magnetic bead solutions of Examples 3 and 4 and the silica magnetic bead solution of Reference Example 2 was added to a microtube containing the stationary purified DNA solution and suspended by pipetting, and the entire amount of each silica magnetic bead solution was mixed with the purified DNA solution. The mixture was then stirred for 5 minutes, allowing the DNA in the microtube to be adsorbed onto the silica magnetic beads. (3) Next, the silica magnetic beads in each microtube were collected by magnetic separation and the supernatant was removed. After that, 1 mL of Wash Solution I provided with the nucleic acid extraction kit was added and stirred for 1 minute, followed by magnetic separation again to remove the supernatant. The same washing step was then performed once more.

[0078] (4) Next, 1 mL of Wash Solution II included with the nucleic acid extraction kit was added, and the same washing procedure as in (3) was repeated twice. After that, 100 μL of Elution Solution included with the diffusion extraction kit was added to the captured silica magnetic beads, and the beads were left to stand at 70°C for 10 minutes with stirring to elute the DNA from the silica magnetic beads. The supernatant containing the DNA was then recovered through magnetic separation, and DNA was extracted from each of the three microtubes.

[0079] The amount of DNA extracted using the silica magnetic beads of Example 3, Example 4, and Reference Example 2 was measured using a spectrophotometer. The DNA concentration in 100 μL of sterilized water was measured using a spectrophotometer (NanoDrop2000, Nucleic Acid mode) to determine the amount of DNA captured by the silica magnetic beads and eluted into the sterilized water. The above steps (1) to (3) were repeated three times, and the average amount of DNA per 100 μL of sterilized water was evaluated based on the DNA concentration obtained. The results of the evaluation of the amount of DNA extracted using the silica magnetic beads of Example 3, Example 4, and Reference Example 2 were all comparable.

[0080] Next, the recovery speed of the silica magnetic beads of Examples 3, 4, and Reference Example 2 using a magnet was evaluated. The recovery speed was determined as follows. A square spectroscopic cell was filled with 2 ml of water and 1 mg of magnetic beads to produce a magnetic bead solution. The square spectroscopic cell containing the magnetic bead solution was then exposed to ultrasonic waves in an ultrasonic bath for 2 minutes and stirred with a vortex mixer. A magnet with a surface magnetic flux density of 309 mT was then placed 6 mm from the inner wall of the square spectroscopic cell, and the absorbance of the magnetic bead solution was measured using a spectrophotometer (V-770, JASCO Corporation). Measurements were continued for 600 seconds until the absorbance reached saturation. The initial absorbance was A0, the absorbance after 600 seconds was A10, and the absorbance after t seconds was At. The magnetic recovery amount after t seconds was evaluated as (A0 - At) / (A0 - A10) × 100 (%). The results are shown in Figure 12. The horizontal axis of Figure 12 represents the elapsed time from the start of magnetic separation, and the vertical axis represents the magnetic recovery amount (%). Furthermore, since almost no change in absorbance was observed after 300 seconds, FIG. 12 shows the period up to 300 seconds. 12, it can be seen that the silica magnetic beads of Examples 3 and 4 have a faster magnetic recovery rate than the silica magnetic beads of Reference Example 2. From this, it can be said that Examples 4 and 5 have the same level of nucleic acid extraction ability as Comparative Example 2, but can perform extraction work with higher time efficiency. [Explanation of symbols]

[0081] 1: Iron-based magnetic metal particles 2: Metal core particles 3: Coating layer (TiO2 (titania))

Claims

1. Iron-based magnetic metal particles for magnetic beads, characterized in that the iron-based magnetic metal particles are metal core particles whose main component is iron and are coated with a coating layer made of an oxide whose Gibbs standard free energy of formation per 1 mol of oxygen is smaller than that of iron oxide, the iron-based magnetic metal particles have an average particle size of 0.1 μm or more and 1 μm or less, a d90 based on the number rate of the circumscribed circle of the metal core particles is 0.7 μm or less, and the mass ratio of the coating layer to the total mass of the metal core particles and the coating layer is 20% or more and 55% or less.

2. 2. The iron-based magnetic metal particles for magnetic beads according to claim 1, wherein the coating layer is an oxide of Ti, Al or Zr.

3. 2. The iron-based magnetic metal particles for magnetic beads according to claim 1, wherein the coating layer is made of titania.

4. 2. The iron-based magnetic metal particle for magnetic beads according to claim 1, wherein the average diameter of the circumscribed circle of the metal core particle is 0.05 μm or more and 0.4 μm or less.

5. Saturation magnetization is 100 Am ２ 2. The iron-based magnetic metal particles for magnetic beads according to claim 1, wherein the iron-based magnetic metal particles have a molecular weight of 1 / kg or more.

6. The iron-based magnetic metal particles for magnetic beads according to claim 1, characterized in that when 25 mg of the iron-based magnetic metal particles are added per mL of a 6 M aqueous solution of guanidine hydrochloride and immersed at 25°C for 24 hours, the amount of iron ions eluted is 17 mg / L or less.

7. 7. Magnetic beads comprising iron-based magnetic metal particles according to claim 1 coated with silica.

Images