magnetic beads

Abstract

Provided is a magnetic bead that can shorten the time for recovery processing and reduce the amount of use. The magnetic bead (1) has a magnetic metal powder (2) and a coating layer (10) that covers the surface of the magnetic metal powder (2), the magnetic metal powder (2) has a magnetic property of 100 to 200 emu / g and a particle size distribution D50 of 1.0 to 25 μm in terms of volume, and the coating layer (10) has a first layer (11) that is the outermost layer and a porous second layer (12) formed between the first layer (11) and the magnetic metal powder (2), the first layer (11) has a film thickness that is thinner than the film thickness of the second layer (12). The coating layer (10) further has a third layer (13) between the second layer (12) and the magnetic metal powder (2).

Description

Technical Field

[0001] This invention relates to a magnetic bead. Background Technology

[0002] In order to recover biological substances such as proteins, nucleic acids, and cells from solutions, magnetic separation treatment using magnetic beads (magnetic microparticles) was carried out.

[0003] The surface of the magnetic beads is covered with a coating that specifically adsorbs the target substance (target) to be recovered (see Patent Document 1).

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2022-154413 Summary of the Invention

[0007] In magnetic beads, reducing the size is an effective way to improve the adsorption efficiency of the target. However, the smaller the size of the magnetic beads, the weaker their magnetization. When the size is below 1 μm, it becomes difficult to overcome the viscous resistance of the liquid and be attracted by the permanent magnet. As a result, the recovery and processing of the target takes a long time. In addition, sometimes the amount of magnetic beads used is increased. However, when the amount of magnetic beads is increased, the amount of liquid carried over (the amount of liquid carried into the next process) increases, which may lead to a decrease in cleaning efficiency or purity. Therefore, it is necessary to control the amount of magnetic beads used.

[0008] The purpose of this invention is to provide a magnetic bead that can shorten the recycling process and reduce the amount of material used.

[0009] The magnetic beads involved in this disclosure are as follows: having magnetic metal powder and a covering layer covering the surface of the magnetic metal powder, wherein the magnetic metal powder has a magnetic property of 100~200 emu / g and a particle size distribution D50 of 1.0~25 μm in volumetric terms, and the covering layer has a first layer as the outermost layer and a porous second layer formed between the first layer and the magnetic metal powder, wherein the thickness of the first layer is thinner than the thickness of the second layer. Attached Figure Description

[0010] Figure 1 This is a cross-sectional view showing a magnetic bead according to a first embodiment of the present invention.

[0011] Figure 2 This is a cross-sectional view showing the cover layer of the described embodiment.

[0012] Figure 3 This is a cross-sectional view showing the cover layer according to a second embodiment of the present invention.

[0013] Explanation of reference numerals in the attached figures

[0014] 1, 1A: Magnetic beads; 2: Magnetic metal powder; 10, 10A: Covering layer; 11: First layer; 12: Second layer; 13: Third layer; 14: Pores. Detailed Implementation

[0015] [First Implementation]

[0016] Figure 1 and Figure 2 A first embodiment based on the present invention is shown.

[0017] Figure 1 In this embodiment, the magnetic bead 1 has magnetic metal powder 2 and a covering layer 10 covering the surface of the magnetic metal powder 2.

[0018] Magnetic metal powder 2 is Fe alloy and other magnetic metal particles used for the magnetic recovery of target substances (targets) such as proteins, nucleic acids, and cells. The magnetic properties are 100~200 emu / g, and the particle size distribution D50 based on volume is 1.0~25μm.

[0019] Furthermore, in the state where the magnetic bead 1 is formed by forming the capping layer 10, the specific surface area of ​​the magnetic metal powder 2 based on nitrogen adsorption is 2~45m². 2 / g, with an average pore size of 2~25nm based on nitrogen adsorption.

[0020] exist Figure 2 In the process, the capping layer 10 has a first layer 11 as the outermost layer and a porous second layer 12 formed between the first layer 11 and the magnetic metal powder 2. The capping layer 10 also has a third layer 13 formed between the second layer 12 and the magnetic metal powder 2.

[0021] The first layer 11 is formed of silicon dioxide as the first oxide material, and its surface has functional groups for increasing the binding affinity with the target.

[0022] The functional groups in the first layer 11 are selected according to the target substance, and can be exemplified by: OH group, COOH group, NH2 group, epoxy group, trimethylsilyl group, NHS group, etc.

[0023] Examples of target biomolecules include: streptavidin, proteins such as protein A and protein G, and antibodies. When nucleic acids are the target, preferred substances include: nucleic acids with properties complementary to the target nucleic acid, specifically oligonucleotide (dT) primers and cDNA. For isolating cells and exosomes, examples include: antibodies against CD3, CD4, CD8, CD9, CD63, and CD81.

[0024] The second layer 12 is formed from a second oxide material different from the first oxide material of the first layer 11, namely, any one of alumina, titanium dioxide, or apatite, and is a porous material with multiple pores 14. Among them, the alkoxides used for forming the alumina or titanium dioxide film in the second layer 12 react faster with water and protic solvents than the raw materials used for forming the silica film in the first layer 11, and the resulting film structure is more prone to pore formation.

[0025] The thickness of the first layer 11 is formed to be sufficiently thin compared to the thickness of the second layer 12. The first layer 11 is formed to the surface inside the pores 14, thereby significantly increasing the surface area of ​​the first layer 11 compared to when the second layer 12 is non-porous.

[0026] The third layer 13 is formed of a material different from the second layer 12 (a second oxide material, namely alumina, titanium dioxide or apatite), namely silicon dioxide, and has the function of suppressing the dissolution of Fe ions from the magnetic metal powder 2 in the water in which the target is dispersed.

[0027] When forming the capping layer 10, firstly, a third layer 13 is coated on the surface of the magnetic metal powder 2, and then a second layer 12 having pores 14 is formed on the surface of the third layer 13. Furthermore, a first layer 11 is coated on the surface of the second layer 12 in such a way that it covers the inner surface of the pores 14. The coating process and the like can utilize existing techniques.

[0028] [Effects of this implementation method]

[0029] The magnetic bead 1 of this embodiment has magnetic metal powder 2 and a cover layer 10 covering the surface of the magnetic metal powder 2. The magnetic properties of the magnetic metal powder 2 are 100~200 emu / g, and the particle size distribution D50 in volumetric terms is 1.0~25 μm. The cover layer 10 has a first layer 11 as the outermost layer and a porous second layer 12 formed between the first layer 11 and the magnetic metal powder 2. The film thickness of the first layer 11 is thinner than that of the second layer 12.

[0030] In this configuration, by making the magnetic metal powder 2 have a magnetic property of 100~200 emu / g and a particle size distribution D50 of 1.0~25 μm by volume, magnetic beads 1 with both high saturation magnetization and high specific surface area can be formed. Therefore, by increasing the saturation magnetization of magnetic beads 1 and simultaneously making them micron-sized, it is possible to rapidly recover magnetic particles attached to a target and to recover the target in a short time.

[0031] Furthermore, by having the cover layer 10 have a first layer 11 as the outermost layer and a porous second layer 12 formed between the first layer 11 and the magnetic metal powder 2, the total surface area of ​​the first layer 11, including the porous inner surface, can be increased, and the target recovery amount can be increased.

[0032] In particular, since the thickness of the first layer 11 is thinner than that of the second layer 12, when the first layer 11 is formed on the outside of the second layer 12, the pores 14 of the second layer 12 will not be blocked or filled by the material of the first layer 11, allowing the first layer 11 to be formed to the inner surface of the pores 14 of the second layer 12. In other words, the pores 14 of the second layer 12 have a depth that will not be filled even when the first layer 11 is formed on the second layer 12. That is, the thickness of the first layer 11 is less than the depth of the pores 14 of the second layer 12. As a result, the total surface area of ​​the first layer 11 utilizing the porous second layer 12 can be maximized.

[0033] In the magnetic bead 1 of this embodiment, the first layer 11 is formed of a first oxide material (silicon dioxide), and the second layer 12 is formed of a second oxide material (alumina, titanium dioxide, or apatite) that is different from the first oxide material.

[0034] In this configuration, by using oxide materials for both the first layer 11 and the second layer 12, stable manufacturing can be achieved without degradation of properties, even after processing through high-temperature and high-pressure processes such as sterilization. Furthermore, the adsorption of the target is ensured in the first layer 11, while a porous structure is formed in the second layer 12.

[0035] In the magnetic bead 1 of this embodiment, the specific surface area based on nitrogen adsorption is 2~45m². 2 / g, with an average pore size of 2~25nm based on nitrogen adsorption.

[0036] In this configuration, the sample size per unit particle of magnetic bead 1 can be increased.

[0037] In the magnetic bead 1 of this embodiment, the first oxide material of the first layer 11 is silicon dioxide.

[0038] In this configuration, by using silica, which has a high adsorption capacity for the target, as the first layer 11, efficient recycling can be achieved.

[0039] In the magnetic bead 1 of this embodiment, the second oxide material of the second layer 12 is any one of alumina, titanium dioxide or apatite.

[0040] This configuration promotes the porosity of the second layer 12 and improves the nucleic acid yield.

[0041] In the magnetic bead 1 of this embodiment, the cover layer 10 has a third layer 13 between the second layer 12 and the magnetic metal powder 2. The third layer 13 is formed of a material (silicon dioxide) that is different from the second oxide material (alumina, titanium dioxide or apatite) of the second layer 12.

[0042] In this configuration, the third layer 13 can suppress the dissolution of Fe ions from magnetic metal powder 2 in an aqueous solution containing the target.

[0043] In the magnetic bead 1 of this embodiment, the third layer 13 is silicon dioxide coated on the surface of the magnetic metal powder 2.

[0044] In this configuration, by using silicon dioxide as the third layer 13, magnetic metal powder 2 can be uniformly coated onto the surface.

[0045] [Second Implementation]

[0046] Figure 3 A second embodiment based on the present invention is shown.

[0047] Similar to the magnetic bead 1 of the first embodiment described above, the magnetic bead 1A of this embodiment has magnetic metal powder 2 and a covering layer 10A covering the surface of the magnetic metal powder 2. In this embodiment, the magnetic metal powder 2 is the same as that in the first embodiment described above.

[0048] The cover layer 10A in this embodiment has the same first layer 11 and second layer 12 as in the first embodiment described above. The second layer 12 is formed directly on the surface of the magnetic metal powder 2, and the third layer 13 in the first embodiment is omitted.

[0049] In this embodiment of the magnetic bead 1A, the effect of the third layer 13 of the magnetic bead 1 of the first embodiment (which can suppress the dissolution of Fe ions from the magnetic metal powder 2 in the aqueous solution containing the target) cannot be obtained, but other than that, the same effect as the magnetic bead 1 of the first embodiment can be obtained.

[0050] Furthermore, since the magnetic bead 1A in this embodiment does not have the third layer 13 as in the magnetic bead 1 of the first embodiment, a film-forming process can be omitted accordingly, thereby simplifying the manufacturing process compared to the magnetic bead 1.

[0051] [Experimental Example]

[0052] Specific tests were conducted on the magnetic bead 1 of the first embodiment and the magnetic bead 1A of the second embodiment described above.

[0053] [Table 1]

[0054] In Table 1, Examples 1 and 10 are magnetic beads 1 with a first layer 11 to a third layer 13 (three-layer structure), Examples 2 to 9 and Comparative Examples 1 to 5 are magnetic beads 1A without a third layer 13 (double-layer structure), and Comparative Example 6 is a magnetic bead with a cover layer 10 having only a first layer 11.

[0055] [Manufacturing of the magnetic bead 1 in the first embodiment]

[0056] The magnetic bead 1 is formed by sequentially forming a third layer 13, a second layer 12, and a first layer 11 on the surface of the magnetic metal powder 2.

[0057] As the material for magnetic metal powder 2, in Examples 1-6, 9, 10 and Comparative Examples 2-6, Fe amorphous particles (Fe...) were used. 73 Si 11 Cr2B 11 C3), in Example 7, Fe was used 87 Cr 2.6 Si 9.8 In Example 8, Fe was used 73 Si 10 B 15 C2. In Comparative Example 1, carbonyl iron (Fe100) was used as magnetic metal powder 2. Except for Comparative Examples 2 and 3, the particle size of magnetic metal powder 2 was set in the range of 1.0~25 μm.

[0058] The formation of the third layer 13 is as follows.

[0059] First, Fe amorphous particles, serving as magnetic metal powder 2, were suspended in an ethanol solvent and stirred for 1 hour while being subjected to ultrasound. Then, NH3 (actually an aqueous NH3 solution) and purified water were added as a catalyst. Next, TEOS (tetraethoxysilane) was slowly added dropwise over a prolonged period, and the silica coating reaction was carried out through the hydrolysis and condensation reaction of TEOS. After film formation, magnetic separation and solvent replacement (ethanol) were repeatedly performed to recover the particles. The recovered particles were dried on a hot plate at 100°C for 1 hour. After confirming solvent evaporation, the particles were calcined at 200°C for 2 hours.

[0060] The formation of the second layer 12 can be carried out as follows.

[0061] Magnetic metal powder 2, with a third layer 13 formed, was suspended in ethanol solvent and stirred for 1 hour while being subjected to ultrasound. Then, AIP (aluminum isopropoxide) and purified water were added, and the reaction was allowed to proceed for 4 hours. After film formation, magnetic separation and solvent replacement (ethanol) were repeatedly performed to recover the particles. The recovered particles were dried on a hot plate at 100°C for 1 hour. After confirming solvent evaporation, the particles were then calcined at 200°C for 2 hours.

[0062] The formation of the first layer 11 can be carried out as follows.

[0063] Magnetic metal powder 2, with a second layer 12 and a third layer 13, was suspended in an ethanol solvent and stirred for 1 hour while being subjected to ultrasound. Then, an aqueous solution of NH3 and purified water were added as a catalyst, followed by the slow, dropwise addition of TEOS over a longer period. The silica coating reaction proceeded through the hydrolysis and condensation reactions of TEOS. After film formation, repeated magnetic separation and solvent replacement (ethanol) were performed to recover the particles. The recovered particles were dried on a hot plate at 100°C for 1 hour. After confirming solvent evaporation, they were calcined at 200°C for 2 hours. The calcined particles were then suspended in purified water and heated at 70°C for 3 hours.

[0064] [Manufacturing of the magnetic bead 1A according to the second embodiment]

[0065] The magnetic bead 1A is formed by sequentially forming a second layer 12 and a first layer 11 on the surface of the same magnetic metal powder 2 as the magnetic bead 1 in the first embodiment.

[0066] The formation process of the second layer 12 and the first layer 11 is the same as that of the magnetic bead 1 in the first embodiment. The formation of the second layer 12 can be carried out directly on the Fe amorphous particles (magnetic metal powder 2), which will not be described in detail.

[0067] [Determination of Nucleic Acid Yield]

[0068] Human genomic DNA was prepared as a nucleic acid model, and lysozyme was prepared as an impurity model. Next, the magnetic beads from Examples 1-10 and Comparative Examples 1-6 shown in Table 1 were dispersed in purified water and stirred to prepare magnetic bead suspensions. The magnetic bead content in the suspensions was 53.17% by mass. Then, after the reagents other than nucleic acids were brought to room temperature, the reagents were added to the tubes in the following order.

[0069] 65μL of purified water

[0070] 20 μL of nucleic acid dispersion with a concentration of 0.1 μg / μL

[0071] Dissolve adsorption solution 750 μL

[0072] 15 μL of lysozyme aqueous solution with a concentration of 10 μg / μL

[0073] 10 μL of magnetic bead suspension

[0074] Next, the contents of the tube were stirred with an eddy current mixer for 10 minutes, then the tube was placed on a magnetic rack and left for 30 seconds. After magnetic capture of the beads, the supernatant was removed.

[0075] Next, 900 μL of cleaning solution was added to the tube, stirred with a vortex mixer for 5 seconds, and then centrifuged. The tube was then placed on a magnetic rack and left for 30 seconds. After magnetic bead capture, the supernatant was removed. Then, the process of adding cleaning solution, magnetic capture, and supernatant removal was repeated.

[0076] Next, 900 μL of 70% ethanol aqueous solution was added to the tube, stirred with a vortex mixer for 5 seconds, and then centrifuged. Then, the contents of the tube were aspirated and returned using a pipette. The tube was then placed on a magnetic rack and left for 30 seconds. After magnetic bead capture, the supernatant was removed. Then, the process of adding ethanol aqueous solution, magnetic capture, and supernatant removal was repeated. Finally, the tube was centrifuged, and the remaining supernatant was removed.

[0077] Next, 100 μL of purified water was added to the tube, and the mixture was stirred with a vortex mixer for 1 minute to dissolve the nucleic acid. Then, the tube was placed on a magnetic rack and left for 30 seconds. After magnetic beads were captured, the dissolution solution containing the nucleic acid was recycled to another tube.

[0078] Next, the recovered dissolution solution was placed in a spectrophotometer, and the concentration of nucleic acid in the dissolution solution was quantified based on the absorbance at a wavelength of 260 nm. Then, the amount of nucleic acid recovered was calculated from the obtained concentration, and the ratio of the amount of nucleic acid recovered to the amount of nucleic acid input was calculated as the nucleic acid yield.

[0079] It should be noted that since nucleic acid bases have maximum absorption around 260 nm, the absorbance at a wavelength of 260 nm is used when quantifying nucleic acid concentration.

[0080] The nucleic acid yield calculated above is determined as follows.

[0081] ◎: Nucleic acid yield is over 80%; ○: Nucleic acid yield is above 60% but below 80%; △: Nucleic acid yield is above 40% but below 60%; ×: Nucleic acid yield is less than 40%.

[0082] [Determination of magnetic separation time]

[0083] Magnetic separation time is determined by measuring the change in absorbance when the magnet is brought close together. Add 2.5 mL of PBS and 5 μL of magnetic bead suspension to a cuvette. After capping and inverting the cuvette, place it in a spectrophotometer (Hitachi High-Tech U-3900H), set the wavelength to 550 nm, and begin absorbance measurement. The time required for the absorbance to reach 10% of its initial value (a 90% reduction) after the permanent magnet is brought into close contact with the side of the cuvette is measured and taken as the magnetic separation time.

[0084] The determination of the magnetic separation time measured above is as follows.

[0085] ◎: Magnetic separation time is less than 10 seconds; 〇: Magnetic separation time exceeds 10 seconds but is less than 60 seconds; △: Magnetic separation time exceeds 60 seconds but is less than 300 seconds; ×: Magnetic separation time exceeds 300 seconds.

[0086] [Evaluation Results]

[0087] In Examples 1-10 of the present invention, good results were obtained in which both nucleic acid yield and magnetic separation time were ○ or ◎.

[0088] In contrast, in Comparative Examples 1-6 (not of this invention), only Δ or × results were obtained for nucleic acid yield or magnetic separation time, which was insufficient.

[0089] Comparative Example 1: Magnetic metal powder 2 is Fe100. In terms of magnetic properties, its saturation magnetization is as high as 220 emu / g, and magnetic separation is early, but the nucleic acid yield cannot be obtained to a sufficient value. This is likely because, since magnetic metal powder 2 does not contain Cr, it will corrode, dissolving Fe ions. These dissolved Fe ions strongly bind to nucleic acids, preventing the nucleic acids from dissolving from the magnetic beads, thus reducing the nucleic acid yield.

[0090] In contrast, in Examples 1-10, the magnetic properties of the magnetic metal powder 2 were in the range of 100-200 emu / g (see Examples 6 and 7), and the nucleic acid yield was sufficiently high.

[0091] Comparative Example 2: The magnetic properties are in the range of 100~200 emu / g, but the particle size is small, at 0.4 μm. Therefore, the magnetization of each particle is small, and the magnetic separation time is long.

[0092] In contrast, in Examples 1-10, the particle size of the magnetic metal powder 2 is in the range of 1.0-25 μm (see Examples 2 and 4), and the magnetic separation time is shortened.

[0093] Comparative Example 3: The magnetic properties are in the range of 100~200 emu / g, but the particle size is as high as 36 micrometers and the specific surface area is small. Therefore, the nucleic acid yield is reduced.

[0094] In contrast, in Examples 1-10, the particle size of the magnetic metal powder 2 is in the range of 1.0-25 μm, and the specific surface area is in the range of 2-45 m². 2 Within the range of / g (see Examples 5 and 6), the nucleic acid yield was obtained to a sufficient value.

[0095] Comparative Example 4: The porous alumina film forming the second layer 12 was as thin as 5 nm, with a pore size distribution reduced to 1 nm, and the specific surface area could not be increased, being as low as 0.45 μm. 2 / g, nucleic acid yield decreased.

[0096] In contrast, in Examples 1-10, the thickness of the second layer 12 is in the range of 30-800 μm, thereby resulting in a specific surface area of ​​2-45 m². 2 Within the range of / g (see Examples 5 and 6), the nucleic acid yield was obtained to a sufficient value.

[0097] Comparative Example 5: The porous alumina film forming the second layer 12 has a film thickness of up to 1000 nm, resulting in poor magnetic separation. Furthermore, the relatively thick second layer 12 leads to larger pores 14, making it impossible to detach the adsorbed nucleic acids and reducing the nucleic acid yield.

[0098] In contrast, in Examples 1-10, the thickness of the second layer 12 is in the range of 30-800 μm, thereby resulting in a specific surface area of ​​2-45 m². 2 Within the range of / g (see Examples 5 and 6), the nucleic acid yield was obtained to a sufficient value.

[0099] Comparative Example 6: Due to the absence of a porous second layer 12 and a specific surface area as small as 0.45 m², 2 / g, therefore the nucleic acid yield is low.

[0100] In contrast, in Examples 1-10, the specific surface area reaches 2-45 m² through the porous second layer 12. 2 Within the range of / g, the nucleic acid yield was obtained to a sufficient value.

[0101] [Summary of this disclosure]

[0102] The first embodiment of this disclosure relates to a magnetic bead that comprises magnetic metal powder and a covering layer covering the surface of the magnetic metal powder. The magnetic metal powder has a magnetic property of 100-200 emu / g and a particle size distribution D50 of 1.0-25 μm in volumetric terms. The covering layer has a first outermost layer and a porous second layer formed between the first layer and the magnetic metal powder. The thickness of the first layer is thinner than that of the second layer.

[0103] In this configuration, by making the magnetic properties of the magnetic metal powder 100~200 emu / g and the particle size distribution D50 by volume standard 1.0~25 μm, magnetic beads with both high saturation magnetization and high specific surface area can be prepared, enabling target recovery in a short time.

[0104] Furthermore, by having the capping layer have a first outermost layer and a porous second layer formed between the first layer and the magnetic metal powder, the total surface area of ​​the first layer, including the porous inner surface, can be increased, thereby increasing the target recovery amount.

[0105] In particular, since the first layer is thinner than the second layer, when the first layer is formed on the outside of the second layer, the pores of the second layer will not be blocked or filled by the material of the first layer, allowing the first layer to be formed to the inner surface of the pores of the second layer. This maximizes the total surface area of ​​the first layer utilizing the porous second layer.

[0106] In the magnetic beads disclosed herein, the first layer is formed of a first oxide material, and the second layer is formed of a second oxide material different from the first oxide material.

[0107] In this configuration, by using oxide materials for both the first and second layers, stable manufacturing can be achieved without performance degradation even after high-temperature and high-pressure treatments such as sterilization. Furthermore, the adsorption capacity of the target is ensured in the first layer, while a porous structure is formed in the second layer.

[0108] In the magnetic beads disclosed herein, the specific surface area based on nitrogen adsorption is preferably 2 to 45 m². 2 / g, with an average pore size of 2~25nm based on nitrogen adsorption.

[0109] This configuration allows for an increase in the amount of sample per unit particle.

[0110] In the magnetic beads disclosed herein, the first oxide material is preferably silicon dioxide.

[0111] In this configuration, efficient recycling can be achieved by using silica, which has a high adsorption capacity for the target.

[0112] In the magnetic beads disclosed herein, the second oxide material is preferably any one of alumina, titanium dioxide, or apatite.

[0113] This configuration promotes porosity and increases nucleic acid yield.

[0114] In the magnetic beads of this disclosure, preferably the covering layer has a third layer between the second layer and the magnetic metal powder, the third layer being formed of a material different from the second layer.

[0115] In this configuration, it is possible to suppress the dissolution of Fe ions from magnetic metal powder in an aqueous solution containing the target.

[0116] In the magnetic beads disclosed herein, the third layer is preferably silicon dioxide coated on the surface of the magnetic metal powder.

[0117] In this configuration, by using silicon dioxide as a third layer, magnetic metal powder can be uniformly coated onto the surface.

Claims

1. A magnetic bead comprising magnetic metal powder and a coating layer covering the surface of the magnetic metal powder. The magnetic metal powder has a magnetic property of 100~200 emu / g and a particle size distribution D50 of 1.0~25 μm by volume. The capping layer has a first outermost layer and a porous second layer formed between the first layer and the magnetic metal powder. The thickness of the first layer is thinner than that of the second layer.

2. The magnetic bead as described in claim 1, wherein, The first layer is formed of a first oxide material, and the second layer is formed of a second oxide material that is different from the first oxide material.

3. The magnetic bead as described in claim 2, wherein, The specific surface area based on nitrogen adsorption is 2~45m². 2 / g, with an average pore size of 2~25nm based on nitrogen adsorption.

4. The magnetic bead as described in claim 3, wherein, The first oxide material is silicon dioxide.

5. The magnetic bead as described in claim 4, wherein, The second oxide material is any one of alumina, titanium dioxide, or apatite.

6. The magnetic bead according to any one of claims 1 to 5, wherein, The covering layer has a third layer between the second layer and the magnetic metal powder, the third layer being formed of a different material than the second layer.

7. The magnetic bead as described in claim 6, wherein, The third layer is silicon dioxide coated on the surface of the magnetic metal powder.

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